JPH046349B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for stabilizing dried Bifidus cells. The present invention utilizes a solution containing Bifidobacterium and a stabilizer.
The present invention relates to a method for stabilizing dried bifidus cells, which comprises adjusting the pH to 7 to 10 and freeze-drying the cells. Bifidobacterium has physiological functions such as assisting in the synthesis, digestion, and absorption of proteins and vitamins, preventing the growth of foreign bacteria, and stimulating immune function, and is said to be closely related to human health. It is used in foods and medicines for the purpose of regulating the intestines and treating bacterial replacement caused by antibiotic administration. Bifidobacterium is sensitive to heat and pressure during processing steps, and is extremely unstable in aqueous solutions in the presence of acidity, light, and oxygen. In order to maintain a viable bacterial state for a long period of time, bacterial cells are dried and stored, and freeze-vacuum drying is used as the drying method. In general, freezing or drying microorganisms causes changes in cell morphology, permselectivity of cell membranes, abnormalities in metabolism, and complex nutritional requirements, pH, and temperature-sensitive damaged bacteria are produced. When the number of such damaged bacteria increases in the dried bacterial cells, the number of surviving bacteria decreases rapidly during storage. Various efforts have been made to reduce these damaging bacteria as much as possible. For example, there are many reports regarding the culture age of bacterial cells, bacterial concentration, types and concentrations of stabilizers and excipients, freeze-drying conditions, etc. There is always a need for superior methods for stabilizing Bifidobacterium. As a result of various studies on methods for stabilizing Bifidobacterium, we found that stabilized dried Bifidobacteria cells can be obtained by adding a stabilizer to a solution containing Bifidobacteria, adjusting the pH to 7 to 10, and then freeze-drying. was discovered. The present invention will be explained in detail below. As the Bifidobacterium used in the present invention, any strain belonging to the genus Bifidobacterium can be used. Specifically, Bifidobacterium infantes ATCC 15697 and Bifidobacterium longum ATCC 15707 are used, and these strains are cultured and concentrated by conventional methods. For example, skim milk, yeast extract, peptone,
This bacterium is cultured anaerobically in a mixed medium containing glucose, cysteine hydrochloride, phosphate, etc., and immediately after the culture is completed, the bacteria are collected by centrifugation. Sterilized water is added to the collected bacterial cells to suspend them, and centrifugation is performed again to obtain washed bacterial cells. As stabilizers, amino acids (glutamic acid,
aspartic acid, lysine, arginine, etc.), sugars (glucose, lactose, sugar, sorbitol, raffinose, etc.), polymeric substances or their decomposition products (albumin, seratin, mucin, peptone, meat extract,
soluble starch, dextrin, alginic acid, pectin, gum arabic, etc.), and others (skimmed milk, vitamin C). The amount of stabilizer used is 1 to 50% of the bacterial cell fluid before drying. The pH of the solution containing Bifidobacterium and the stabilizer is 7 to 10, preferably 8 to 9.5. As the PH adjuster, sodium hydroxide, potassium hydroxide, etc. are used. Freeze-drying is performed at a shelf temperature of 30°C or lower (the quality should not exceed 30°C) and a vacuum level of around 0.1 Torr. Next, the storage stability of dried Bifidus cells due to pH changes will be explained. Bifidobacterium infantes ATCC15697 or Bifidobacterium infantes by conventional methods.
Longum ATCC15707 was cultured anaerobically, washed with water, and the concentrated viable bacterial solution was dissolved in skim milk, dextrin, and sodium glutamate, mixed with a pH-adjusted stabilizer solution, and freeze-dried. The dried bacterial cells were packed into a moisture-proof film, and the changes in the number of surviving bacteria after storage at 45°C for 10 and 30 days were measured in Tables 1 (Bifidobacterium infantes ATCC 15697) and 2 (Bifidobacterium infantes).・Longham ATCC15707).

【table】

【table】

[Table] As is clear from Tables 1 and 2, the storage stability of dried bacterial cells with an alkaline pH was significantly improved. Examples are shown below. Example 1 Preparation method of Bifidobacterium concentrate Bifidobacterium infantes
ATCC15697 was incubated at 37°C in a mixed medium containing skim milk.
Culture was carried out for 18 hours while blowing carbon dioxide gas. The obtained culture was washed with water and centrifuged to obtain a concentrated solution containing 4×10 ¹¹ viable Bifidus cells/ml. Preparation method of stabilizer solution The pH of the stabilizer solution containing 40% skim milk powder, 40% dextrin, and 10% monosodium glutamate was 6.4. A 10% sodium hydroxide solution was added to this stabilizer solution to make it alkaline to pH 8.0. Add 100 ml of the Bifidobacteria concentrate to 200 ml of this stabilizer solution, mix, freeze at -30°C, and keep the shelf temperature at 30°C.
It was dried for 18 hours at a vacuum degree of 0.07 Torr. Drying loss of dried bacterial cells (vacuum drying at 70℃ for 16 hours) is 1.5%.
The number of viable bacteria was 2×10 ¹¹ cells/g, and the amount of bacteria obtained was 200 g. The number of viable bacteria and loss on drying of dried bacterial cells without pH adjustment (6.4) were also exactly the same. After filling 1 g of these dried bacteria into an aluminum laminate film and storing it at 45℃ for 1 month, the number of viable Bifidus bacteria is 6.5 x 10 ⁸ cells/g for PH-unadjusted bacteria and 5.0 x 10 ¹⁰ for PH 8.0-adjusted bacteria. It was ko/g. Example 2 In Example 1, Bifidobacterium concentrate (number of viable bacteria 1.5×10 ¹¹ /ml) was obtained. Meanwhile, a stabilizer solution containing 20% skim milk powder, 2% soluble starch, and 2% sodium glutamate was prepared. The pH of this solution was 6.6. Next, the pH of the stabilizer solution was adjusted to 9.0 with 15% potassium hydroxide solution.
ml, mixed and lyophilized as in Example. 55 g of dried bacterial cells with a drying loss of 1.60% and a viable bacterial count of 2×10 ¹¹ cells/g were obtained. The loss on drying of dried bacterial cells without pH adjustment was 1.50%, and the number of viable bacteria was 1.5×10 ¹¹ cells/g.
After filling 1 g of these dried bacterial cells into an aluminum laminate film and storing it at 45℃ for 1 month, the number of surviving bacteria is as follows:
1.5 x ^{108 cells/g for non-PH adjusted bacteria cells, 1.5 x 108} cells/g for PH 9.0 adjusted bacteria cells
It was 4.5×10 ¹⁰ pieces/g.

Claims (1)

[Claims] 1 The pH of the solution containing Bifidobacterium and stabilizer is 7.
A method for stabilizing dried Bifidus cells, which comprises adjusting the concentration to ~10 and freeze-drying.