JPWO2014084340A1 - Yogurt and method for producing the same, method for producing lactic acid bacteria extracellular functional product, and lactic acid bacteria extracellular functional product production agent - Google Patents

Abstract

The subject of this invention is providing the new method which can increase the production amount of the functional product derived from lactic acid bacteria efficiently. The yogurt production method according to the present invention includes a pH buffer addition step and a fermentation step. In the pH buffer addition step, a pH buffer is added to the yogurt raw material. In the fermentation process, the yogurt raw material to which the pH buffer is added is fermented by lactic acid bacteria.

Description

  The present invention relates to a yogurt production method for increasing the production amount of a functional product derived from lactic acid bacteria, and a yogurt produced by the yogurt production method. In addition, the present invention relates to a method for producing a lactic acid bacterium extracellular functional product that increases the production amount of the lactic acid bacterium extracellular functional product, and a lactic acid bacterium extracellular functional product enhancer used in the method.

  It is known that by taking lactic acid bacteria and functional products derived from lactic acid bacteria, the bacterial flora in the gastrointestinal tract is improved and has beneficial effects on the host (maintenance of health and reduction of disease risk). For example, Lactobacillus delbruechii subsp. Bulgaricus (hereinafter also referred to as “Bulgaria bacterium”) is one of lactic acid bacteria used as a yogurt starter in the production of fermented milk. Among these Bulgarian bacteria, there are many strains that produce exopolysaccharide (EPS). And this EPS is known not only to contribute to the physical properties and stability of fermented milk products, but also to enjoy the probiotic effect. For example, Lactobacillus delbruechii subsp. It is known that EPS produced by Bulgaricus OLL1073R-1 strain (hereinafter also referred to as “OLL1073R-1 strain”) has an effect of preventing autoimmune diseases. In addition, it is known that fermented milk produced using this strain has effects such as activation of NK cells, reduction of common cold, and anti-influenza. (For example, refer to Patent Documents 1-3). Thus, functional foods and supplements that contribute to health can be provided by using lactic acid bacteria that produce EPS or EPS that is produced by lactic acid bacteria or the like.

  By the way, in order to efficiently manufacture such functional foods and supplements, it is necessary to increase the EPS production amount in the functional foods.

  In order to increase EPS derived from lactic acid bacteria as well as Bulgaria bacteria, various methods have been proposed in the past. For example, Patent Document 4 discloses that a high concentration of EPS is produced in a culture solution by culturing lactic acid bacteria having kefiran-producing ability such as Lactobacillus kefiranofaciens in a medium containing peptone, yeast extract, and unsaturated fatty acid or ester thereof. It is disclosed that it can be produced. Patent Document 5 discloses that EPS derived from lactic acid bacteria is remarkably increased by adding whey protein or soybean protein to milk and fermenting with lactococcus lactis subspecies cremolith.

JP 2000-247895 A JP 2005-194259 A International Publication Pamphlet WO2011 / 065300 JP 2011-250756 A JP 2011-51268 A

  The subject of this invention is providing the new method which can increase the production amount of the functional product derived from lactic acid bacteria efficiently.

  The yogurt manufacturing method which concerns on 1 aspect of this invention is equipped with a pH buffer addition process and a fermentation process. In the pH buffer addition step, a pH buffer is added to the yogurt raw material. In the fermentation process, a yogurt raw material to which a pH buffer is added (hereinafter referred to as “pH buffer added yogurt raw material”) is fermented by lactic acid bacteria. In addition, the addition of lactic acid bacteria to the yogurt raw material may be before the addition of the pH buffering agent or after the addition.

  As a result of intensive studies by the inventors of the present application, as described in the above-mentioned yogurt production method, by adding a pH buffer to the yogurt raw material and then fermenting the yogurt raw material, the time in the pH region where lactic acid bacteria can grow is extended. As a result, it was revealed that the production amount of functional products derived from lactic acid bacteria in yogurt can be increased. That is, this yogurt manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria.

  By the way, in this yogurt manufacturing method, it is preferable that lactic acid bacteria contain at least lactic acid bacteria which have the ability to produce lactic acid bacteria extracellular polysaccharides. And it is preferable that the lactic acid bacteria which have the ability to produce lactic acid bacteria exopolysaccharides include at least Lactobacillus delbruecki subspice bulgaricus lactic acid bacteria. In such a case, the lactic acid bacteria having the ability to produce lactic acid bacteria exopolysaccharides may be Lactobacillus delbruecky subspice bulgaricus lactic acid bacteria alone, or Lactobacillus delbruecki subspice bulgaricus sp. And lactic acid bacteria mixture containing other lactic acid bacteria.

  Moreover, in this yogurt manufacturing method, it is preferable that a pH buffer is a phosphate. When a mixture of a lactic acid bacterium of Lactobacillus delbruecki subspice bulgaricus species and a lactic acid bacterium of Streptococcus thermophilus species (hereinafter also referred to as “thermophilus bacterium”) is used as the lactic acid bacterium, This is because it is possible to promote the growth of Lactobacillus delbruecki, subspice bulgaricus lactic acid bacteria while suppressing the growth of the bacteria. In addition, it is preferable that this phosphate is an alkali metal salt. Furthermore, the phosphate is preferably at least one phosphate selected from the group consisting of disodium hydrogen phosphate and dipotassium hydrogen phosphate.

  Furthermore, in this yogurt manufacturing method, it is preferable that the yogurt raw material contains non-fat milk solid content in the range of 8 wt% or more and 20 wt% or less. Here, the yoghurt raw material means that before adding the pH buffering agent.

  In addition, in this yogurt production method, in the fermentation step, it is preferable that the pH buffer-added yogurt raw material is fermented by lactic acid bacteria at a temperature within the range of 30 ° C. or higher and 40 ° C. or lower.

  The yogurt produced by the above-mentioned yogurt production method contains lactic acid bacteria and a pH buffer. In this yogurt, the pH buffer is uniformly dissolved. Here, the lactic acid bacteria and the pH buffer are as described above. In addition, the pH buffer in this yogurt is, for example, a chromatography technique such as high performance liquid chromatography, or a nuclear magnetic resonance (NMR) technique (for example, Szlyk E, Hrynczyszyn P, “Phosphate additives determination in meat products by 31- It can be detected by, for example, phosphorus nuclear magnetic resonance using new internal reference standard: hexamethylphosphoroamide phosphate solution, Talanta. 2011 Mar 15; 84 (1): 199-203. doi: 10.1016 / j.talanta.2010.12.046).

  Moreover, it is preferable that the above-mentioned yogurt further contains a lactic acid bacterial extracellular functional product (for example, a lactic acid bacterial extracellular polysaccharide). In such a case, it is preferable that the lactic acid bacterial extracellular functional product is contained in an amount within the range of 40 mg / kg to 100 mg / kg with respect to the total amount of yogurt.

  The method for producing an extracellular functional product of lactic acid bacteria according to another aspect of the present invention includes a pH buffer addition step and a fermentation step. In the pH buffer addition step, a pH buffer is added to the milk raw material. In the fermentation process, the milk raw material to which the pH buffer is added is fermented by a lactic acid bacterium that produces a lactic acid bacteria extracellular functional product (hereinafter also referred to as “extracellular functional product-producing lactic acid bacteria”). The lactic acid bacterium may be added to the milk raw material before or after the addition of the pH buffering agent.

  As a result of intensive studies by the inventors of the present application, the extracellular functional product is produced by adding a pH buffer to the milk raw material and then fermenting the milk raw material in accordance with the above-described method for producing an extracellular functional product of lactic acid bacteria. It has been clarified that the time in the pH region where the produced lactic acid bacteria can grow can be extended, and as a result, the production amount of the functional product derived from the lactic acid bacteria can be increased. That is, this lactic acid bacteria extracorporeal functional product manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria. That is, in this aspect, the pH buffer functions as an active ingredient of the lactic acid bacteria extracellular functional product increaser.

  In addition, the above-mentioned method for producing an extracellular functional product of a lactic acid bacterium has a different viewpoint: “A lactic acid bacterial extracellular functional product in which a lactic acid bacterium is fermented by adding (adding) a pH buffer to the milk material. "Production method", "Production method of lactic acid bacteria functional product by adding (adding) a pH buffer to milk material", "Combination (addition of pH buffering agent to dairy material to increase production of lactic acid bacteria functional product" ) "And" use of pH buffering agent for milk raw material to increase production of lactic acid bacteria extracorporeal functional product (method of use) ".

  In the yogurt production method according to one aspect of the present invention, by adding a pH buffer to the yogurt raw material and then fermenting the yogurt raw material, the time in the pH region where the lactic acid bacteria can grow can be extended. The production amount of functional products derived from lactic acid bacteria in yogurt can be increased. That is, this yogurt manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria.

