JP2021019576A - Bacterial group - Google Patents

Abstract

To provide a method for producing fermented solution whose acidity is pH 3 or more and 4 or less, containing colloidal particles whose particle size does not exceed 50 nm and short chain fatty acid.SOLUTION: Provided is a fermentation apparatus including a plurality of sets of fermentation vessels 10 whose temperature is controlled in each multi-stage fermentation process, and a method for producing fermented solution by the multi-stage fermentation processes uses: soft water W which is a starter; original bacteria solution b comprising 7 types of fermentation bacteria, that is, accession numbers NITEp-02945 to NITEp-02951, deposited and registered in the NPMD including ascospore Clostridium genus bacteria to be controlled in a low-temperature state; and 3 types of fermentation media obtained by separately fermenting and generating a first medium m1 of dry soybeans which are natural materials, a second medium m2 which is a mixed medium comprising Ziziphus jujuba, lycium fruit and turmeric which are dry plants, and a third medium m3 which is a honey raw material.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for producing a fermented liquid containing short-chain fatty acids by fermenting a natural material. The fermented liquor produced by the present invention is a fermented liquor having an acidity of pH 3 or more and 4 or less, which contains colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids of butyric acid, propionic acid and lactic acid. The content of colloidal particles in the fermentation broth is 6.5% to 7.5%, and the mass ratio of butyric acid is 0.5 g to 0.6 g per 100 mL of the fermentation broth.

For example, there are numerous fermented foods on the market as daily foods, such as vinegar, miso, soy sauce, and brewed sake. In addition, there are many fermented foods in which fermented liquids of functional foods and fermented liquids are commercially available as undiluted solutions of functional foods. Conventionally, it has been various since prehistoric times to put raw materials such as crushed legumes such as fruits, vegetables and soybeans and nuts such as chestnuts and peaches into natural water and produce liquid fermented extracts by selected fermenting bacteria. It is a well-known method for producing processed foods that have been devised in the above, and it is a well-known processed food.

Regarding short-chain fatty acids such as butyric acid, propionic acid, and lactic acid according to the present invention, for example, according to Non-Patent Document 1, the short-chain fatty acid produced in the anaerobic fermentation process of plant fibers by intestinal bacteria is butyric acid, which is a butyric acid. When the action was investigated, it was confirmed that butyric acid promotes only the development of regulatory T cells, and the same document states that "when butyric acid starch feed was given to chronic enteritis model mice, regulatory T cells from transferred cells. The outbreak was promoted in the colon and improvement of intestinal inflammation was observed. "
Furthermore, in Non-Patent Document 2, two types of regulatory T cells (Treg), thymus-derived tTreg and peripherally induced pTreg, were confirmed, and pTreg is associated with RORγt, which is a master transcription factor of TH17 lymphocytes. It has been reported that butyric acid produced by cluster IV and XIVa of the order Clostridium is involved in the induction of pTreg, and that short-chain fatty acids, especially butyric acid, are important for the induction of pTreg in the large intestine.
Furthermore, Non-Patent Document 3 states, "Short-chain fatty acids are used as an energy source for hosts, and also play an indispensable role in maintaining energy homeostasis of hosts, such as suppressing weight gain, ingestion, improving glucose metabolism, and increasing insulin sensitivity. Recent studies have revealed that this is the case, "he said, pointing out the importance of ingesting plant fiber containing indigestible polysaccharides, which are sources of short-chain fatty acids. Various molecular mechanisms mediated by fatty acid receptors GPR41, GPR43, GPR109a and 01fr78, especially due to chain fatty acids, butyric acid, are introduced in detail.
In addition, Non-Patent Document 4 reports that Shigella and butyrate-producing bacteria (Clostridium butyricum MIYAIRI 588 strain) were mixed-cultured in an anaerobic environment, and suppression of the growth of Shigella was observed even with a small amount of bacteria. In recent years, it has been reported that the anti-inflammatory effect of butyric acid or butyrate-producing bacteria in vivo has been attracting attention.
In Non-Patent Document 5, of the short-chain fatty acids produced in the intestinal flora resident in the large intestine, butyric acid is an essential nutrient for physical condition and is not only consumed as energy by epithelial cells but also of butyric acid. It has been reported that metabolic disorders contribute to ulcerative colitis, and the physiological effects of short-chain fatty acids include promotion of absorption of minerals such as calcium, suppression of cholesterol synthesis, and suppression of the onset of colorectal cancer by butyric acid. The results are presented.

Regarding the conventional method for producing a fermented liquid, for example, in Patent Document 1, a raw material obtained by crushing natural products of legumes and hard fruits is put into a lactose solution, and at least two fermenters and a breeder for culturing microorganisms are used. , A fermentation product having immunological activity in which fresh microorganisms and fermented extracts are uniformly mixed and mixed with them, and a method for producing the same are described. Further, as a method for producing such an acidic substance, Patent Document 2 describes a multi-step fermentation step including nanofiltration, and Patent Document 3 describes a multi-step fermentation process in which natto bacteria are added to ashitaba leaves and soybean powder. The method for producing the fermented liquid produced by the process is described.

Looking at Patent Document 4, a liquid of a substance produced by lactic acid bacteria is described, in which the liquid forms a plurality of groups using 16 kinds of lactic acid bacteria and maintains a coexisting state of subculture for each group. It is further described that the lactic acid bacterium culture solution produced thereby is filtered, and the filtered lactic acid bacterium culture solution has an action of activating benign bacteria in the intestine.

Regarding the promotion of calcium metabolism to a living body, Patent Document 5 describes a physiological activator having calcium absorption promoting activity and antioxidant activity containing a peptide or a peptide mixture obtained by decomposing casein with a lactic acid bacterium-producing proteinase. , Patent Document 6 describes a dietary supplement for beverages, which is a fermented polymer substance containing folic acid and fermented by adding soybeans to purified water and having a calcium absorption promoting function.

Regarding fermented foods using soybeans as raw materials, Patent Documents 7 to 9 disclose fermented foods produced by a production method using soybeans as raw materials and bifidobacteria or lactic acid bacteria strains as starters.

Regarding the acidity of the fermented liquid to be produced, Patent Document 10 describes a method for producing a fermented soymilk whose acidity is adjusted to less than pH 4.5 and soymilk having an acidity of less than pH 4.5 and a viscosity of 5.9 mPa or more. Fermented beverages are described, and Patent Document 11 describes enzyme-containing health foods having an acidity of about pH 3.7 to 3.9 obtained by adding several kinds of fermented liquids to wild grass extract and fermenting them, and the health thereof. It describes how to make food.

"Intestinal bacterial metabolites that control the development of regulatory T cells" Morikei Hayakawa Biopharmacy Vol. 50 No. 8 2014 Pharmacia "Allergic diseases and intestinal flora" Naoki Shimojo Experimental Medicine Special Edition Vol. 37-No. 2 2019 "Gut flora" edited by Hiroshi Ohno Yodosha Published February 1, 2019 "Host Metabolism Control and Intestinal Bacterial flora" Ikuo Kimura Experimental Medicine Special Edition Vol. 37-No. 2 2019 "Gut flora" edited by Hiroshi Ohno Yodosha Published February 1, 2019 "Suppressive effect of butyrate-producing bacteria (Clostridium butyricum MIYAIRI 588 strain) on intestinal pathogens" Toyoaki Kuroiwa Kazumine Kobari Masaaki Iwanaga Infectious Diseases Journal Vol. 64, No. 3, March 20, 1990 "Physiological effects of short-chain fatty acids acidified in the large intestine from prebiotex" Hiroshi Hara (Graduate School of Agriculture, Hokkaido University) Journal of Gut Bacteriology 16: 35-42, 2002

Special Table 2013-524791 Japanese Unexamined Patent Publication No. 2016-0000039 Japanese Unexamined Patent Publication No. 2013-132290 Japanese Patent No. 4540376 Japanese Unexamined Patent Publication No. 5-304889 Special Table 2010-540623 Japanese Unexamined Patent Publication No. 2001-120180 Japanese Unexamined Patent Publication No. 2005-218390 Japanese Unexamined Patent Publication No. 2011-167190 Japanese Unexamined Patent Publication No. 2014-168441 Japanese Unexamined Patent Publication No. 2009-178084

The present invention is a method for producing a fermented liquid containing short-chain fatty acids by fermenting a natural material. More specifically, the present invention is a method for producing a fermentation broth having an acidity of pH 3 or more and pH 4 or less, which contains colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids.

The fermented liquor produced by the present invention contains colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids of butyric acid, propionic acid and lactic acid, and has an acidity of pH 3 or more and 4 or less. Incidentally, the content of colloidal particles in the fermentation broth is 6.5% to 7.5%, and the mass ratio of butyric acid is 0.5 g to 0.6 g per 100 mL of the fermentation broth.

As a functional food, the fermented liquor doubles lactic acid bacteria and bifidobacteria that promote intestinal regulation, while halving Clostridium perfringens, which is harmful to intestinal regulation, and further increases the rate of calcium absorption into the body and calcium metabolism. It has been confirmed that it promotes lactic acid and significantly affects the bone strength and metabolism of the living body. This is considered to be a result consistent with various molecular mechanisms of short-chain fatty acids introduced in detail in Non-Patent Document 3, especially via the fatty acid receptors GPR41, GPR43, GPR109a and 01fr78 by butyric acid.

The present invention has been completed through many years of steady practical research by the inventors, and contains colloidal particles that ferment natural materials and do not exceed 50 nm, and short-chain fatty acids of butyric acid, propionic acid, and lactic acid. A method for producing a fermentation broth having a pH of 3 or more and 4 or less is provided.

More specifically, it is also a fermented bacterium containing spore-forming Clostridium spp. (Independent Administrative Institution Product Evaluation Technology Base) that produces organic acids including short-chain fatty acids that are coexisted in advance and controlled in a stable low temperature state. Mechanism: Colloids and butyric acid that do not exceed 50 nm are produced by a multi-step fermentation process in which 7 types of fermenting bacteria with a deposit number NITEp-02945-NITEp-02951, which are registered as deposits at the Patented Microbial Deposit Center [NPMD], act coexistently. , Propionic acid, and lactic acid, which provide a method for producing a fermentation broth having an acidity of pH 3 or more and 4 or less.

The method of the present invention
Each of the multi-stage fermentation processes is equipped with a fermentation apparatus containing multiple sets of temperature-controlled fermentation vessels.
Soft water w as a starter and
The original bacterial solution b composed of seven fermenting bacteria of the accession number NITEp-02945-NITEp-02951 registered as a deposit with NPMD containing spore-forming Clostridium spp. Managed in a low temperature state.
Three types produced by separately fermenting the first medium pm1 of dried soybeans, which is a natural material, the second medium pm2, which is a mixed medium consisting of dried plants taiso, kukoshi, and turmeric, and the third medium pm3, which is a raw material for honey. Fermentation medium m1, m2, m3,
Using
By multi-step fermentation process
It is characterized by producing a fermentation broth 1 having an acidity of pH 3 or more and 4 or less, which contains colloidal particles not exceeding 50 nm and short-chain fatty acids.

The non-patent documents and patent documents are reminiscent of fermented liquids having an acidity of pH 3 or more and 4 or less, which contains colloidal particles having a particle size not exceeding 50 nm and three types of short-chain fatty acids of butyric acid, propionic acid, and lactic acid. The description cannot be found. Furthermore, there is also a description of a method for producing a fermentation broth having an acidity of pH 3 or more and 4 or less, which contains colloidal particles not exceeding 50 nm and three types of short-chain fatty acids of butyric acid, propionic acid and lactic acid by a multi-step fermentation step. It cannot be found. In addition, the technical elements described in these non-patent documents and patent documents contain colloidal particles not exceeding 50 nm and three types of short-chain fatty acids of butyric acid, propionic acid, and lactic acid, no matter how they are combined by those skilled in the art. It is also clear that there is no conceivable description of the method of the present invention for producing a fermented liquor having an acidity of pH 3 or more and 4 or less.

The first feature constituting the method according to one aspect of the present invention for producing a fermentation broth having an acidity of pH 3 or more and 4 or less, which contains colloidal particles and short-chain fatty acids not exceeding 50 nm, is each multi-step fermentation step. It is on the first fermentation line f1 of.

The first fermentation line f1
A plurality of (4 or more) pottery fermentation bottles 10, a first medium tank 20 provided with a heating pot 30, and two first fermentation tanks 100, as shown in FIGS. 1 [1] to [5]. First fermentation system s1 equipped with
One second fermentation tank 200 shown in FIGS. 1 [6] to [8], and an extraction device for the first fermentation tank 100 connected to the second fermentation tank 200 shown in FIG. 4 [b]. Using the second fermentation system s2 equipped with 110,
The first step p1 ₁ for producing the preliminary first fermented liquid pn1 and the fermented soybean fs shown in FIG. 2 (2), and
And FIG. 2 [b] ~ [c] and the second step _{p 1} 2 the acidity to produce a first fermented liquid n1 of pH 5.3 ± indicated in Stage 1 of FIG. 16,
And Figure 1 [8], and the third step _{p 1} 3 for generating a pH5.0 second fermentation liquor n2 of ± shown in stage 1 of FIG. 16,
Consists of.

The pH value ± is a symbol indicating that the fluctuation range of the acidity pH value, which indicates the average value of the annual lots of each fermentation broth in the fermentation process shown in FIG. 16, is within 0.3, and the same applies hereinafter. is there.

More specifically
The first fermentation line f1 whose entire process is shown in FIGS. 1 [1] to [8] is
In each of the plurality (4 or more) ceramic fermentation bottles 10 shown in FIG. 2 (1), soft water w having an acidity of pH 7.3 ± as a starter and dried soybean ds as a raw material of the first medium pm1 When,
Using a part of the original bacterial solution b that promotes fermentation of the dried soybean ds that was immersed in the soft water w of the fermentation bottle 10 shown in step pp _{10 of} FIG. 2 and returned with water, and the sugar chain s.
By fermentation for 3 days (68 hours to 74 hours) while maintaining the fermentation state of 37 ° C to 40 ° C shown in stage 1 of FIG.
It includes a first step p1 ₁ for producing a preliminary first fermented liquid pn1 having a pH of 4.5 ± and fermented soybean fs shown in FIG. 2 (2).

The first step p ₁ 1 of the first fermentation line f1 shown in steps pp _{1 1 to} pp ₁ 3 of FIGS. 1 [3] to [4] and FIG. 2 is further described.
Fermented soybean fs is ground by a crushing means (not shown) to generate soybean paste gfs, which is transferred to a plurality of (4 or more) containers 11 shown in FIG. 2 (3) corresponding to each fermentation bottle 10 and prepared. The step of mixing with the first fermentation liquid pn1 and transferring from each container 11 to the heating pot 30 and
Transferred to Figure 2 [a] Step pp ₁ 4 first medium tank 20 in advance soft water w is introduced for the soybean paste gfs was heated to 55 to 60 ° C. cooling the heating pot 30 shown in the, pH 4.5 ± Includes a first preliminary step of producing the first fermentation medium m1 of the above.

Step 2 _{p 1} 2 of the first fermentation line f1 shown in Figure 1 [5],
A step of evenly transferring the first fermentation medium m1 shown in FIGS. 2 (4) and [b] to two first fermentation tanks 100, and further adding soft water w and the original bacterial solution b to each first fermentation tank 100. When,
The first fermentation medium m1, soft water w, and the original bacterial solution b charged into each of the first fermentation tanks 100 shown in step pp ₁₅ of FIG. 2 [c] were stirred and mixed to have a pH of 6.4 ±. It includes a second preliminary step of producing a preliminary second fermentation broth pn2.
Incidentally, the preliminary second fermentation liquid pn2 having a pH of 6.4 ± produced in the second preliminary step is a turbid liquid pn2 in which the first fermentation medium m1, soft water w, and the original bacterial solution b are mixed, and has a pH of 50 nm. It corresponds to the fermentation stock solution θ for producing a fermentation solution having an acidity of pH 3 or more and 4 or less, which contains colloidal particles and short-chain fatty acids that do not exceed the limit. The reason why the turbid liquid pn2 becomes the fermentation stock solution θ is that soft water w and the original bacterial solution b are not newly added in the subsequent multi-step fermentation steps.

Step 2 _{p 1} 2 of the first fermentation line f1 shown in Figure 1 [6] In addition, the
Preliminary second fermentation liquor pn2 of pH6.4 generated in the first fermentation tank 100 shown in Step pp ₁ 6 of Figure 2 [d],
FIG. 16 By fermentation for 50 to 100 days while maintaining the fermentation state of 37 ° C to 40 ° C shown in stage 1.
The third preliminary step for producing the first fermentation broth n1 having a pH of 5.3 ± is included.

In each first fermentation tank 100 of the first fermentation line f1 shown in FIG. 1 [6],
As shown in FIG. 4 [a] and the details will be described later.
A sponge layer sp due to fermentation gas is formed in the supernatant, and a first precipitation layer dep1 containing the fiber of the first fermentation medium m1 is deposited in the lower layer, and pH 5 is formed between the sponge layer sp and the first precipitation layer dep1. .3 ± 1st fermentation broth n1 is formed.

The first fermentation line f1 as shown in FIG. 1 [8] may include a third step _{p 1} 3. Third Step _{p 1} 3, depending 4 second step _{p 1} 2 pH 5.3 only the first fermentation liquid n1 of ± generated in, draw-off device 110 of the first fermentation tank 100 shown in [b] 2 It is taken out from the first fermentation tank 100 of the group, transferred to one second fermentation tank 200 in which a fermentation environment is formed without using a fermentation medium, stirred and mixed, and while maintaining the fermentation state of 37 ° C to 40 ° C. , FIG. 16 is a step of producing a second fermented liquid n2 having a pH of 5.0 ± by fermenting the original bacterial liquid b contained in the first fermented liquid n1 shown in stage 1 with the fermenting bacteria b1 for 3 to 5 days. ..

The second feature that constitutes a method according to one aspect of the present invention for producing a fermentation broth containing colloidal particles not exceeding 50 nm and short-chain fatty acids and having an acidity of pH 3 or more and 4 or less is that of each multi-step fermentation step. It is on the second fermentation line f2.

