JPS6143032B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for culturing microorganisms, particularly Lactobacillus sanfrancisco, and a method for preserving freeze-dried bacterial cells. Lactobacillus sanfrancisco is known as a sour dough strain. Sourdough bacteria is a useful microorganism used in the production of sourdough French bread, and its culture and freeze-drying preservation techniques are covered in U.S. Patent No. 3734743.
No. 3891773 and No. 4021581. According to the conventional method, when culturing sourdough bacteria, it is essential to add fresh yeast extract, that is, a hot water extract of fresh yeast cells, to the medium.
This cannot be replaced with commercially available yeast extract. Sourdough culture solution 10 i.e. as dried bacterial cells
To obtain 30 to 50 g, 2 kg of fresh yeast cells (pack weight) is required. Since it is difficult to constantly supply fresh yeast extract, it would be industrially advantageous if there was a substance that could be substituted for fresh yeast extract and could be constantly supplied. The present inventors have discovered that L-cysteine can be substituted for fresh yeast extract. L-cysteine is an amino acid that can be supplied industrially at low cost. In addition, in order to constantly supply sourdough bacteria, it is convenient to use a freeze-drying preservation method. It is conventionally known to use polymeric substances such as skim milk and serum, amino acids such as glutamic acid, and sugars such as glucose and sucrose as protective substances in the freeze-drying of microbial cells. Regarding sourdough bacteria, US Pat. No. 4,021,581 discloses a freezing method using glycerol, skim milk, sucrose, unsaturated fatty acids, monosodium glutamate, etc. The present inventors conducted various studies to further improve the preservability of sourdough bacteria during freeze-drying storage, and found that the preservability was significantly improved by adding maltose to conventional skim milk and monosodium glutamate. I found that it can be improved. The present invention will be explained in detail below. The cultivation of the microorganism in the present invention is carried out by culturing a strain belonging to Lactobacillus San Francisco in the presence of a carbon source, a nitrogen source, inorganic nutrients and other nutrients, as well as L-cysteine or a substance containing L-cysteine. Any strain can be used as long as it belongs to Lactobacillus San Francisco. Specifically, Lactobacillus San Francisco KY3689 (Feikoken Bacteria No. 4466)
can be given. Applied for the mycological properties of Lactobacillus San Francisco
Microbiology Vol.21, No.3, p.459−465, 1971
There is a description in . Carbon sources for the culture medium include maltose, nitrogen sources include organic nitrogen sources such as peptone, yeast extract, corn steep liquor, and casein hydrolyzate, and inorganic nutrients include magnesium sulfate, manganese sulfate, and monosodium phosphate. , dibasic sodium phosphate, ferrous sulfate, etc. are used. Lactobacillus San Francisco requires unsaturated fatty acids for growth, so the culture medium includes oleic acid, linoleic acid, linolenic acid, ricinoleic acid or their sodium or calcium salts, olive oil, cottonseed oil, linseed oil, soybean oil, safflower oil,
Add corn oil etc. Other partial esters of unsaturated fatty acids and sugars or polyhydric alcohols, such as sucrose monooleate, sucrose dioleate, glycerin monooleate, ethylene glycol monooleate, pentaerythritol monooleate, mannite monooleate, sorbitan monooleate , sorbitan polyoxyethylene monooleate, polyoxyethylene monooleate, etc. can be used. The content of L-cysteine or L-cysteine-containing substances in the medium is 100 to 500% as L-cysteine.
Good culture results can be obtained with mg/. Examples of L-cysteine-containing substances include casamino acids. The culture is carried out at 25 to 35°C, preferably about 28 to 32°C, until the early stationary phase, while adjusting the pH of the medium to 5.0 or higher using sodium hydroxide, potassium hydroxide, aqueous ammonia, or the like. After culturing, the culture is centrifuged to collect the bacterial cells,
Subject to the next freeze-drying process. The freeze-drying process in the present invention is carried out as follows. The bacterial cells collected as described above were washed with physiological saline, and 10 ⁹ to 10 ¹¹ cells were placed in a dispersion medium consisting of 10 to 30% skim milk, 1 to 3% monosodium glutamate, and 0.5 to 9% maltose. Suspend at a concentration of /ml. After freezing the suspension at -25°C to -70°C, dry it at room temperature to reach a product temperature of 25 to 30°C. The dried bacterial cells thus obtained are preferably placed in a plastic bag, sealed with a vacuum packaging machine, and stored at 0 to 10°C. The survival rate of freeze-dried bacterial cells obtained by the method of the present invention during freeze-drying was 70% or more, and the survival rate after storage at 5°C for 6 months was 60% or more at the start of storage. . The values when maltose is not used are as follows:
It was 20%, 25%. According to the present invention, the production cost of Lactobacillus sanfrancisco has been significantly reduced and storage has become easier, making the production of sourdough bread industrially very advantageous. Example 1 Lactobacillus San Francisco KY3689 was mixed with maltose 40g/, peptone 10g/, yeast extract 15g/, MgSO ₄ 7H ₂ O 200mg/, MnSO ₄ .
4H ₂ O 40mg/, Tween 80 (trade name of surfactant sorbitan monooleate, manufactured by Kanto Kagaku Co., Ltd.) 300mg/
, 300 mg/L-cysteine/20 g/agar/slant culture medium (PH5.6 before sterilization) at 30°C for 24 hours. This bacterial loop was inoculated into a 300 ml Erlenmeyer flask containing 150 ml of a liquid medium (seed medium) obtained by removing the agar from the above medium, and incubated at 30°C.
Reciprocal shaking culture was performed at 70 rpm for 15 hours. This seed culture solution was transferred to a 5-jar fermenter containing medium 3 having the same composition as the seed medium, and cultured. Culture is
The mixture was stirred at 300 rpm and heated at 30° C. for 15 hours while adjusting the pH to around 5 with 3NKOH. The bacterial cell concentration at the end of the culture was 3.8 g/dry bacterial cell. This culture solution 1 was centrifuged at 5000 rpm for 20 minutes.
After suspending in 300 ml of dispersion medium containing dl and freezing at -30℃,
After drying for 16 hours, 36 g of lyophilized bacterial cells were obtained at a final product temperature of 30°C. The survival rate was 72% of the number of viable bacteria before freeze-drying. The freeze-dried cells were placed in plastic bags, sealed in a vacuum packaging machine, and stored at 5°C. The survival rate after 6 months of storage was 80% compared to the time at the start of freeze-drying. Example 2 The procedure was carried out in the same manner as in Example 1, except that the amount of L-cysteine in the medium was adjusted to 0 to 800 mg/, or the culture was performed with fresh yeast extract added instead of L-cysteine. The results shown in Table 1 were obtained.
Fresh yeast extract is obtained by culturing baker's yeast in a conventional manner, suspending it in water at a concentration of 200g/ml, autoclaving it at 120℃ for 30 minutes,
After being left at 5°C for 12 hours, centrifugation was performed at 5000 rpm for 20 minutes, and the supernatant was used to dissolve the medium components.

[Table] Example 3 Same as Example 1 except that the maltose during freeze-drying was replaced with the sugars listed in Table 2 below, and the freeze-dried product was stored at 5°C in a vacuum desiccator.
Samples were taken after 1 month, 3 months, and 6 months to examine the survival rate. The results are shown in Table 2.

【table】

Claims (1)

[Claims] 1. Freeze-dried cells of Lactobacillus San Francisco, characterized by using skim milk, monosodium glutamate, and maltose in an amount of 5 to 20% by weight based on the weight of the skim milk as protective agents when storing cultured cells of Lactobacillus San Francisco by freeze-drying. preservation method.