  In addition, in the method for producing a lactic acid bacteria extracellular functional product according to another aspect of the present invention, by adding a pH buffer to the milk material and then fermenting the milk material, the time in the pH region where the lactic acid bacteria can grow is extended. As a result, the production amount of the functional product derived from lactic acid bacteria can be increased. That is, this lactic acid bacteria extracorporeal functional product manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria.

  Hereinafter, although the form for implementing this invention is demonstrated in detail, this invention is not limited to each embodiment described below.

-First embodiment-
The yogurt according to the present embodiment is manufactured through a pH buffer addition process and a fermentation process. In the pH buffer addition step, a pH buffer is added to the yogurt raw material. In the fermentation process, a yogurt raw material to which a pH buffer is added (hereinafter referred to as “pH buffer added yogurt raw material”) is fermented by lactic acid bacteria.

  In the present embodiment, yogurt refers to “fermented milk” and “lactic acid bacteria beverage” as defined by a ministerial ordinance such as milk. “Fermented milk” in the ministerial ordinance of milk, etc. means “milk or milk containing non-fat milk solids equal to or higher than this, fermented with lactic acid bacteria or yeast to make paste or liquid” or “frozen Defined as "thing". Fermented milk is “hard yogurt fermented and solidified after filling in container (solid fermented milk, set type yogurt)”, “soft yogurt (fermented fermented milk) filled with container after fermentation and filled into containers”, “ Soft yogurt is roughly classified into drink yogurt (liquid fermented milk) that is further crushed with a homogenizer to enhance liquid properties. In addition, “lactic acid bacteria beverage” in a ministerial ordinance such as milk means “a beverage (excluding fermented milk) obtained by processing or fermenting milk or the like with lactic acid bacteria or yeast”.

  The yogurt raw material includes at least one of milk, dairy products, and milk protein. Examples of yogurt ingredients include animal milk such as milk, sheep and goats, processed products thereof, sterilized milk, skim milk, full fat milk powder, partially skimmed milk, skim milk powder, full fat concentrated milk, skim concentrated milk, cream, Butter, buttermilk, whey, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), α-casein, β- Casein, κ-casein, non-protein nitrogen and other milk ingredients, sugar, sugar, modified starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, sweetener, organic Examples include acids (malic acid, citric acid, lactic acid, tartaric acid, etc.), fragrances, and water. The yogurt raw material can be obtained by mixing conventional raw materials as described above and dissolving them while heating. Gelling agents such as gelatin, agar, pectin, and carboxymethylcellulose (CMC), thickeners, and stabilizers may be added to the yogurt raw material. In such a case, a raw material for yogurt is prepared by preliminarily heating and dissolving a stabilizer such as gelatin in a solvent such as water and mixing this aqueous stabilizer solution with other components.

  Moreover, it is preferable that non-fat milk solid content (henceforth "SNF") of this yogurt raw material exists in the range of 8 weight% or more and 20 weight% or less. In the yogurt production method according to the present embodiment, when Bulgarian bacteria and Thermophilus bacteria that produce EPS are employed as lactic acid bacteria and phosphate is employed as a pH buffering agent, while maintaining the flavor of yogurt in a good manner, This is because the exopolysaccharide content (hereinafter referred to as “EPS”) can be increased. The SNF of the yogurt raw material is more preferably in the range of 8.5% by weight to 18.5% by weight, still more preferably in the range of 9% by weight to 15% by weight. It is particularly preferable that the amount be in the range of 5% by weight to 14% by weight.

In this embodiment, the pH buffer is not particularly limited, but is particularly preferably a phosphate. In the present embodiment, the phosphate includes a hydrate. Examples of the phosphate include monosodium dihydrogen phosphate (NaH ₂ PO ₄ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ), dipotassium dihydrogen phosphate (KH ₂ PO ₄ ), and dihydrogen phosphate. potassium _(K 2 HPO _4), and the like. In addition, these phosphates may be used independently and may be used together. In the yogurt production method according to the present embodiment, it is preferable to use disodium hydrogen phosphate (Na ₂ HPO ₄ ) and dipotassium hydrogen phosphate (K ₂ HPO ₄ ) alone or in combination, and disodium hydrogen phosphate. It is particularly preferred to use (Na ₂ HPO ₄ ) alone.

  In addition, in the pH buffer addition step of the yogurt production method according to this embodiment, an amount of the pH buffer in the range of 0.05 wt% or more and 0.55 wt% or less is added to the yogurt raw material. preferable. In the yogurt production method according to this embodiment, when Bulgarian bacteria and Thermophilus bacteria that produce EPS are employed as lactic acid bacteria and phosphate is employed as the pH buffering agent, the added amount of the pH buffering agent is within this range. This is because, while maintaining good yogurt card strength (card tension) and flavor, the growth of Bulgarian bacteria can be promoted and the production of EPS derived from Bulgarian bacteria can be enhanced. The addition amount of the pH buffering agent is more preferably in the range of 0.08 wt% or more and 0.45 wt% or less, and is preferably in the range of 0.1 wt% or more and 0.35 wt% or less. Particularly preferred.

  In the present embodiment, the lactic acid bacterium is an extracellular functional product such as lactic acid bacterium that produces EPS (hereinafter referred to as “extracellular functional product-producing lactic acid bacterium”), such as Lactobacillus delbruecki subspice bulgari. Lactobacillus species of Lactobacillus delbruechii subsp. Bulgaricus (hereinafter also referred to as “Bulgaria bacterium”). In the present embodiment, the lactic acid bacterium may be any strain as long as it is an extracellular functional product-producing lactic acid bacterium, but Lactobacillus delbruechii subsp. bulgaricus OLL1073R-1 strain (FERM BP-10741) is preferred. In this embodiment, it is preferable to mainly use a Bulgarian bacterium, but other lactic acid bacteria that produce EPS (hereinafter referred to as “EPS-producing lactic acid bacteria”), such as Lactobacillus bulgaricus, Lactococcus, etc. -Lactococcus lactis ssp. Cremoris etc. may be used in combination with Bulgarian bacteria.

  In addition, lactic acid bacteria other than EPS-producing lactic acid bacteria, such as Thermophilus bacteria, Lactobacillus gasseri (Lactobacillus gasseri), bifidobacteria, propionic acid bacteria and other lactic acid bacteria generally used for the production of fermented milk, You may use together. Moreover, you may add yeast with the said lactic acid bacteria. In addition, in the yogurt manufacturing method which concerns on this embodiment, it is especially preferable to use together the Bulgarian bacterium and Thermophilus bacterium which produce EPS as a lactic acid bacterium.

  In the fermentation process of the yogurt manufacturing method according to the present embodiment, it is preferable that the fermentation temperature be in the range of 30 ° C. or higher and 40 ° C. or lower. This is because, when Bulgarian bacteria and Thermophilus bacteria that produce EPS are employed as lactic acid bacteria and phosphate is employed as a pH buffering agent, the EPS content in yogurt can be increased. The fermentation temperature is more preferably in the range of 32 ° C. or more and 39 ° C. or less, and further preferably in the range of 34 ° C. or more and 38 ° C. or less.

  The extracellular functional product-producing lactic acid bacterium according to this embodiment preferably produces an extracellular functional product in an amount within the range of 40 mg / kg to 100 mg / kg with respect to the total amount. For example, when Bulgarian bacteria and Thermophilus bacteria producing EPS are used as lactic acid bacteria and phosphate is used as a pH buffering agent, when an amount of EPS within the above range is produced, the pH buffering agent is not added. This is because it is recognized that the ratio of the EPS production amount when the pH buffer agent is added to the EPS production amount is increased, that is, the effect of adding the pH buffer agent is enhanced. The extracellular functional product-producing lactic acid bacterium preferably produces an extracellular functional product in an amount in the range of 42 mg / kg to 100 mg / kg, and more than 44 mg / kg to 100 mg. It is more preferable to produce an extracellular functional product in an amount within the range of / kg or less, and to produce an extracellular functional product in an amount within the range of 48 mg / kg or more and 100 mg / kg or less. More preferably, it is particularly preferable to produce an extracellular functional product in an amount in the range of 50 mg / kg to 100 mg / kg.

  In the yogurt production method according to the present embodiment, the production amount of the extracellular functional product is preferably 1.05 times or more, 1.1 times or more of the production amount when no pH buffer is added. Is more preferably 1.2 times or more, and particularly preferably 1.25 times or more. For example, when Bulgarian bacteria and Thermophilus bacteria producing EPS are used as lactic acid bacteria, and phosphate is used as a pH buffering agent, the EPS production amount when adding a pH buffering agent is the EPS production when no pH buffering agent is added. It may be 1.25 times or more of the amount.

