JP2012056921A - Probiotic preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a probiotic preparation having a superior storage stability and a method of selecting bacterial cells used for the probiotic preparation.SOLUTION: The storage-stable probiotic preparation is obtained by conducting at least once a series of steps comprising (1) a step to culture microbial cells, (2) a step to harvest the microbial cells, (3) a step to dry the microbial cells, (4) a step to culture the dried microbial cells in a solid medium, and (5) a step to select large colonies.

Description

  The present invention relates to a viable bacterial preparation excellent in storage stability.

  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 been revealed that some of these microorganisms have physiological functions such as intestinal regulation, infection prevention, immunostimulation, cancer prevention, allergy improvement, and microbial bacteria such as lactic acid bacteria, bifidobacteria and yeast. Research and development is being conducted to use killed bacteria or their cultures as materials for health foods and pharmaceuticals. In particular, it has also been found that the above-mentioned physiological activity 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 need to improve the taste of the live bacterial preparation, and are not satisfactory in the storage stability improving effect of the live bacteria, and a live bacteria preparation with better storage stability is desired. Yes.

  In addition, the viable cell preparation is produced by collecting and drying previously cultured cells, and adding an appropriate excipient, but the cells are killed in the drying process during the production of the live cell preparation. There is a practical problem that productivity decreases.

  In order to solve this problem, a method of adding sodium glutamate, L-ascorbic acid, whey and glucose at the time of producing a viable bacterial preparation (Patent Document 4), aspartic acid, arginine, glutamic acid, proline, lysine, leucine, and methionine Examples include a method of adding a mixture of at least three kinds of amino acids (Patent Document 5).

  However, even if these methods are used, the effect of preventing the killing of the cells in the drying process during the production of the viable preparation is not satisfactory, and the death of the cells in the drying process during the production of the viable preparation is truly prevented. A method is desired.

JP-A-61-265085 JP 2003-219862 A JP 2009-284820 A JP-A-2-86769 JP-A-8-205857

  The present invention relates to a viable preparation excellent in survival rate in a drying process during production of a viable preparation, a viable preparation excellent in storage stability of viable bacteria, and a method for selecting a cell used in the viable preparation. The issue is to provide.

  As a result of intensive studies to solve the above problems, the present inventors have (1) a step of culturing microbial cells, (2) a step of collecting bacteria, (3) a step of drying, and (4) a dried state. Bacteria in the drying process at the time of producing a live cell preparation by using a microbial cell selected by repeating a series of steps of culturing cells in a solid medium and (5) selecting a large colony at least once. It was found that the killing of the body can be reduced, and that the prepared viable bacterial preparation has excellent storage stability, and the present invention has been completed.

  That is, the present invention includes (1) a step of culturing microbial cells, (2) a step of collecting bacteria, (3) a step of drying, (4) a step of culturing the dried cells in a solid medium, (5) A viable preparation excellent in storage stability, prepared from a microorganism obtained by repeating a series of steps including the steps of selecting large colonies and the steps (1) to (5) at least once.

  The present invention also relates to a method for producing a microbial cell preparation which is prepared by drying microbial cells by adding an excipient as necessary. (1) A step of culturing microbial cells, A series of steps including 2) a step of collecting bacteria, (3) a step of drying, (4) a step of culturing dried cells in a solid medium, and (5) a step of selecting large colonies are repeated at least once. The method is characterized in that the microorganism obtained in this way is used.

  Furthermore, the present invention is a method for selecting a microbial cell having excellent storage stability and / or survival rate in the drying step when preparing a viable cell preparation, wherein (1) a microorganism A series of steps including a step of culturing cells, (2) a step of collecting cells, (3) a step of drying, (4) a step of culturing dried cells in a solid medium, and (5) a step of selecting large colonies. The method is characterized by repeating the step at least once.

  According to the present invention, there are provided a viable preparation having excellent survival rate in a drying step during production of a viable preparation, and excellent storage stability of the viable microorganism, and a method for selecting microbial cells used in the viable preparation. Can do.

  In the present invention, (1) a step of culturing microbial cells, (2) a step of collecting bacteria, (3) a step of drying, (4) a step of culturing the dried cells in a solid medium, (5) large By repeating a series of steps of selecting colonies at least once, microbial cells having excellent storage stability are selected.

  The microorganism used in the present invention is not particularly limited as long as it can be cultured and dried by a usual method, and includes microorganisms such as bacteria and yeast.

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

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

  Examples of the yeast include yeast belonging to the genus Saccharomyces such as Saccharomyces cerevisiae.