The second fermentation line f2
As shown in FIGS. 1 [9] to [12] and FIG.
With the second medium tank 40 shown in FIG. 1 [9] or FIG. 7 [a],
A third fermentation tank 300 equipped with a bag-shaped medium filter 320 and a circulation pump device 330 installed so as to be suspended inside, which is shown in FIG. 1 [11] or details shown in FIGS. 8 and 9.
The extraction device 210 of the second fermentation tank 200 connected to the third fermentation tank 300 shown in FIG. 1 [12] (not shown by the device having the same principle as the extraction device 110 of FIG. 4 [b]).
Using the third fermentation system s3 consisting of
The first step p ₂ 1 of producing the second fermentation medium m2 having a pH of 4.8 ± and
It is composed of a second step p ₂ 2 for producing a third fermentation broth n3 having a pH of 4.5 ±.

More specifically, the first step p ₂ 1 for producing the second fermentation medium m2 having a pH of 4.8 ± on the second fermentation line f2 shown in FIGS. 1 [9] and [10] is as follows. 1 Preliminary step pp ₂ 1 and 2nd preliminary step pp ₂ 2 are included.

The first preliminary step pp ₂ 1 is
In the second medium tank 40 shown in FIG. 1 [9] or FIG. 7 [a],
Either the second fermented liquid n2 having a pH of 5.0 ± or the first fermented liquid n1 having a pH of 5.3 ± as a starter.
Add pm2, a mixed medium consisting of sterilized dried plants such as taiso, kukoshi, and turmeric.
The sterilized mixed medium pm2 is immersed in a part of either the second fermented liquid n2 or the first fermented liquid n1, mixed, stirred, and stirred.
FIG. 16 By fermentation for 2 to 3 days while maintaining the fermentation state of 37 ° C to 40 ° C shown in stage 2.
The preliminary fermentation mixed medium pfm2 and the preliminary third fermentation broth pn3 shown in FIG. 1 [10] or FIG. 7 [b] are produced.

In the first preliminary step pp 2 _1, when mixed immersed in a part of either the second fermentation liquor n2 or first fermentation liquid n1 charged with mixed media pm2 the second medium tank 40, preferably, mixed Each of the medium pm2, Taisou, Kukoshi, and Ukon, is used in a weight ratio of 7: 4: 1. For example, the total amount of the mixed medium pm2 per 35 liters of either the second fermented liquid n2 or the first fermented liquid n1. On the other hand, 200 to 210 g of dried culture medium, 110 to 120 g of dried kukoshi, and 25 to 30 g of dried corn are mixed at a weight ratio of 7: 4: 1.

The second preliminary step pp ₂ 2 is
Preliminary fermentation mixed medium pfm2 is taken out from the second medium tank 40 shown in FIG. 1 [10] or FIG. 7 [b], and the seeds of Taisou are crushed to the extent that they are not ground using a crushing means (not shown). A preliminary third fermentation liquid pn3 having a pH of 4.8 ± and a second fermentation medium m2 are produced.

More specifically, the second step p ₂ 2 for producing the third fermentation broth n3 having a pH of 4.5 ± on the second fermentation line f2 includes the following preliminary steps.

The second step p ₂ 2 is
Figure 1 [11] in the first step _{p 2} hung 1 second fermentation medium m2 generated by the bag-like medium filter 320 enclosed in a third fermentation tank 300 shown, pH 4.8 Preliminary third ± The process of transferring the fermentation medium pn3 to the third fermentation tank 300,
The second fermentation liquid n2 having a pH of 5.0 ± was taken from the second fermentation tank 200 to the third fermentation tank 300 by the extraction device 210 of the second fermentation tank 200 shown in FIG. 1 [8] connected to the third fermentation tank 300. And the process of moving to
The circulation pump device 330 shown in FIG. 9 is operated to prevent the second fermentation medium m2 from being mixed with the preliminary third fermentation liquid pn3 having a pH of 4.8 ± and the first fermentation liquid having a pH of 5.0 ± via the bag-shaped medium filter 320. 2 The process of integrally circulating the fermented liquid n2 and
Including
FIG. 16 A third fermentation broth n3 having a pH of 4.5 ± is produced by fermentation for 8 to 9 days while finally maintaining the fermentation state of 37 ° C. to 40 ° C. shown in stage 2.

The third feature that constitutes the method according to one aspect of the present invention for producing a fermentation broth containing colloidal particles not exceeding 50 nm and short-chain fatty acids and having an acidity of pH 3 or more and 4 or less is that of each multi-step fermentation step. It is on the third fermentation line f3.

The third fermentation line f3
As shown in FIGS. 1 [13] to [16] and FIGS. 12 [a] to [[g].
With the third medium tank 50 shown in FIGS. 1 [13] to [14] and FIG. 12 [b],
With the extraction device 310 of one fourth fermentation tank 400 shown in FIGS. 1 [15] and 12 [d] and the third fermentation tank 300 connected to the fourth fermentation tank 400 shown in FIG. 12 [e]. Using the 4th fermentation system s4 consisting of
The first step p ₃ 1 to generate the third fermentation medium m3 having a pH value of 4.4 ±, and
pH3.7 composed of the second step _{p 3} 2 for generating a fourth fermentation liquor n4 of ±.

More specifically, the first step p ₃ 1 for producing the third fermentation medium m3 having a pH value of 4.4 ± on the third fermentation line f3 includes the following preliminary steps.

The first step p ₃ 1 is
In the third medium tank 50 shown in FIGS. 12 [a] to 12 [c],
A third fermented liquor having a pH of 4.5 ±, which is a starter corresponding to four times the amount of the added honey raw material pm3 by adding the honey raw material pm3 which is preferably composed of encyclopedia honey and acacia honey in a ratio of 1: 4. The process of adding a part of n3 and
A part of the honey raw material pm3 charged into the third medium tank 50 and a part of the third fermented liquid n3 corresponding to four times the amount of the honey raw material pm3 charged, and the third fermented liquid produced in the third fermentation tank 300. A step of adding and stirring so that the amount corresponds to 3 to 5% of n3 to produce a preliminary fermentation medium pfm3, and
Including
Finally, the preliminary fermentation medium pfm3 of step pp _{31 of} FIG. 12 shown in stage 2 of FIG. 16 was fermented for 2 to 3 days while maintaining the fermentation state of 37 ° C to 40 ° C.
A third fermentation medium m3 having a pH value of 4.4 ± is produced.

More specifically, the second step p ₃ 2 for producing the fourth fermentation broth n4 having a pH of 3.7 ± on the third fermentation line f3 includes the following preliminary steps.

The second step p ₃ 2 is
The step of transferring the third fermentation medium m3 shown in FIG. 12 [d] to the fourth fermentation tank 400, and
The third fermentation liquid n3 having a pH of 4.5 ± of the starter shown in FIGS. 12 [e] to [f] is extracted from the third fermentation tank 300 by the extraction device 310 of the third fermentation tank 300 connected to the fourth fermentation tank 400 from the third fermentation tank 300. The process of further transferring to the fourth fermentation tank 400 and stirring,
Including
By fermentation for 30-60 days, finally maintaining the fermentation state of 37 ° C.-40 ° C. shown in FIG. 12 [g] and FIG. 16 Stage 2.
A fourth fermentation broth n4 having a pH of 3.7 ± is produced.

The fourth characteristic of the method constituting the method according to one aspect of the present invention for producing a fermentation broth containing colloidal particles not exceeding 50 nm and short-chain fatty acids and having an acidity of pH 3 or more and 4 or less is that of each multi-step fermentation step. It is on the fourth fermentation line f4.

The fourth fermentation line f4
The extraction device 410 (of FIG. 12 [e]) of the fourth fermentation tank 400 connected to the one fifth fermentation tank 500 shown in FIG. 1 [17] and the fifth fermentation tank 500 shown in FIG. 12 [g]. Using the fifth fermentation system s5 consisting of the extraction device 310 and the device of the same principle (not shown),
Step of producing the fourth fermented liquid n4 having a pH of 3.7 ± shown in FIG. 12 [g] into the fifth fermented liquid n5 having a pH of 3.6 ± p ₄ 1
Consists of.

Step _{p 4} 1 to generate a fifth fermentation liquid n5 of pH 3.6 ± fourth fermentation lines f4, more specifically, includes the following preliminary steps.

Step p ₄ 1 is
As shown in FIGS. 1 [16] to [17].
With the extraction device 410 of the fourth fermentation tank 400 (not shown by the device of the same principle as the extraction device 310 of FIG. 12) in which the fourth fermentation liquid n4 having a pH of 3.7 ± as a starter is connected to the fifth fermentation tank 500. The process of transferring from the 4th fermentation tank 400 to the 5th fermentation tank 500,
A step of forming a fermentation environment without using a fermentation medium by stirring the fourth fermentation broth n4 having a pH of 3.7 ± transferred to the fifth fermentation tank 500 multiple times.
Including
FIG. 16: By fermentation of the fourth fermented liquid n4 with the fermenting bacterium b1 for 30 to 60 days while finally maintaining the fermentation state at 37 ° C to 40 ° C shown in stage 2.
A fifth fermentation broth n5 having a pH of 3.6 ± is produced.

Fifth of the features constituting the method according to one aspect of the present invention for producing a fermentation broth containing colloidal particles not exceeding 50 nm and short-chain fatty acids and having an acidity of pH 3 or more and 4 or less is each of the multi-step fermentation steps. It is on the fifth fermentation line f5.

The fifth fermentation line f5
As shown in FIGS. 1 [18]-[19] or 15 [a]-[c],
One sixth fermentation tank 600 shown in FIG. 1 [19] or FIG. 15 [c], and a cooling device 610 shown in FIG. 1 [18] or FIG. 15 [b].
Using the sixth fermentation system s6 including the spare tank 620 shown in FIG. 15 [a] and the extraction device 510 of the fifth fermentation tank 500 connected to the spare tank 620.
The first step p ₅ 1 for rapidly cooling the fifth fermentation liquid n5 having a pH of 3.6 ± at 35 to 40 ° C. shown in FIG. 1 [18] or FIG. 15 [b] to a liquid temperature of 4 to 5 ° C. or lower.
The rapidly cooled fifth fermentation broth n5 having a pH of 3.6 ± is returned to a normal temperature state, and the sixth fermentation having an acidity of 3.3 ± while maintaining the normal temperature state shown in FIG. 1 [19] or FIG. 15 [c]. composed of the second step _{p 5} 2 to generate a liquid n6.

More specifically, the first step p ₅ 1 for rapidly cooling the fifth fermentation liquid n5 of the fifth fermentation line f5 at a pH of 3.6 ± at 35 to 40 ° C. to a liquid temperature of 4 to 5 ° C. or lower is prepared as follows. Including the process.

The first step p ₅ 1 is
A fifth fermentation broth n5 having a liquid temperature of 35 to 40 ° C. and a pH of 3.6 ± is supplied from the fifth fermentation tank 500 to a spare tank 620 to which the extraction device 510 of the fifth fermentation tank 500 shown in FIG. 15 [a] is connected. The process of transferring and
Including the step of immersing the spare tank 620 shown in FIG. 15 [b] in the cooling water of the cooling device 610.
Finally, the fifth fermentation liquid n5 at 35 to 40 ° C. is rapidly cooled to a liquid temperature of 4 to 5 ° C. or lower.

15 Second Step _{p 5} 2 to generate a sixth fermentation liquor n6 retention while pH 3.3 ± normal temperature state of the fifth fermentation line f5 of pH 3.6 ± shown in [c] are more specifically Includes the following preliminary steps.

The second step _{p 5} 2 is,
A step of taking out the spare tank 620 shown in FIG. 15 [c] from the cooling device 610, returning the rapidly cooled fifth fermentation liquid n5 to a room temperature state, and then transferring it to the sixth fermentation tank 600.
The process of forming a fermentation environment that does not use a fermentation medium,
Including
Finally, by fermenting the 5th fermented liquid n5 with a pH of 3.6 ± for 180 to 240 days by the fermenting bacterium b1 of the 5th fermented liquid n5 while maintaining the normal temperature state.
A sixth fermentation broth n6 having a pH of 3.3 ± is produced.

As described above, the present invention comprises colloidal particles not exceeding 50 nm, which are generally composed of the first fermentation line f1 to the fifth fermentation line f5, and three types of short-chain fatty acids of butyric acid, propionic acid and lactic acid. This is a method for producing a fermented liquor having an acidity of pH 3 or more and 4 or less.

The amount of soft water w used as a starter is estimated at each stage assuming that the amount of the two first fermentation tanks 100 charged in FIG. 1 [5], that is, the preliminary second fermentation liquid pn2 is 1800 L. The amount of fermented liquid produced is roughly as follows.

When four pottery fermentation bottles 10 are used, 20 L of soft water w for immersing 6.5 kg of dried soybeans in each fermentation bottle 10 shown in FIGS. 2 (1) to 2 (2) is used. The amount of soft water w for four bottles is 80 L. Next, the fermented soybean fs in each fermentation bottle 10 was crushed into a paste while adding about 5 L of soft water w, and the finished soybean paste gfs was transferred to the four separate containers 11 shown in FIG. 2 (3). When mixed with the preliminary first fermentation broth pn1, about 10 L of soft water w is further added. The soybean paste gfs to which 10 L of soft water w corresponding to 10 L is added from each container 11 and the preliminary first fermentation liquid pn1 are transferred to the heating pot 30 and heated. The soybean paste gfs corresponding to each fermentation bottle 10 and the preliminary first fermentation liquid pn1 (gfs + pn1 + w) corresponding to 35 L of the heated soybean paste are transferred to the first medium tank 20.

In the heating pot 30, the soybean paste gfs equivalent to 35 L of each fermentation bottle 10 and the preliminary first fermentation liquid pn1 are stirred and heated while adding 10 L equivalent of soft water w. Next, the soybean paste gfs and the preliminary first fermentation broth pn1 are cooled to the fermentation temperature with 100 L of soft water w prepared in the first medium tank 20 for cooling. As a result, the soft water w required for the production of the first fermentation medium m1 is 180 L (80 L + 20 L + 40 L + 40 L). Since the soft water w used for cooling in the first medium tank is 100 L, the first fermentation medium m1 produced is equivalent to 240 L [35 L × 4 + 100 L].

In the two first fermentation tanks 100, soft water w is further added to 240 L of the first fermentation medium m1 and 270 L to 450 L of the original bacterial solution b, which serve as starters.

The amount of soft water w charged into the two first fermentation tanks 100 shown in FIG. 2 [c] is 111 to 1290 L, assuming that the amount charged in the first fermentation tank 100, that is, the preliminary second fermentation liquid pn2 is 1800 L. It becomes the usage amount. Therefore, the amount of soft water w required for the production of the first fermentation broth n1 is 1290 (1110 + 180) L to 1470 (1290 + 180) L. In the method of the present invention for producing a fermentation broth containing colloidal particles not exceeding 50 nm and short-chain fatty acids and having an acidity of 3 or more and 4 or less, there is no other step in which soft water w is used.

Each of the two first fermentation tanks 100 that have undergone 50 to 100 days of fermentation in FIG. 16 Stage 1 shown in FIG. 2 [d] or FIG. 4 [a] has an air blocking action formed by the fermentation gas. A translucent first intermediate layer liquid is formed between the sponge layer sp having the sponge layer sp, the first precipitation layer dep1 containing the fiber of the first fermentation medium m1, and these two layers. The translucent first intermediate layer liquid is the first fermentation liquid n1 having a pH of 5.3 ±.

Assuming that the amount charged for two units of the first fermentation tank 100, that is, the preliminary second fermentation liquid pn2 is 1800 L, 30% or more is absorbed by the sponge layer sp and the first sedimentation layer dep1, so that one unit The first fermentation broth n1 having a pH of 5.3 ± produced in the first fermentation tank 100 is equivalent to 600 to 650 L.

Therefore, the first fermentation liquid n1 having a pH of 5.3 ± is extracted from the two first fermentation tanks 100 and transferred to the second fermentation tank 200 by the extraction device 110 of the first fermentation tank 100 connected to the second fermentation tank 200. Is equivalent to 1200 to 1300 L. The first fermentation broth n1 having a pH of 5.3 ± is the first fermentation broth 1 of the fermentation broth 1 having an acidity of pH 3 or more and 4 or less, which contains colloidal particles and short-chain fatty acids not exceeding 50 nm produced by the present invention. ..

In the second fermentation tank 200, a fermentation environment that does not use a medium is formed, and the fermentation of the first fermentation broth n1 having a pH of 5.3 ± by the fermenting bacterium b1 contained in the first fermentation broth n1 causes a pH of 5.0 ±. The second fermented liquid n2 is produced. In the second fermentation tank 200, the first upper surface membrane layer sf1 formed in the supernatant by the bubbles of the fermentation gas by the fermentation of the first fermentation liquid n1 having a pH of 5.3 ± and the second precipitation layer dep2 deposited in the lower layer , A translucent second intermediate layer liquid is formed between these two layers.

The second intermediate layer liquid is a second fermentation liquid n2 having a pH of 5.0 ±. Since the second fermentation broth n2 is absorbed by the first upper surface membrane layer sf1 and the second precipitation layer dep2 by about 5% during fermentation, the second fermentation broth n2 produced is about 1140 to 1240 L.

The first fermentation line f1 ends at this stage. It corresponds to the stage 1 of FIG. 16, and the time required for fermentation during that period is 55 days or more and does not exceed 108 days.

In the second fermentation line f2 shown in FIGS. 1 [9] to [12], the second fermentation liquid n2 having a pH of about 1140 to 1240 L and a pH of 5.0 ± is a starter. A part of the second fermentation broth n2 is added to the second medium tank 40. The remaining second fermentation broth n2 is transferred to the third fermentation tank 300. Based on many years of steady practical experience of the inventors, the finished amount of the third fermented liquid n3 having a pH of 4.5 ± is 8% to 10% of that of the third fermented liquid n3 produced in the third fermentation tank 300. Since there is a reduction of about%, it is about 1050 to 1100 L.

In the third fermentation line f3 to the fifth fermentation line f5 shown in FIGS. 1 [13] to [19], the amount of loss due to vaporization or the like during fermentation of the fourth fermentation liquid n4 to the sixth fermentation liquid n6 is 2% to 3 The finished amount of the colloidal fermented liquid that does not exceed 50 nm of the sixth fermented liquid n6 having a pH of 3.3 ±, which is as small as%, is about 1000 to 50 L.

