JPS62239A - Fermented milk, lactic acid bacterial beverage and production thereof - Google Patents

Abstract

PURPOSE:To preserve fermented milk or lactic acid bacterial beverage without causing deterioration in flavor, etc., due to an increase in acid taste during refrigeration, by fermenting a low temperature-sensitive microorganism, belonging to the genus Lactobacillus and slightly producing lactic acid at low temperatures. CONSTITUTION:A low temperature-sensitive microorganism belonging to Lactobacillus bulgaricus variant strain giving <=0.1% increase in lactic acid when microbial cells are inoculated into a cow's culture medium, cultivated at 43 deg.C until the amount of lactic acid becomes 0.23-0.28%, cooled and kept at 10 deg.C for 7 days, e.g. Lactobacillus bulgaricus OLL1074 (FERM-P No.1041) obtained from Lactobacillus bulgaricus ATCC11842 as a parent strain or a mixed microorganism of the above-mentioned microorganism with another lactic acid bacterium, e.g. Streptococcus thermophilus, is used as a starter, inoculated into a milky raw material and fermented.

Description

[Detailed Description of the Invention] [Object of the Invention] The present invention relates to a novel fermented milk or lactic acid bacteria beverage and a method for producing the same, and more specifically, to a low-temperature-sensitive Lactobacillus bulgaricus OL L whose lactic acid production is weak at low temperatures.
1074 (Lactobucillus bulgari
The present invention relates to fermented milk or lactic acid bacteria beverages obtained using L.cus OL L1074) and a method for producing the same.

[Conventional technology]

Yogurt, which has been produced since ancient times as a typical fermentation method, is produced by Lactobacillus bulgaricus.
illus bulgaricus) and Streptococcus thermophilus
hermophilus) as the main starter. That is, a yogurt mix is inoculated with these lactic acid bacteria starters, fermented at 40 to 46°C for several hours, and when a suitable acidity is reached, it is refrigerated to stop the fermentation and sold in a refrigerated state.

Although lactic acid fermentation of yogurt is considerably suppressed by storing it at low temperatures, it is not completely suppressed. Even during low-temperature storage, lactic acid is gradually produced and the sourness of yogurt increases, so it is extremely difficult to maintain the desired sourness immediately after production even during storage and distribution.

Therefore, a method is known that sterilizes yogurt immediately after production to effectively increase its shelf life. characteristics are lost. As another method for preventing increases in acidity during yogurt storage, the yogurt disclosed in JP-A-50-6745 is heated at a temperature and for a period of time that is above the growth termination limit temperature on the high-temperature side of lactic acid bacteria but does not completely kill them. Lactobacillus nigriti (Lactbacillus nigriti) was used as a starter in Japanese Patent Publication No. 54-38187, which describes a heating method under the conditions of
There is a method of using mutant strain M-13, which is obtained by artificially mutating M. jugurti and making it non-lactic acid fermentable. However, the method disclosed in Japanese Patent Application Laid-open No. 50-6745 requires heating the material left at a low temperature and then cooling it further.
The process is complicated and uneconomical in terms of heat energy, and it is difficult to control temperature and viable bacteria count. Special Public Service No. 54-38187
In the method of the publication, it is essential to add the fermentable saccharides of Jugurti mutant strain to the medium, so it is complicated to set the amount added to the target acidity, and consumer demands have recently increased. Brain type yogurt cannot be manufactured.

The present inventor focused on the mechanism of increase in lactic acid amount during storage of yogurt, isolated a cold-sensitive Lactobacillus bulgaricus mutant strain that exhibits an extremely low rate of increase in lactic acid under low temperature, and completed the present invention. It has arrived.