-Second Embodiment-
The lactic acid bacterial extracellular functional product according to the present embodiment is produced through a pH buffer addition step and a fermentation step. In the pH buffer addition step, a pH buffer is added to the milk raw material. In the fermentation process, the milk raw material to which the pH buffer is added is fermented by lactic acid bacteria that produce lactic acid bacteria extracorporeal functional products.

  In addition, the lactic acid bacteria extracellular functional product which concerns on this embodiment is the same as the yogurt manufacturing method which concerns on 1st Embodiment except a yoghurt raw material being used as a milk raw material. Moreover, the “lactic acid extracellular functional product” referred to in the present embodiment is the same as the “extracellular functional product” referred to in the first embodiment. And the “milk raw material” here means animal milk such as cow's milk, sheep, goat, etc., processed products, sterilized milk, skim milk, whole milk powder, partially skimmed milk, skimmed milk powder, whole fat concentrated milk, skimmed milk Concentrated milk, cream, butter, buttermilk, whey, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), α -Casein, β-casein, κ-casein, non-protein nitrogen and the like. The non-fat milk solid content (hereinafter referred to as “SNF”) of the milk raw material is preferably in the range of 8% by weight to 20% by weight, and in the range of 8.5% by weight to 18.5% by weight. More preferably, it is more preferably in the range of 9% by weight to 15% by weight, and particularly preferably in the range of 9.5% by weight to 14% by weight.

  Further, the production amount of the lactic acid bacteria extracellular functional product according to the present embodiment is preferably 1.05 times or more, 1.1 times the production amount of the lactic acid bacteria extracellular functional product when no pH buffer is added. More preferably, it is 1.2 times or more, more preferably 1.25 times or more.

<Example>
Hereinafter, preferred examples of the present invention will be described, but the present invention is not limited to the above-described embodiments and the following examples, and various modifications are made within the technical scope grasped from the description of the scope of claims. Is possible. Note that Lactobacillus delbruechii subsp. bulgaricus OLL1073R-1 strain (hereinafter referred to as OLL1073R-1 strain) was issued on November 29, 2006 (contract date), National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1-1 Tsukuba, Tsukuba City, Ibaraki Prefecture) In the center center 6), it is a lactic acid bacterium that has been deposited internationally under the Budapest Treaty under the accession number FERM BP-10741. Streptococcus thermophilus OLS3059 strain was consigned to the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Tsukuba Center 1-1-1 Tsukuba Center Central 6th, Ibaraki Prefecture) on December 15, 2006 (contract date). It is a lactic acid bacterium that has been deposited internationally under the Budapest Treaty as FERM BP-10740 by number. In addition, the patent microorganisms deposit work of the National Institute of Advanced Industrial Science and Technology patent biological depository center was succeeded by the National Institute of Technology and Evaluation on April 1, 2012. As of April 1, 2013, the Biological Depositary Center has been relocated to 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan.

(Production example)
(1) Preparation of yogurt raw material mix A (SNF = 9.4% by weight) 50 kg of raw milk, 5.49 kg of skim milk powder, 1.5 kg of unsalted butter and 43.01 kg of water were mixed, and the mixture was mixed at 95 ° C. for 5 After sterilization by heating for minutes, the mixture was cooled to around 37 ° C. to prepare a yogurt raw material mix A.

(2) Preparation of yogurt raw material mix B (SNF = 10.2% by weight) 25 kg raw milk, 18.4 kg nonfat concentrated milk, 0.96 kg nonfat dry milk, 1.31 kg unsalted butter and 45.75 kg water are mixed, The mixture was sterilized by heating at 95 ° C. for 5 minutes and then cooled to around 37 ° C. to prepare a yogurt raw material mix B having a non-fat milk solid content (SNF) higher than that of the yogurt raw material mix A.

-Verification of the effect of phosphate addition on EPS production-
It is a mixed culture of Thermophilus OLS3059 strain (Streptcoccus thermophilus OLS3059, hereinafter also referred to as OLS3059 strain) and 1073R-1 strain which is a lactic acid bacterium of Lactobacillus delbruecki subspice bulgaricus species that produces polysaccharides After inoculating the lactic acid bacteria starter (yoghurt starter) into the yogurt raw material mix A so that the lactic acid bacterium starter is contained in 2% by weight with respect to the total amount, dispense 20 mL of the yoghurt raw material mix A containing the lactic acid bacteria starter into 5 test tubes. Then, five samples 1-1, 1-2, 1-3, 1-4, and 1-5 were prepared. Then, the sample 1-1 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.5 wt% of the total amount. The sample _{1-2, Na} 2 Na 2 HPO as HPO ₄ comprises 0.4% by weight based on the total amount and _NaH 2 PO ₄ is contained 0.1% by weight based on the total amount ₄ and _NaH 2 PO ₄ Was added. The Sample _1-3, was added _Na 2 HPO ₄ and _NaH 2 PO ₄ as Na 2 HPO ₄ and _NaH 2 PO ₄ is contained 0.25 wt% respectively of the total amount. The sample 1-4 _was added _Na 2 HPO ₄ and _NaH 2 PO ₄ as Na 2 HPO ₄ and _NaH 2 PO ₄ is contained 0.5 wt% respectively of the total amount. In addition, phosphate was not added to Sample 1-5 (control). Then, each said sample was immersed in a 43 degreeC thermostat and fermented. When the pH of each sample reached 4.4 to 4.5, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. About each sample, time to fermentation stop (fermentation time), acidity, EPS content, the number of Bulgaria bacteria, and the number of thermophilus bacteria were measured (refer Table 1).

  The pH was measured with a pH meter (TOA-HM50V, manufactured by Toa DKK Corporation) using a glass electrode. The acidity was measured by the following procedure. 9.00 g was sampled from each sample, and 500 μL of phenolphthalein was added to the sample. Then, the preparative sample was titrated with 0.1N sodium hydroxide, and the end point was a time point at which the faint red color did not disappear for 30 seconds.

Moreover, the EPS content of yogurt was measured by the following procedure. First, 10 g of each sample was weighed and placed in a 50 mL tube, and 1 mL of 100% trichloroacetic acid was added thereto. Next, after the contents were stirred, the contents were allowed to stand at about 4 ° C. for about 10 minutes. The contents were then centrifuged at 12,000 × g relative centrifugal force for 20 minutes at a temperature of 4 ° C., and the supernatant was transferred to a new 50 mL tube. Then, while stirring this supernatant, cold ethanol twice the amount of the supernatant was gradually added to the supernatant, and the supernatant and cold ethanol were mixed thoroughly. Subsequently, the mixture was allowed to stand overnight at a temperature of about 4 ° C., and then the mixture was centrifuged under the same conditions as described above. Thereafter, the supernatant of the mixture was discarded, and 10 mL of purified water was added to the precipitate to completely dissolve the precipitate in purified water. 150 μL of the aqueous solution was injected into the HPLC using a syringe with a filter having a diameter of 0.45 μm. The ratio of the “peak area of a single peak detected by the RI detector around 16 minutes after the start of injection” to the “total peak area” was taken as the EPS content. The analytical operation conditions of HPLC are as follows.
HPLC system: Aquity H-class (Waters)
Column: OHpak 806HQ (Shodex) + SB-G (Shodex)
Column temperature: 40 ° C
Solvent: 0.2 M NaCl aqueous solution Flow rate: 0.5 mL / min
Detector: RI detector 2414 (Waters), detection temperature 40 ° C
Sample injection: 150 μL
Analysis time: 50 min

Table 1 shows the influence of the phosphate addition amount on the EPS content. In Samples 1-1, 1-2, 1-3, and 1-4 to which phosphate was added, the fermentation time was longer than that of Control Sample 1-5, and the acidity at the end of fermentation was also 1 or more. . The addition of phosphate resulted in increased EPS production of yogurt. Incidentally, in the case of adding only _Na 2 HPO ₄ as a phosphate (Sample 1-1), EPS content was the highest. In addition, the number of Bulgarian bacteria increased with the addition of phosphate, whereas the number of Thermophilus decreased with increasing Na ₂ HPO ₄ concentration.

From these results, the fermentation time in the pH range where lactic acid bacteria can grow can be extended by the addition of phosphate, and it is considered that the number of Bulgarian bacteria increased due to this effect. Furthermore, since a decrease in growth of Thermophilus was observed by the addition of phosphate, particularly Na ₂ HPO ₄ , it is considered that the decrease in growth of Thermophilus also contributed to an increase in EPS derived from Bulgaria.