The amount of fermentation broth produced by each stage of fermentation is estimated as follows. In the two first fermentation tanks 100, the soft water w equivalent to 1290 to 1470 L of the starter and the first fermentation liquid n1 having a pH of 5.3 ± produced by using the first fermentation medium m1 in an amount equivalent to 1800 L are as follows. It is used as a starter of the above, and about 1140 to 1240 L of the second fermentation broth n2 having a pH of 5.0 ± is produced. Further, using the second fermentation liquid n2 and the second fermentation medium m2, about 1050 to 1100 L of the third fermentation liquid n3 having a pH of 4.5 ± is produced. Subsequent fermentation produces a fourth fermentation broth n4 with a pH of 3.7 ± using a third fermentation broth n3 of about 1050 to 1100 L and a third fermentation medium m3, but subsequent fermentation uses the fermentation medium. An unused fermentation environment is formed, and the generated fourth fermentation broth n4 is used to generate a fifth fermentation broth n5 having a pH of 3.6 ±, and finally the produced fifth fermentation broth n5 is used to produce pH 3. About 1000 to 150 L of 3 ± 6th fermentation broth n6 is produced.

It is a schematic diagram which shows the multi-step fermentation process including the 1st fermentation line to the 5th fermentation line for producing the colloidal fermentation liquid which does not exceed 50 nm of pH 3 or more and 4 or less. It is an enlarged schematic diagram which shows the 1st process which constitutes the 1st fermentation line, and the 2nd process which consists of 1st preliminary process, 2nd preliminary process and 3rd preliminary process. It is a perspective view and the enlarged schematic view of the lid mechanism which represent the fermentation bottle made of ceramics which the capacity which the lid having a valve function is installed in the central part does not exceed 60L. By opening and closing the on-off valve of the extraction device attached to the bottom of the first fermentation tank, Pascal's principle and the principle of hydrostatic equilibrium parallelism are applied, and only the first fermentation liquid of the generated first intermediate layer liquid is discharged from the first fermentation tank. It is a schematic diagram which shows the state which takes out and transfers to the 2nd fermentation tank. A first fermentation broth A produced by fermentation for 6 days using a sample A of a first fermentation medium obtained by grinding pre-fermented soybeans, and a fermentation medium obtained by grinding dried soybeans soaked in water without pre-fermentation. It is Photo A and Photo B of the comparative test with the first fermentation liquid B produced by the fermentation for 6 days in the same manner as the sample A using the sample B. It is a list of 7 kinds of fermenting bacteria including spore-forming Clostridium spp. That produce organic acids including short-chain fatty acids. The strains in the list were registered as deposits at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation, an independent administrative agency of the International Depositary Organization under the Budapest Convention. Deposit number: NITE p-02945-NITE p-02951 (Deposit date: May 16, 2019). It is a schematic diagram of the 2nd culture medium tank which arranged the drop lid which made a hole through which a fermentation gas passes. Schematic diagram of a bag-shaped medium filter installed so as to hang inside a third fermentation tank used in a second fermentation line that produces a third fermentation liquid using a second fermentation medium, and a third fermentation tank provided with a bag-shaped medium filter. It is a perspective view and a plan view of. It is a schematic diagram of the 3rd fermentation tank equipped with the circulation pump device used in the 2nd fermentation line which produces the 3rd fermentation liquid by the 2nd fermentation medium. By fermentation for 5 days using sample A of the second fermentation medium, which was pre-fermented using the second fermentation broth and ground and crushed with a weight ratio of 7: 4: 1 so that the seeds of taiso remained. A fermented medium obtained by grinding and crushing the produced third fermentation broth A and taisou, kukoshi, and corn with a weight ratio of 7: 4: 1 soaked in the second fermentation broth without pre-fermentation so that the seeds of taiso remain. It is Photo A and Photo B of the comparative test with the third fermentation liquid B produced by the same 5 days fermentation as the sample A using the sample B. It is a graph and the comparison table which show the transition of the acidity pH value of the 3rd fermentation liquid A and the 3rd fermentation liquid B for 5 days from the start of fermentation. With the second fermentation broth transferred to the third fermentation tank, the second fermentation medium produced in the second fermentation tank is charged into the bag-shaped medium filter installed inside the third fermentation tank, and the circulation pump device. Step [a], which represents a second fermentation line that produces a third fermentation broth by fermentation while circulating, and a part of the third fermentation broth and an appropriate amount of honey raw material are put into the third medium tank and stirred. Steps [b] and [c] of producing a third fermentation medium by fermentation while allowing the fermentation, and the third fermentation broth and the third fermentation medium transferred from the third fermentation tank to the fourth fermentation tank are charged and stirred. It is a partially enlarged view which consists of steps [d] to [g] which represent the 3rd fermentation line which produces the 4th fermentation liquid by fermentation while letting. Using the 4th fermented liquid A produced by fermentation for 4 days using the 3rd fermentation medium of the honey raw material pre-fermented with the 3rd fermented liquid, and the medium of the honey raw material not pre-fermented with the 3rd fermented liquid. It is Photo A and Photo B of the comparative test with the 4th fermentation liquid B produced by the fermentation for 4 days. It is a graph and the comparison table which show the transition of the pH value of the acidity of the 4th fermentation liquid A and the 4th fermentation liquid B for 4 days from the start of fermentation. The 5th fermentation broth is transferred from the 5th fermentation tank to the spare tank, rapidly cooled by a cooling device, the liquid temperature is controlled at room temperature according to the season, and the cooled 5th fermentation broth is transferred to the 6th fermentation tank. FIG. 5 is an enlarged schematic view of a fifth fermentation line in which a fermentation environment without using a fermentation medium is formed and a sixth fermentation broth is produced by fermentation of the fifth fermentation broth with fermenting bacteria. The transition between the fermentation time required from the first fermentation line to the fifth fermentation line and the pH value of the acidity of the first fermentation broth to the sixth fermentation broth and the first fermentation medium to the third fermentation medium is changed from stage 1 to stage 3. It is a figure and the table which expressed the change of the degree of fermentation by the pH value of the acidity. It is a graph and table which shows the result of the cumulan analysis which measures the size of the colloidal particles contained in the 4th fermented liquid after the sample heat treatment / sterilization treatment of the supernatant of the 4th fermented liquid as a sample. 6 is a graph and a table showing the results of a cumulan analysis in which the size of colloidal particles contained in the 6th fermentation broth is measured after heat treatment / sterilization treatment of the supernatant of the 6th fermentation broth as a sample. It is a table of the analysis result of the sugar mass contained in 100 g [mL] of the fermentation broth by the modified Somogie method of the Japan Food Research Laboratories. It is a graph of the analysis result of the amount of short-chain fatty acids contained in 100 g [mL] of the sixth fermentation liquid by high performance liquid chromatography of the Japan Food Research Laboratories. It is a bar graph showing the amount of calcium increase and the causium absorption rate based on the result of the "calcium absorption examination test by the inverted intestinal tract method" by administration of the test substance (sixth fermented liquid) using SD and male rats as model mice. Impact test based on "Gla / Glu-osteocalcin ratio" and "bone weight, bone strength, and bone density" data by administration of test substance (sixth fermented solution) using estrogen-deficient osteoporosis model mice by bilateral ovariectomy It is a graph and a bone density photograph based on the result of. Aitec Lab Co., Ltd. (Investigator Masaki Matsuura) orally administered 0.5 mL / sample to the test substance (6th fermented solution) administration group and control group of cc57BL / 6 mice (♂ 7 weeks old) for 28 days continuously. Later, the colon contents of the sample were collected, the bacterial DNA of the colon contents was extracted, and the test results were tested for the effect on the intestinal flora. It is a graph showing the expression level of the Krt6b gene in the tumor tissue of the test substance (sixth fermented liquid) administration group and the water injection group administration group mouse.

Aspects for carrying out the invention

The following is an embodiment of the present invention. One embodiment of the present invention provides a method for producing a fermentation broth having a pH of 3 or more and 4 or less, which contains colloidal particles and short-chain fatty acids not exceeding 50 nm by a multi-step fermentation step. The fermented liquor produced by the method of the present invention has a colloidal particle content of 6.5% to 7.5%, and the mass ratio of butyric acid is 0.5 g to 0. per 100 mL of the fermented liquor. It weighs 6 g and is itself a functional food or a functional food and its undiluted solution.

Details of the first fermentation line f1

Fermentation process of the multiple stages, and more specifically, starting from the first step p _{1 1} constituting the first fermentation line f1 shown in Figure 1 [1] of the conceptual diagram representing the method of the present invention.

Assuming that the amount charged for producing the first fermentation liquid n1 of the first fermentation line f1, that is, the preliminary second fermentation liquid pn2 is 1800 L, the amount charged as a starter for each step and the amount of finished product produced are approximate. It becomes as follows.

The first step p ₁ 1 of the first fermentation line f1 shown in FIGS. 2 (1) and 2 (2) is
In each of the four pottery fermentation bottles 10 provided with a lid 12 having a valve function 11 for letting the fermentation gas escape in the central portion having a capacity not exceeding 60 L shown in FIG.
Soak the dried soybean ds equivalent to 6.5 kg rehydrated in 20 L of soft water w having a pH of 7.3 ± with 0.25 kg of sugar chains s in an amount corresponding to the amount of each dried soybean ds equivalent to 6.5 kg. The first pretreatment step of adding 0.14 L, which is a part of the original bacterial solution b shown in FIG. 6, and
Each fermentation bottle 10 treated in the first pretreatment step is fermented for 3 days (68 to 74 hours) to generate 35 L of the first preliminary fermented liquid pn1 and fermented soybean fs in combination with each fermentation bottle 10. The second pretreatment process and
Includes a first preliminary step pp ₁₁ consisting of.

The first step p ₁ 1 of the first fermentation line f1 shown in FIG. 2 (3) further includes a second preliminary step pp ₁ 2. In the second preliminary step pp ₁ 2 the fermented soybean fs of the second pretreatment step constituting the first preliminary step pp ₁ 1 is taken out from each fermentation bottle 10 and pasted while adding a small amount of soft water w by a crushing means (not shown). The fermented soybean fs that has been ground into a shape and ground is transferred to the four containers 11 corresponding to the fermentation bottle 10, mixed with the first preliminary fermented liquid pn1 in the second pretreatment step, and combined for each container 11. A first preliminary fermentation broth pn1 equivalent to 35 L and soybean paste gfs are produced.

In the second step p ₁ 1 of the first fermentation line f1 shown in FIG. 2 (3), 35 L of the first preliminary fermentation liquid pn 1 and soybean paste gfs generated in each container 11 are heated from each container 11. Transfer to 30, gradually heat to 55-60 ° C., stir, and combine with the heating pot 30 to produce the first preliminary fermentation liquor pn1 equivalent to 180 L [(35 L + 10 L) × 4] and soybean paste gfs. 3 Preliminary step pp ₁ 3 is included.

Step 2 _{p 1} 2 of the first fermentation line f1 further comprises a fourth preliminary step _pp 1. 4 to 6 preliminary step pp ₁ 6 shown in FIG. 2 [a] ~ [d] .

The fourth preliminary step pp ₁ 4 of the second step p ₁ 2 is the first preliminary fermented liquid pn 1 equivalent to 180 L and soybeans produced in the heating pot 30 in the third preliminary step pp ₁ 3 of the first step p ₁ 1. The paste gfs and the paste gfs are transferred to a first medium tank 20 in which 100 L of cooling soft water w is charged in advance so as not to be exposed to the outside air, and stirred to generate a first fermentation medium m1 having a pH of 4.5 ± equivalent to 280 L. To do.

2 (4) in the second step _{p 1} 2 4 preliminary step pp ₁ 4 of further shown, the first fermentation tank 100 of the 2 groups, without being exposed to the outside air, pH 4 the first medium tank 20. Equally equalize 280 L of the first fermentation medium m1 of 5 ±, 270 L to 450 L of the original bacterial solution b in which the fermented bacterium b1 shown in FIG. 6 coexists and stabilizes in advance, and 1100 to 1300 L of soft water w. Sort and throw in. As shown in FIG. 2 [b], in each first fermentation tank 100, the first fermentation medium m1 and the original bacterial solution b are stirred together with the soft water W, and the preliminary second fermentation medium having a pH of 6.4 ± equivalent to 1800 L is combined. Fermentation liquid pn2 is produced.

Figure 2 [c] and fifth preliminary step pp ₁ 5 and sixth preliminary step pp ₁ 6 of the second step _{p 1} 2 shown in [d], the preliminary second pH 6.4 ± each fermentation tank 100 The fermentation broth pn2 is sealed so as not to come into contact with the outside air, and the fermentation is carried out for 50 to 100 days while maintaining the fermentation state at 37 ° C. to 40 ° C., as shown in FIG. 4A, with bubbles of fermentation gas. While forming the sponge layer sp in the supernatant, it prevents the dissolution of oxygen into the liquid, promotes the secretion and fermentation of acidic substances by the fermenting bacteria b1 of the original bacterial solution b, and the fibrous material of the first medium m1 and the fermenting bacteria b1. The paste-like first settling layer dep1 on which the syrup is deposited is formed in the lower layer, and a translucent first intermediate layer liquid is formed between the sponge layer sp and the first settling layer dep1. The translucent first intermediate layer liquid formed is the first fermentation liquid n1 having a pH of 5.3 ±.

In each fermentation tank 100, the sponge layer sp expanded by long-term fermentation and the paste-like first precipitation layer dep1 occupy equivalent to 300 L, so that the pH is 5.3, which is a translucent first intermediate layer liquid. The ± first fermented liquid n1 is equivalent to 600 L. Incidentally, the sponge layer sp of the supernatant corresponding to 300 L and the paste-like first precipitation layer dep1 serve as the basic materials of the original bacterial solution b.

The first fermentation line f1 can further comprise a third step _{p 1} 3. Third Step _{p 1} 3 of the first fermentation line f1 is 4 as shown in [b], pH 5.3 to ± a first intermediate layer solution of 600L corresponds generated in the first fermentation tank 100 The first fermentation broth n1 is transferred to one second fermentation tank 200.

Third Step _{p 1} 3 is
Using the first fermentation broth n1 having a pH of 5.3 ± equivalent to 1200 L transferred to one second fermentation tank 200 as the first starter,
By fermentation of the original bacterial solution b contained in the first fermented solution n1 with the fermenting bacteria b1 for 3 to 5 days,
A second fermentation broth n2 having a pH of 5.0 ± is produced.

Technical Issues for Generation of Second Fermented Liquid n2

Fermentation in the third step p _{1 3} is in an environment that does not use media or fermentation medium is characterized in that ferment first fermentation liquor n1 only by fermentative bacteria b1 of Motokin'eki b. Here, worth mentioning for the technical problem of the third step p _{1 3} of the first fermentation line f1, the first intermediate layer solution n1 which is separately generated by the first fermentation tank 100 of the 2 groups, Motokin'eki b The fermentation environment due to the coexistence of the fermenting bacteria b1 is different, and the original bacteria contained in the first fermentation liquid n1 are transferred to one second fermentation tank 200, mixed, and integrated. The coexistence antagonism of the fermenting bacterium b1 in the liquid b will be further stimulated. It is estimated from the fact that the first fermentation broth n1 having a pH of 5.3 ± is acidified to the second fermentation broth n2 having a pH of 5.0 ±.

Accordingly, multi-step fermentation process of the present invention, without passing through the third step p _{1 3} of the first fermentation line f1 shown in two routes 1 [6] or [8], the first fermentation line a second step _{p 1} 2 of f1, it is possible to directly proceed to the second fermentation line f2.

When proceeding to the second step p _{1 2} directly from the second fermentation line f2 fermentation line f1 is the 1 group of the first fermentation tank having a size of the second fermentation tank 200 (not shown) is used become. We years of practical experience, Force antagonism in the third step p _{1 3} without going through the fermentative bacteria b1 of the first fermentation line f1 stops in low, i.e. sufficient secretion and fermentation of acidic substances by fermenting bacteria b1 It was found that it is not easy to stably produce the first fermentation broth n1 having a pH of 5.3 ± equivalent to 1200 L in one first fermentation tank. Based on such findings, the inventors need further ingenuity to ferment the first fermentation broth n1 with a pH of 5.3 ± in advance until the second fermentation broth n2 with a pH of 5.0 ± is reached, and more fermentation days are required. problem becomes to solved the recall to challenge the idea to first fermentation line f1 incorporating a third step p _{1 3.}

In fact, the third step _{p 1} 3 of the first fermentation line f1 is, 37 ° C. to 40 ° C. while retaining the fermentation state of pH 5.3 ± first fermentation liquid n1 fermenters b1 only by 3 days to 5 days By fermentation, the bubbles of fermentation gas form the first upper surface layer sf1 of the supernatant, which further promotes the secretion and fermentation of the fermenting bacterium b1 while preventing the dissolution of oxygen into the liquid, and the residue of the first medium m1 is deposited. The pasty second precipitation layer dep2 is formed in the lower layer, and a translucent second intermediate layer liquid equivalent to 1140 to 1240L is formed between the first upper surface layer sf1 and the second precipitation layer dep2. The translucent second intermediate layer liquid is the second fermentation liquid n2 having a pH of 5.0 ±. Whether incorporate third step p _{1 3} to the first fermentation line f1, a matter of choice.

Technical features of the first fermentation line f1

The technical features of the first fermentation line f1 are that, unlike normal soybean fermentation, the first preliminary fermentation liquid pn1 obtained by fermenting dried soybean ds in a fermentation bottle 10, soybean paste gfs, soft water w, and original bacterial solution b A part of the first fermentation medium m1 is produced in advance, and the pre-fermented first fermentation medium m1 having a pH of 4.5 ± and soft water w are used to produce a first fermentation liquid n1 having a pH of 5.3 ±. That's what I did.

Usually, a fermentation product using soybean is produced in a state where soybean ds is immersed in a liquid such as a fermented product or water. However, the present inventors have decided to produce a first fermentation broth n1 having a pH of 5.3 ± by a fermentation step of producing a fermentation broth through a step of pre-fermenting the first fermentation medium m1 using dried soybeans. Focusing on it, the present invention was completed.

