JPS62268A - Mutant lactobacillus bulgaricus and selection thereof - Google Patents

Abstract

PURPOSE:To separate a novel mutant of Lactobacillus bulgaricus producing little lactic acid at low temperature and having high low-temperature sensitivity, by culturing activated bacterial strain in milk medium and cooling the culture product. CONSTITUTION:Lactobacillus bulgaricus ATCC 11842 is inoculated in a defatted milk medium containing yeast extract and cultured twice. The activated bacterial strain is inoculated in a milk medium and cultured at 43 deg.C until the amount of lactic acid reaches 0.23-0.28%. The obtained culture product is cooled without delay and stored for 7 days at 10 deg.C. The bacterial strain having a lactic acid increment of <=0.1% is selected from the culture product to obtain Lactobacillus bulgaricus OLL 1070 (FERM-BP 1041) which is a mutant of Lactobacillus bulgaricus having low-temperature sensitivity.

Description

[Detailed Description of the Invention] [Object of the Invention] The present invention relates to a novel microorganism and a method for selecting the same, and more specifically, the present invention relates to a novel microorganism and a method for selecting the same.
actobacillus bulgaricus O
LL 1074) and its selection method.

[Conventional technology]

Yogurt, which has been produced for a long time as a typical fermentation method, is produced by Lactobacillus bulgaricus (Lactobacillus bulgaricus).
illus bulgaricus) and Streptococcus thermophilus (Streptococcus tococcu).
sthermoph i Ius) 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 nigritii (Lactobacillus nigriti) was used as a starter in Japanese Patent Publication No. 54-38187.
There is a method of using mutant strain M-13, which is obtained by artificially mutating S. jugurti and making it non-lactic acid fermentative.

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, making the process complicated and uneconomical in terms of thermal energy.
Difficult to control temperature and number of viable bacteria. Tokuko Sho 54-3818
In the method of Publication No. 7, it is essential to add the fermentable saccharide of jugurti mutant strain to the medium, so it is complicated to set the amount to be added to the target acidity. It is not possible to produce brain-type yogurt.

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 is 23% to 0.28%, it is cooled.
Lactobacillus bulgaricus was added to a skim milk medium containing yeast extract and microorganisms belonging to a cold-sensitive Lactobacillus bulgaricus mutant strain characterized by an increase in lactic acid of 0.1% or less when stored at 10°C for 7 days. B. bulgaricus strain was inoculated and cultured twice, and this activated strain was inoculated into a milk culture medium, and the amount of lactic acid was raised to 0.23% to 0.2% at 43°C.
A low-temperature-sensitive lactobacillus that consists of culturing until the lactic acid concentration reaches 28%, immediately cooling the resulting culture, storing it at 10°C for 7 days, and selecting from the culture a strain with an increase in lactic acid of 0.1% or less. This is a method for selecting microorganisms belonging to Bacillus bulgaricus mutants.

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. Figure 1 shows the increases in the amount of D (-) lactic acid, the amount of L (+) lactic acid, and the amount of total lactic 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, Lactobacillus bulgaricus AT
After inoculating CC11842 into MR3 medium and culturing, a portion of this culture solution is added to MRS 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. Bacterial strains accounting for 1% or less were selectively isolated.

In addition, MR8 Danchi culture medium peptone 10g, meat extract 10g
, yeast extract 5g, glucose 20g, Tween 801.
0rBl, K2HPO42,Og, CHzCOO
Na'3H, 05g, ammonium citrate 2g
1MgSO4・7H, 0200mg, Mn5Oa
44Hz 050mg and distilled water 1000+nl were mixed, the pH was adjusted to 6.0 to 6.5, and sterilized at 120℃ for 15 minutes.The milk medium was a mixture of milk, skim milk powder, and water at a ratio of 95=2:3. , dissolved 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. Named °. 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 (1) Cell shape: Bacillus (2) Motility: None 3) Presence of spores: None 4) Durham staining: Positive (5) B with metachromatic bodies, on medium Growth Condition 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.

