JPH0330668A - Stabilization of preserving lactic acid bacteria, material, solution and fat and oil composition contained in the stabilized lactic acid bacteria - Google Patents

Abstract

PURPOSE:To improve stabilization of preserving lactic acid bacteria and to obtain simply usable lactic acid bacteria having high activity and stability by blending and dispersing lactic acid bacteria into squeezed yeast. CONSTITUTION:For example, lactic acid bacteria of genus Lactobacillus or Leuconostoc are blended and dispersed into squeezed yeast such as baker's yeast or wine yeast belonging to the genus Saccharomyces cerevisiae to stabilize the lactic acid bacteria. 10<6>-10<11> lactic acid bacteria are blended and dispersed into 1g squeezed yeast to give a stabilized lactic acid bacteria-containing material. As another stabilization of preservation, 105-1011 lactic acid bacteria are blended with a solution containing 0.1-30wt.% protein substance at pH4.0-6.0 to give a stabilized solution containing lactic acid bacteria and further 5-70 pts.wt. of the stabilized solution containing lactic acid bacteria as an aqueous phase part is blended with 95-30 pts.wt.. fat and oil part and 0.1-0.5 pts.wt. emulsifying agent to give a stabilized lactic acid bacteria-containing fat and oil composition.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to storage stabilization of lactic acid bacteria, and more specifically, to mixing and dispersing lactic acid bacteria in compressed yeast and adjusting the pH of a proteinaceous storage solution to an appropriate level. The present invention relates to a method for improving storage stability of lactic acid bacteria by adjusting the storage stability to a certain value, and to a lactic acid bacteria-containing product whose storage stability is improved by the above method.

[Prior art and problems]

Currently, lactic acid bacteria are used in the production of many foods such as fermentation tubes, yogurt, cheese, and bread, but it is generally said that the storage stability of lactic acid bacteria is considerably worse than that of yeast and the like.

For this reason, many studies have been conducted to date on improving the storage stability of lactic acid bacteria. For example, there are a low-temperature cryopreservation method in which the material is frozen and preserved at a low temperature of -80°C, a freeze-drying method in which it is dried after freezing, and a method in which bacteria are mixed with a suitable carrier and then dried at a low temperature to form lactic acid bacteria powder.

However, the above methods that have been considered so far are
Although it is true that it has the effect of greatly improving the storage stability of bacteria, when producing the above-mentioned lactic acid bacteria-based foods (especially bread) using lactic acid bacteria obtained by these methods, there are various disadvantages shown below. There is.

■The above storage stabilization method for lactic acid bacteria (hereinafter referred to as the conventional method)
Because operations such as freezing and drying are performed, the bacteria that survive after this operation remain stable for a long time, but this operation kills a considerable number of bacteria, resulting in a poor yield of bacteria that can be used effectively.

■When using lactic acid bacteria produced using conventional methods as a starter (including addition to bread dough), it is necessary to pre-culture the bacteria in an appropriate medium before use.
Its usage is very complicated.

■If the above-mentioned lactic acid bacteria are used as they are without activation, the activity of the bacteria is not only weak but also variable, making it difficult to stably produce high-quality products.

■Conventional methods require costly operations such as freezing and drying, which not only increases the price of the lactic acid bacteria products, but also
Frozen products and freeze-dried products need to be stored at low temperatures, and the expensive lactic acid bacteria described above are difficult to use in the production of inexpensive foods.

For the reasons mentioned above, even though the function of lactic acid bacteria has become quite clear in bread manufacturing, etc., where the product price is relatively low and stable expression of lactic acid bacteria activity is required, industrial Currently, lactic acid bacteria are hardly used in mass production on a commercial scale.

(Means for Solving the Problems) In view of the above circumstances, the present inventors conducted intensive research to solve the above problems, especially from the viewpoint of storage stabilization of lactic acid bacteria used in bread production. By storing the lactic acid bacteria by mixing and dispersing them in compressed yeast, or by storing the lactic acid bacteria in a solution containing a protective proteinaceous substance and with the pH adjusted to an appropriate value, the storage stability of the bacteria can be dramatically improved. The present invention has been completed based on the discovery that when such lactic acid bacteria-containing materials, solutions, or O/W emulsion-type oil and fat compositions are used in bread production, they stably exhibit lactic acid production activity immediately after addition.