-Verification of the effect of phosphate type on EPS production-
Lactic acid bacteria starter (yoghurt starter), which is a mixed culture of OLS 3059 and 1073R-1 strains, was inoculated into 500 g of yoghurt raw material mix A so that the lactic acid bacteria starter was contained at 2% by weight with respect to the total amount, and then entered into the lactic acid bacteria starter The yogurt raw material mix A was dispensed into 6 test tubes each 20 mL to prepare 6 samples 2-1, 2-2, 2-3, 2-4, 2-5, 2-6. Then, the sample 2-1 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.5 wt% of the total amount. The sample 2-2 _was added _K 2 HPO ₄ as K 2 HPO ₄ is contained 0.61 wt% of the total amount. The molar concentration of K ₂ HPO ₄ in sample 2-2 is the same as the molar concentration of Na ₂ HPO ₄ in sample 2-1. In Sample 2-3, NaCl was added so that NaCl was contained in an amount of 0.21% by weight based on the total amount. The molar concentration of NaCl in Sample 2-3 is the same as the molar concentration of Na ₂ HPO ₄ in Sample 2-1. The sample 2-4 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.3 wt% of the total amount. The sample 2-5 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.1% by weight based on the total amount. In addition, phosphate was not added to Sample 2-6 (control). Then, each said sample was immersed in a 43 degreeC thermostat and fermented. When the pH of each sample reached 4.4 to 4.5, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. About each sample, time to fermentation stop (fermentation time), acidity, EPS content, the number of Bulgaria bacteria, and the number of thermophilus bacteria were measured (refer Table 2). These physical property values were measured in the same manner as in Example 1.

Table 2 shows the influence of the type of phosphate on the EPS content. In Samples 2-1, 2-4, and 2-5 to which only Na ₂ HPO ₄ was added, the fermentation time was increased and the EPS content was increased as the amount of Na ₂ HPO ₄ added was increased. In addition, the number of Bulgarian bacteria increased and the number of Thermofilus bacteria decreased as the amount of Na ₂ HPO ₄ added increased. Here, the factors affecting the decrease in the number of thermophilus bacteria were examined. Specifically, the numbers of thermophilus bacteria were compared for sample 2-1, sample 2-2, and sample 2-3. The number and EPS content of Thermophilus bacteria in Sample 2-2 were almost the same as the number and EPS content of Thermophilus bacteria in Sample 2-1. In Sample 2-3, as in Sample 2-6 (control), an increase in EPS content and a decrease in the number of Thermophilus bacteria were not observed. This seems to be because the added NaCl did not exhibit the pH buffering action. Therefore, it was suggested that the addition of phosphate may suppress the growth of Thermophilus bacteria.

-Verification of the effect of phosphate addition on EPS production and card tension-
100 kg of yogurt raw material mix A was divided into 20 kg each, and five samples 3-1, 3-2, 3-3, 3-4, 3-5 were prepared. No phosphate was added to Sample 3-1 (control). The sample 3-2 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.1 wt%. Na ₂ HPO ₄ was added to Sample 3-3 so that 0.3% by weight of Na ₂ HPO ₄ was contained. In Sample 3-4, Na ₂ HPO ₄ was added so that 0.5 wt% of Na ₂ HPO ₄ was contained. The sample 3-5 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 1.0 wt%. Then, each sample was inoculated with a lactic acid bacteria starter which is a mixed culture of Thermophilus bacteria and 1073R-1 strain generally used in the production of yogurt so that the lactic acid bacteria starter was contained at 2% by weight with respect to the total amount. Thereafter, 85 g of each sample containing the lactic acid bacteria starter was filled in an 85 g capacity container and fermented in a thermostatic chamber at 43 ° C. When the pH of each sample reached 4.4, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped.

  And about each sample, time to fermentation stop (fermentation time), EPS content, and card tension (CT) were measured (refer Table 3). The EPS content in each sample was measured in the same manner as in Example 1. CT was measured by the following procedure. A 100 g weight was attached to each sample, and the elasticity when each sample reached breakage was measured by a card meter max (ME-500, manufactured by Asuka Kikai Co., Ltd.). This elastic force was used as the CT index value.

Table 3 shows the fermentation time, EPS content, and CT results of each sample. As the amount of Na ₂ HPO ₄ added increased, the EPS content in the sample increased and the CT value decreased (ie the sample became softer).

-Verification of influence of SNF content of yogurt raw material mix on EPS production-
Lactic acid bacteria starter (yogurt starter), which is a mixed culture of OLS-3059 strain and 1073R-1 strain, was inoculated into yogurt raw material mix A and yogurt raw material mix B so that the lactic acid bacteria starter was included at 2% by weight with respect to the total amount. Thereafter, 85 g of each yoghurt raw material mix A and B containing lactic acid bacteria starter was dispensed into a 85 g capacity container, and two types of samples 4-1 (SNF = 9.4 wt%), 4-2 (SNF = 10 2% by weight). And these samples 4-1 and 2 were fermented in a 43 degreeC thermostat. When the acidity of each sample reached 0.8, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. The EPS content in each sample was measured. The EPS content was measured by the same method as in Example 1.

  The EPS content of Sample 4-1 was 46.0 mg / kg. On the other hand, the EPS content of Sample 4-2 was 48.6 mg / kg. That is, the EPS content of Sample 4-2 was 1.1 times that of Sample 4-1. Therefore, it became clear that the EPS content in yogurt can be increased by using a high SNF yogurt raw material.

-Verification of the effect of fermentation temperature on EPS production-
After inoculating the lactic acid bacteria starter (yogurt starter), which is a mixed culture of OLS-3059 and 1073R-1 strains, into the yogurt raw material mix A so that the lactic acid bacteria starter is contained in 2% by weight, the lactic acid bacteria starter is contained. The yoghurt raw material mix A was dispensed by 85 g into a 85 g capacity container to prepare two types of samples 5-1, 5-2. And sample 5-1 was fermented at 43 ° C., and sample 5-2 was fermented at 37 ° C. When the acidity of each sample reached 0.8, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. The EPS content in each sample was measured. The EPS content was measured by the same method as in Example 1.

  The EPS content of Sample 5-1 was 43.4 mg / kg. On the other hand, the EPS content of Sample 5-2 was 51.1 mg / kg. That is, the EPS content of Sample 5-2 was 1.2 times that of Sample 5-1. Therefore, it became clear that the EPS content in yogurt can be increased by lowering the fermentation temperature.

First, a yogurt raw material mix A was prepared as sample 6-1 (control). Further, the yogurt raw material mix B was divided into three to prepare three samples 6-2, 6-3, and 6-4. Then, each sample was inoculated with a lactic acid bacteria starter which is a mixed culture of Thermophilus bacteria and 1073R-1 strain generally used in the production of yogurt so that the lactic acid bacteria starter was contained at 2% by weight with respect to the total amount. Further, the sample 6-4 was _further added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.3 wt% of the total amount. Samples 6-1 and 6-2 were fermented at 43 ° C., and samples 6-3 and 6-4 were fermented at 37 ° C. When the acidity of each sample reached 0.8, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. The EPS content in each sample was measured. The EPS content was measured by the same method as in Example 1.

Table 4 shows SNF, fermentation temperature, phosphate (Na ₂ HPO ₄ ) addition amount, and EPS content in yogurt for each sample. In sample 6-2 (high SNF content), the EPS content is increased 1.15 times that in the control sample 6-1, and in sample 6-3 (high SNF content, low fermentation temperature), the EPS content is increased in the control sample. It was increased to 1.19 times 6-1. Furthermore, in sample 6-4 (high SNF content, low fermentation temperature, phosphate addition), the EPS content of yogurt was increased to 1.37 times that of control sample 6-1.

  Since the method for producing yogurt according to the present invention can efficiently increase the content of functional products derived from lactic acid bacteria, it enhances the health promoting action of functional products derived from lactic acid bacteria (polysaccharides, etc.). Yogurt and a small volume of yogurt sufficiently containing an effective amount of a functional product derived from lactic acid bacteria can be efficiently produced.

  The present invention relates to a yogurt production method for increasing the production amount of a functional product derived from lactic acid bacteria, and a yogurt produced by the yogurt production method. In addition, the present invention relates to a method for producing a lactic acid bacterium extracellular functional product that increases the production amount of the lactic acid bacterium extracellular functional product, and a lactic acid bacterium extracellular functional product enhancer used in the method.

