JP2012055288A - Stabilized viable bacterial preparation, and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a viable bacterial preparation and a method for producing the viable bacterial preparation excelling in storage stability of viable bacteria.SOLUTION: The bacteria are washed with distilled water and dispersed in the distilled water, and then 2 mL of an aqueous solution with sucrose comprising trehalose, sodium glutamate, histidine and apple is added to prepare a cell dispersion. After freeze-drying the cell dispersion, the internal temperature of a freeze-dryer is raised to 40°C for ageing for 24 hours to obtain the viable bacterial preparation.

Description

  The present invention relates to a viable bacterial preparation excellent in storage stability and a method for producing the live bacterial preparation.

  Microorganisms such as lactic acid bacteria and yeast are useful microorganisms that have long been deeply involved in fermented foods. In recent years, it has become clear that some of these microorganisms have physiological effects such as intestinal regulation, infection prevention, immune activation, cancer prevention, allergy improvement, microbial bacteria such as lactic acid bacteria and yeast, In addition, research and development are being conducted to use the culture as a material for health foods and pharmaceuticals. In particular, it has also been found that the above-mentioned physiological action is improved by allowing microorganisms to reach the intestine in a living state, so that a viable preparation capable of stably and easily ingesting more viable bacteria is required. became. However, the viable preparation has a practical problem that the viable count decreases during storage.

  As a method for improving the storage stability of a live bacterial preparation, a method of adding silica gel or skim milk powder is generally used. In addition, a method of adding a compound selected from the group consisting of phenylalanine, histidine, citric acid, succinic acid, tartaric acid and salts thereof and an alkali carbonate (Patent Document 1), arginine, ornithine or serine, or Examples thereof include a method of adding these salts (Patent Document 2) and a method of adding theanine to a lyophilized powder (Patent Document 3). However, these methods make it difficult to prepare a taste of a viable bacterial preparation, and the effect of improving the storage stability of the live bacteria is not satisfactory, and a viable bacterial preparation having more excellent storage stability is desired. Yes.

JP-A-61-26508 JP 2003-219862 A JP 2009-284820 A

  An object of the present invention is to provide a viable bacterial preparation excellent in storage stability of viable bacteria and a method for producing the viable bacterial preparation.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a microbial cell body excellent in storage stability can be obtained by further aging after drying microbial cells. The present invention has been completed.

  That is, the present invention is a viable cell preparation obtained by further aging after drying microbial cells. In addition, the present invention is a method for producing a viable microorganism preparation, which includes a step of keeping the dried cells at 40 ° C. or higher.

  INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a viable bacterial preparation excellent in storage stability of viable bacteria and a method for producing the viable bacterial preparation.

Hereinafter, the present invention will be described in more detail.
In the present invention, a viable cell preparation excellent in storage stability can be obtained by culturing cells and then drying and further aging.

  The microbial cells used in the present invention are not particularly limited as long as they can be cultured in a commonly used medium and can be dried by a commonly used drying method, and include microorganisms such as bacteria and yeasts.

  Examples of the bacterium include bacteria such as lactic acid bacteria. For example, lactic acid bacteria belonging to the genus Pediococcus such as Pediococcus acidilactici and Pediococcus pentosaceus; Lactobacillus brevis (Lactobacillus brevis), Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei and other lactic acid bacteria belonging to the genus Lactobacillus; Enterococcus faecalis Lactic acid bacteria belonging to the genus; Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium lactis, etc. Examples include lactic acid bacteria belonging to the genus Dobacterium.

  Examples of other bacteria include bacteria belonging to the genus Escherichia such as Escherlchia coli, and bacteria belonging to the genus Bacillus such as Bacillus subtilis. Examples of yeast include yeast belonging to the genus Saccharomyces such as Saccharomyces cerevisiae.

  Among these microorganisms, lactic acid bacteria are preferable because they have physiological activity such as intestinal regulation and have high industrial utility value. Among the lactic acid bacteria, lactic acid bacteria belonging to the genus Pediococcus, particularly Pediococcus acidilactici R037 strain (NITE BP-900) having a strong neutral fat reducing action and an antiallergic action. Lactobacillus belonging to the genus Lactobacillus, particularly Lactobacillus brevis Kaneka-01 strain (NITE P-558) having a strong intestinal and immunostimulatory action or having a strong antiglycemic action Lactobacillus delbrueckii KLAB-4 strain (NITE BP-394) is preferred. These microorganisms are deposited with the independent administrative corporation Product Evaluation Technology Infrastructure (2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan) under the above-mentioned deposit numbers.

  The culture method is not particularly limited as long as the desired cells can be cultured, and there are no particular limitations on medium components, culture methods, etc., and any known medium components and culture methods that are usually performed may be liquid culture or solid culture. Liquid culture is preferred over solid culture due to the need to culture and recover the body. Hereinafter, the case of liquid culture will be described as an example.