That is, the first aspect of the present invention is a method for storage stabilizing lactic acid bacteria, which is characterized by improving the storage stability of lactic acid bacteria by mixing and dispersing lactic acid bacteria in compressed yeast. A third aspect of the present invention provides a method for storage stabilizing lactic acid bacteria, which is characterized in that lactic acid bacteria are present in a proteinaceous substance-containing solution adjusted to a concentration of 4.0 to 6.0.
The fourth aspect of the present invention is a lactic acid bacteria-containing material in which 106 to 1011 lactic acid bacteria are mixed and dispersed per 1 g of compressed yeast, and the fourth aspect of the present invention is a product containing 0.1 to 30% by weight of proteinaceous substances and 1011 to 10'' lactic acid bacteria/
The fifth aspect of the present invention is a lactic acid bacteria-containing solution containing 5 to 70 parts by weight of the lactic acid bacteria-containing solution according to claim 7 as an aqueous phase part and 95 to 30 parts by weight of an oil and fat part. Department,
Each content is an O/W emulsion type oil and fat composition containing 0.1 to 5 parts by weight of an emulsifier.

The present invention will be explained in detail below.

First, as a method for mixing and dispersing lactic acid bacteria in compressed yeast, for example, commercially available baker's yeast (bacterial cells containing 65 to 75% water) is dissolved in water to form a milky yeast suspension, or cultured in the yeast manufacturing process. Lactic acid bacteria if separated and washed from the culture solution into yeast milk whose bacterial bodies were separated and washed after the process
! A method may be mentioned in which a certain amount of liquid III is added, mixed, and then vacuum filtered, but there is no particular limitation on methods other than the above, as long as the lactic acid bacteria are uniformly mixed and dispersed in the compressed yeast.

The number of lactic acid bacteria in compressed yeast is 101 per gram of compressed yeast.
The number is preferably 1 to 10, and if the number is larger than this, the stability tends to decrease slightly. Examples of the lactic acid bacteria to be added include strains belonging to the genera Lactobacillus, Leuconostoc, Streptococcus, and Pediocoscus, but the strains whose storage stability is particularly improved by the method of the present invention are Lactobacillus and Leuconostoc. It is a lactic acid bacterium of the genus.

These lactic acid bacteria may be used alone or in combination of two or more.

These lactic acid bacteria can be isolated from type cultures in various preservation institutions or from natural products and foods such as dairy products, fruits, brewed foods, baker's yeast products, and grains such as flour, or from mutated strains of these bacteria. Breeding strains such as cell fusion strains and the like can also be used. A typical strain is Lactobacillus bulgaricus.
us bulgaricus) IFO13953, Lactobacillus casei (Lactobacillus casei)
asei) ATCC393, Lactobacillus brevis (L″″actobacillus brsvis)
IFO3960, Lactobacillus fermentum (
Lactobacillus fermentum) I
FO3956, Leuconostoc Oianus (Leu
co-nostoc oenos) ATCC232
79, Leuconostoc 9 methucentiroides (Leu
conostoc 5esenteroid-es)
IAM 1046, Streptococcus bovis (St
r-eptococcus bovis) IPO12
057, Streptococcus thermophilus (Str
aptococcus thermophi-Ius)
IFO13957, Pediococcus halophilus
Examples include JCM 2014.

Types of yeast used as compressed yeast include yeast belonging to the genus Romyces cerevisiae, such as baker's yeast, sake yeast, beer yeast, and wine yeast, but yeast that is not harmful to the human body and has fermentation power in the dough If so, there are no particular limitations.

The reason why the stability of lactic acid bacteria is improved by mixing and dispersing lactic acid bacteria in compressed yeast is not clear, but it is thought that the coexistence of yeast and lactic acid bacteria causes the yeast to exert some kind of stabilizing effect. The stability of the lactic acid bacteria in this compressed yeast is determined by the pH of the yeast suspension to which the lactic acid bacteria are added. 4.0-6.0 in ll regulation substance
The synergistic effect can be further improved by adjusting the pH of the yeast or by using a lactic acid bacteria solution containing a pH-adjusting proteinaceous substance, which will be described later, in place of the water used for dissolving the yeast.