C0 physiological properties (1) Reduction of nitrate: negative (2) Formation of indole: negative (3) Liquefaction of gelatin: negative (4) Catalase: negative (5) Decomposition of starch: negative (6) Two sets of attitudes toward oxygen Anaerobe (7) Produces D(-) lactic acid from glucose by homolactic acid fermentation,
No gas is produced.

(8) No CO2 is produced from malate.

(Proliferation is impossible or very weak at 25°C in 91MR3 medium, but it grows vigorously at 45°C.

Degradability of various carbohydrates of α ■ Glucose: + ■ Lactose: + ■ Fructose: + ■ Mannose: + ■ Galactose nii ■ Sucrose nii ■ Maltose nii ■ Cellobiose nii ■ Trehalose nii [phase] Melibiose: - ■ Raffinose nii ■ Meletetose 21 ■ Starchney ■ Mannitolney [phase] Sorbitolney [phase] Nice scrine @ salicin 21 [phase] Amygdalin
74)), the parent strain Lactobacillus
Although the mycological properties were consistent with those of bulgaricus ATCC 11842, there was a clear difference in the ability to grow at 25° C. in MR3 medium, indicating that the strain was a mutant strain with low temperature sensitivity.

This low-temperature 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 Lactobacillus bulgaricus 0LL1074 (I Koken Bacteria 2nd Edition) obtained by subculture in skim milk medium was inoculated at 2% into milk medium, cultured at 43°C, and the amount of lactic acid was reduced to about 0. .25%
The increase in lactic acid when the sample was stored at 10℃ for 7 days.

In order to further clarify the difference between the cold-sensitive mutant Lactobacillus bulgaricus OL L1074 (Feikoken Bulgaricus No. 2322) 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 the amount of lactic acid produced is considerably suppressed when the fermentation hole using Lactobacillus bulgaricus OL L LO74 is stored at a low temperature. 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 10oC 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 OL L 1074 is used as a starter for yogurt, the increase in acidity during low temperature storage is low and the amount of lactic acid produced is suppressed, resulting in a desirable sour taste immediately after fermentation holes are produced. can be retained even during distribution and storage.

Next, examples of the present invention will be shown.

<Example> Lactobacillus bulgaricus ATCC11842
It is inoculated into R5 medium 30- and cultured at 37°C for 16 hours.

20 mZ of this culture solution was mixed with 0.1 penicillin G potassium salt.
500 mt of MR3 medium containing 0 unit/rnl
and cultured at 25°C for 48 hours. This culture solution is centrifuged at 5000 rpm for 15 minutes to collect bacteria, and the resulting bacterial cells are suspended in sterile physiological saline. A 0.1 ml portion of this was 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 to 0.
．． After culturing at 43°C until the concentration reaches 28%, it is immediately cooled. From these cultured seedling 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 selected a strain of cold-sensitive Lactobacillus bulgaricus 0LL1.
074 was isolated.

Next, a case where yogurt and a lactic acid bacteria drink were prepared using Lactobacillus bulgaricus 0LLIO74 will be shown as a reference example.

Reference 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 prepare a starter for fermented milk. Add this starter to sterilized yogurt mix (sterilized milk enriched with skim milk powder).
% inoculation, 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 at 43°C for 3.5 hours. It was exactly the same as when Lactobacillus bulgaricus ATCC 11842 was used as a control, but when stored at a low temperature below 25°C, the increase in acidity was less than 2 compared to the control product, and the increase in acidity was low. ,10℃
The proportion of D(-) lactic acid in the total lactic acid after 14 days of storage was 20.8%, which was clearly lower than that of the control product, which was 50.4%.

<Reference example 2> Low temperature sensitive Lactobacillus bulgaricus 0LLI0
74 and Streptococcus 7cus thermophilus f A
M-1047 was 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, vine, etc.) was added with stirring, and the mixture was filled into containers to obtain a fruit yogurt product. The fruit yogurt thus obtained was fermented at 43°C for 4 hours, and the control Lactobacillus bulgaricus ATCC1
Although it was exactly the same as when using 1842, the increase in lactic acid when stored at 10°C for 7 days was 0.08%, and the increase in acidity during low temperature storage was considerably suppressed.