  After collecting and washing the cells from the culture solution, a cell dispersion is prepared with a solution to which distilled water and / or a protective agent or the like is added. It is preferable to adjust the cell dispersion using a solution to which a protective agent is added because the storage stability of the viable cell preparation is improved.

  The protective agent is not particularly limited, and examples thereof include trehalose, bovine serum albumin, skim milk powder, sodium glutamate, L-ascorbic acid, whey, glucose, aspartic acid, methionine, starch, dextran, sucrose, and lactose. It is done.

  In addition, since the storage stability of the viable bacterial preparation is improved, when the pH of the bacterial cell dispersion is acidic, a pH adjuster such as sodium hydroxide, sodium carbonate, potassium hydroxide or the like is used. Desirably, it is preferable to adjust the pH to be almost neutral. In addition, as long as the objective of this invention is not prevented, the said protective agent may contain another substance.

  The method for drying the bacterial cell dispersion is not particularly limited, and known methods such as freeze drying, spray drying, drum drying, and vacuum drying can be used alone or in combination. Using these known methods, dried bacterial cells can be obtained. From the viewpoint of minimizing the killing of cells during drying, it is usually preferable to perform drying at 40 ° C. or less, and freeze drying is particularly preferable. However, it can be dried at 40 ° C. or higher depending on the drying method and conditions, such as when the time of exposure to high temperature is short, or when the cells whose moisture content has been reduced by preliminary drying or the like are dried. Not too long.

  The viable cell preparation excellent in storage stability of the present invention can be produced, for example, by aging the above-mentioned dried cell body. The aging of the present invention refers to holding the dried microbial cell product for a certain period of time under a temperature condition of 40 ° C. or higher.

  The upper limit of the aging temperature is 70 ° C. or lower, preferably 60 ° C. or lower. When the aging temperature exceeds 70 ° C., the cells themselves are not suitable because they are damaged and killed.

  The time required for the aging can be appropriately set according to the type of microorganism and the aging temperature, and is not particularly limited. However, the higher the aging temperature, the shorter the required time.

  For example, in the case of lactic acid bacteria, when the aging temperature is 40 ° C., the lower limit of the aging time is 6 hours or more, preferably 12 hours or more, more preferably 24 hours or more. When the aging time is less than 6 hours, aging does not proceed and it is difficult to obtain a viable bacterial preparation excellent in storage stability. The upper limit of the aging time can be 90 days or less, preferably 60 days or less, more preferably 30 days or less. Even if the aging time exceeds 90 days, a viable bacterial preparation excellent in storage stability cannot be obtained any more, which is not preferable from the viewpoint of productivity.

  When the aging temperature is 50 ° C., the lower limit of the aging time is 3 hours or more, preferably 6 hours or more, more preferably 12 hours or more. If the aging time is less than 3 hours, aging does not proceed and it is difficult to obtain a viable bacterial preparation excellent in storage stability. The upper limit of the aging time is 60 days or less, preferably 30 days or less, more preferably 15 days or less. Even if the aging time exceeds 60 days, a viable microbe preparation excellent in storage stability cannot be obtained.

  When the aging temperature is 60 ° C., the lower limit of the aging time is 1 hour or longer, preferably 3 hours or longer, more preferably 6 hours or longer. When the aging time is less than 1 hour, aging does not proceed and it is difficult to obtain a viable bacterial preparation excellent in storage stability. The upper limit of the aging time can be 30 days or less, preferably 15 days or less, more preferably 7 days or less. Even if the aging time exceeds 30 days, a viable bacterial preparation excellent in storage stability cannot be obtained.

  When the aging temperature is 70 ° C., the lower limit of the aging time is 0.3 hours or more, preferably 1 hour or more, more preferably 3 hours or more. When the aging time is less than 0.3 hours, aging does not progress and it is difficult to obtain a viable preparation having excellent storage stability. The upper limit of the aging time is 15 days or less, preferably 7 days or less, more preferably 3 days or less. If the aging time exceeds 15 days, the microbial cells themselves may be damaged and a viable bacterial preparation excellent in storage stability may not be obtained.

  The pressure at the time of aging may be a normal pressure, a negative pressure state, or a pressurized state. Needless to say, drying and aging can be performed as a series / integrated operation. For example, after freeze-drying, the pressure in the chamber may be continuously maintained, or the inside of the apparatus may be returned to normal pressure and aged. In this case, the drying may be performed at 40 ° C. or lower, and the temperature may be 40 ° C. or higher after the drying is completed. The drying may be performed at 40 ° C. or higher, and after the drying is finished (after the water content is reduced to a desired value). It may be maintained at a temperature of not lower than ° C. and subsequently ripened. Drying is preferably carried out at 40 ° C. or lower, and then aged at 40 ° C. or higher. In particular, it is preferable to ripen lyophilized or vacuum-dried cells at 40 ° C. or lower at 40 ° C. or higher.