A lactic acid bacteria solution with excellent storage stability is made of proteinaceous substances expected to have a protective effect on lactic acid bacteria, such as natural products such as soy protein, milk protein, blood plasma protein, wheat protein, corn protein, egg protein, vegetable proteins, or these. pH of a solution containing an appropriate amount (0.1 to 30% by weight) of one or more hydrolysates or enzymatically decomposed products
It can be obtained by adding lactic acid bacteria to a solution adjusted to 4.0 to 6.0, preferably 4.25 to 5.5. The optimal pH value for storage varies slightly depending on the type of lactic acid bacteria, storage conditions, etc., but if the pH is adjusted to the above range, the stability of the strain will be considerably improved. The reason why stability is improved by adjusting p[ljl is unknown, but by storing the bacteria at a slightly lower pit value than the neutral pH value that is optimal for growth, it is a kind of hibernation state in which the activity of the bacteria is suppressed to an appropriate level. This is thought to be due to the fact that it is placed in

The appropriate concentration of lactic acid bacteria to be added is 1011 to 101 cells/d.
There is a time.

Substances for adjusting the pH of the solution include, for example, acidic substances such as citric acid, succinic acid, malic acid, gluconic acid,
Organic acids such as tartaric acid, acetic acid, fumaric acid, and lactic acid, amino acids such as aspartic acid and glutamic acid, various phosphates, inorganic salts and acids such as phosphoric acid, hydrochloric acid, various carbonates, and carbonic acid, apple juice, tangerine juice On the other hand, examples of alkaline substances include inorganic alkaline substances such as sodium hydroxide and calcium hydroxide, but these substances have no negative effect on the preservation of lactic acid bacteria and are permitted as food additives. There is no particular limitation, and it is only necessary to use a substance that is not assimilated by the lactic acid bacteria to be stored.It is even more effective if you use appropriate amounts of acidic and alkaline substances to give the storage solution a buffering effect. It is true.

The target lactic acid bacteria are the same as those mixed and dispersed in compressed yeast, and may be type cultures, naturally isolated strains, mutant strains, cell fusion strains, etc., but the above method particularly improves storage stability. The bacteria that do this are strains of the genus Lactobacillus and Leuconostoc. When preserving lactic acid bacteria in a solution, the preservation property is further improved by adding compressed yeast to the solution, although the reason is unknown.

In addition, as another form, p) IAII-treated lactic acid bacteria containing proteinaceous substances? It can also be stored as an O/W emulsion type oil and fat composition in which the 8 liquid is used as an aqueous phase.

In this case, the emulsified oil and fat composition includes the aqueous phase [105-
10” lactic acid bacteria I8 solution (pH adjusted)] 55-7
A composition consisting of 0 parts by weight, 95 to 30 parts by weight of fats and oils, and 0.1 to 5 parts by weight of an emulsifier is exemplified. In addition, when storing lactic acid bacteria as the above-mentioned oil and fat composition, the lactic acid bacteria may be added to the aqueous phase of the oil and fat composition whose pH has been adjusted in advance. Also, by adding some compressed yeast to the aqueous phase, the storage stability is further improved.

In the present invention, the storage stability of lactic acid bacteria was evaluated by the viability of bacteria over time when stored at a constant temperature, lactic acid growth activity from glucose, and lactic acid growth activity in dough.

To measure the viable bacteria rate, take a portion of the lactic acid bacteria solution and compressed yeast containing lactic acid bacteria (hereinafter referred to as lactic acid bacteria yeast) after storage, dilute it appropriately with physiological saline, and add a portion of it to B for lactic acid bacteria detection.
This was carried out by inoculating the cells into a CP medium (CBCP Cycloplate Count Agar (manufactured by Hinaga Pharmaceutical Co., Ltd.)), performing flat culture, and counting the colonies that appeared. In the case of lactic acid bacteria yeast, 100 pp of cycloheximide was added to the medium to control yeast growth.