(Reference example 3) Low temperature sensitive Lactobacillus bulgaricus OL L
1074 is inoculated into sterilized skim milk and cultured at 37°C for 4 hours. Separately, Lactobacillus acidophilus ΔTCC-4
356 was inoculated into sterilized skim milk and cultured at 35°C for 20 hours. pl+3 consisting of sucrose, CMC, fragrance, and organic acid.
60 kg of sterilizing mixture (sucrose 14.0. CMC
O, 4, fragrance 0.05. Citric acid 0.20°, Ascorbic acid 0.05 kg, Water 46.0: Units are kg
), and add 40k of equal amounts of the above two types of bacterial liquid to this.
g and homogenize to make 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〕

Yogurt and lactic acid bacteria beverages prepared using the cold-sensitive mutant strain of Lactobacillus bulgaricus according to the present invention suppress the increase in sourness when stored at low temperatures, and retain the favorable sourness immediately after production even during distribution and storage. This has the extremely useful effect of being retained.

[Brief explanation of drawings]

Figure 1 shows the amount of increase in lactic acid when yogurt is stored at 10°C, Figure 2 shows the bacterial growth curve in MR3 medium, and Figure 3' shows the change in the amount of lactic acid when fermented milk is stored at 10°C. , 4th
The figure shows Am using a mixed starter! The changes in the amount of lactic acid when the yogurt was stored at 10°C are shown. Agent Patent Attorney Yusuke Hiraki M I Figure: Increase in lactic acid when yogurt is stored at 1σC Number of storage days at 10℃ M3 Figure: Change in lactic acid amount when fermented milk is stored at 106C 2) C) ←) Lactic Acid (vomit) Procedural Amendment June 19, 1988 Michibe Uga, Commissioner of the Patent Office1, Indication of Case Patent Application No. 137831-1982, Name of Invention Mutant strain Lactobacillus bulgaricus and its selection method 3. Relationship with the case of the person making the amendment Patent applicant address 2-3-6 Kyobashi, Chuo-ku, Tokyo Name (6
13) Meiji Dairies Co., Ltd. Representative: Hazo Shimamura 4, Agent address: 3-20-4-7 Toranomon, Minato-ku, Tokyo Contents of amendment (11 Specification, page 8, lines 6-7 Kenbokuyori No. 83
No. 22 (FEI?M-PNo. 8322) J and the text “Feikoken Jyoyori No. 1041 (Feikoken Bichiyori No. 8322)
” he corrected. (2) Page 11, line 5 of the specification:
2)” should be amended to read “(Feikoken Joyori No. 1041).” (3) Specification, page 12, line 1
22)” should be amended to read “(Feikoken Jokyo No. 1041).” Notification of change in accession number 7゜June 19, 1985 8゜Mr. Michibe Uga, Commissioner of the Patent Office
9゜1, Indication of the case Japanese Patent Application No. 137831/1986 2, Name of the invention Mutant strain Lactobacillus bulgaricus and its selection method 3, Person who carried out the procedure Relationship with the case Patent applicant address Kyobashi, Chuo-ku, Tokyo 2-3-6 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, Agency of Industrial Science and Technology, Institute of Biological Technology 6, former accession number: No. 8322 Name of new depositary institution Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Innovative research on microbial technology

Claims (3)

[Claims] (1) Inoculate the bacterial cells into milk culture medium and reduce the amount of lactic acid to 0.2 at 43℃
After culturing until the concentration is 3% to 0.28%, cool it and incubate at 10°C.
A microorganism belonging to a cold-sensitive Lactobacillus bulgaricus mutant characterized by an increase in lactic acid of 0.1% or less when stored for 7 days. (2) The microorganism is Lactobacillus bulgaricus strain OL
Claim 1 characterized in that it is L1074.
Microorganisms listed in section. (3) Lactobacillus in skim milk medium containing yeast extract
Bulgaricus strain is inoculated and cultured twice, and this activated bacterial strain is inoculated into a milk medium and cultured at 43°C until the amount of lactic acid reaches 0.23% to 0.28%, resulting in a culture. A method for selecting a microorganism belonging to a cold-sensitive Lactobacillus bulgaricus mutant strain, which comprises immediately cooling and storing at 10°C for 7 days, and selecting from the culture a strain with an increase in lactic acid of 0.1% or less. .