[Structure of the invention]

In the present invention, bacterial cells are inoculated into a milk medium and the amount of lactic acid is reduced to 0 at 43°C.
．． After culturing until the concentration reached 23-0.28%, it was cooled and
A microorganism belonging to a cold-sensitive Lactobacillus bulgaricus mutant strain characterized by an increase in lactic acid of 0.1% or less when stored at ℃ for 7 days is used, and if necessary, other microorganisms such as Streptococcus thermophilus etc. are used. The present invention relates to a fermented milk or a lactic acid bacteria drink obtained by using a mixture of lactic acid bacteria, and a method for producing fermented milk or a lactic acid bacteria drink by using the above-mentioned microorganism as a starter, inoculating it into a milk raw material, and fermenting it.

The increase in acidity when yogurt is stored refrigerated is mainly due to D(-) produced by Lactobacillus bulgaricus.
) Due to lactic acid.

This is a yogurt mix that contains Lactobacillus bulgaricus and Streptococcus thermophilus.
This can be determined from the increase in the amount of lactic acid when the resulting yogurt is stored at low temperature by inoculating the yogurt with the following: (us) and carrying out lactic acid fermentation at 40 to 46°C. FIG. 1 shows the increase in the amount of D(-) lactic acid, the amount of L(+) lactic acid, and the amount of total floral acid when yogurt was stored at 10° C. for about two weeks. In addition, D (-)
Lactic acid is produced by Lactobacillus bulgaricus and also produced by L(
+) Lactic acid is produced by Streptococcus thermophilus. It can be seen from FIG. 1 that most of the lactic acid produced during low temperature storage is D(-) lactic acid. This indicates that the increase in sourness of yogurt during low-temperature storage is mainly caused by Lactobacillus bulgaricus.

The present inventors focused on this point and conducted intensive research, and as a result,
We have succeeded in isolating a cold-sensitive Lactobacillus bulgaricus mutant strain that exhibits active lactic acid fermentation at around 43°C but produces extremely low amounts of lactic acid during low-temperature storage, and has completed the present invention.

The present inventors have discovered that Lactobacillus bulgaricus ATCC
11842 as a parent strain, it was cultured in an isolation medium, and the cold-sensitive Lactobacillus bulgaricus mutant of the present invention was isolated. That is, to Lactobacillus bulgaricus
After inoculating CC11842 into MRS medium and culturing, a portion of this culture solution is added to MR3 medium containing penicillin G potassium salt and cultured. Bacterial cells are separated from this culture solution, smeared on a blood liver agar plate, and cultured under anaerobic conditions. Colonies appearing on blood liver agar are subcultured twice in sterile skim milk. This culture solution was inoculated into a milk medium and cultured at 43°C until the amount of lactic acid reached 0.23 to 0.28%, immediately cooled, and when stored at 10°C for 7 days, the increase in lactic acid was 0.1%. % or less of bacterial strains were selectively isolated.

In addition, MR3 medium contains 10 g of peptone, meat extract Log
, yeast extract 5g, glucose 20g, Tween 801.
Od, K2HPO42, Og, CHzCOON
a'3Hz05g, ammonium citrate 2g,
MgSO4・7Hz0200mg, Mn5On・4
Mix 050 mg of Hz and 1000 mZ of distilled water,
It was prepared to a pi of 6.0 to 6.5 and sterilized at 120°C for 15 minutes, and the milk medium was prepared by mixing milk, skim milk powder, and water at
The mixture was mixed and dissolved in a ratio of 5:2:3, and heat sterilized at 95°C for 5 minutes.

Although this newly isolated microorganism has the following mycological properties and belongs to Lactobacillus bulgaricus for the reasons described below, it was identified as a mutant strain of Lactobacillus bulgaricus and was designated as Lactobacillus bulgaricus OL L 1074. I named it. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as Fiber Science and Technology Research Institute No. 8322 (FERM-P No. 8322).

Its mycological properties are as follows.

A, Morphological characteristics (11II spore shape: Bacillus (2) Motility: None 3) Presence of spores: None 4) Durham staining: Positive (5) B with true east corpuscles B, Growth on medium This strain was spread on a BL agar medium (Eiken) plate and cultured at 37°C for 48 hours using the steel wool method, showing an opaque amorphous R-type colony morphology.