FIG. 5 shows the first fermentation broth A produced by fermentation for 6 days using the sample A of the first fermentation medium obtained by grinding the pre-fermented soybeans, and the dried soybeans soaked in water without being pre-fermented. Photograph A and the first fermentation broth B of the first fermentation broth A produced by a comparative test with the first fermentation broth B produced by fermentation for 6 days in the same manner as the sample A using the sample B of the crushed fermentation medium. Photo B.

Photo A shows a soybean paste obtained by grinding 60 g of pre-fermented soybeans assuming the production of a first fermented liquid n1 equivalent to 4 L, soft water w, and 0.8 L of the original bacterial solution b heated to 55 ° C. Later, the sample A cooled to 37 ° C. and stirred is the first fermentation broth A produced by fermentation for 6 days. As is clear from Photo A, in the produced first fermentation broth A, the supernatant sponge layer sp, the precipitate layer dep, and the highly transparent intermediate layer liquor n1 equivalent to 4 L between the two layers are clean. It is formed in three layers.

In Photo B, in order to compare with Photo A, 60 g of the same amount of soybean is immersed in water as it is for 18 hours, ground to prepare a soybean paste that is not pre-fermented, and the soybean paste and soft water w thus prepared. And 0.8 L of the original bacterial solution b are heated to 55 ° C., then cooled to 37 ° C., and the stirred sample B is a fermented solution B produced by fermentation for 6 days. As is clear from Photo B, the produced fermented liquid B is a soybean paste that is not pre-fermented, which is different from the conditions of sample A. As a result, a clear liquid having low transparency and a precipitation layer are formed. , The supernatant sponge layer sp required for the fermentation environment of the fermenting bacterium b1 is hardly formed.

The results of the comparative test shown in FIG. 5 reveal that there is a decisive difference in the fermentation environment between the fermented liquid A in which the dried soybeans in the first medium are pre-fermented and the fermented liquid B in which the dried soybeans are not pre-fermented. did. In this comparative test, more specifically, the technical matter that it is essential that the fermentation environment using the fermenting bacteria group b1 contained in the original bacterial solution b forms a supernatant sponge layer sp and blocks air. It will be clarified. In fact, it is presumed that the fermented liquid B, in which the supernatant sponge layer sp is not produced, has a fishy odor and the fermenting bacteria b1 is exposed to the outside air, so that the decrease in the number of bacteria and the progress of oxidation and putrefaction are the result of superior fermentation. To.

In order to realize the production of the first fermentation liquid n1 having a pH of 5.3 ± by the method of the present invention, a step of producing a first fermentation medium m1 for pre-fermenting the first medium, which forms the supernatant sponge layer sp, is indispensable. This is clear from the test results of Photo A and Photo B of FIG.

Details of the second fermentation line f2

The second fermentation line f2
The first step p ₂ 1 of preliminarily producing the second fermentation medium m2 having a pH of 4.8 ± in the second medium tank 40 shown in FIGS. 1 [9] and [10].
In the third fermentation tank 300 and the third fermentation system s3 shown in Figure 1 [11], a second fermentation of the second fermentation medium m2 and pH 5.0 ± of pH 4.8 ± produced in the first step _{p 2} 1 The second step p ₂ 2 for producing the third fermentation liquid n3 having a pH of 4.5 ± using the liquid n2 as the second starter.
Consists of.
Here, the starter means a raw material.

As a preliminary step, the first step p ₂ 1 of the second fermentation line f2 is closed by a sheet 42 provided with a drop lid 41 having a hole through which the fermentation gas passes as shown in the enlarged view 7 [a]. 2 Prepare the medium tank 40.

The first step p ₂ 1 is further added to the second medium tank 40.
With the pre-prepared mixed medium pm2 shown in FIG. 7 [a],
With either a part of the second fermentation broth n2 or a part of the second fermentation broth n2 in the fermentation process after about 1 to 2 days,
In total, add 35 L, stir, and stir.
By fermentation for 2 to 3 days while maintaining the fermentation state of 37 ° C to 40 ° C,
The preliminary fermentation mixed medium pfm2 and the preliminary third fermentation liquid pn3 shown in FIG. 7 [b] were produced.
By a process not shown
The generated preliminary fermentation mixed medium pfm2 was ground by a pulverizing means to the extent that the Tyson seeds were not crushed, and returned to the second medium tank 40.
A second fermentation medium m2 having a pH of 4.8 ± is produced by blending the ground preliminary fermentation mixed medium pfm2 with the preliminary third fermentation liquid pn3.

The mixed medium pm2 prepared in advance contains 200 to 210 g of taiso, 110 to 120 g of kukoshi, and 25 g of turmeric with respect to the total amount of dried plants composed of sterilized taiso, kukoshi, and turmeric, which is equivalent to 35 L of the second fermented liquid n2. It is a mixed medium mixed in a weight ratio of ~ 30 g. By the way, the dried plant consisting of taiso, wolfberry, and turmeric is a naturally occurring product with medicinal properties, and the fruit of jujube listed in the "list of crude drugs" is taiso, wolfberry fruit, and turmeric of the family Zingiberaceae. .. These were selected from many herbal medicines from the steady practice of the inventors over many years.

Technical features of the second fermentation line f2

The inventors of the practice of many years of trial and error, as shown in Figure 7 [a] and [b], in a first step _{p 2} 1 of the second fermented line f2, considerable 35L of the second fermentation liquor n2 A mixed medium pm2 was formed in which 200 to 210 g of taiso, 110 to 120 g of kukoshi and 25 to 30 g of corn (weight ratio 7: 4: 1) were mixed with respect to the total amount, and the mixed medium pm2 was prepared in advance in the second medium tank 40. To produce a preliminary fermentation mixed medium pfm2 and a preliminary third fermentation liquid pn3, and by a step (not shown), the preliminary fermentation mixed medium pfm2 is ground to the extent that the seeds of Taisou are not crushed by a pulverizing means. The process of returning to the medium tank 40 and acclimating the ground preliminary fermentation mixed medium pfm2 with the preliminary third fermentation liquid pn3 was completed to generate a second fermentation medium m2 having a pH of 4.8 ±.

The first step p ₂ 1 of the second fermentation line f2 preferably includes the fermentation step p ₁ 3 of the second fermentation broth n ₂ when the third step p ₁ 3 is incorporated in the first fermentation line f _1. A partially overlapping process is adopted.

The first technical reason for making the first step p ₂ 1 parallel to the fermentation step p ₁ 3 of the second fermented liquid n ₂ so as to partially overlap the fermentation step p ₁ 3 is that the fermenting bacteria b 1 in the fermentation step It is to change the fermentation environment. By changing the fermentation environment, the secretion and fermentation of the acidic substance of the fermenting bacterium b1 were further promoted, and the second fermentation medium m2 was produced more rapidly.

Second technical reasons, the start of the fermentation process p _{2 2} of the third fermentation liquor n3 of the third fermentation tank 300, in order to match at the end of the fermentation process p _{1 3} of the second fermentation liquor n2. Thereby, the first step _{p 2} 1 of the second fermented line f2 to generate a second fermentation medium m2 is fermentation step _{p 1} 3 of the second fermentation liquor n2 in the third step _{p 1} 3 of the first fermentation line f1 You can start without waiting for the end. As a result, the second step p ₂ 2 of the second fermentation line f2 for producing the third fermentation liquid n3 is the end of the fermentation step (third step p ₁₃ ) of the second fermentation liquid n2 of the first fermentation line f1. It will be started without delay.

In a first step p _{2 1} of the second fermented line f2, the technical significance of the second fermentation line f2 using a second fermentation medium m2 obtained by pre-fermenting dried plant pm2 a three crude drugs selected, FIG. It becomes clear from the result of the comparative test of the sample A and the sample B of the fermented liquid model of the third fermented liquid n3 shown in No. 10 and the analysis result of the progress of different fermentations of the sample A and the sample B shown in FIG.

The second fermentation line f2 includes a step p ₂ 1 for producing the second fermentation medium m ₂ . This step _{p 2} 1 in the total equivalent 35L of the second fermentation liquor n2, gymnastics 200～210G, Kukoshi ranging between 110 to 120 g, turmeric 25-30 g (weight ratio 7: 4: 1) mixed in equivalent the mixed medium the pm2, together with a portion of the second fermentation liquor n2, pre fermented in a second culture medium tank 40, it includes a preliminary step pp ₂ 1 to generate a preliminary third fermentation liquor pn3 and preliminary fermentation mixture medium Pfm2, generated The prepared preliminary fermentation mixed medium pfm2 was ground using a crushing means to the extent that Tyson seeds were not crushed and returned to the second medium tank 40, and the prepared preliminary fermentation mixed medium pfm2 was subjected to a preliminary third. the fermentation broth pn3 adapt, a preliminary step pp ₂ 2 for generating a second fermentation medium m2 of pH 4.8 ±, further comprising.

The second fermentation line f2 further includes a step p ₂ 2 for producing a third fermentation broth n 3 having a pH of 4.5 ±. Figure 1 [11] to step _{p 2} 2 shown in [12], hung a second fermentation medium m2 produced in step _{p 2} 1 to the third fermentation tank 300 in a state of being sealed in the bag-like medium filter 320, A preliminary step pp ₂ 1 of transferring the preliminary third fermentation liquid pn3 from the second medium tank 40 to the third fermentation tank 300 and further transferring the second fermentation liquid n2 having a pH of 5.0 ± from the second fermentation tank 200 is performed. Including. Step _{p 2} 2 further so immiscible second fermentation medium m2 of the bag-like medium filter 320, circulated a preliminary third fermentation liquor pn3 the second fermentation liquor n2, fermentation conditions of 37 ° C. to 40 ° C. by fermentation of while 8th to 9th hold, including a preliminary step pp ₂ 2 to generate a third fermentation liquor n3 of 1050L considerable pH 4.5 ±.

FIG. 10 is Photograph A and Photograph B showing the results of a comparative test of Sample A and Sample B of the fermentation liquid model of the third fermentation liquid n3.

Specimen A is prepared by pre-fermenting pm2, which is a mixed medium consisting of 1 L of the second fermented liquid n2 and 5.71 g of taiso, 3.14 g of kukoshi, and 0.85 g of corn (weight ratio 7: 4: 1) corresponding to 1 L. The pre-fermented second fermentation medium m2 is sealed in a substitute filter for raw medicine and hung in the test container A, and the second fermented liquid n2 of the starter equivalent to 1 L and the preliminary third fermented liquid pn3 are put into the test container A. It is a fermentation broth A composed of two layers of a supernatant fermentation gas layer and a turbid third fermentation broth n3 by fermentation for 5 days while maintaining a fermentation state of 38 ° C. to 40 ° C. with stirring (Photo A in FIG. 10). ).

Specimen B contains 1 L of the second fermented liquid n2 and 5.71 g of Taisou corresponding to 1 L.
A mixed medium pm2 consisting of 3.14 g of kukoshi and 0.85 g of turmeric (weight ratio 7: 4: 1) was sealed in a substitute filter for raw medicine without pre-fermentation as the mixed medium pm2, and hung in test container B. The second fermentation broth n2 and the preliminary third fermentation broth pn3 of the starter equivalent to 1 L are put into the test container B, stirred, and fermented for 5 days while maintaining the fermentation state at 38 ° C. to 40 ° C. to obtain the supernatant. It is a fermentation broth B composed of a third fermentation broth n3 in which a fermentation gas layer is hardly formed (Photo B in FIG. 10). The fermentation broth B is decisively different from the fermentation broth A in that the fermentation gas layer of the supernatant is hardly formed.

The fermented liquid A and the fermented liquid B are due to the difference in the degree of fermentation due to the secretion and metabolism of the acidic substance of the fermented bacterium b1, and more specifically, the fermented liquid A is more like the fermented bacterium b1 than the fermented liquid B. It is a result of a comparative test showing that fermentation by secretory metabolism is progressing.

FIG. 11 shows the difference in the degree of fermentation between the fermentation broth A and the fermentation broth B for 5 days by the change in acidity. The fermented liquid A always shows a pH value of lower acidity than the fermented liquid B. Until the third day, the acidity of the fermented liquid A changed from pH 5.11 to pH 5.09, the acidity of the fermented liquid B changed from pH 5.13 to pH 5.14, and the acidity was fermented liquid A>. The acidity is B, but there is no significant change in either. However, it is clear that the acidity becomes stronger on the 4th to 5th days, the acidity of the fermentation broth A ranges from pH 4.76 to pH 4.68, and the acidity of the fermentation broth B ranges from pH 4.87 to pH 4.87. Acidification progresses to pH 4.78, and the acidity is fermentation broth A> acidity B, both of which confirm the degree of fermentation by secretory metabolism of anaerobic fermenting bacterium b1.

FIG. 11 also changes the fermentation environment of the fermenting bacterium b1 by using the pre-fermented second fermentation medium m2 when producing the third fermented liquid n3, and enhances the secretory metabolism of the acidic substance of the fermenting bacterium b1 to promote fermentation. It is a graph which shows the result which works to encourage. Here is the technical significance of producing the third fermentation broth n3 using the pre-fermented second fermentation medium m2.

The first preliminary step pp ₂ 1 of the second step p ₂ 2 of the second fermentation line f2 is
Figure 1 [11] First Step p _{2 1} a pH4.8 second fermentation medium m2 of ± produced in the third bag-shaped medium of a plurality the installed already steam sterilization as hanging in the fermentation tank 300 shown in Enclose evenly in the filter 320 and
Preliminary third fermentation broth pn3 generated in the first step _{p 2} 1 transferred from the second medium tank 40 to the third fermentation tank 300,
The second fermentation liquid n2 having a pH of 5.0 ± from the second fermentation tank 200 is transferred to the third fermentation tank 300 via the extraction device 210 of the second fermentation tank 200 connected to the third fermentation tank 300.

The second preliminary step pp ₂ 2 of the second step p ₂ 2 of the second fermentation line f2 is
Operate the circulation pump device 330 shown in FIG.
In the third fermentation tank 300,
A second fermentation medium m2 having a pH of 4.8 ± enclosed in a bag-shaped medium filter 320 suspended inside so as not to be mixed with others.
Preliminary fermented liquid pn3 with pH 4.8 ± and second fermented liquid n2 with pH 5.0 ±
Is circulated integrally as the second starter,
By fermentation for 8 to 9 days while maintaining the fermentation state of 37 ° C to 40 ° C,
A third fermentation liquid n3 having a pH of 4.5 ± is produced, which is a turbid liquid in which the second upper surface film layer sf2 is formed in the supernatant.

The second fermentation medium m2 is because it is enclosed in a fine bag-like medium filter 320 textured, in the second step p _{2 2} of the second fermentation line f2, the bottom residue of the second fermentation medium m2 is the fermentation tank 300 Does not deposit in. It has also been confirmed that when the second fermented liquid n2 changes to the third fermented liquid n3, the acidity of the third fermented liquid n3 advances to pH 5 or less.

Details of the third fermentation line f3

In the third fermentation line f3 shown in FIGS. 1 [13] to [16], the first step p ₃ 1 in which the third fermentation medium m3 having a pH of 4.4 ± is produced and the fourth fermentation liquid n 4 having a pH of 3.7 ± It consists of the second step p ₃ 2 in which is generated.

The overall picture of the third fermentation line f3 is shown in FIG. In FIG. 12, in a state where the second fermentation liquid n2 generated in the second fermentation line f2 is transferred to the third fermentation tank 300, the second fermentation medium filter 320 installed inside the third fermentation tank 300 is used for the second fermentation. The step [a] of charging the second fermentation medium m2 produced in the medium tank 40, operating the circulation pump device 330, and fermenting for 8 to 9 days while circulating the medium to produce the third fermentation liquid n3. Shown.

In the third fermentation line f3, an appropriate amount of a part of the third fermentation liquid n3 of the third medium tank 300 and the honey raw material pm3 is put into the third medium tank 50 and stirred to bring the fermentation state to 37 ° C to 40 ° C. The step [c] is included in which a third fermentation medium m3 is produced by fermentation for 2 to 3 days while holding the medium.

In the third fermentation line f3, the third fermentation liquid n3 and the third fermentation medium m3 transferred from the third fermentation tank 300 are further charged into the fourth fermentation tank 400, and the mixture is stirred to bring the fermentation state to 37 ° C to 40 ° C. The steps [d] to [g] for producing the fourth fermentation broth n4 are included by fermentation for 30 to 60 days while maintaining the above.

More specifically, the first step p ₃ 1 of the third fermentation line f3 is 3 to 5% of the generated third fermentation liquid n3 corresponding to 1050 to 1100 L shown in FIGS. 12 [a] to [b]. Touch the honey raw material pm3, which is composed of a considerable amount of encyclopedia honey and acacia honey in a ratio of 1: 4, and a part of the third fermented liquid n3, which is four times the amount of the honey raw material pm3, to the outside air. The first preliminary step pp ₃₁ is included in which the mixture is transferred to the third medium tank 50 and stirred so as not to cause the preliminary fermentation medium pfm3 shown in FIG. 12 [c].

The first step _{p 3} 1 further preliminary fermentation medium pfm3 generated by the first preliminary step pp ₃ 1, by fermentation of 2 days to 3 days while maintaining the fermentation state of 37 ° C. to 40 ° C., honey material It includes a second preliminary step pp ₃ 2 in which a third fermentation medium m 3 having a pH of 4.4 ± by pm 3 is produced.

The second step p ₃ 2 of the third fermentation line f3 is
The third fermentation medium m3 having a pH of 4.4 ± was charged into the fourth fermentation tank 400 shown in FIGS. 12 [d] to [f].
The third fermentation liquid n3 having a pH of 4.5 ± was transferred to the fourth fermentation tank 400 by the extraction device 310 of the third fermentation tank 300 connected to the fourth fermentation tank 400.
The third fermentation medium m3 having a pH of 4.4 ± and the third fermentation liquid n3 having a pH of 4.5 ± were mixed and stirred as a fourth starter.
By fermentation for 30 to 60 days while maintaining the fermentation state at 37 ° C to 40 ° C,
The third upper surface layer sf3 of the fermentation gas layer is formed in the supernatant, and the third precipitation layer dep3 containing a precipitate is formed in the lower layer.
A translucent third intermediate layer liquid equivalent to 1000 to 150 L is generated between the formed third upper surface layer sf3 and the third settling layer dep3. The translucent third intermediate layer liquid is the fourth fermentation liquid n4 having a pH of 3.7 ±.