  It is known that by taking lactic acid bacteria and functional products derived from lactic acid bacteria, the bacterial flora in the gastrointestinal tract is improved and has beneficial effects on the host (maintenance of health and reduction of disease risk). For example, Lactobacillus delbruechii subsp. Bulgaricus (hereinafter also referred to as “Bulgaria bacterium”) is one of lactic acid bacteria used as a yogurt starter in the production of fermented milk. Among these Bulgarian bacteria, there are many strains that produce exopolysaccharide (EPS). And this EPS is known not only to contribute to the physical properties and stability of fermented milk products, but also to enjoy the probiotic effect. For example, Lactobacillus delbruechii subsp. It is known that EPS produced by Bulgaricus OLL1073R-1 strain (hereinafter also referred to as “OLL1073R-1 strain”) has an effect of preventing autoimmune diseases. In addition, it is known that fermented milk produced using this strain has effects such as activation of NK cells, reduction of common cold, and anti-influenza. (For example, refer to Patent Documents 1-3). Thus, functional foods and supplements that contribute to health can be provided by using lactic acid bacteria that produce EPS or EPS that is produced by lactic acid bacteria or the like.

  By the way, in order to efficiently manufacture such functional foods and supplements, it is necessary to increase the EPS production amount in the functional foods.

  In order to increase EPS derived from lactic acid bacteria as well as Bulgaria bacteria, various methods have been proposed in the past. For example, Patent Document 4 discloses that a high concentration of EPS is produced in a culture solution by culturing lactic acid bacteria having kefiran-producing ability such as Lactobacillus kefiranofaciens in a medium containing peptone, yeast extract, and unsaturated fatty acid or ester thereof. It is disclosed that it can be produced. Patent Document 5 discloses that EPS derived from lactic acid bacteria is remarkably increased by adding whey protein or soybean protein to milk and fermenting with lactococcus lactis subspecies cremolith.

JP 2000-247895 A JP 2005-194259 A International Publication Pamphlet WO2011 / 065300 JP 2011-250756 A JP 2011-51268 A

  The subject of this invention is providing the new method which can increase the production amount of the functional product derived from lactic acid bacteria efficiently.

  The yogurt manufacturing method which concerns on 1 aspect of this invention is equipped with a pH buffer addition process and a fermentation process. In the pH buffer addition step, a pH buffer is added to the yogurt raw material. In the fermentation process, a yogurt raw material to which a pH buffer is added (hereinafter referred to as “pH buffer added yogurt raw material”) is fermented by lactic acid bacteria. In addition, the addition of lactic acid bacteria to the yogurt raw material may be before the addition of the pH buffering agent or after the addition.

  As a result of intensive studies by the inventors of the present application, as described in the above-mentioned yogurt production method, by adding a pH buffer to the yogurt raw material and then fermenting the yogurt raw material, the time in the pH region where lactic acid bacteria can grow is extended. As a result, it was revealed that the production amount of functional products derived from lactic acid bacteria in yogurt can be increased. That is, this yogurt manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria.

  By the way, in this yogurt manufacturing method, it is preferable that lactic acid bacteria contain at least lactic acid bacteria which have the ability to produce lactic acid bacteria extracellular polysaccharides. And it is preferable that the lactic acid bacteria which have the ability to produce lactic acid bacteria exopolysaccharides include at least Lactobacillus delbruecki subspice bulgaricus lactic acid bacteria. In such a case, the lactic acid bacteria having the ability to produce lactic acid bacteria exopolysaccharides may be Lactobacillus delbruecky subspice bulgaricus lactic acid bacteria alone, or Lactobacillus delbruecki subspice bulgaricus sp. And lactic acid bacteria mixture containing other lactic acid bacteria.

  Moreover, in this yogurt manufacturing method, it is preferable that a pH buffer is a phosphate. When a mixture of a lactic acid bacterium of Lactobacillus delbruecki subspice bulgaricus species and a lactic acid bacterium of Streptococcus thermophilus species (hereinafter also referred to as “thermophilus bacterium”) is used as the lactic acid bacterium, This is because it is possible to promote the growth of Lactobacillus delbruecki, subspice bulgaricus lactic acid bacteria while suppressing the growth of the bacteria. In addition, it is preferable that this phosphate is an alkali metal salt. Furthermore, the phosphate is preferably at least one phosphate selected from the group consisting of disodium hydrogen phosphate and dipotassium hydrogen phosphate.

  Furthermore, in this yogurt manufacturing method, it is preferable that the yogurt raw material contains non-fat milk solid content in the range of 8 wt% or more and 20 wt% or less. Here, the yoghurt raw material means that before adding the pH buffering agent.

  In addition, in this yogurt production method, in the fermentation step, it is preferable that the pH buffer-added yogurt raw material is fermented by lactic acid bacteria at a temperature within the range of 30 ° C. or higher and 40 ° C. or lower.

  The yogurt produced by the above-mentioned yogurt production method contains lactic acid bacteria and a pH buffer. In this yogurt, the pH buffer is uniformly dissolved. Here, the lactic acid bacteria and the pH buffer are as described above. In addition, the pH buffer in this yogurt is, for example, a chromatography technique such as high performance liquid chromatography or a nuclear magnetic resonance (NMR) technique (for example, Szlyk E, Hrynczyszyn P, “Phosphate additives determination in meat products by 31- for example, phosphorus nuclear magnetic resonance using new internal reference standard: hexamethylphosphoroamide phosphate solution ”, Talanta. 2011 Mar 15; 84 (1): 199-203. doi: 10.1016 / j.talanta.2010.12.046).

  Moreover, it is preferable that the above-mentioned yogurt further contains a lactic acid bacterial extracellular functional product (for example, a lactic acid bacterial extracellular polysaccharide). In such a case, it is preferable that the lactic acid bacterial extracellular functional product is contained in an amount within the range of 40 mg / kg to 100 mg / kg with respect to the total amount of yogurt.

  The method for producing an extracellular functional product of lactic acid bacteria according to another aspect of the present invention includes a pH buffer addition step and a fermentation step. In the pH buffer addition step, a pH buffer is added to the milk raw material. In the fermentation process, the milk raw material to which the pH buffer is added is fermented by a lactic acid bacterium that produces a lactic acid bacteria extracellular functional product (hereinafter also referred to as “extracellular functional product-producing lactic acid bacteria”). The lactic acid bacterium may be added to the milk raw material before or after the addition of the pH buffering agent.

  As a result of intensive studies by the inventors of the present application, the extracellular functional product is produced by adding a pH buffer to the milk raw material and then fermenting the milk raw material in accordance with the above-described method for producing an extracellular functional product of lactic acid bacteria. It has been clarified that the time in the pH region where the produced lactic acid bacteria can grow can be extended, and as a result, the production amount of the functional product derived from the lactic acid bacteria can be increased. That is, this lactic acid bacteria extracorporeal functional product manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria. That is, in this aspect, the pH buffer functions as an active ingredient of the lactic acid bacteria extracellular functional product increaser.

  In addition, the above-mentioned method for producing an extracellular functional product of a lactic acid bacterium has a different viewpoint: “A lactic acid bacterial extracellular functional product in which a lactic acid bacterium is fermented by adding (adding) a pH buffer to the milk material. "Production method", "Production method of lactic acid bacteria functional product by adding (adding) a pH buffer to milk material", "Combination (addition of pH buffering agent to dairy material to increase production of lactic acid bacteria functional product" ) "And" use of pH buffering agent for milk raw material to increase production of lactic acid bacteria extracorporeal functional product (method of use) ".

  In the yogurt production method according to one aspect of the present invention, by adding a pH buffer to the yogurt raw material and then fermenting the yogurt raw material, the time in the pH region where the lactic acid bacteria can grow can be extended. The production amount of functional products derived from lactic acid bacteria in yogurt can be increased. That is, this yogurt manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria.

  In addition, in the method for producing a lactic acid bacteria extracellular functional product according to another aspect of the present invention, by adding a pH buffer to the milk material and then fermenting the milk material, the time in the pH region where the lactic acid bacteria can grow is extended. As a result, the production amount of the functional product derived from lactic acid bacteria can be increased. That is, this lactic acid bacteria extracorporeal functional product manufacturing method can efficiently increase the production amount of functional products derived from lactic acid bacteria.

  Hereinafter, although the form for implementing this invention is demonstrated in detail, this invention is not limited to each embodiment described below.

-First embodiment-
The yogurt according to the present embodiment is manufactured through a pH buffer addition process and a fermentation process. In the pH buffer addition step, a pH buffer is added to the yogurt raw material. In the fermentation process, a yogurt raw material to which a pH buffer is added (hereinafter referred to as “pH buffer added yogurt raw material”) is fermented by lactic acid bacteria.

  In the present embodiment, yogurt refers to “fermented milk” and “lactic acid bacteria beverage” as defined by a ministerial ordinance such as milk. “Fermented milk” in the ministerial ordinance of milk, etc. means “milk or milk containing non-fat milk solids equal to or higher than this, fermented with lactic acid bacteria or yeast to make paste or liquid” or “frozen Defined as "thing". Fermented milk is “hard yogurt fermented and solidified after filling in container (solid fermented milk, set type yogurt)”, “soft yogurt (fermented fermented milk) filled with container after fermentation and filled into containers”, “ Soft yogurt is roughly classified into drink yogurt (liquid fermented milk) that is further crushed with a homogenizer to enhance liquid properties. In addition, “lactic acid bacteria beverage” in a ministerial ordinance such as milk means “a beverage (excluding fermented milk) obtained by processing or fermenting milk or the like with lactic acid bacteria or yeast”.