C1 Physiological properties (1) Nitrate reduction: Negative (2) Indole formation: Negative (3) Gelatin liquefaction: Negative (4) Catalase: Negative (5) Starch decomposition: Negative (6) Attitude toward oxygen: General Sexual anaerobes (7)
D(-) lactic acid is produced from glucose by homolactic acid fermentation, and no gas is produced.

(8) No CO2 is produced from malate.

(9) Growth is impossible or very weak at 25°C in MR3 medium, but it grows vigorously at 45°C.

α・ Degradability of various carbohydrates ■ Glucose: + ■ Lactose: + ■ Fructose: + ■ Mannose: + ■ Galactose nii ■ Sucrose nii ■ Maltose nii ■ Cellobiose nii ■ Trehalose nii [phase] Melibiose: - ■ Raffinose nii ■ Meletetose 210 Starchney ■ Mannitolney ■ Sorbitonil: 1 [Phase] Eustarin 21 ■ Salicin: - [Phase] Amygdalin: -
974) '), the mycological properties matched the parent strain Lactobacillus bulgaricus ATCC 11842, but they were clearly different in that their growth ability at 25°C in MR3 medium was extremely low, making it a mutant strain with low temperature sensitivity. One thing was recognized.

This cold-sensitive Lactobacillus bulgaricus OL
L 1074 was subcultured and tested for 10 generations, but the results are shown in Table 1, and the lactic acid production ability at low temperatures was always low.

Table 1: Increase in lactic acid when Lactobacillus lactic acid obtained by subculturing in skim milk medium and containing approximately 0.25% was stored at 10°C for 7 days.

In order to further clarify the difference between the cold-sensitive mutant Lactobacillus bulgaricus OL L1074 (Huikoken Bacteria Collection No. 3322) and the parent strain Lactobacillus bulgaricus ATCC11842, both strains were inoculated into MR3 medium. , the growth curves when cultured at 25°C and 43°C were investigated. The results are shown in Figure 2. As is clear from FIG. 2, the growth curves of the parent strain and this strain show no difference at 43°C, but are clearly different at 25°C.

Next, Lactobacillus bulgaricus 0LLIO74 was inoculated into sterilized skim milk and cultured at 43°C. 2% of the obtained starter was inoculated into the milk and cultured at 43°C for 4 hours to prepare fermentation holes. When this fermentation hole was stored at 10° C. for 14 days, changes in the amount of lactic acid were investigated.

The results are shown in Figure 3. From FIG. 3, it can be seen that when the fermentation chamber using Lactobacillus bulgaricus OL L 1074 is stored at low temperature, the amount of lactic acid produced is considerably suppressed. Furthermore, Lactobacillus bulgaricus OL L
1074 and Streptococcus thermophilus IA
Yogurt was prepared using a starter mixed with M-1047. When this yogurt was stored at 10° C. for 14 days, changes in lactic acid acidity (%) and D(-) lactic acid amount (%) were investigated. The results are shown in Figure 4. Figure 4 shows that when Lactobacillus bulgaricus 0LL1074 is used as a starter for yogurt, the increase in acidity during low temperature storage is low and the amount of D (-) lactic acid produced is suppressed, so the desirable sour taste immediately after fermentation pores are produced is distributed. It can be retained even during storage.

Other lactic acid bacteria used in the present invention include Streptococcus thermophilus (IAM 1047) (Streptococcus thermophilus).
ptococcus thermophilus), Lactobacillus lactis
Iactis), Bifidobacterium (Bifid
obacterium), Lactobacillus acidophyllus
illus), Lactobacillus casei (Lactob
acillus casei).

Reference example: Lactobacillus bulgaricus ATCC11842
It is inoculated into R3 medium 30- and cultured at 37°C for 16 hours.