  The aging in the present invention can also be performed in an environment in which the dried bacterial cell product is packaged and packed with a glass, plastic and / or metal material and is shielded from the external environment.

  Examples of the glass material include soft glass and hard glass.

  Examples of the plastic material include high density polyethylene, medium density polyethylene, low density polyethylene, polypropylene, polyethylene terephthalate, polyvinyl alcohol, polyvinyl chloride, polyvinylidene chloride, and nylon. Needless to say, a film in which the plastic material is laminated, a film in which a metal such as aluminum is laminated on a plastic material such as aluminum laminate, and a film in which alumina, silica, etc. are vapor-deposited on a sub-plastic material (alumina vapor deposition) Film and silica vapor deposition film) are also included in the plastic material.

  Examples of the metal material include iron, aluminum, zinc, nickel, cobalt, copper, tin, titanium, chromium, and alloys thereof (stainless steel, brass, etc.).

  The above-mentioned material is preferably molded into bottles, bags, cans, drums, boxes, etc., and dried products are packed and packed. In addition, when a material having a relatively high gas flow property such as polyethylene is used, it is preferable to pack or pack two or more layers. At this time, a vapor deposition film such as aluminum laminate, alumina, or silica, glass, metal, etc. It is particularly preferable to use a material having a relatively high gas barrier property and moisture resistance.

  In the packaging and packaging, a desiccant or oxygen scavenger may be used in combination, and it is particularly preferable to use a desiccant in combination. Examples of the desiccant include silica gel, calcium chloride, and synthetic zeolite.

  In this invention, you may age | ripen what added the excipient | filler etc. to the microbial cell dried material. The excipient herein is not particularly limited, and examples thereof include lactose, starch, dextrin, and sucrose.

  In the present invention, even when an excipient, a flavor improver, a fragrance, a coloring agent, or the like is further added to the dried microbial cell product obtained by the above aging method and the dried microbial cell product, a viable cell preparation is used in the present invention.

  The viable bacterial preparation obtained by the present invention is used as it is or after being processed for food, health food, nutritional supplement, nutritional functional food, food for specified health use, pharmaceutical, quasi-drug, feed, pet food, etc. it can. Examples of the processing form include tablets, powders, chewable tablets, pills, hard capsules, soft capsules and the like. Moreover, the obtained living microbe formulation can be used as starters, such as fermented milk and fermented soymilk.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention does not receive a restriction | limiting at all by these Examples.

(Preparation of bacterial cell dispersion)
The cell body was 1% by weight of tryptone, 0.5% by weight of yeast extract, 0.5% by weight of glucose, 0.5% by weight of lactose, 0.1% by weight of Tween 80, and 0.1% of L-cysteine hydrochloride. Inoculated into 1 L of a sterilized liquid medium containing 02% by weight, followed by stationary culture at 37 ° C. for 20 hours. After culturing, the bacterial cells obtained by centrifuging the culture solution at 5000 rpm for 10 minutes at 4 ° C. were washed twice with 500 ml of distilled water and dispersed in 20 ml of distilled water. Add 2 ml of an aqueous solution containing 4% by weight, trehalose 0.2% by weight, sodium glutamate 0.2% by weight, histidine 0.2% by weight and malic acid 0.2% by weight to prepare a cell dispersion. did.

(Dry)
The cell dispersion was frozen at −25 ° C. and then vacuum dried.

(Method for measuring survival rate during storage)
A dried bacterial cell product or a dried bacterial cell product obtained by adding an excipient to the dried bacterial cell product, a live bacterial product obtained by aging the dried bacterial cell product, and a live bacterial product after storage The number of bacteria was measured according to the multiplication method, and the survival rate was determined from the following equation.
Survival rate during storage (%) = (Number of viable bacteria in 1 g of sample after storage / Number of viable bacteria in 1 g of sample before storage) × 100

Example 1
After freeze-drying the cell suspension of Pediococcus acidilactici R037, the internal temperature of the freeze-dryer is raised to 40 ° C. and aged for 24 hours to obtain a viable cell preparation It was. The survival rate of the viable preparation after storage at 40 ° C. for 1 month was measured and shown in Table 1.

(Comparative Example 1)
A bacterial cell dispersion composed of Pediococcus acidilactici R037 was freeze-dried to obtain a dried bacterial cell product. The survival rate of the dried microbial cell product after storage at 40 ° C. for 1 month was measured and shown in Table 1.