Technical features of the third fermentation line f3

After many years of steady trial and error, the inventors conceived the third fermentation line f3, repeated trial and error, supplied a certain amount of sugar to the fermenting bacterium b1, and increased the growth and secretory metabolic capacity of the fermenting bacterium b1. It was realized that fermentation by the secretion and metabolism of further acidic substances could be continued.

In order to maintain the secretory and metabolic capacity of the fermenting bacterium b1 determined from the water activity (if it is too low, the bacterium cannot grow due to the suppression of the aquatic activity of the bacterium) through repeated trial and error. It was discovered that the honey raw material pm3 had a sugar mass corresponding to 3 to 5% of the third fermentation broth n3, and this was stirred with a quadruple amount of the third fermentation broth n3 to generate a preliminary fermentation medium pfm3. The produced preliminary fermentation medium pfm3 was fermented for 2 to 3 days while maintaining the fermentation state at 37 ° C to 40 ° C to generate a third fermentation medium m3.

The inventors further mixed the produced third fermentation medium m3 with a third fermentation liquid n3 equivalent to 20 times, stirred, and fermented for 30 to 60 days while maintaining the fermentation state at 37 ° C to 40 ° C. This promoted fermentation of the fermenting bacterium b1 by secretory metabolism of an acidic substance, and succeeded in producing a fourth fermentation broth n4 having a pH of 3.7 ±. This is the third fermentation line f3. Its technical features are shown in FIGS. 13 and 14.

FIG. 13 is Photograph A and Photograph B showing the results of a comparative test of Sample A and Sample B of the fermentation liquid model of the fourth fermentation liquid n4.

In the sample A, the third fermentation medium m3 obtained by pre-fermenting the honey raw material pm3 is mixed with a third fermentation liquid n3 equivalent to 20 times so as to become 1 L, and the mixture is stirred to maintain the fermented state at 38 ° C to 40 ° C. However, it is the fermentation broth A of the fourth fermentation broth n4 produced by fermentation for 4 days. Specimen B is mixed with a 20-fold equivalent third fermentation broth n3 so as to have 1 L of the honey raw material pm3 (third medium pm3), stirred, and fermented with the fourth fermentation broth n4 produced by fermentation for 4 days. Liquid B.

For sample A, a third fermentation medium m3 obtained by pre-fermenting the honey raw material pm3 was placed in one of the test containers, stirred, and fermented for 4 days to form a supernatant of the third upper surface layer sf3. It is a fermentation broth A that forms three layers of a translucent third intermediate layer liquid between the third precipitation layer dep3 containing the precipitate accumulated in the lower layer and the third upper surface layer sf3 and the third precipitation layer dep3. The translucent third intermediate layer liquid corresponds to the fourth fermentation liquid n4.

Specimen B is a fermentation broth B produced by pouring the honey raw material pm3 into the other test container as it is, stirring the mixture, and fermenting for 4 days. From Photo B, it is confirmed that the third upper surface layer sf3 of the fermentation gas layer is formed in the supernatant, but it is an extremely thin layer as compared with the sample A. This is a manifestation of the fact that fermentation by secretory metabolism of the acidic substance of fermenting bacterium b1 is not as active as that of sample A. That is confirmed from FIG.

FIG. 14 shows changes in the acidity of the fermentation broth A and the fermentation broth B due to fermentation for 4 days. The acidity of the fermented liquid A and the fermented liquid B on the first day is pH 4.6. The change in acidity increased together on the second day, with fermented liquid A having a pH of 4.7 and fermented liquid B having a pH of 4.73. The degree of fermentation progresses on the 3rd to 4th days, and as a result, both the acidity decreases, and the fermentation broth A has a pH of 4.6 to 4.52, while the fermentation broth B has a pH of 4.68 to 4.6. .. The acidity of both on the 2nd to 4th days is always fermented liquid A> acidity B.

FIG. 14 is a graph showing the transition of the acidity of the fermentation broth A and the fermentation broth B for 4 days from the start of fermentation. The difference between the two is that the degree of fermentation is so high that the fermentation broth A is always lower than the fermentation broth B at the pH value of acidity for 4 days. This is proof that the fermenting bacterium b1 of the fermented liquid A secretes an acidic substance more actively. That is, when the fourth fermented liquid n4 is produced, the inventors use a third fermentation medium m3 in which the honey raw material pm3 is pre-fermented, instead of the third medium in which the honey raw material pm3 remains as it is. We succeeded in making the fermenting bacterium b1 work so as to change the fermentation environment of b1 and enhance the secretion and metabolism of acidic substances.

Details of the 4th fermentation line f4

The fourth fermentation line f4 shown in FIG. 1 [17] is
Using the 4th fermented liquid n4 with pH 3.7 ± as the 5th starter,
By fermentation for 30 to 60 days using only the fermenting bacterium b1 contained in the fourth fermentation broth n4 while maintaining the fermentation state of 37 ° C to 40 ° C.
A fifth fermentation broth n5 having a pH of 3.6 ± is produced.
This is a step _{p 4} 1 of the fourth fermentation lines f4, since there is little ullage during the fermentation process, the fifth fermentation liquid n5 generated will correspond 1000～1050L.

Step p _{4 1} of the fourth fermentation lines f4 is subjected to the following steps, a step of generating a fifth fermentation liquid n5.

It contains one first fermentation broth n1 produced in two first fermentation tanks 100 in the first step p ₁ 1 and the second step p _{12 of} the first fermentation line f1 using the first fermentation medium m1. second transferred to the fermentation tank 200, using the third step p _{1 3} second fermentation medium m2 generated by the second fermentation liquid n2 and the second medium tank 40 generated by the fermentation environment that does not use the fermentation medium in the , The third fermentation broth n3 was produced at the second fermentation line f2, the third fermentation broth n3 produced at the second fermentation line f2, and the third fermentation medium m3 produced at the third fermentation medium tank were used. The fermentation line f3 produces the fourth fermentation broth n4, and the fourth fermentation line f4 produces the fifth fermentation broth n5 in a fermentation environment that does not use a fermentation medium. Fifth starter step p _{4 1} of the fourth fermentation lines f4 is only fourth fermentation broth n4, is not a new medium or fermentation medium is used.

Technical features of the 4th fermentation line f4

At first glance, there seems to be no change in the acidity between the acidity pH 3.7 ± of the fourth fermentation broth n4 and the acidity pH 3.6 ± of the fifth fermentation broth n5. However, as is clear from FIG. 16, the acidity representing the change in the fermentation degree of the fifth fermentation liquid n5, which expresses the change in the fermentation degree in each step by the pH value of the acidity based on the actual measurement, is the fourth fermentation liquid n4. During the fermentation period, which took 30 to 60 days, the pH was gradually increased. This proves that fermentation by secretory metabolism of fermenting bacterium b1 contained in the fourth fermentation broth n4 is proceeding.

Regarding the technical intention of the 4th fermentation line f4, in the process of the inventors' steady trial and error for many years, the method of producing the fermentation broth that the inventors aimed at was completed by the 3rd fermentation line f3 for a while. It was regarded as. At that time, the technical need for the fourth fermentation line f4 following the third fermentation line f3 was not envisioned. However, the inventors still took various measures such as heat sterilizing the fermenting bacterium b1 because the secretory and metabolic activity of the acidic substance of the fermenting bacterium b1 did not stop even at the end of the third fermentation line f3. Many attempts were made to stably produce the fourth fermentation broth n4 of the third fermentation line f3. As a result, the technical findings obtained by the inventors are that the fermenting bacterium b1 including the spore-forming fermenting bacterium forms spores even under severe conditions such as high temperature, dryness, and deterioration of nutrition, and survives in a dormant state. It was said that it was difficult to process by heat sterilization.

The inventors who noticed this technical knowledge, although it takes time, technically whether it is really necessary to end the active secretion and metabolism of the acidic substance of the fermenting bacterium b1 at the end stage of the third fermentation line f3. Focusing on the problems, we have discovered the technical value of the secretion and metabolism of the acidic substance of the fermenting bacterium b1 in the fermentation step below the fourth fermentation line f4, and succeeded in completing the present invention.

Details of the 5th fermentation line f5

The fifth fermentation line f5 shown in FIGS. 1 [18] to [19] is
The first step p in which the fifth fermentation liquid n5 produced in the fifth fermentation tank 500 in the fourth fermentation line f4 was once rapidly cooled, and the rapidly cooled fifth fermentation liquid n5 was returned to room temperature to be in a normal temperature state. ₅ 1 and
At a liquid temperature while managing at ambient temperature conditions, by fermentation of fermenting bacteria b1 contained in the fifth fermentation liquid n5, the second step _{p 5} 2 to generate a sixth fermentation liquor n6 of 1000~1050L considerable pH 3.3 ± ,
including.

The sixth fermentation broth n6 is produced by fermentation with only the fermenting bacterium b1 contained in the fifth fermentation broth n5. It is the same as when the second step p _{5 2} in the fifth fermentation liquid n5 of the fifth fermentation line f5 is generated by fermentation with only fermenting bacteria b1 included in the fourth fermentation liquor n4. However, the fermentation environment when the sixth fermentation liquid n6 is produced is decisively different from the fermentation environment when the fifth fermentation liquid n5 is produced in the following two points. That is the technical feature of the fifth fermentation line f5.

Technical features of the 5th fermentation line f5

The first technical feature of the fifth fermentation line f5 is shown in FIG. 15 [a], which is the fifth fermentation broth n5 held in the fifth fermentation tank 500 in a fermentation state of 37 ° C. to 40 ° C. This is a step of transferring 1000 to 1050 L of the above to a spare tank 620 provided in the cooling device 610 of the sixth fermentation system s6.

Its technical intent is shown in FIG. 15 [b], which is to quench to 4-5 ° C or less where the activity of secreting the acidic substance of the fermenting bacterium b1 contained in the fifth fermented liquid n5 is stopped. It is in. This means switching from the fermentation environment in which the fermenting bacterium b1 is most easily secreted and metabolized to the environment in which the secretory and metabolism is most difficult to be secreted and metabolized. The fermenting bacterium b1 in this state becomes dormant and stops secretory metabolism.

The fifth fermentation broth n5 containing the fermenting bacterium b1 whose secretory metabolism was stopped by the rapid cooling shown in FIG. 15 [c] was transferred from the reserve tank 620 to the sixth fermentation tank 600 and returned to a normal temperature state according to the season. In a fermentation trans-environment at room temperature, fermentation for 180 to 240 days gradually promotes the activity of fermenting bacteria b1 and activates the secretion and metabolism of acidic substances.

The second technical feature of the fifth fermentation line f5 is not the fermentation state of 37 ° C. to 40 ° C. in which the fermentation bacterium b1 is easy to act, but the normal temperature state in which the fermentation bacterium b1 is hard to act, specifically, 5 ° C. or higher. By creating a fermentation environment according to the season of 28 ° C or lower and performing long-term fermentation (June to August) of 180 days to 240 days shown in stage 3 of FIG. 16, secretory metabolism of the acidic substance of fermenting bacterium b1 is performed. It was made to converge the metabolic activity while promoting it. As a result, the fermenting bacterium b1 that has finally stopped secretory metabolism goes into a dormant state, and the sixth fermented liquid n6 having a pH of 3.3 ± is produced in the sixth fermentation tank 600.

Action effect of the invention

In principle, the first fermentation broth n1 having a pH of 5.3 ± produced according to the present invention is the first fermentation broth for producing the sixth fermentation broth n6 having a pH of 3.3 ± of the final product. The first fermentation broth n1 contains soft water w, a fermentation medium m1 of soybean paste, and a deposit number NITE p-02945-NITE p-02951 registered with the Patent Microorganisms Depositary Center (NPMD) in an appropriate volume ratio. Date: May 16, 2019) A turbid solution (preliminary second fermented solution pn2) mixed with a source bacterial solution b consisting of seven types of fermented bacteria b1 including spore-forming fermenting bacteria, that is, does not exceed 50 nm. As shown in Stage 1 of FIG. 16, fermentation at an appropriate fermentation temperature of 37 ° C. to 40 ° C. using a stock solution θ for producing a fermentation broth containing colloidal particles and short-chain fatty acids having a pH of 3 or more and 4 or less as a starter. It is produced by fermentation over 50 to 100 days while maintaining the condition.

The reason why the turbid liquid (preliminary second fermented liquid pn2) that serves as the starter of the first fermented liquid n1 is the undiluted solution θ is that the original bacterial solution b containing soft water w and the spore-forming fermenting bacteria b1 is used. It is derived from the fact that it is limited to the production of the first fermentation broth n1. Specifically, the origin is that soft water w and the original bacterial solution b are not newly used when the sixth fermented solution n6 is produced from the second fermented solution n2. All of the steps of producing the sixth fermentation broth n6 from the second fermentation broth n2 are the fermentation of seven kinds including the spore-forming fermenting bacteria contained in the first fermentation broth n1 in the appropriate fermentation environment formed in each. The acidity of each fermentation broth is increased by activating the secretion and metabolism of acidic substances by the bacterium b1 and advancing each fermentation in each step.

In the present invention relating to a method for producing a fermented solution having a pH of 3 or more and 4 or less containing colloidal particles not exceeding 50 nm and short-chain fatty acids, the original bacterial solution b composed of 7 kinds of fermenting bacteria b1 including spore-forming fermenting bacteria is used. , Is an essential component. The original bacterial solution b contains a spore-forming fermenting bacterium having a deposit number NITE p-02945-NITE p-02951 (consignment date: May 16, 2019) registered as a deposit in (NPMD) shown in FIG. It is composed of 7 kinds of fermenting bacteria b1. The present invention cannot be established unless the original bacterial solution b can be stably supplied.

The inventors repeated trial and error in a long-term practice process, and as a result, seven kinds including spore-forming fermenting bacteria that produce organic acids including butyric acid, propionic acid, and short-chain fatty acids of lactic acid bacteria shown in FIG. We found and selected fermenting bacteria b1 and succeeded in producing a source bacterial solution b that stably produces them.

The production of the original bacterial solution b depends on the three layers of sponge layer sp formed in the two first fermentation tanks 100, the translucent intermediate layer solution, and the first precipitation layer dep. More specifically, the original bacterial solution b is taken out from the first fermentation tank 100 with the supernatant sponge layer sp and the first sedimentation layer dep deposited on the bottom, mixed, and made into a paste. Seven kinds of fermenting bacteria b1 including spore-forming fermenting bacteria constituting the original bacterial solution b are contained in any of the three layers formed in the two first fermentation tanks 100.

The reason why the sponge layer sp and the first settling layer dep formed in the first fermentation tank 100 excluding the translucent intermediate layer liquid become the original bacterial solution b is that the charge amount of each first fermentation tank 100 is assumed to be 900 L. Then, one-third of 300 L is used as the raw material for the original bacterial solution b, and when the original bacterial solution b finished in a considerable amount of paste is used as the original bacterial solution b for the next preparation, the first fermentation tank is used. Three layers of the same quality are formed in 100, and the first fermentation broth n1 having a pH of 5.3 ± is produced.

Further, the original bacterial solution b finished in a paste form can be stored frozen. Even if the required equivalent amount of the cryopreserved original bacterial solution b is returned to the fermentation state of 37 ° C. to 40 ° C. and used as the paste-like original bacterial solution b to be prepared next, the same applies to the first fermentation tank 100. The next three layers of quality are formed, and the acidity of the first fermentation broth n1 of the translucent intermediate layer liquor formed there is always pH 5.3 ±, which can be confirmed.

While the paste-like original bacterial solution b can be stored for a long period of time in a large freezing device, it can also be used in the next fermentation line f1 at the fermentation temperature immediately after fermentation for 50 to 100 days. As is clear from FIG. 16, the production method for producing the fermentation broth containing the short-chain fatty acid of the present invention is at least 303 days (10 months) until the final product, the sixth fermentation broth n6, is produced. Although it takes 477 days (16 months), continuous production can be carried out at intervals of about 100 days at the latest when the production of the first fermentation broth n1 is completed, without waiting for the production of the final product. In short, the sixth fermentation broth n6 having a pH of 3.3 ± of the final product produced according to the present invention can be continuously produced in units of 100 days at the latest.

Fermented liquid produced by the present invention

The fermented liquor produced by the present invention includes soft water w as a starter, a source bacterium liquor b consisting of seven types of fermenting bacteria b1 including spore-forming fermenting bacteria, and dried soybeans used in three types of fermentation media. It is a fermented liquid fermented with a dried plant consisting of three kinds of raw materials such as soybean, kukoshi, and turmeric, and a natural material made of honey raw material.

The following analysis was performed on the sixth fermented liquid n6 having a pH of 3.3 ± of the final product produced by the present invention, and it was determined by the secretory metabolism of the fermenting bacterium b1 in the fermentation from the first fermented liquid n1 to the sixth fermented liquid n6. Assuming the action of acidic substances, the elements constituting the fermentation broth produced by the present invention are estimated.
(1) Analysis of acidity pH value of 1st to 6th fermented liquid (2) Particle size analysis of 6th fermented liquid n6 (3) Analysis of sugar concentration of 4th fermented liquid n4 to 6th fermented liquid n6 ( 4) Analysis of short-chain fatty acids from the second fermented liquid n2 to the sixth fermented liquid n6

(1) Transition of acidity pH value from the first fermented liquid to the sixth fermented liquid

The inventors are engaged in the production of the fermentation medium and the fermentation broth produced in the present invention on a daily basis while monitoring the pH value of the acidity of the fermentation medium and the fermentation broth produced in each step described above. FIG. 16 is a graph showing the average value of acidity for each lot, which is constantly measured and monitored. Each pH value is given a ±, which is the fluctuation range of the pH value (usually 0.3) due to the temperature difference and humidity difference in the measurement room, which always occurs in the fermentation process, and the slight deviation in the measurement timing. It expresses the swing width).

The transition of the acidity of the first fermentation line f1 starting from the soft water w of pH 7.3 ± shown in FIG. 16 includes the acidity of the first fermentation medium m1 and the first fermentation liquid n1 and the second fermentation liquid n2. Including, it was classified as stage 1. The fermentation period of the category is 55 days at the shortest and 108 days at the longest.