Add 1 Wi 20 rnl of this culture medium to 0% penicillin G potassium salt.
．． MR3 medium 500 containing 10 units/rri
mZ and cultured at 25°C for 48 hours. This culture solution is centrifuged at 500 rpm for 15 minutes to collect bacteria, and the resulting bacterial cells are suspended in sterile physiological saline. This 0.1d
The cells are smeared onto a blood liver agar (BL agar) plate and cultured anaerobically at 37°C for 48 hours.

These operations are repeated, and each colony that appears on the BL agar plate is inoculated into sterilized skim milk containing 0.1% yeast extract, and subcultured twice at 37° C. for 16 hours. This culture solution was inoculated into milk culture medium at 2%, and the amount of lactic acid was 0.23~
After culturing at 43°C until the concentration reaches 0.28%, it is immediately cooled. From these cultured strains, we selected a strain with an increase in lactic acid of 0.1% or less when stored at 10°C for 7 days, and extracted the cold-sensitive Lactobacillus bulgaricus OL.
L 1074 was isolated.

Next, the present invention will be explained in detail.

<Example 1> Low temperature sensitive Lactobacillus bulgaricus OL L
1074 and Streptococcus thermophilus IA
M-1047 was inoculated into sterilized skim milk and cultured at 43°C to obtain a starter for fermented milk. Add this starter to sterilized yogurt mix (sterilized milk enriched with skim milk powder) at 2%
The cells were inoculated and immediately cultured at 43° C. until the lactic acid acidity reached 0.8%, and immediately cooled to obtain yogurt. The yogurt thus obtained was fermented for 3.5 hours at 43°C and was fermented with Lactobacillus bulgaricus AT as a control.
It was exactly the same as when using CC11842, but 25
The increase in acidity when stored at a low temperature below ℃ is higher than that of the control product.
The increase in acidity was low. Also, 10℃ 14
The proportion of D(-) lactic acid in the total lactic acid after storage for 1 day was 20.8%, which was clearly lower than that of the control product, which was 50.4%.

<Example 2> Low temperature sensitive Lactobacillus bulgaricus OL L
1074 and Streptococcus thermophilus IAM
-1047 is inoculated into sterilized skim milk and cultured at 43°C to obtain a starter for fermented milk. This starter was inoculated at 2% into sterilized yogurt mix (sterilized sweetened milk with skimmed milk powder and sucrose added), immediately cultured at 43°C, and immediately cooled when the lactic acid acidity reached 1.0%, and sterilized. 7.0% of fruit with pulp (orange, strawberry, pineapple, etc.) was added with stirring, and the mixture was filled into containers to obtain a fruit yogurt product. The fruit yogurt obtained in this way takes 4 hours to ferment at 43℃.
Lactobacillus bulgaricus ATCC11 used as a control
It was exactly the same as when using 842, but at 10℃
The increase in lactic acid when stored for days was 0.08%,
The increase in acidity during low-temperature storage was considerably suppressed.

(Example 3) Cold-sensitive Lactobacillus bulgaricus OL L1
074 is inoculated into sterilized skim milk and cultured at 37°C for 4 hours. Separately Lactobacillus acidophilus ATCC-43
56 inoculated into sterile skimmed milk at 35°C.

Incubate for 20 hours. po3 consisting of sucrose, CMC, fragrance, and organic acid. 60 kg of sterilizing preparation of s (1!14.
0. CMCO, 4, fragrance 0.05. Citric acid 0.2
0.05 kg of 0° ascorbic acid and 46.0 kg of water (unit: kg) are prepared, and 40 kg of the above-mentioned two types of bacterial solutions in equal parts are blended therein and homogenized to obtain a lactic acid bacteria drink. When this lactic acid bacteria drink was stored at 10°C for 7 days, no change in acidity was observed, the number of the two types of lactic acid bacteria used did not change, and the flavor was good.

〔Effect of the invention〕

In the present invention, fermented milk or lactic acid bacteria beverages obtained by using a cold-sensitive Lactobacillus bulgaricus mutant strain as a starter were significantly inhibited from increasing in acidity when they were stored at low temperatures. As a result, fermented milk or lactic acid bacteria beverages of excellent quality were obtained that were free of flavor deterioration due to increased acidity during refrigerated storage.