To measure lactic acid production activity from glucose (hereinafter referred to as GLF activity), a certain amount of the above lactic acid bacteria solution was added to 1% glucose in 0.2M phosphate buffer (PIT 5.0), and the mixture was incubated at 30°C. After a certain period of reaction, the amount of lactic acid produced was measured using high-performance liquid chromatography (LC-3A manufactured by Kinsei Seisakusho).
).

To measure the lactic acid production activity in the dough, we added a portion of the above lactic acid bacteria solution and lactic acid bacteria yeast, performed a 1.5-hour fermentation straight bread making experiment (up to fermentation), and measured the amount of lactic acid in the dough at the end of the fermentation. This was carried out by measuring with the high performance liquid chromatography described above. In addition, the quantitative determination of lactic acid in the dough is raw 11! ! The test was carried out by adding room water to 20 g, homogenizing it, centrifuging it, and measuring the amount of lactic acid in the obtained supernatant. The bread making operation and basic recipe are shown below.

(Dough mixture) Wheat flour (strong flour) 100 parts by weight Sugar
5〃Table salt
2 Shortening 5 Yeast (pressed yeast) 2.2 Yeast food O1l Skimmed milk powder
2 Water 70 (Bread making operation) Add raw ingredients 1 other than shortening, knead on low speed for 1 minute, medium speed for 1 minute, and high speed for 5 minutes, add shortening, and then knead for another 1 minute on low speed, 1 minute on medium speed, and 5 minutes on high speed. The mixture is kneaded, kneaded at 27°C, fermented at 30°C for 1.5 hours, then divided and rounded, and then molded using a molder after a 20-minute hench time. Next, at 38°C and 80% humidity,
Perform 0 minute fermentation. In addition, when lactic acid bacteria solution and lactic acid bacteria yeast were used, the bread-making experiment was conducted by subtracting the amounts of water and yeast from the basic formulation by the respective added amounts.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to examples, but it goes without saying that the present invention is not limited to these examples.

Example 1 In order to clarify that storage stability is improved when lactic acid bacteria are stored in compressed yeast, Lactobacillus casei (ATCC393), Leuconostoc metsucentiloides (IAM1046), Streptococcus bovis ( IFO12057) and Pedeiococcus halophilus (JCM2O14), the storage stability of lactic acid bacteria in sterilized water and compressed yeast was compared. Note that hereinafter, a liquid obtained by suspending lactic acid bacteria in sterilized water will be abbreviated as lactic acid bacteria liquid. The method for preparing lactic acid yeast is shown below.

The above lactic acid bacteria were cultured for 24 hours in MR3 medium for lactic acid bacteria (M, R, S, AGAR medium manufactured by OXID■ in the UK),
This was then washed with sterilized water) to obtain a concentrated lactic acid bacteria solution (bacteria concentration of approximately 1.0×101°/d). In the case of lactic acid bacteria solution: Add a certain amount of agricultural shrinkage solution to sterilized water and use 31
If using lactic acid yeast, use commercially available raw yeast)7
Dissolve 0 g in 30 d of sterilized water, and add 5 d of the above concentrated solution to this solution.
After mixing thoroughly, prepare by vacuum filtration through a mensier with filter paper, and reduce the number of bacteria to 1.0 to 2.0.
XIO'/g of lactic acid bacteria yeast was obtained.

The storage stability of lactic acid bacteria was evaluated based on the survival rate over time when stored at 5°C and the lactic acid production activity in the dough.

FIG. 1 shows the viability of the four strains of lactic acid bacteria and lactic acid yeast, with the value immediately after preparation taken as 100%.

In addition, FIG. 2 shows the lactic acid production activity in the dough (the amount of lactic acid produced after fermentation), with the value immediately after preparation taken as 100%. 1st
As is clear from the figure and FIG. 2, when lactic acid bacteria are stored in the form of lactic acid yeast, their stability is significantly improved compared to when they are stored in lactic acid bacteria solution, and in particular, Lactobacillus casei (ATCC393), Leuconobacterium spp. The storage stability of both stock metsucentroides (IAM1046) images was significantly improved.

Example 2 Lactobacillus casei (ATCC) prepared in Example 1
393), Leuconostoc mesicenthroides (
A storage stability experiment was conducted in the same manner as in Example 1, except that the storage temperature was 10°C for the lactic acid bacteria solution and lactic acid bacteria yeast (lAM1046).