  The yogurt raw material includes at least one of milk, dairy products, and milk protein. Examples of yogurt ingredients include animal milk such as milk, sheep and goats, processed products thereof, sterilized milk, skim milk, full fat milk powder, partially skimmed milk, skim milk powder, full fat concentrated milk, skim concentrated milk, cream, Butter, buttermilk, whey, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), α-casein, β- Casein, κ-casein, non-protein nitrogen and other milk ingredients, sugar, sugar, modified starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, sweetener, organic Examples include acids (malic acid, citric acid, lactic acid, tartaric acid, etc.), fragrances, and water. The yogurt raw material can be obtained by mixing conventional raw materials as described above and dissolving them while heating. Gelling agents such as gelatin, agar, pectin, and carboxymethylcellulose (CMC), thickeners, and stabilizers may be added to the yogurt raw material. In such a case, a raw material for yogurt is prepared by preliminarily heating and dissolving a stabilizer such as gelatin in a solvent such as water and mixing this aqueous stabilizer solution with other components.

  Moreover, it is preferable that non-fat milk solid content (henceforth "SNF") of this yogurt raw material exists in the range of 8 weight% or more and 20 weight% or less. In the yogurt production method according to the present embodiment, when Bulgarian bacteria and Thermophilus bacteria that produce EPS are employed as lactic acid bacteria and phosphate is employed as a pH buffering agent, while maintaining the flavor of yogurt in a good manner, This is because the exopolysaccharide content (hereinafter referred to as “EPS”) can be increased. The SNF of the yogurt raw material is more preferably in the range of 8.5% by weight to 18.5% by weight, still more preferably in the range of 9% by weight to 15% by weight. It is particularly preferable that the amount be in the range of 5% by weight to 14% by weight.

In this embodiment, the pH buffer is not particularly limited, but is particularly preferably a phosphate. In the present embodiment, the phosphate includes a hydrate. Examples of the phosphate include monosodium dihydrogen phosphate (NaH ₂ PO ₄ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ), dipotassium dihydrogen phosphate (KH ₂ PO ₄ ), and dihydrogen phosphate. potassium _(K 2 HPO _4), and the like. In addition, these phosphates may be used independently and may be used together. In the yogurt production method according to the present embodiment, it is preferable to use disodium hydrogen phosphate (Na ₂ HPO ₄ ) and dipotassium hydrogen phosphate (K ₂ HPO ₄ ) alone or in combination, and disodium hydrogen phosphate. It is particularly preferred to use (Na ₂ HPO ₄ ) alone.

  In addition, in the pH buffer addition step of the yogurt production method according to this embodiment, an amount of the pH buffer in the range of 0.05 wt% or more and 0.55 wt% or less is added to the yogurt raw material. preferable. In the yogurt production method according to this embodiment, when Bulgarian bacteria and Thermophilus bacteria that produce EPS are employed as lactic acid bacteria and phosphate is employed as the pH buffering agent, the added amount of the pH buffering agent is within this range. This is because, while maintaining good yogurt card strength (card tension) and flavor, the growth of Bulgarian bacteria can be promoted and the production of EPS derived from Bulgarian bacteria can be enhanced. The addition amount of the pH buffering agent is more preferably in the range of 0.08 wt% or more and 0.45 wt% or less, and is preferably in the range of 0.1 wt% or more and 0.35 wt% or less. Particularly preferred.

  In the present embodiment, the lactic acid bacterium is an extracellular functional product such as lactic acid bacterium that produces EPS (hereinafter referred to as “extracellular functional product-producing lactic acid bacterium”), such as Lactobacillus delbruecki subspice bulgari. Lactobacillus species of Lactobacillus delbruechii subsp. Bulgaricus (hereinafter also referred to as “Bulgaria bacterium”). In the present embodiment, the lactic acid bacterium may be any strain as long as it is an extracellular functional product-producing lactic acid bacterium, but Lactobacillus delbruechii subsp. bulgaricus OLL1073R-1 strain (FERM BP-10741) is preferred. In this embodiment, it is preferable to mainly use a Bulgarian bacterium, but other lactic acid bacteria that produce EPS (hereinafter referred to as “EPS-producing lactic acid bacteria”), such as Lactobacillus bulgaricus, Lactococcus, etc. -Lactococcus lactis ssp. Cremoris etc. may be used in combination with Bulgarian bacteria.

  In addition, lactic acid bacteria other than EPS-producing lactic acid bacteria, such as Thermophilus bacteria, Lactobacillus gasseri (Lactobacillus gasseri), bifidobacteria, propionic acid bacteria, and other lactic acid bacteria generally used for the production of fermented milk are used. You may use together. Moreover, you may add yeast with the said lactic acid bacteria. In addition, in the yogurt manufacturing method which concerns on this embodiment, it is especially preferable to use together the Bulgarian bacterium and Thermophilus bacterium which produce EPS as a lactic acid bacterium.

  In the fermentation process of the yogurt manufacturing method according to the present embodiment, it is preferable that the fermentation temperature be in the range of 30 ° C. or higher and 40 ° C. or lower. This is because, when Bulgarian bacteria and Thermophilus bacteria that produce EPS are employed as lactic acid bacteria and phosphate is employed as a pH buffering agent, the EPS content in yogurt can be increased. The fermentation temperature is more preferably in the range of 32 ° C. or more and 39 ° C. or less, and further preferably in the range of 34 ° C. or more and 38 ° C. or less.

  The extracellular functional product-producing lactic acid bacterium according to this embodiment preferably produces an extracellular functional product in an amount within the range of 40 mg / kg to 100 mg / kg with respect to the total amount. For example, when Bulgarian bacteria and Thermophilus bacteria producing EPS are used as lactic acid bacteria and phosphate is used as a pH buffering agent, when an amount of EPS within the above range is produced, the pH buffering agent is not added. This is because it is recognized that the ratio of the EPS production amount when the pH buffer agent is added to the EPS production amount is increased, that is, the effect of adding the pH buffer agent is enhanced. The extracellular functional product-producing lactic acid bacterium preferably produces an extracellular functional product in an amount in the range of 42 mg / kg to 100 mg / kg, and more than 44 mg / kg to 100 mg. It is more preferable to produce an extracellular functional product in an amount within the range of / kg or less, and to produce an extracellular functional product in an amount within the range of 48 mg / kg or more and 100 mg / kg or less. More preferably, it is particularly preferable to produce an extracellular functional product in an amount in the range of 50 mg / kg to 100 mg / kg.

  In the yogurt production method according to the present embodiment, the production amount of the extracellular functional product is preferably 1.05 times or more, 1.1 times or more of the production amount when no pH buffer is added. Is more preferably 1.2 times or more, and particularly preferably 1.25 times or more. For example, when Bulgarian bacteria and Thermophilus bacteria producing EPS are used as lactic acid bacteria, and phosphate is used as a pH buffering agent, the EPS production amount when adding a pH buffering agent is the EPS production when no pH buffering agent is added. It may be 1.25 times or more of the amount.

-Second Embodiment-
The lactic acid bacterial extracellular functional product according to the present embodiment is produced through a pH buffer addition step and a fermentation step. In the pH buffer addition step, a pH buffer is added to the milk raw material. In the fermentation process, the milk raw material to which the pH buffer is added is fermented by lactic acid bacteria that produce lactic acid bacteria extracorporeal functional products.

In addition, the manufacturing method of the lactic acid bacteria extracorporeal functional product which concerns on this embodiment is the same as the yogurt manufacturing method which concerns on 1st Embodiment except a yoghurt raw material being used as a milk raw material. Moreover, the “lactic acid extracellular functional product” referred to in the present embodiment is the same as the “extracellular functional product” referred to in the first embodiment. And the “milk raw material” here means animal milk such as cow's milk, sheep, goat, etc., processed products, sterilized milk, skim milk, whole milk powder, partially skimmed milk, skimmed milk powder, whole fat concentrated milk, skimmed milk Concentrated milk, cream, butter, buttermilk, whey, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), α -Casein, β-casein, κ-casein, non-protein nitrogen and the like. The non-fat milk solid content (hereinafter referred to as “SNF”) of the milk raw material is preferably in the range of 8% by weight to 20% by weight, and in the range of 8.5% by weight to 18.5% by weight. More preferably, it is more preferably in the range of 9% by weight to 15% by weight, and particularly preferably in the range of 9.5% by weight to 14% by weight.