[Brief explanation of drawings]

Figure 1 shows the increase in lactic acid when yogurt is stored at 10°C, Figure 2 shows the growth curve of bacteria in MR3 medium, and Figure 3 shows the change in lactic acid when fermentation holes are stored at 10°C. Figure 4 shows yogurt prepared using a mixed starter at 10°C.
The changes in the amount of lactic acid when stored are shown below. Agent Patent attorney Yusuke Hiraki 1cic@Existence days M 3 Figure, complete taxi 1oc T: Maltization of lactic acid amount during storage LJ/Pi 14
June 19, 1988 Amendment to the proceedings by Michibe Uga, Commissioner of the Patent Office1, Indication of the case, Japanese Patent Application No. 137832-1982, Name of the invention: Fermentation hole, lactic acid bacteria beverage and its manufacturing method, 3. Amendments are made. Relationship with the Patent Case Patent Applicant Address 2-3-6 Kyobashi, Chuo-ku, Tokyo Name (6
13) Meiji Dairies Co., Ltd. Representative: Hazo Shimamura 4, Agent address: 3-20-4-6, Toranomon, Minato-ku, Tokyo, Detailed explanation of the invention column 7 of the specification subject to amendment, Contents of amendment + 11 specifications Book, page 8, lines 5-6
No. 22 (PERM-PNo. 8322)
Microtechnical Research Institute No. 1041 (Microtechnology Research Institute No. 8322)
and correct it. (2) Page 11, line 4 of the specification:
2)" should be amended to read "(Feikoken Joyori No. 1041)." (3) In the first line from the bottom of page 11 of the specification, it says “(Feikoken Bikki No. 8322)”
” he corrected. Notification of change in accession number June 19, 1988 Mr. Michibe Uga, Commissioner of the Patent Office ■, Indication of the incident, Patent Application No. 1988-137832 2, Name of the invention: Fermentation hole, lactic acid bacteria beverage and its manufacturing method 3, Procedures were carried out. Relationship with the Patent Case Patent Applicant Address 2-3-6 Kyobashi, Chuo-ku, Tokyo Name (6
13) Meiji Dairies Co., Ltd. Representative Hazo Shimamura 4, Agent address 3-20-4 Toranomon, Minato-ku, Tokyo, Ministry of International Trade and Industry, Institute of Microbial Technology, Agency of Industrial Science and Technology, 6, former accession number Huikoken Bacteria No. 8322 No. 7, Name of the new depositary institution: Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Institute of Microbial Technology 8, New deposit number: FAIKEN JOKYO No. 1041 No. 9, List of attached documents

Claims (3)

[Claims] (1) Inoculate the bacterial cells into milk culture medium and reduce the amount of lactic acid to 0.2 at 43℃
Using a microorganism belonging to a cold-sensitive Lactobacillus bulgaricus mutant strain that exhibits an increase in lactic acid of 0.1% or less when cultured until the concentration of lactic acid is 3 to 0.28%, cooled, and stored at 10°C for 7 days. , if necessary Streptococcus
Fermented milk or lactic acid bacteria drink obtained by mixing and using other lactic acid bacteria such as thermophilus. (2) The microorganism is Lactobacillus bulgaricus strain OL
Claim 1 characterized in that it is L1074.
Fermented milk or lactic acid bacteria beverage as described in Section 1. (3) Inoculate the bacterial cells into milk culture medium and reduce the amount of lactic acid to 0.2 at 43℃.
A microorganism belonging to a cold-sensitive Lactobacillus vulgaris mutant strain or a microorganism that exhibits an increase in lactic acid of 0.1% or less when it is cultured until the concentration is 3 to 0.28% and then cooled and stored at 10°C for 7 days. A method for producing fermented milk or lactic acid bacteria beverages, which comprises using a mixed microorganism of microorganisms and other lactic acid bacteria such as Streptococcus thermophilus as a starter, inoculating this into a milk raw material and fermenting it.