The results are shown in FIGS. 3 and 4.

These figures show that although the overall stability of lactic acid bacteria decreases considerably when stored at 10°C, the stability of lactic acid bacteria in lactic acid bacteria yeast is considerably better than that in lactic acid bacteria liquid.

Example 3 Storage stability of lactic acid bacteria
In order to clarify that Htl adjustment improves the effect, Lactobacillus casei (AT
CC393) and Leuconostoc metsucentiloides (IAM1046) were mixed into various pt+
The storage stability of the obtained lactic acid bacteria solution at 5°C was compared with that of the lactic acid bacteria solution, and the viability and GLF activity were evaluated. The ρ11 of the skim milk medium was adjusted using DL-lactic acid for food additives and 4N sodium hydroxide. Lactobacillus casei (ATCC393)
The results for bacteria are shown in Figures 5 and 6, and the results for Leuconostoc mencentiloides (IAM1046) are shown in Figure 7.
These figures shown in Figure 8 show that, although there are slight differences depending on the strain, storage stability is improved by adjusting the pH of the storage solution in the range of pH 4.0 to pH 6.0 in both images; It can be seen that the pH value is further improved in the range of pH 25 to 5.5.

Example 4 A storage stability experiment at 5°C was conducted on the above two strains in the same manner as in Example 3, except that a wheat flour supernatant containing wheat protein was used instead of the 10% by weight skim milk medium. Ta. In addition, the wheat flour supernatant liquid here refers to commercially available wheat flour 10
This is a liquid obtained by adding lOOd of water to g and mixing the mixture for 5 minutes using a household mixer, followed by centrifugation at 3000 rpm. Lactobacillus casei (ATC
C393) bacteria are shown in Figures 9 and 10, and the results for Leuconostoc metsucentiloides (IAM1046) bacteria are shown in Figures 11 and 12.From these figures, both images show pH 4, It can be seen that storage stability is greatly improved by adjusting the pB of the storage solution to a range of 0 to pH 6.0.

Example 5 Skim milk culture medium lactic acid bacteria solution prepared in Example 3 (pH 5°0)
(hereinafter referred to as defatted lactic acid bacteria solution), the wheat supernatant lactic acid bacteria solution (all 5.0) prepared in Example 4 was evaluated for the stability of the lactic acid bacteria based on the lactic acid production activity in the dough. For comparison, lactic acid bacteria solution pl+non-adjusted skim milk medium lactic acid bacteria solution (all6.8) was also evaluated in the same way. FIG. 13 shows the results for Lactobacillus casei (ATCC393), and FIG. 14 shows the results for Leuconostoc metsucentroides (IAM1046). From these figures, both images contain p)! It can be seen that the stability is considerably improved in an unadjusted skim milk medium, and that the storage stability is further improved by storing in a pH-adjusted skim milk medium or wheat supernatant.

Example 6 Preservation of lactic acid bacteria using emulsified oil prepared by adjusting the aqueous phase PL+ of an acid-resistant O/W type oil composition containing whey protein (TL Fuchi Kagaku FXOO5) to pH 5.0 with citric acid and sodium hydroxide. An experiment similar to Example 5 was conducted except that the solution was used as a solution. For comparison, the stability of the lactic acid bacteria in the lactic acid bacteria solution and the above oil and fat composition without ρ■ adjustment (pH 17,0) was also evaluated. Figure 15 shows the results for Lactobacillus casei (ATCC393), and Figure 16 shows the results for Leuconostonk metsucentroides (IAM1046).
) From these figures showing the results for W, it can be seen that even in emulsified fats and oils containing proteinaceous substances, the storage stability of lactic acid bacteria can be greatly improved if the pH of the aqueous phase is adjusted to an appropriate value.

Example 7 In the production of lactic acid bacteria yeast, p prepared in Example 4
Lactic acid yeast was prepared in the same manner as in Example 1 except that H5.0 wheat supernatant lactic acid bacteria solution (Lactobacillus casei (ATCC393)) was used, and the storage stability at 10°C was tested in the dough. The lactic acid production activity was evaluated. For comparison, lactic acid bacteria yeast produced using the lactic acid bacteria of Example 1 was also evaluated in the same manner. The results are shown in FIG.