It should be noted that a large amount of the first fermentation medium m1 having a pH of 4.5 ±, the original bacterial solution b, and the soft water w produced in the first medium tank 20 was put into the two first fermentation tanks 100. The preliminary second fermentation broth pn2 having a pH of 6.4 ± of the turbid liquid produced by stirring becomes the so-called starting point fermentation broth which is the starting point of the fermentation broth produced by the present invention. After that, the original bacterial solution b and the soft water w are not re-injected. Therefore, the preliminary second fermented liquid pn2 having a pH of 6.4 ± corresponds to the stock solution θ of the sixth fermented liquid n6 having a pH of 3.3 ± produced by the present invention. Then, the preliminary second fermentation broth pn2 having a pH of 6.4 ± is produced into the first fermentation broth n1 having a pH of 5.3 ± by fermentation at an appropriate fermentation temperature for 50 to 100 days.

The other notable one is pH 5.0 by fermentation in the second fermentation tank 200, which is the final division of stage 1, at an appropriate fermentation temperature only by the fermenting bacterium b1 of the first fermentation broth n1 for 3 to 5 days. The second fermentation broth n2 of ± is produced. Further, within the same category, the steps of producing the second fermentation medium m2 having a pH of 4.8 ± by fermentation for 2 to 3 days constituting the second fermentation line f2 are overlapped.

The transition of acidity from the second fermentation line f2 where the second fermentation broth n2 of pH 5.0 ± starts to the fourth fermentation line f4 of the fifth fermentation broth n5 of pH 3.6 ± shown in FIG. 16 is the second fermentation. The third fermentation medium m3 and the third fermentation medium m3 of the third fermentation line f3, including the acidity of the third fermentation liquid n3 having a pH of 4.5 ± generated by using the second fermentation medium m2 of the line f2 and the second fermentation liquid n2 as a starter. The fifth of pH 3.6 ± produced using only the fourth fermentation broth n4 of the fourth fermentation line f4 as a starter, further including the fourth fermentation broth n4 of pH 3.7 ± produced using the three fermentation broth n3 as a starter. It was classified as stage 2 including the fermented liquid n5. The fermentation period at an appropriate fermentation temperature within the same category is 68 days at the shortest and 129 days at the longest.

Notable transitions are the following two changes. The first change is that in the second fermentation line f2 and the third fermentation line f3 using the second fermentation medium m2 and the third fermentation medium m3, the secretion and metabolism of the acidic substance of the fermenting bacterium b1 is activated, and the third fermentation broth The acidity progressed from pH 5.0 ± to pH 3.7 ± up to n3 and the fourth fermentation broth n4. The second change is the fifth fermentation produced by fermentation over 30 to 60 days while maintaining the fermentation state of 37 ° C to 40 ° C, using only the fourth fermentation liquid n4 of the fourth fermentation line f4 as a starter. Although it is the liquid n5, the acidity is almost unchanged at pH 3.6 ± as compared with the pH 3.7 ± of the fourth fermentation liquid n4.

The inventors take advantage of the fact that the secretory and metabolic activity of the acidic substance of the fermenting bacterium b1 does not subside even at the end of the third fermentation line f3, and the acidic substances of the fermenting bacterium b1 at the end stage of the third fermentation line f3. This is due to the discovery of the technical value of the fermentation process of the fourth fermentation line f4, which promotes the secretion and metabolism of the acidic substance of the fermenting bacterium b1 even in slow fermentation without terminating the active secretion and metabolism of. It is because fermenting bacteria b1 containing spore-forming fermenting bacteria that form spores form spores and survive in a dormant state even under harsh conditions such as high temperature, drying, and deterioration of nutrition, so they are treated by heat sterilization. This is because the inventors have noticed the merit of accelerating the fermentation of the fourth fermentation broth n4, which is difficult and rather time-consuming.

The transition of the acidity of the fifth fermentation line f5 starting from the fifth fermentation liquid n5 having a pH of 3.6 ± shown in FIG. 16 was classified as stage 3. Stage 3 is an environment in which the fifth fermentation broth n5 held in a fermentation state of 37 ° C. to 40 ° C. at an appropriate fermentation temperature is once rapidly cooled to 4 to 5 ° C. or lower, and then the fermenting bacterium b1 is most difficult to secrete and metabolize. It is a process of switching from fermentation to a normal temperature fermentation environment, which is a gradual secretion and metabolism, and producing by fermentation for 180 to 240 days in a normal temperature fermentation environment according to the season, and the fermentation period within the same category is naturally long. The shortest is 180 days (June) and the longest is 240 days (August).

The fifth fermentation line f5 technically seems to be a process close to the aging of the fifth fermentation broth n5 having a pH of 3.6 ±, but in reality, the metabolic activity is converged while promoting the secretion and metabolism of the acidic substance of the fermenting bacterium b1. Finally, the secret metabolism of the fermenting bacterium b1 is stopped, and the spore-forming fermenting bacterium b1 is put into a dormant state to produce a sixth fermented liquid n6 having a pH of 3.3 ±.

The change in the acidity pH value during each fermentation period is that the acidity at the start of full-scale fermentation of the two first fermentation tanks 100 is the pH 6.4 ± of the preliminary second fermentation liquid pn2, which is the undiluted solution θ. The acidity at the end of the first stage progresses to pH 5.0 ±, and from the end of the first stage, fermentation progresses due to the secretory activity of the fermenting bacterium b1 contained in the second fermentation broth n2, and at the end of the second stage. While maintaining the proper fermentation temperature of 37 ° C to 40 ° C, the acidity progresses to 3.6 ±, during which fermentation bacteria that do not form buds contained in the original bacterial solution b, and aerobic germs However, even if it is mixed in or during the fermentation process, the highly acidic environment suppresses the growth of such bacteria, and the dead bacteria are deposited as a sedimentation layer in the lower layer of each fermentation tank. ..

In the third stage, which is a long-term fermentation period in a fermentation environment at room temperature according to the season, fermentation proceeds while promoting the secretion activity of spore-forming fermenting bacteria that form spores contained in fermenting bacteria b1, and finally there is. The secretory activity of fermenting bacterium b1 including spore-forming fermenting bacterium is led to a state close to the end, further fermentation does not proceed, and the acidity enters a stable state at pH 3.3 ±.

(2) Particle size analysis of the sixth fermented liquid n6

Through many years of steady practical research, the inventors have noticed that the fermented liquid produced by the present invention has an extremely excellent absorption rate in the body of mammals, etc., and requested a specialized agency to analyze the composition of the fermented liquid. From the analysis results, it has been confirmed that the fermentation broth is a product containing about 6.5% to 7.5% of colloidal particles. The specialized agency is the Gifu Prefectural Industrial Technology Center, and the inventors requested the center to perform a particle size distribution test for the next two types of fermented liquids produced by the present invention, and the inspection results were obtained. Is getting.

The two types of fermented liquids produced by the present invention are a fourth fermented liquid n4 having a pH of 3.7 ± and a sixth fermented liquid n6 having a pH of 3.3 ±. Preparations for these particle size distribution tests by cumulan analysis are performed as follows, and the result of cumran analysis for the fourth fermentation broth n4 is shown in FIG. The result of the cumulan analysis for the sixth fermentation broth n6 is shown in FIG. The cumulan analysis is a measurement of colloidal particles contained in the fermentation broth at about 6.5% to 7.5%.

Therefore, the sample submitted to the center is a supernatant obtained by heat-sterilizing a fourth fermented liquid n4 having a pH of 3.7 ± fermented by fermenting bacteria b1 using fermentation media m1 to m3, cooling it, and precipitating it for one day. The first sample (Calbio-5), which was sterilized by 200 mL and separated into two 100 mL bottles, and the fifth fermented liquid n5 with a pH of 3.6 ± fermented only by the fermenting bacterium b1 without using a fermentation medium were placed at room temperature. The sixth fermentation broth n6 having a pH of 3.3 ± produced by long-term fermentation for 180 days (June) to 240 days (August) is heat-sterilized and then cooled and precipitated for 1 day. This is a second sample (Calbio-7) obtained by sterilizing 200 mL of the supernatant and separating it into two 100 mL bottles.

The first sample (Calbio-5) uses the final fermentation medium m3 (pH 4.4 ±) and the third fermentation broth n3 (pH 4.5 ±) as starters, and keeps the fermentation state at 37 ° C to 40 ° C for 30. By fermentation for 1 to 60 days, the third upper surface layer sf3 of the fermentation gas layer was formed in the supernatant, the third precipitation layer dep3 containing the precipitate was formed in the lower layer, and the third upper surface layer sf3 and the third precipitation layer dep3 The translucent third intermediate layer liquid formed between the above is the fourth fermentation liquid n4 having a pH of 3.7 ± as the first sample. It corresponds to the fermentation broth produced in the final fermentation step using the fermentation medium.

The second sample (Calbio-7) is maintained in a fermentation state of 37 ° C. to 40 ° C. at an appropriate fermentation temperature, using the fifth fermentation broth n5 having a pH of 3.6 ± as a starter, without using any medium or fermentation medium. The fifth fermentation broth n5 is once rapidly cooled to 4 to 5 ° C. or lower, and the environment in which the fermentation bacterium b1 is most difficult to secrete and metabolize is switched to the fermentation environment at room temperature where the fermentation bacterium b1 is slowly secreted and metabolized. Fermentation proceeds while promoting the secretory activity of the spore-forming fermenting bacteria that form the spores, and finally the secretory activity of the fermenting bacteria b1 including the spore-forming fermenting bacteria is brought to a state near the end, and further fermentation. The acidity that does not progress is the sixth fermentation broth n6 of the final product having a pH of 3.3 ±. This is the second sample.

The particle size distribution of the fourth fermentation broth n4 is shown in FIG. FIG. 17 is a graph and a table showing the results of a cumulan analysis for measuring the size of colloidal particles contained in the fourth fermentation broth n4 after heat treatment / sterilization treatment of the first sample in an amount of about 6.5% to 7.5%. Is. According to the analysis result, the fourth fermentation broth n4 was found to be colloidal particles on the order of microns. Specifically, the graph of the particle size (μm) x represented by the frequency (%) y on the vertical axis and the logarithm on the horizontal axis shows that the particle size is in the micron unit. In addition, specific numerical values can be grasped from the table shown by detailed numerical values. The frequency exceeds 0% from the serial number CH55 and becomes 0% from CH101.
The conclusion is that the particle size x of the colloidal particles contained in the first sample is
x = 0.375 μm (y = 0.24%) to 18.5 μm (y = 0.17%)
Is.

The particle size distribution of the sixth fermentation broth n6 is shown in FIG. FIG. 18 is a graph and a table showing the results of a cumulan analysis for measuring the size of colloidal particles contained in the sixth fermentation broth n6 at about 6.5% to 7.5% after the heat treatment / sterilization treatment of the second sample. is there. According to the analysis results, the sixth fermentation broth n6 was found to be nano-unit colloidal particles. Specifically, the particle size (nm) x represented by the volume distribution (%) y on the vertical axis and the logarithm on the horizontal axis is the particle size in nanometers in the upper left graph, and the average particle size is 21.4 nm from the graph. Is shown to be. In addition, specific numerical values can be grasped from the table shown by detailed numerical values. The volume distribution frequency depends on the numerical value in the range from 0% or more to 0%.
The conclusion is that the particle size x of the colloidal particles contained in the second sample is
x = 1.1 nm (y = 5.7%) to 6.6 nm (y = 0.1%)
Is.

The difference between the analytical data in FIGS. 17 and 18 is due to the difference between the micron unit and the nano unit of 1/1000 μm. The second sample in FIG. 18 is not an analysis of the micron-level particle size distribution of the first sample, but an analysis of the nano-scale particle size distribution based on the volume distribution frequency.

For a period of time, the inventors recognized that the method for producing the fermented liquid aimed at by the inventors was completed by the third fermentation line f3, and are trying to end the metabolic activity of the fermenting bacterium b1 that secretes an acidic substance. The inventors also noticed that the spore-forming fermenting bacteria contained in the fermenting bacterium b1 are a difficult-to-treat group of bacteria that form spores and survive in a dormant state even under harsh conditions. Through various trial and error practices, the metabolic activity of fermenting bacterium b1 including active spore-forming fermenting bacterium is utilized, and the fourth fermented liquid n4 having a particle size of micron unit is used as a starter for 30 to 60 days. A fifth fermentation broth n5 having a pH of 3.6 ± was produced by the fermentation of.

Using the produced fifth fermentation broth n5 as the final starter, the secretory activity of fermenting bacterium b1 was gradually converged by long-term fermentation for 180 days (June) to 240 days (August) in a fermentation environment at room temperature. Thus, we succeeded in gradually increasing the acidity and stabilizing it at the final acidity of pH 3.3 ± by natural fermentation by fermenting bacteria b1 containing spore-forming fermenting bacteria. Moreover, an unexpected result is the realization of the sixth fermentation broth n6 containing nano-unit colloidal particles. Needless to say, the obvious fact is that there is a marked difference in the absorption rate in the living body when used as a fermented food between a fermented liquid containing micron-sized colloidal particles and a fermented liquid containing nano-sized colloidal particles. Needless to say.

The second of the performance analysis of the fourth fermented liquid n4 and the sixth fermented liquid n6 is to measure the sugar mass contained in each. The inventors generate a third fermentation medium m3 from the honey raw material pm3, mix it with a third fermentation liquid n3 equivalent to 20 times, and stir to secrete fermenting bacteria b1 having a sugar concentration of 3 to 5%. It forms a fermentation environment in which the activity is most easily activated, and by fermentation for 30 to 60 days while maintaining the fermentation state of 37 ° C to 40 ° C, the secretion and metabolism of the acidic substance of the fermenting bacterium b1 is further promoted, and the pH is 3.7 ±. Succeeded in producing the fourth fermented liquid n4.

(3) Sugar concentration analysis from the 4th fermented liquid n4 to the 6th fermented liquid n6

FIG. 19 shows the measurement results of the sugar mass c1 of the fourth fermented liquid n4, the sugar mass c2 of the fifth fermented liquid n5, and the sugar mass c3 of the sixth fermented liquid n6 per 100 mL.
Specifically, it is as follows.
c1 = 3.39g / 100mL
c2 = 2.88g / 100mL
c3 = 1.19g / 100mL

From many years of steady trial and error, the inventors conceived the third fermentation line f3, repeated trial and error, and supplied fermenting bacteria b1 with a certain amount of sugar fermentation medium obtained by fermenting honey raw materials. Both the growth of the fermenting bacterium b1 and the secretory and metabolic capacity were enhanced, and fermentation by further secretory and metabolism of acidic substances was realized.

The inventors have found that the constant mass of sugar for maintaining the secretory and metabolic capacity of the fermenting bacterium b1 determined from the water activity is pm3, which is a honey raw material corresponding to 3 to 5% of the third fermented liquid n3. Was stirred with a 4-fold amount of the third fermentation broth n3 to produce a preliminary fermentation medium pfm3, and the produced preliminary fermentation medium pfm3 was fermented for 2 to 3 days to produce a third fermentation medium m3. The inventors further mixed the produced third fermentation medium m3 with a third fermentation liquid n3 equivalent to 20 times, stirred, and fermented for 30 to 60 days while maintaining the fermentation state at 37 ° C to 40 ° C. This promoted fermentation of the fermenting bacterium b1 by secretory metabolism of an acidic substance, and succeeded in producing a fourth fermentation broth n4 having a pH of 3.7 ±. This is the third fermentation line f3.

The technical features of supplying a certain amount of the fermented medium of fermented sugar to the third fermented liquid n3 to enhance the growth and secretory metabolic capacity of the fermenting bacterium b1 are shown in FIGS. 13 and 14. FIG. 13 is Photograph A and Photograph B showing the results of a comparative test of Sample A and Sample B of the fermentation liquid model of the fourth fermentation liquid n4. Specimen A was produced by fermenting a third fermented medium m3 pre-fermented using honey raw material pm3 equivalent to 5% of the third fermented liquid n3 and a third fermented liquid n3 equivalent to 20 times to 1 L. Fermentation liquid A (corresponding to the fourth fermentation liquid n4). Specimen B is a fermented liquid B produced by putting the honey raw material pm3 into 1 L of the third fermented liquid n3 equivalent to 20 times and fermenting it.

In the sample A, three layers of a translucent third intermediate layer liquid are formed between the third upper surface layer sf3 and the third precipitation layer dep3 in the test container, and the translucent third intermediate layer liquid is the fourth fermentation. Corresponds to liquid n4. According to Photo B, the upper surface layer sf3 is formed in the test container, but the sample B is an extremely thin layer as compared with the sample A, which means that the fermentation by the secretory metabolism of the acidic substance of the fermenting bacterium b1 is as much as the sample A. It is a sign of inactivity, which can be confirmed from FIG.

FIG. 14 is a proof that the fermenting bacterium b1 of the fermented liquid A secretes an acidic substance more actively. The inventors have created a fermentation environment for the fermenting bacterium b1 by using a third fermentation medium m3 in which the honey raw material pm3 is pre-fermented, instead of the third medium in which the honey raw material pm3 remains when the fourth fermentation liquid n4 is produced. Was changed, and a device was devised to proliferate the fermenting bacterium b1 and work to increase the secretion and metabolism of acidic substances again.

The pH 3 produced by fermentation of only the fermenting bacterium b1 without using a fermentation medium using the fourth fermented liquid n4 having a pH of 3.7 ± and the fourth fermented liquid n4 having a pH of 3.7 ± as a starter for 30 to 60 days. 180 days (June) of only fermenting bacteria b1 that does not use a fermentation medium in a fermentation environment at room temperature, using the fifth fermentation liquid n5 with a pH of 3.6 ± and the fifth fermentation liquid n5 with a pH of 3.6 ± as starters. The sixth fermentation broth n6 with a pH of 3.3 ± produced by long-term fermentation for ~ 240 days (August) was cooled after heat sterilization treatment, and 200 mL of the supernatant obtained by precipitating each was sterilized. After the treatment, they were separated into two 100 mL bottles, and the Japan Food Research Laboratories requested the analysis of sugar concentration by the modified Somogie method.