  Further, the production amount of the lactic acid bacteria extracellular functional product according to the present embodiment is preferably 1.05 times or more, 1.1 times the production amount of the lactic acid bacteria extracellular functional product when no pH buffer is added. More preferably, it is 1.2 times or more, more preferably 1.25 times or more.

<Example>
Hereinafter, preferred examples of the present invention will be described, but the present invention is not limited to the above-described embodiments and the following examples, and various modifications are made within the technical scope grasped from the description of the scope of claims. Is possible. Note that Lactobacillus delbruechii subsp. bulgaricus OLL1073R-1 strain (hereinafter referred to as OLL1073R-1 strain) was issued on November 29, 2006 (contract date), National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1-1 Tsukuba, Tsukuba City, Ibaraki Prefecture) In the center center 6), it is a lactic acid bacterium that has been deposited internationally under the Budapest Treaty under the accession number FERM BP-10741. Streptococcus thermophilus OLS3059 strain was consigned to the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Tsukuba Center 1-1-1 Tsukuba Center Central 6th, Ibaraki Prefecture) on December 15, 2006 (contract date). It is a lactic acid bacterium that has been deposited internationally under the Budapest Treaty as FERM BP-10740 by number. In addition, the patent microorganisms deposit work of the National Institute of Advanced Industrial Science and Technology patent biological depository center was succeeded by the National Institute of Technology and Evaluation on April 1, 2012. As of April 1, 2013, the Biological Depositary Center has been relocated to 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan.

(Production example)
(1) Preparation of yogurt raw material mix A (SNF = 9.4% by weight) 50 kg of raw milk, 5.49 kg of skim milk powder, 1.5 kg of unsalted butter and 43.01 kg of water were mixed, and the mixture was mixed at 95 ° C. for 5 After sterilization by heating for minutes, the mixture was cooled to around 37 ° C. to prepare a yogurt raw material mix A.

(2) Preparation of yogurt raw material mix B (SNF = 10.2% by weight) 25 kg raw milk, 18.4 kg nonfat concentrated milk, 0.96 kg nonfat dry milk, 1.31 kg unsalted butter and 45.75 kg water are mixed, The mixture was sterilized by heating at 95 ° C. for 5 minutes and then cooled to around 37 ° C. to prepare a yogurt raw material mix B having a non-fat milk solid content (SNF) higher than that of the yogurt raw material mix A.

-Verification of the effect of phosphate addition on EPS production-
It is a mixed culture of Thermophilus OLS3059 strain (Streptcoccus thermophilus OLS3059, hereinafter also referred to as OLS3059 strain) and 1073R-1 strain which is a lactic acid bacterium of Lactobacillus delbruecki subspice bulgaricus species that produces polysaccharides After inoculating the lactic acid bacteria starter (yoghurt starter) into the yogurt raw material mix A so that the lactic acid bacterium starter is contained in 2% by weight with respect to the total amount, dispense 20 mL of the yoghurt raw material mix A containing the lactic acid bacteria starter into 5 test tubes. Then, five samples 1-1, 1-2, 1-3, 1-4, and 1-5 were prepared. Then, the sample 1-1 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.5 wt% of the total amount. The sample _{1-2, Na} 2 Na 2 HPO as HPO ₄ comprises 0.4% by weight based on the total amount and _NaH 2 PO ₄ is contained 0.1% by weight based on the total amount ₄ and _NaH 2 PO ₄ Was added. The Sample _1-3, was added _Na 2 HPO ₄ and _NaH 2 PO ₄ as Na 2 HPO ₄ and _NaH 2 PO ₄ is contained 0.25 wt% respectively of the total amount. The sample 1-4 _was added _Na 2 HPO ₄ and _NaH 2 PO ₄ as Na 2 HPO ₄ and _NaH 2 PO ₄ is contained 0.5 wt% respectively of the total amount. In addition, phosphate was not added to Sample 1-5 (control). Then, each said sample was immersed in a 43 degreeC thermostat and fermented. When the pH of each sample reached 4.4 to 4.5, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. About each sample, time to fermentation stop (fermentation time), acidity, EPS content, the number of Bulgaria bacteria, and the number of thermophilus bacteria were measured (refer Table 1).

  The pH was measured with a pH meter (TOA-HM50V, manufactured by Toa DKK Corporation) using a glass electrode. The acidity was measured by the following procedure. 9.00 g was sampled from each sample, and 500 μL of phenolphthalein was added to the sample. Then, the preparative sample was titrated with 0.1N sodium hydroxide, and the end point was a time point at which the faint red color did not disappear for 30 seconds.

Moreover, the EPS content of yogurt was measured by the following procedure. First, 10 g of each sample was weighed and placed in a 50 mL tube, and 1 mL of 100% trichloroacetic acid was added thereto. Next, after the contents were stirred, the contents were allowed to stand at about 4 ° C. for about 10 minutes. The contents were then centrifuged at 12,000 × g relative centrifugal force for 20 minutes at a temperature of 4 ° C., and the supernatant was transferred to a new 50 mL tube. Then, while stirring this supernatant, cold ethanol twice the amount of the supernatant was gradually added to the supernatant, and the supernatant and cold ethanol were mixed thoroughly. Subsequently, the mixture was allowed to stand overnight at a temperature of about 4 ° C., and then the mixture was centrifuged under the same conditions as described above. Thereafter, the supernatant of the mixture was discarded, and 10 mL of purified water was added to the precipitate to completely dissolve the precipitate in purified water. 150 μL of the aqueous solution was injected into the HPLC using a syringe with a filter having a diameter of 0.45 μm. The ratio of the “peak area of a single peak detected by the RI detector around 16 minutes after the start of injection” to the “total peak area” was taken as the EPS content. The analytical operation conditions of HPLC are as follows.
HPLC system: Aquity H-class (Waters)
Column: OHpak 806HQ (Shodex) + SB-G (Shodex)
Column temperature: 40 ° C
Solvent: 0.2 M NaCl aqueous solution Flow rate: 0.5 mL / min
Detector: RI detector 2414 (Waters), detection temperature 40 ° C
Sample injection: 150 μL
Analysis time: 50 min

Table 1 shows the influence of the phosphate addition amount on the EPS content. In Samples 1-1, 1-2, 1-3, and 1-4 to which phosphate was added, the fermentation time was longer than that of Control Sample 1-5, and the acidity at the end of fermentation was also 1 or more. . The addition of phosphate resulted in increased EPS production of yogurt. Incidentally, in the case of adding only _Na 2 HPO ₄ as a phosphate (Sample 1-1), EPS content was the highest. In addition, the number of Bulgarian bacteria increased with the addition of phosphate, whereas the number of Thermophilus decreased with increasing Na ₂ HPO ₄ concentration.

From these results, the fermentation time in the pH range where lactic acid bacteria can grow can be extended by the addition of phosphate, and it is considered that the number of Bulgarian bacteria increased due to this effect. Furthermore, since a decrease in growth of Thermophilus was observed by the addition of phosphate, particularly Na ₂ HPO ₄ , it is considered that the decrease in growth of Thermophilus also contributed to an increase in EPS derived from Bulgaria.

-Verification of the effect of phosphate type on EPS production-
Lactic acid bacteria starter (yoghurt starter), which is a mixed culture of OLS 3059 and 1073R-1 strains, was inoculated into 500 g of yoghurt raw material mix A so that the lactic acid bacteria starter was contained at 2% by weight with respect to the total amount, and then entered into the lactic acid bacteria starter The yogurt raw material mix A was dispensed into 6 test tubes each 20 mL to prepare 6 samples 2-1, 2-2, 2-3, 2-4, 2-5, 2-6. Then, the sample 2-1 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.5 wt% of the total amount. The sample 2-2 _was added _K 2 HPO ₄ as K 2 HPO ₄ is contained 0.61 wt% of the total amount. The molar concentration of K ₂ HPO ₄ in sample 2-2 is the same as the molar concentration of Na ₂ HPO ₄ in sample 2-1. In Sample 2-3, NaCl was added so that NaCl was contained in an amount of 0.21% by weight based on the total amount. The molar concentration of NaCl in Sample 2-3 is the same as the molar concentration of Na ₂ HPO ₄ in Sample 2-1. The sample 2-4 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.3 wt% of the total amount. The sample 2-5 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.1% by weight based on the total amount. In addition, phosphate was not added to Sample 2-6 (control). Then, each said sample was immersed in a 43 degreeC thermostat and fermented. When the pH of each sample reached 4.4 to 4.5, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. About each sample, time to fermentation stop (fermentation time), acidity, EPS content, the number of Bulgaria bacteria, and the number of thermophilus bacteria were measured (refer Table 2). These physical property values were measured in the same manner as in Example 1.