  As shown in Table 1, the survival rate after storage at 40 ° C. for 1 month of the dried microbial cell product of Comparative Example 1 was 58%, whereas the inside of the freeze dryer was As shown in Example 1, the survival rate after storage at 40 ° C. for one month of the viable bacterial preparation aged at 24 ° C. by raising the temperature to 40 ° C. was 83%, and the storage stability was improved by aging. .

(Example 2)
90 g of starch was added to 10 g of lyophilized product of lactic acid bacterium Pediococcus acidilactici R037 to obtain a dried microbial cell product. Dispensing 10 g of the dried bacterial cells into a polyethylene bag with a chuck is packed in an aluminum pouch containing 1 g of silica gel, sealed by heat sealing, and aged at 40 ° C for 1 month, 2 months, or 3 months. A viable bacterial preparation was obtained.

(Comparative Example 2)
90 g of starch was added to 10 g of the lyophilized product of lactic acid bacterium Pediococcus acidilactici R037 to obtain an unripened microbial cell product.

Example 3
A lactic acid bacterium of Example 2 was treated in the same manner except that the lactic acid bacterium was replaced with Lactobacillus brevis kaneka-01 to obtain a matured bacterium preparation.

(Comparative Example 3)
The lactic acid bacteria of the comparative example 2 were processed similarly except having replaced with the Lactobacillus brevis (Lactobacillus brevis) kaneka-01, and the microbial cell dried material which was not matured was obtained.

Example 4
A lactic acid bacterium of Example 2 was treated in the same manner except that the lactic acid bacterium was replaced with Lactobacillus delbrueckii KLAB-4 to obtain a matured bacterium preparation.

(Comparative Example 4)
The lactic acid bacteria of the comparative example 2 were processed similarly except having replaced with the Lactobacillus delbrueckii (Lactobacillus delbrueckii) KLAB-4, and the microbial cell dried material which was not matured was obtained.

The survival rate of the viable cell preparations of Examples 2 to 4 and the dried cell product of Comparative Examples 2 to 4 after storage at 40 ° C. for 1 month was measured and shown in Table 2.

  As shown in Table 2, the survival rate after storage at 40 ° C. for 1 month for a viable bacterial preparation aged at 40 ° C. for 1 month, 2 months, and 3 months was aged in any of the examples. It was revealed that the storage stability was improved as compared with each comparative example that was not.

(Example 5)
90 g of starch was added to 10 g of a lyophilized product of Lactobacillus brevis kaneka-01 to obtain a dried product of the cells. Dispensing 10 g of the dried bacterial cells into a polyethylene bag with a chuck is packed in an aluminum pouch containing 1 g of silica gel, sealed by heat sealing, 0.5 days, 1 day, 7 days at a temperature of 50 ° C. , 14 days, 30 days, 60 days, and aged to obtain a viable bacterial preparation. The survival rate of the viable preparation after storage at 40 ° C. for 1 month was measured and shown in Table 3.

(Comparative Example 5)
Lactobacillus brevis (Lactobacillus brevis) Kaneka-01 freeze-dried product was added with 90 g of starch to obtain an unripened microbial cell product. The survival rate of the dried microbial cells after storage at 40 ° C. for 1 month was measured and shown in Table 3.

  As shown in Table 3, the survival rate after storage at 40 ° C. for 1 month of the viable bacterial preparation aged at a temperature of 50 ° C. is the survival rate of Comparative Example 5 that was not aged at any aging time It was revealed that the storage stability was improved by aging.

Claims (10)

A viable cell preparation obtained by further aging after drying microbial cells. The viable preparation according to claim 1, wherein the microorganism is a bacterium or a yeast. The viable preparation according to claim 2, wherein the bacterium is a lactic acid bacterium. The live bacterium preparation according to claim 3, wherein the lactic acid bacterium is a lactic acid bacterium belonging to the genus Pediococcus or Lactobacillus. The lactic acid bacteria are Pediococcus acidilactici R037 (NITE BP-900), Lactobacillus brevis kaneka-01 (NITE P-558), and Lactobacillus delbruii (Lactobacillus delbrueck) The viable preparation according to claim 4, which is at least one selected from the group consisting of -4 (NITE BP-394). A method for producing a viable microorganism preparation, the method comprising a step of maintaining the dried cells at 40 ° C or higher. The production method according to claim 6, wherein the microorganism is a bacterium or a yeast. The production method according to claim 7, wherein the bacterium is a lactic acid bacterium. The method according to claim 8, wherein the lactic acid bacterium is a lactic acid bacterium belonging to the genus Pediococcus or the genus Lactobacillus. The lactic acid bacteria are Pediococcus acidilactici R037 (NITE BP-900), Lactobacillus brevis kaneka-01 (NITE P-558), and Lactobacillus delbrueck The production method according to claim 9, which is at least one selected from the group consisting of KLAB-4 (NITE BP-394).