The analysis results are as described above. Since the 4th fermented liquid n4 having a pH of 3.7 ± uses 5% of the honey raw material pm3, 5 g of sugar mass per 100 mL is added to the 3rd fermented liquid n3. The fermented fourth fermented liquor n4 was finally reduced to a sugar mass of 3.93 g. It is the result of being consumed by the action of the fermenting bacterium b1, and similarly, 2.88 g of the fifth fermented liquid n5 and 1.19 g of the sixth fermented liquid n6 are also the result of being consumed by the action of the fermenting bacterium b1.

From these results, the sugar concentration of the 4th fermented liquid n4 is close to 4%, and in the 3rd fermentation line f3, the metabolic activity of the fermenting bacterium b1 that secretes the acidic substance that the inventors have experienced does not stop, and the fermenting bacterium b1 is produced. It is understood that various treatments such as heat sterilization are difficult.

Further, 2.88 g / 100 mL representing the sugar concentration of the fifth fermented liquid n5 and 1.19 g / 100 mL representing the sugar concentration of the sixth fermented liquid n6 are subjected to forcible treatment such as heat sterilization over time. It shows that the sugar concentration can be lowered so that the secretory activity can be easily brought to convergence while promoting the active secretory metabolism of the fermenting bacterium b1.

The step of producing the fifth fermentation broth n5 by fermentation of the fourth fermentation line f4 for 30 to 60 days and the sixth fermentation of the fifth fermentation line f5 for 180 days (June) to 240 days (August). In both steps of producing the fermented liquid n6, spores are formed while promoting the secretory activity of the spore-forming fermenting bacteria contained in the fermenting bacteria b1, and finally the fermenting bacteria b1 is provided with an environment close to a dormant state. It is a process.

(4) Analysis of short-chain fatty acids from the second fermented liquid n2 to the sixth fermented liquid n6

Seven kinds of original bacterial solution b composed of fermenting bacteria of accession number NITEp-02945 to NITEp-02951 deposited and registered in NPMD containing spore-forming Clostridium spp., Which is a constituent requirement of the present invention. The acidic substance produced by the secretory activity of the fermenting bacterium b1 is a short-chain fatty acid containing so-called butyric acid, which forms an intestinal ecology in the living body, as assumed from the acidic substance shown in FIG. This is clarified by a comparative analysis of short-chain fatty acids contained in the second fermentation broth n2 to the sixth fermentation broth n6.

The second fermentation broth n2 is the final product of the first fermentation line f1. The first fermentation line f1, as shown in a conceptual diagram of FIG. 4, a second fermentation tank of the first fermentation liquid n1 2 group first fermentation tank 100 from 1 group of which is generated in the second step p _{1 2} Transfer to 200 to form a fermentation environment that does not use a fermentation medium, and while maintaining the fermentation state at 37 ° C to 40 ° C, the fermentation bacteria b1 of the original bacterial solution b contained in the first fermentation solution n1 having a pH of 5.3 ± It is a second fermentation broth n2 having a pH of 5.0 ± produced by fermentation for 3 to 5 days. The third fermentation broth n3 to the sixth fermentation broth n6 are the final products from the second fermentation line f2 to the fifth fermentation line f5.

Each fermented liquor of the final product of each fermentation line is an original composed of fermenting bacteria of accession number NITEp-02945-5NITEp-02951 deposited and registered with NPMD containing spore-forming Clostridium spp. Observe how the changes are caused by the acidic substances produced by the secretory activity of the seven fermenting bacteria b1 of the bacterial solution b.

Therefore, (1) the second fermentation broth n2 having a pH of 5.0 ± and the second fermentation broth n2, which were produced in the first fermentation line over 55 to 108 days, were produced by fermentation for 8 to 9 days using the second fermentation broth n2 as a starter. (2) A third fermentation broth n3 having a pH of 4.5 ± and a fourth fermentation broth n4 having a pH of 3.7 ± produced by fermentation using the third fermentation broth n3 as a starter for 30 to 60 days. 4 The fifth fermentation broth n5 having a pH of 3.6 ± produced by fermentation using the fermentation broth n4 as a starter for 30 to 60 days is the final fermentation while maintaining the fermentation state at 37 ° C. to 40 ° C. It is a product. (5) The sixth fermentation broth n6 having a pH of 3.3 ± produced by fermentation for 180 to 240 days using the fifth fermentation broth n5 as a starter is in a fermentation environment at room temperature different from (1) to (4). It is the final product of fermentation in.

The fermented liquids (1) to (5) above were both heat-sterilized and then cooled, and 200 mL of the supernatant, which had been precipitated for one day, was sterilized and then separated into two 100 mL bottles. We requested the analysis of short-chain fatty acids by high performance liquid chromatography, and obtained the measurement results of the short-chain fatty acids content of lactic acid, propionic acid, and butyric acid contained in the second fermentation broth n2 to the sixth fermentation broth n6.

The measurement result is shown in FIG. Specifically, it is as follows.
Types of short-chain fatty acids Lactic acid Propionic acid Butyric acid
(Unit / 100 mL)
2nd fermented liquid n2 No detection 0g 0.01g 0.18g
Third fermented liquid n3 0.08g 0.01g 0.30g
4th fermented liquid n4 0.20g 0.02g 0.54g
Fermentation solution n5 0.13g 0.02g 0.59g
6th fermented liquid n6 0.21g 0.02g 0.58g

Lactic acid contained in 100 mL of the second fermentation broth n2 was not detected. Probion is 0.01 g / 100 mL and butyric acid is 0.18 g / 100 mL. The fermented liquor produced by the present invention is fermented using a natural material. A fermented liquid made of a natural material, the content of short-chain fatty acids, especially butyric acid, is 0.30 g / 100 mL for the third fermented liquid n3, 0.54 g / 100 mL for the fourth fermented liquid n4, and a medium or a fermentation medium is used. The evaluation of the butyric acid content that is about to reach 0.60 g / 100 mL in the fifth fermented liquid n5 or the sixth fermented liquid n6 using only the fermenting bacterium b1 contained in the fourth fermented liquid n4 or the fifth fermented liquid n5 is evaluated. However, the content of 6 g of butyric acid per 1000 mL has a great effect on the generation of controlled T cells related to at least anti-inflammatory effect, bone strength improvement, anti-obesity, anti-cancer effect, etc. It is a numerical value that is fully predicted to give.

Finally, the sixth fermentation broth n6 has various natural fermentation substances such as extraction of fermentation metabolites containing accumulated short-chain fatty acids by filtering with a 0.2 μm mesh and using them as food raw materials and foods. There is a possibility of use. Some of the possibilities are shown below.

Action effect of fermented liquid produced by the present invention

The fermented liquor produced by the present invention is a fermented liquor of a natural material having an acidity pH of 3 or more and 4 or less, which contains 5 g or more of colloidal particles and short-chain fatty acid butyric acid not exceeding 50 nm per 1000 mL. Calcium metabolism to the living body is activated by daily intake of an appropriate amount of the fermented liquid, and bone strength and bone metabolism are significant. Osteoporosis model mice were used for the "calcium absorption study test by reversal intestinal test" by the Institute for Natural Materials Exploration Co., Ltd. and the physiology of Gifu University School of Medicine for the functional food test based on the test shown below. It has been confirmed by detailed tests.

Natural Material Exploration Research Institute Co., Ltd. (Representative Director Miko Aoyama) submitted a report on "Calcium absorption study test by inverted intestinal test" in November 2015. In the test, the test animals were male (♂) rats of strain SD with free intake of solid feed CRF-1 (Oriental Yeast Co., Ltd.) during the preliminary breeding period, and distilled water was freely ingested with a water bottle. went. The test object is the sixth fermented liquid n6 (Calbio-7).

The test group of the test animals was set to 5 animals in the Control group (9 intestinal tracts 30, 60, 90 minutes reaction each 3 intestinal tracts) and 5 animals in the 0.1% addition group of fermented liquid n6 (Calbio-7) ( 9 intestinal tracts 30, 60, 90 minutes reaction 3 intestines each) and 5 animals in the 0.5% addition group of fermented liquid n6 (Calbio-7) (9 intestinal tracts 30, 60, 90 minutes reaction 3 intestines each) 3 Depends on the set.

The test items and methods were as follows: (1) The preliminary breeding period was set to 6 days, and the preliminary breeding was performed by free intake of the feed CRF-1 until the start of the main breeding to acclimatize. Next, (2) an absorption test by the inverted intestinal method was performed. It consists of a) adjustment of internal and external fluids, reagent adjustment, external fluid adjustment and internal fluid adjustment, and b) an inverted intestinal tract is created, and c) the reaction time of the inverted intestinal tract is 30 minutes. , 60 minutes and 90 minutes, and finally, 2) calcium measurement was performed. Furthermore, e) it was confirmed by statistical processing that the test result had a risk rate of 5% or less, and it was judged to be significant.
The experimental results of the calcium absorption test by the inverted intestinal method are shown in FIG.

FIG. 21 shows the results of a calcium absorption test by the reversal intestinal method in which the test substance Calbio-7 was administered to a model mouse of an SD male (♂) rat. It shows the "calcium absorption amount" in the upper figure and the "calcium increase amount" in the lower figure. According to the results of a comparative study on the calcium absorption effect by the inverted intestinal method in the upper figure, the amount of calcium increase in the lower figure was significantly higher in the Calbio-7-added group than in the Control group at each calcium source. The calcium absorption rate in the above figure was higher in the calcium lactate source than in the Control group, and was significantly higher in the calcium citrate and calcium carbonate sources in the 0.5% addition group.

To summarize the results of this test, in terms of calcium increase, each concentration in the Calbio-7 group showed a significantly higher value than that in the Control group at all reaction times, and the transition from the external solution to the internal solution for 30 minutes was the maximum. Met. On the other hand, regarding the calcium absorption rate, each concentration of the Calbio-7 group showed a significantly higher value than that of the Control group at all reaction times, and the absorption rate at a reaction time of 60 minutes was the highest value.

Gifu University School of Medicine Physiology confirmed a test report based on detailed test results using osteoporosis model mice in February 2017 by Shinya Minatoguchi, Dean of the Graduate School of Medicine, Gifu University, who was involved in the creation of Abe power of Gifu University School of Medicine physiology. It was stamped and stamped. This report is a bone strength and bone metabolism marker of Calbio-7 administration of the test substance to osteoporosis model mice, which was conducted on the premise of the above-mentioned test report of "Calcium absorption study test by reversal intestinal test" in November 2015. It is about the effect on. A part of the contents is excerpted below.

The test task concerns the effects of administration of the test substance on osteoporosis model mice on bone mineral density and bone metabolism markers. The outline of the test was to examine the preventive effect of the test substance on osteoporosis prevention by continuous administration of the test substance for 8 weeks using estrogen-deficient osteoporosis model mice by bilateral ovariectomy (OVX).

The experimental method is as follows.
The laboratory animal is a 12-week-old rat C57BL / 6 (female ♀, n = 24). In the test group, 6 12-week-old rats C57BL / 6 (female ♀, n = 24) were used in the following 4 groups, usually having rat Sham and bilateral ovariectomized rats OVX.
・ Sham + water (SWW), n = 6
-Sham + Calbio-7 (0.5 mL / day) (SCC), n = 6
・ OVX + water (OWW), n = 6
-OVX + Calbio-7 (0.5 mL / day) (OCC), n = 6
The test substance administration plan is as follows.
C7: Test substance Calbio-7 (6th fermented liquid n6)
The concentration of C7 was adjusted so as to ingest 0.5 mL per day based on the drinking water data.
The test items are data collection of the ratio of the bone formation marker Gla-osteocalcin in the blood and the bone resorption marker Glu-osteocalcin for bone tissue, bone weight and bone strength, and data of the degree of influence test on bone density. ..

The results of the Gla / Glu-osteocalcin ratio, bone weight and bone strength, and the degree of influence on bone density in the test results using estrogen-deficient osteoporosis model mice are shown in FIG. 22.

FIG. 22 shows the ratio of the test group of “Gla / Glu-osteocalcin ratio” by administration of the test substance using an osteoporosis model mouse in an estrogen-deficient state by bilateral ovariectomy (OVX), and further, “bone weight and bone strength”. It is a bar graph and a photograph showing the degree of influence on and the degree of influence on "bone density". By the way, in the "Gla / Glu-osteocalcin ratio", since Gla-osteocalcin in blood is a bone formation marker and Glu-osteocalcin is a bone resorption marker, when the ratio is high, it indicates promotion of bone formation or suppression of bone resorption, and the ratio is low. If the case indicates suppression of bone formation or promotion of bone resorption.

The left figure showing the degree of influence on "bone weight and bone strength" in FIG. 22 is the dry bone weight of the femur (weight corrected). The figure on the right also shows the bone strength of the femur by the (weight-corrected) three-point bending test. Bilateral ovariectomy (OVX) reduced dry bone weight. Although no recovery of this value was observed with Calbio-7 intake, in the bone strength test, Calbio-7 intake was able to significantly prevent the decrease in bone strength due to bilateral ovariectomy (OVX).

The left figure showing the degree of influence on the “bone density” in FIG. 22 is a typical example of the bone mass of the cancellous bone in the femur, which is related to the bone density. The figure on the right shows the bone mass density of cancellous bone (cancellous bone trabecula weight / cancellous bone cavity volume). Bilateral ovariectomy (OVX) resulted in a significant reduction in cancellous trabecular density. This value did not recover with Calbio-7 intake. On the one hand, a high number was shown in the SCC group, although it was not significant.

The above is a part of the excerpt of this report. The experimenters summarized the following evaluation results (1) to (3).

(1) In this experiment, ingestion of Calbio-7 improved the bone strength of the OCC group. However, since there was no difference between the OCC group and the OWW group in the quantitative evaluation of dry bone weight and cancellous trabecular bone, it is considered that factors other than bone tissue (trabecula density) influence the recovery of bone strength. Be done. Regarding the state of bone formation and bone resorption, the value of the OCC group tended to be higher than the value of the OWW group at the 8th week. It is considered that calcium metabolism was activated for some reason.

(2) Since the fluctuation of female hormone balance due to bilateral ovariectomy (OVX) is very strong, it seems that the amount of calcium contained in the normal diet is not sufficient to supplement the consumption. As a result of testing the calcium absorption effect using the inverted intestinal method, the amount of calcium increase and the absorption rate at each calcium source were measured.
The value was significantly higher than that of the Control group.

(3) From (1) and (2), especially ("Reversal" in November 2015, because the decrease in bone strength due to bilateral ovariectomy (OVX) could be significantly prevented by ingestion of Calcio-7. (Refer to "Calcium absorption study test by intestinal test") An additional study was conducted using a diet containing calcium lactate, which had a remarkable rate of absorption and increase, including an increase in bone resorption by bilateral ovariectomy (OVX). It is desirable to study the improvement of fluctuations in each numerical value.

Regarding the function of a fermented liquid of a natural material having an acidity of pH 3 or more and 4 or less, which is produced by the present invention and contains 5 g or more of colloidal particles not exceeding 50 nm and butyric acid of a short-chain fatty acid per 1000 mL, (1) intestine. A test on the effect of ingesting Calbio-7 on the gut flora and a test on (2) anti-cancer effect were conducted in May 2014 at Aitec Lab Co., Ltd. in Kaizu City, Gifu Prefecture (Investigator Masaki Matsuura, Report Creator) It was conducted by Shin Nakamura) and the test was reported. The test substance at this time is Calbio-7 as in the above test.

In study (1), C57BL / 6 mice (male ♂ 7 weeks old) were divided into a Control group and a Calbio-7 administration group (n = 10), water for injection in the Control group, and Calbio- in the Calbio-7 administration group. 7 was orally administered at 0.5 mL / body for 28 consecutive days.

In this study (1), the colon contents were collected after the end of the administration period and analyzed or cryopreserved. Furthermore, bacterial DNA was extracted from the colon contents using a special kit for extracting bacterial nucleic acids. The concentration of the obtained DNA was measured and its purity was confirmed. At the time of DNA extraction, samples of 3 to 4 individuals in the same group were pooled and extracted in equal amounts. Furthermore, using specific primers of lactic acid bacteria, bifidobacteria, and Clostridium bacteria, and universal primers, bacterial DNA was quantified to calculate the relative number of copies of bacterial DNA, and the ratio to the Control group was calculated. ..

The test results regarding the effect of ingesting Calbio-7 on the intestinal flora by the method of test (1) are shown in FIG. FIG. 23 shows the test results on the effect on the intestinal flora when the fermented solution (Calbio-7) produced by the present invention was administered to mice.

The upper figure of FIG. 23 shows that the number of “good bacteria” of benign intestinal bacteria including at least lactic acid bacteria and bifidobacteria in the intestinal flora has doubled. The lower figure of FIG. 23 shows that the number of "bad bacteria" of intestinal malignant bacteria including at least Clostridium-Urelsch in the intestinal flora has been halved.

In test (2), as a result of gene expression analysis by real-time PCR method by gene expression analysis in tumor tissue, specifically, among the genes whose expression fluctuation was observed in DNA microarray (ITL-13-MO-049), Krt6b This is the result of designing a specific primer for (Ketatin6b) and quantitatively analyzing its gene expression by a real-time PCR method.

In this study (2), the tumor tissue of Calbio-7-administered mice and the tumor tissue used were Lewis Lung carcinoma cells of epithelial cell lineage cancer, and injection was performed on the gene expression of Krt6b, which is considered to be a tumor-increasing factor. A comparative analysis was performed with the water administration group. The results showed expression of keratinocyte (keratinocyte) genes such as keratin. In short, as shown in FIG. 24, Krt6b has become a marker for differentiating squamous epithelial cancer among lung cancers, and suppression of the expression of this Krt6b gene was observed.

Summary of test results

Fermentation of a natural material having an acidity of pH 3 or more and 4 or less, which is produced by the present invention and contains about 6.5% to 7.5% of colloidal particles not exceeding 50 nm and 5 g or more of butyric acid, which is a short-chain fatty acid, per 1000 mL. Regarding the function that the liquid gives to the living body, it becomes a functional food or its undiluted solution. The first summary is the data of "increase and absorption" of the calcium absorption test results by the inverted intestinal method shown in FIG. 21, and the test using the estrogen-deficient osteoporosis model mouse shown in FIG. 22. As is clear from the resulting data of "Gla / Glu-Osteocalcin ratio", "effect of bone weight and bone strength", and "effect of bone density", it activates calcium metabolism in the living body and bones. It has a significant effect on strength and bone metabolism.