Table 2 shows the influence of the type of phosphate on the EPS content. In Samples 2-1, 2-4, and 2-5 to which only Na ₂ HPO ₄ was added, the fermentation time was increased and the EPS content was increased as the amount of Na ₂ HPO ₄ added was increased. In addition, the number of Bulgarian bacteria increased and the number of Thermofilus bacteria decreased as the amount of Na ₂ HPO ₄ added increased. Here, the factors affecting the decrease in the number of thermophilus bacteria were examined. Specifically, the numbers of thermophilus bacteria were compared for sample 2-1, sample 2-2, and sample 2-3. The number and EPS content of Thermophilus bacteria in Sample 2-2 were almost the same as the number and EPS content of Thermophilus bacteria in Sample 2-1. In Sample 2-3, as in Sample 2-6 (control), an increase in EPS content and a decrease in the number of Thermophilus bacteria were not observed. This seems to be because the added NaCl did not exhibit the pH buffering action. Therefore, it was suggested that the addition of phosphate may suppress the growth of Thermophilus bacteria.

-Verification of the effect of phosphate addition on EPS production and card tension-
100 kg of yogurt raw material mix A was divided into 20 kg each, and five samples 3-1, 3-2, 3-3, 3-4, 3-5 were prepared. No phosphate was added to Sample 3-1 (control). The sample 3-2 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.1 wt%. Na ₂ HPO ₄ was added to Sample 3-3 so that 0.3% by weight of Na ₂ HPO ₄ was contained. In Sample 3-4, Na ₂ HPO ₄ was added so that 0.5 wt% of Na ₂ HPO ₄ was contained. The sample 3-5 _was added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 1.0 wt%. Then, each sample was inoculated with a lactic acid bacteria starter which is a mixed culture of Thermophilus bacteria and 1073R-1 strain generally used in the production of yogurt so that the lactic acid bacteria starter was contained at 2% by weight with respect to the total amount. Thereafter, 85 g of each sample containing the lactic acid bacteria starter was filled in an 85 g capacity container and fermented in a thermostatic chamber at 43 ° C. When the pH of each sample reached 4.4, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped.

  And about each sample, time to fermentation stop (fermentation time), EPS content, and card tension (CT) were measured (refer Table 3). The EPS content in each sample was measured in the same manner as in Example 1. CT was measured by the following procedure. A 100 g weight was attached to each sample, and the elasticity when each sample reached breakage was measured by a card meter max (ME-500, manufactured by Asuka Kikai Co., Ltd.). This elastic force was used as the CT index value.

Table 3 shows the fermentation time, EPS content, and CT results of each sample. As the amount of Na ₂ HPO ₄ added increased, the EPS content in the sample increased and the CT value decreased (ie the sample became softer).

-Verification of influence of SNF content of yogurt raw material mix on EPS production-
Lactic acid bacteria starter (yogurt starter), which is a mixed culture of OLS-3059 strain and 1073R-1 strain, was inoculated into yogurt raw material mix A and yogurt raw material mix B so that the lactic acid bacteria starter was included at 2% by weight with respect to the total amount. Thereafter, 85 g of each yoghurt raw material mix A and B containing lactic acid bacteria starter was dispensed into a 85 g capacity container, and two types of samples 4-1 (SNF = 9.4 wt%), 4-2 (SNF = 10 2% by weight). And these samples 4-1 and 2 were fermented in a 43 degreeC thermostat. When the acidity of each sample reached 0.8, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. The EPS content in each sample was measured. The EPS content was measured by the same method as in Example 1.

  The EPS content of Sample 4-1 was 46.0 mg / kg. On the other hand, the EPS content of Sample 4-2 was 48.6 mg / kg. That is, the EPS content of Sample 4-2 was 1.1 times that of Sample 4-1. Therefore, it became clear that the EPS content in yogurt can be increased by using a high SNF yogurt raw material.

-Verification of the effect of fermentation temperature on EPS production-
After inoculating the lactic acid bacteria starter (yogurt starter), which is a mixed culture of OLS-3059 and 1073R-1 strains, into the yogurt raw material mix A so that the lactic acid bacteria starter is contained in 2% by weight, the lactic acid bacteria starter is contained. The yoghurt raw material mix A was dispensed by 85 g into a 85 g capacity container to prepare two types of samples 5-1, 5-2. And sample 5-1 was fermented at 43 ° C., and sample 5-2 was fermented at 37 ° C. When the acidity of each sample reached 0.8, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. The EPS content in each sample was measured. The EPS content was measured by the same method as in Example 1.

  The EPS content of Sample 5-1 was 43.4 mg / kg. On the other hand, the EPS content of Sample 5-2 was 51.1 mg / kg. That is, the EPS content of Sample 5-2 was 1.2 times that of Sample 5-1. Therefore, it became clear that the EPS content in yogurt can be increased by lowering the fermentation temperature.

First, a yogurt raw material mix A was prepared as sample 6-1 (control). Further, the yogurt raw material mix B was divided into three to prepare three samples 6-2, 6-3, and 6-4. Then, each sample was inoculated with a lactic acid bacteria starter which is a mixed culture of Thermophilus bacteria and 1073R-1 strain generally used in the production of yogurt so that the lactic acid bacteria starter was contained at 2% by weight with respect to the total amount. Further, the sample 6-4 was _further added _Na 2 HPO ₄ as Na 2 HPO ₄ is contained 0.3 wt% of the total amount. Samples 6-1 and 6-2 were fermented at 43 ° C., and samples 6-3 and 6-4 were fermented at 37 ° C. When the acidity of each sample reached 0.8, the sample was cooled to 10 ° C. or lower, and the fermentation of the sample was stopped. The EPS content in each sample was measured. The EPS content was measured by the same method as in Example 1.

Table 4 shows SNF, fermentation temperature, phosphate (Na ₂ HPO ₄ ) addition amount, and EPS content in yogurt for each sample. In sample 6-2 (high SNF content), the EPS content is increased 1.15 times that in the control sample 6-1, and in sample 6-3 (high SNF content, low fermentation temperature), the EPS content is increased in the control sample. It was increased to 1.19 times 6-1. Furthermore, in sample 6-4 (high SNF content, low fermentation temperature, phosphate addition), the EPS content of yogurt was increased to 1.37 times that of control sample 6-1.

  Since the method for producing yogurt according to the present invention can efficiently increase the content of functional products derived from lactic acid bacteria, it enhances the health promoting action of functional products derived from lactic acid bacteria (polysaccharides, etc.). Yogurt and a small volume of yogurt sufficiently containing an effective amount of a functional product derived from lactic acid bacteria can be efficiently produced.

Claims (12)

A pH buffering agent adding step of adding a pH buffering agent to the yogurt raw material;
A yogurt production method comprising: a fermentation step of fermenting the yogurt material to which the pH buffer is added (hereinafter referred to as “pH buffer added yogurt material”) with lactic acid bacteria. The method for producing yogurt according to claim 1, wherein the lactic acid bacterium includes at least a lactic acid bacterium having an exopolysaccharide-producing ability. The method for producing yogurt according to claim 2, wherein the lactic acid bacteria having the ability to produce exopolysaccharides include at least a lactic acid bacterium of Lactobacillus delbruecky subspices bulgaricus species. The yogurt production method according to any one of claims 1 to 3, wherein the pH buffer is a phosphate. The method for producing yogurt according to claim 4, wherein the phosphate is at least one phosphate selected from the group consisting of disodium hydrogen phosphate and dipotassium hydrogen phosphate. The yogurt manufacturing method according to any one of claims 1 to 5, wherein the yogurt raw material contains a non-fat milk solid content in a range of 8 wt% to 20 wt%. The method for producing yogurt according to any one of claims 1 to 6, wherein, in the fermentation step, the yogurt raw material to which the pH buffer is added is fermented by the lactic acid bacteria at a temperature within a range of 30 ° C or higher and 40 ° C or lower.   The yogurt obtained by the yogurt manufacturing method of any one of Claim 1 to 7. Lactic acid bacteria,
Yogurt containing a pH buffer. The lactic acid bacterium is a lactic acid bacterium that produces a lactic acid bacterium extracorporeal functional product,
The yoghurt of Claim 9 which further contains the lactic-acid-bacteria extracellular functional product of the quantity within the range of 40 mg / kg or more and 100 mg / kg or less with respect to the whole quantity. A pH buffering agent adding step of adding a pH buffering agent to the milk raw material;
A method for producing a lactic acid bacterial extracellular functional product, comprising a fermentation step of fermenting the milk material to which the pH buffer is added with a lactic acid bacterium producing a lactic acid bacterial extracellular functional product.   A lactic acid bacterium extracorporeal functional product enhancer comprising a pH buffer as an active ingredient.