From the figure, it can be seen that by using the wheat supernatant lactic acid bacteria whose pH has been adjusted to 414, the stability of the lactic acid bacteria in the lactic acid bacteria yeast is further improved compared to the lactic acid bacteria yeast of Example 1. This is considered to be due to the synergistic effect of the protective effect of the yeast and the stabilizing effect of the pH-adjusted protein substance solution.

Example 8 In order to compare the lactic acid production activity in dough of a freeze-dried product of lactic acid bacteria and a solution containing lactic acid bacteria, which is one form of the present invention, a defatted lactic acid bacteria solution (pH 5.0) prepared in Example 3 was used. Bacillus casei (ATCC393)) and a freeze-dried product obtained by freeze-drying this solution were added to bread dough so that the number of lactic acid bacteria (viable bacteria) was the same, and the lactic acid production activity in the dough was measured. As a result, although the number of bacteria added was the same, it took longer for the bacteria to activate in the freeze-dried product, and the amount of lactic acid produced after boiling was about 5 times lower than in the defatted lactic acid bacteria solution. For this reason, the storage-stable lactic acid bacteria-containing products and solutions of the present invention are extremely effective for applications that require stable and immediate activity development after addition of lactic acid bacteria, such as the use of lactic acid bacteria in bread production. It is thought that. In addition, for freeze-drying the lactic acid bacteria solution, the above-mentioned defatted lactic acid bacteria solution is placed in a bicelle for approximately 50 minutes.
This was done by freezing the cells at -20'C (about 2 hours) and then vacuuming them for about 24 hours.

[Action/Effect]

As mentioned above, according to the present invention, lactic acid bacteria, which were conventionally said to have poor storage stability, can be mixed and dispersed in compressed yeast or stored in a proteinaceous substance-containing solution with pt+ adjusted to an appropriate range. Therefore, its storage stability can be dramatically improved. Furthermore, when such lactic acid bacteria-containing materials, solutions, or oil and fat compositions with improved storage stability are used, for example, in bread production, conventional freeze-dried lactic acid bacteria,
Unlike frozen lactic acid bacteria, it does not need to be pre-cultured and activated before use, and it stably produces highly active lactic acid in the dough, making it easy to use and requiring only a small amount of bacteria to be added. It has these excellent characteristics and is extremely useful. Furthermore, it goes without saying that the lactic acid bacteria-containing material, solution, or oil/fat composition of the present invention is extremely useful for purposes other than bread production due to the above-mentioned excellent properties.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 to 17 are graphs showing the results of Examples. 5th figure spring PH 150 people 6th figure 8 0-I take away ir I day cup L 1501- 9th figure 11 ＋'fB pier ('/,)

Claims (1)

[Scope of Claims] 1. A method for storage stabilizing lactic acid bacteria, which comprises improving the storage stability of lactic acid bacteria by mixing and dispersing lactic acid bacteria in compressed yeast. 2. The method according to claim 1, wherein the pressed yeast is yeast belonging to Saccharomyces cerevisiae. 3. Claim 2, wherein the pressed yeast is one or more selected from baker's yeast, sake yeast, beer yeast, and wine yeast.
Method described. 4. The method according to claim 1, wherein the lactic acid bacteria are one or more selected from strains belonging to the genus Lactobacillus and the genus Leuconostoc. 5. A method for storage stabilizing lactic acid bacteria, which comprises allowing lactic acid bacteria to exist in a proteinaceous substance-containing solution whose pH is adjusted to 4.0 to 6.0. 6. 10^6-10^1^ of lactic acid bacteria per 1g of compressed yeast
A substance containing lactic acid bacteria mixed and dispersed. 7. Protein substances 0.1-30% by weight, lactic acid bacteria 10^
Contains 5-10^1^1 piece/ml, pH is 4.0-6.0
A solution containing lactic acid bacteria. 8. Lactic acid bacteria-containing solution 5-7 according to claim 7 as the aqueous phase part
0 parts by weight, fats and oils part 95-30 parts by weight, emulsifier 0.1-5
An O/W emulsion type oil and fat composition consisting of parts by weight.