The second summary is intestinal in the gut flora, as evidenced by the data showing an increase in "lactic acid bacteria" and "bifidobacterium" and a decrease in "Crostridium urerus" shown in FIG. It acts to at least double the lactic acid bacteria and bifidobacteria, which are called "good bacteria" of benign bacteria, and at least halve the crotriduum urellus bacteria, which are called "bad bacteria" of gut flora. Furthermore, as shown in FIG. 24, suppression of Krt6b gene expression in the tumor tissue (Lewis Lung carcinoma-singraft model) of Calbio-7-administered mice was confirmed.

Auxiliary material

Seven types of fermenting bacteria registered as deposits with NPMD (Consignment date: May 16, 2019)

As a result of the isolation culture of the fermenting bacteria used in the fermentation product manufactured by Hiermount Co., Ltd. and the partial base sequence analysis (about 1500 bp) method of 16srDNA (16srRNA gene) for determining the bacterial name, the following bacteria were found. It has been confirmed that it is harmless to humans and animals and can be restored to dryness or freeze-drying.

NITE p-02945
Type of microorganism: Bacteria Cell shape: Bacillus Features: Negative spore formation (non-spore-forming)
Taxonomic position Aneurinibacillus sp. (Genus Anurinibacillus)
Culture conditions: Standard agar medium Culture temperature: 30 ° C Culture period: 48 hours Culture method: Aerobic

NITE p-02946
Type of microorganism: Bacteria Cell shape: Bacillus Features: Negative spore formation (non-spore-forming)
Taxonomic position Brevibacillus sp. (Genus Brevibacillus)
Culture conditions: Standard agar medium Culture temperature: 30 ° C Culture period: 48 hours Culture method: Aerobic

NITE p-02947
Type of microorganism: Bacteria Cell shape: Bacillus Features: Negative spore formation (non-spore-forming)
Taxonomic position Pseudoclavibacter sp. (Genus Pseudoclavibacter)
Culture conditions: Standard agar medium Culture temperature: 30 ° C Culture period: 72 hours Culture method: Aerobic

NITE p-02948
Type of microorganism: Bacteria Cell shape: Bacillus Features: Positive spore formation (spore-like)
Taxonomic position Paenibacillus sp. (Panibacillus sp.)
Culture conditions: Standard agar medium Culture temperature: 30 ° C Culture period: 72 hours Culture method: Aerobic

NITE p-02949
Type of microorganism: Bacteria Cell shape: Bacillus Features: Positive spore formation (spore-like)
Taxonomic position Clostridium sp. (Clostridium sp.)
Culture conditions: GAM Broth "Nissui" agar Culture temperature: 30 ° C Culture period: 48 hours Culture method: Anaerobic

NITE p-02950
Type of microorganism: Bacteria Cell shape: Bacillus Features: Positive spore formation (spore-like)
Taxonomic position Clostridium sp. (Clostridium sp.)
Culture conditions GAM Broth "Nissui" agar Culture temperature: 30 ° C Culture period: 48 hours Culture method: Anaerobic

NITE p-02951
Type of microorganism: Bacteria Cell shape: Bacillus Features: Positive spore formation (spore-like)
Taxonomic position Clostridium sp. (Clostridium sp.)
Culture conditions GAM Broth "Nissui" agar Culture temperature: 30 ° C Culture period: 48 hours Culture method: Anaerobic

Contains colloidal particles and short-chain fatty acids not exceeding 150 nm, acidity
3 or more and 4 or less fermented liquid n1 to n6 1st fermented liquid to 6th fermented liquid pn1 to pn3 Preliminary first fermented liquid to preliminary 3rd fermented liquid w Soft water

m Fermentation medium m1 to m3 1st fermentation medium to 3rd fermentation medium ds Dried soybean fs Fermented soybean gfs Soybean paste pm2 Mixed medium pfm2 Preliminary fermentation mixed medium pm3 Honey raw material pfm3 Preliminary fermentation medium

b Fermentative bacterium solution b1 Fermentative bacterium contained in the original bacterium solution (trust number NITEp-02945-NITEp-02951 registered in NPMD)

sp sponge layer sf1 to sf4 1st upper surface layer to 4th upper surface layer dep1 to dep4 1st sedimentation layer to 4th sedimentation layer

f Fermentation stage f1 to f5 1st fermentation line to 5th fermentation line p _{1 1 to} p ₁ 3 f1 1st to 3rd steps p ₂ 1 / p ₂ 2 f2 1st step / 2nd step p ₃ 1 / P ₃ 2 f3 1st step / 2nd step p ₄ 1 f4 step p ₅ 1 f5 step

10 Fermentation bottle (made of pottery)
20 f1 first medium tank 30 f1 heating pot 40 f2 second medium tank 50 f3 third medium tank 100 f1 first fermentation tank (2 units) with a capacity equivalent to 1000 liters
101 On-off valve using Pascal principle of the first fermentation tank and parallel hydrostatic pressure 110 Extraction device with on-off valve of the first fermentation tank 200 f1 Second fermentation tank with capacity equivalent to 2000 liters 201 Pascal principle of the second fermentation tank On-off valve 210 using parallel hydrostatic pressure 210 Third fermentation tank 301 with a capacity equivalent to 2000 liters of the extraction device 300 f2 having an on-off valve for the second fermentation tank 301 On-off valve 310 using the Pascal principle of the third fermentation tank and parallel hydrostatic pressure Extraction device 320 with on-off valve of the third fermentation tank F2 bag-shaped medium filter 330 f2 circulation pump device 400 f3 2000 liters equivalent capacity of the fourth fermentation tank 401 Pascal principle of the fourth fermentation tank and hydrostatic pressure parallel On-off valve 410 Used An on-off valve with an on-off valve for the 4th fermentation tank 500 f4 5th fermentation tank with a capacity equivalent to 2000 liters 501 An on-off valve using the Pascal principle of the 5th fermentation tank and parallel hydrostatic pressure 510 5th fermentation tank 6th fermentation tank 610 f5 cooling device 620 f5 spare tank with capacity equivalent to 2000 liters of extraction device 600 f5

In this study (1), the colon contents were collected after the end of the administration period and analyzed or cryopreserved. Furthermore, bacterial DNA was extracted from the colon contents using a special kit for extracting bacterial nucleic acids. The concentration of the obtained DNA was measured and its purity was confirmed. At the time of DNA extraction, samples of 3 to 4 individuals in the same group were pooled and extracted in equal amounts. Furthermore lactic acid bacteria, bifidobacteria, specific primers Kurosutori di um bacteria, and, using universal primers, to calculate the number of copies relative bacterial DNA perform quantification of bacterial DNA, it was calculated the ratio Control group.

The upper figure of FIG. 23 shows that the number of “good bacteria” of benign intestinal bacteria including at least lactic acid bacteria and bifidobacteria in the intestinal flora has doubled. The lower portion of FIG. 23 indicates that the "bad bacteria" in intestinal malignant bacteria including at least a Kurosutori di Um U E Arche bacteria of the intestinal flora has been halved.

The second summary, as is clear from the data representing the decrease in shown in Figure 23, "lactic acid bacteria" and increased "Bifidobacteria" and "Kurosutori di Um U E Arche bacteria", intestinal the intestinal flora the lactic acid bacteria and bifidobacteria called "beneficial bacteria" internal benign bacteria is at least doubled, and to act to at least half the Kurosutori di um U E Arche bacteria called "bad bacteria" intestinal malignant bacteria. Further, as shown in FIG. 24, suppression of Krt6b gene expression in the tumor tissue (Lewis Lung carcinoma-singraft model) of Calbio-7-administered mice was confirmed.

Claims (8)

With soft water of pH 7.0-7.6,
A first fermentation medium having a pH of 4.2 to 4.8 produced from dried soybeans, a second fermentation medium having a pH of 4.5 to 5.1 produced from a dried plant containing corn in addition to kuco and taiso, and a honey raw material. Three types of fermentation media consisting of a third fermentation medium having a pH of 4.1 to 4.7 produced from
A source bacterial solution having a pH of 5.0 to 5.6 containing seven fermenting bacteria (accession number NITE p-02945-NITE p-02951 registered as a deposit with NPMD) that produces organic acids containing short-chain fatty acids, and
Using
The soft water, the first fermentation medium, and the original bacterial solution were added to the first fermentation tank at a volume ratio of 1100 L to 1300 L of the soft water, 280 L of the first fermentation medium, and 270 L to 450 L of the original bacterial solution, and mixed to pH 6. By the step of producing the turbid liquid of 1 to 6.7 and the fermentation of the turbid liquid at a fermentation temperature of 37 ° C. to 40 ° C. for 50 to 100 days, a sponge layer containing fermentation gas in the supernatant and a first precipitate in the lower layer. A step of forming three layers composed of a layer and a translucent fermented liquid having a pH of 5.0 to 5.6 between the sponge layer and the first settling layer, and the translucent fermented liquid from the three layers. Step A for producing the first fermentation broth having a pH of 5.0 to 5.6, which includes a step of taking out, and
The step of transferring the first fermentation broth of pH 5.0 to 5.6 to the second fermentation tank and 3 to 5 days depending on the fermentation temperature of the fermentation broth at 37 ° C. to 40 ° C. in a fermentation environment without using a fermentation medium. By fermentation, a first upper surface layer is deposited in the supernatant with bubbles of fermentation gas, a second precipitation layer in which the residue of the first medium is deposited on the lower layer, and a pH of 4.7 to between the first upper surface layer and the second precipitation layer. A second fermented liquid having a pH of 4.7 to 5.3 is produced, which comprises a step of forming a three-layer composed of the translucent fermented liquid of 5.3 and a step of taking out the translucent fermented liquid from the three layers. Step B and
The step of transferring the second fermentation broth having a pH of 4.7 to 5.3 to the third fermentation tank, and the step of enclosing the second fermentation medium in a bag-shaped filter and suspending it in the third fermentation tank to circulate the second fermentation broth. By fermentation for 8 to 9 days at a fermentation temperature of 37 ° C to 40 ° C, a second upper layer of fermentation gas and a two-layer consisting of a turbid fermentation liquid having a pH of 4.2 to 4.8 are formed in the supernatant. A step C for producing a third fermented liquid having a pH of 4.2 to 4.8, which comprises a step of taking out the turbid fermented liquid from the two layers.
A step of transferring the third fermentation broth having a pH of 4.2 to 4.8 to a fourth fermentation tank and mixing it with the third fermentation medium at a volume ratio of 20: 1 to generate a turbid liquid, and a step of producing a turbid liquid from 37 ° C. to the turbid liquid. By fermentation for 30 to 60 days at a fermentation temperature of 40 ° C., the supernatant has a third upper surface layer, the lower layer has a third precipitation layer, and the pH between the third upper surface layer and the third precipitation layer is 3.4 to 4-4. A fourth fermented liquor having a pH of 3.4 to 4.0 is produced, which comprises a step of forming a three-layer composed of a translucent fermented liquor of .0 and a step of taking out the translucent fermented liquor from the three layers. Step D and
30 days to 60 depending on the step of transferring the fourth fermentation broth of pH 3.4 to 4.0 to the fifth fermentation tank and the fermentation temperature of the fourth fermentation broth at 37 ° C. to 40 ° C. in a fermentation environment without using a fermentation medium. A step of producing a translucent fermented liquid having a pH of 3.3 to 3.9 and a fourth precipitation layer by daily fermentation, and a step of taking out the translucent fermented liquid of pH 3.3 to 3.9. Step E to generate the fifth fermented liquid and
A step of transferring the fifth fermentation liquid having a pH of 3.3 to 3.9 to a spare tank of a cooling device and temporarily rapidly cooling the fifth fermentation liquid having a fermentation temperature of 37 ° C. to 40 ° C. to 5 ° C. or lower with the cooling device. By the step of returning the rapidly cooled fifth fermented liquid to a normal temperature state to generate a fermented liquid at room temperature and fermenting the fermented liquid at room temperature for 180 to 240 days, the pH is 3.0 to 3.6. A step F for producing a sixth fermented liquid having a pH of 3.0 to 3.6, which comprises a step of producing a fermented liquid and a step for taking out the transparent fermented liquid.
including,
A method for producing a fermentation broth having an acidity of pH 3 to 4, which contains colloidal particles having a particle size not exceeding 50 nm and short-chain fatty acids of butyric acid, propionic acid, and lactic acid.
In the step A, two first fermentation tanks are used in the step A, and the soft water, the first fermentation medium, and the original bacterial solution are added to each of the first fermentation tanks in 1100 L to 1300 L of the soft water. 1 Fermentation medium 280 L, the original bacterial solution 270 L to 450 L, evenly charged and mixed to produce a turbid solution with a pH of 6.1 to 6.7, and the fermentation temperature of the turbid solution at 37 ° C to 40 ° C. After 50 to 100 days of fermentation, a sponge layer containing fermentation gas in the supernatant, a first precipitation layer in the lower layer, and a translucent pH of 5.0 to 5.6 between the sponge layer and the first precipitation layer. A step A'for producing a first fermented liquid having a pH of 5.0 to 5.6, which comprises a step of forming a three-layered fermented liquid and a step of taking out the translucent fermented liquid from the three layers.
The step B is a step of transferring each of the first fermentation broths having a pH of 5.0 to 5.6 to one second fermentation tank and mixing them to produce a fermentation broth having uniform acidity, and no fermentation medium is used. By fermentation of the fermentation broth at a fermentation temperature of 37 ° C. to 40 ° C. for 3 to 5 days in a fermentation environment, a second precipitate in which the residue of the first medium was deposited on the first upper layer and the lower layer by the bubbles of fermentation gas. A step of forming a three layer composed of a translucent fermentation liquid having a pH of 4.7 to 5.3 between the layer and the first upper surface layer and the second precipitation layer, and the translucent fermentation from the three layers. The method according to claim 1, wherein the step B'is to produce a second fermentation broth having a pH of 4.7 to 5.3, which comprises a step of taking out the liquor.
The method according to claim 1 or 2, wherein the mass ratio of the butyric acid to the short-chain fatty acids is 0.5 g to 0.6 g per 100 mL of the fermentation broth.
Assuming that the first fermentation broth formed in three layers in the first fermentation tank is 1200 L, the step of producing the first fermentation medium having a pH of 4.2 to 4.8 is
In each of four or more fermentation bottles equipped with a valve function lid, 6.5 kg of dried soybeans rehydrated in at least 20 L of the soft water was immersed, and 250 g of sugar chains and 140 cc of the original bacteria were immersed. The process of adding liquid and
A step of producing a first preliminary fermented liquid and fermented soybean equivalent to 35 L in each of the fermented bottles by fermentation for 3 days (68 hours to 74 hours) at a fermentation temperature of 37 ° C. to 40 ° C. in the fermented bottles.
A step of taking out the fermented soybean from each of the fermentation bottles, grinding the fermented soybean into a paste by a pulverizing means, and mixing with the first preliminary fermented liquid to produce 35 L of the first preliminary fermented liquid and soybean paste.
The 35 L equivalent of the first preliminary fermented liquid and the soybean paste produced in the fermented bottle are transferred to a heating kettle and gradually heated to 55 to 60 ° C., and 180 L equivalent of the first preliminary fermented liquid and the soybean are combined. The process of producing the paste and
The first preliminary fermentation broth equivalent to 180 L and the soybean paste are transferred from the heating kettle to a first medium tank in which 100 L equivalent of soft water is previously charged for cooling, and stirred so as not to be exposed to the outside air, which is equivalent to 280 L. The method according to any one of claims 1 to 3, which comprises a step of producing the first fermentation medium of the above.
Assuming that the first fermentation broth formed in three layers in the first fermentation tank is 1200 L, the turbid liquid having a pH of 6.1 to 6.7 produced in the first fermentation tank has a pH of 4. The equivalent of 280 L of the first fermentation medium of 2 to 4.8, the equivalent of 270 L to 450 L of the original bacterial solution having a pH of 5.0 to 5.6, and the equivalent of 1000 L to 1300 L of soft water having a pH of 7.0 to 7.6 are described above. The method according to any one of claims 1 to 4, wherein the preliminary second fermentation broth has a pH of 6.1 to 6.7, which is charged into the first fermentation tank, stirred and mixed to be equivalent to 1800 L.
Assuming that 1050 to 1100 L of the third fermentation broth is produced, the method for producing the second fermentation medium having a pH of 4.5 to 5.1 is as follows.
A mixed medium in which each of turmeric in addition to the dried plant kukoshi and turmeric is mixed in a weight ratio of 200 to 210 g of taiso, 110 to 120 g of kukoshi, and 25 to 30 g of turmeric with respect to the total amount equivalent to 35 L of the second fermentation broth. And the process of generating
35 L equivalent of the mixed medium and the second fermented liquor is put into a second medium tank, and the mixed medium and the second fermented liquor are fermented for 2 to 3 days to prepare a pH of 4.5 to 5.1. The step of producing the fermentation mixture medium and the preliminary third fermentation liquid, and
A step of taking out the preliminary fermentation mixed medium, crushing it by a pulverizing means, returning it to the second medium tank, blending it with the preliminary third fermentation solution, and producing a fermentation mixed medium having a pH of 4.5 to 5.1 equivalent to 35 L. The method according to any one of claims 1 to 5, which comprises.
Assuming that 1000 to 150 L of the fourth fermentation broth is produced, the method for producing the third fermentation medium is as follows.
The honey raw material corresponding to 3 to 5% of the third fermented liquid and the third fermented liquid corresponding to four times as much as the honey raw material are transferred to a third medium tank so as not to be exposed to the outside air, and stirred to prepare. The process of producing the fermentation medium and
Claims 1 to 6 include a step of producing a third fermentation medium having a pH of 4.1 to 4.7 by fermentation of the preliminary fermentation medium at a fermentation temperature of 37 ° C. to 40 ° C. for 2 to 3 days. The method described in either.
According to any one of claims 1 to 7, the mixed solution of the step D for producing the fourth fermentation broth is mixed with the third fermentation broth equivalent to 20 times the amount of the third fermentation medium. The method described.