  Among these microorganisms, bacteria and yeasts that are used as viable preparations are preferable, and lactic acid bacteria are preferable because they have physiological activity such as intestinal regulation and have high industrial utility value. Among lactic acid bacteria, lactic acid bacteria belonging to the genus Pediococcus or Lactobacillus are preferable. Among these, Pediococcus acidilactici, Lactobacillus brevis, or Lactobacillus delbrueckii is preferable.

  In particular, Pediococcus acidilactici R037 strain (BP-900) because of its strong neutral fat reducing action and antiallergic action, and Lactobacillus because of its strong intestinal action and immunostimulatory action -Lactobacillus brevis kaneka-01 strain (NITE P-558) or Lactobacillus delbrueckii KLAB-4 strain (NITE BP-394) having a strong antiglycemic action is preferable. It is deposited with the above-mentioned trust number at the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashika, Kisarazu City, Chiba Prefecture, Japan).

  The culture method is not particularly limited as long as the desired cells can be cultured, and any known culture method that is usually performed may be liquid culture or solid culture. Liquid culture is preferred over solid culture depending on the need for recovery. Hereinafter, the case of liquid culture will be described as an example.

  There is no particular limitation on the method of collecting the bacteria in the step (2). If the cells can be separated from the culture after culturing, a known method such as filtration (filter press, screw press, etc.), centrifugation, etc. is appropriately selected and separated. To do. For the purpose of removing medium components, the cells may be suspended in sterilized water and then washed and collected again.

  The drying method in the step (3) 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. Further, a dispersion medium may be added during drying. The dispersion medium refers to a substance that can be added to the collected cells, and is not particularly limited. Examples thereof include physiological saline, phosphate physiological saline, and a solution containing a protective agent. 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, as long as the objective of this invention is not prevented, the said protective agent may contain another substance suitably.

  As a drying method for selecting lactic acid bacteria, freeze-drying is preferable, and it is preferably performed in the absence of a dispersant.

  As the culture method in the solid medium in the step (4), the dried cells obtained in the step (3) are dispersed in physiological saline or the like, applied to a known solid medium such as an agar plate and cultured. There are no particular limitations on the medium components, solidifying agents, etc., as long as the desired cells can grow.

  As a method for selecting a large colony in the step (5), among the colonies grown after culturing in the step (4), for example, a bacterial solution diluted so that the number of growing colonies becomes 100 ± 10 is 90Φ × Apply to a 20 mm plate and at the end of the culture, select colonies with a diameter larger than the average colony on the plate. More specifically, a colony having a diameter of 2 mm or more, preferably 3 mm or more, more preferably 4 mm or more is selected.

  Here, the term “culture end” refers to, for example, lactic acid bacteria lyophilized in the absence of a dispersant, 1% by weight of tryptone, 0.5% by weight of yeast extract, 0.5% by weight of glucose, 0.5% by weight of lactose, When cultured at 37 ° C. on a solid medium having a composition of Tween 80 0.1% by weight, L-cysteine hydrochloride 0.02% by weight, agar 2% by weight, pH 6.2 ± 0.2, it represents about 72 hours .

  The series of steps including step (1) to step (5) is preferably repeated at least once. The microbial cells isolated from the large colonies are directly or once cultured on an agar slope or the like, then inoculated into a liquid medium, and again subjected to a series of selection steps from step (1). The series of sorting steps is preferably repeated 3 times or more, more preferably 5 times or more, still more preferably 7 times or more, and most preferably 10 times or more. The greater the number of repetitions, the easier it is to obtain a viable cell or viable cell preparation excellent in survival rate and a viable cell or viable cell formulation excellent in storage stability in the drying step during production of the viable cell preparation.

  In addition, the storage stability of a living microbe formulation can be represented by a survival rate. The survival rate referred to here is the percentage of the number of viable bacteria in the viable cell preparation after storage with respect to the viable cell count at the time of producing the viable cell preparation.

  The cells selected by this method are cells excellent in storage stability, and the cells are cultured by a known method, the cells are collected from the culture solution, and an appropriate dispersion medium is added, Drying can be performed to obtain a viable bacterial preparation. In addition, you may add an excipient | filler, a flavor improving agent, a fragrance | flavor, a coloring agent, etc. to the said living microbe formulation.

  The viable preparation obtained by the present invention can be used as it is or after being processed as a food, health food, nutritional supplement, functional nutrition food, food for specified health use, pharmaceutical, quasi-drug, feed, pet food, etc. . Examples of the processing form include tablets, powders, chewable tablets, pills, hard capsules, soft capsules and the like. For the purpose of preparing foods such as fermented milk and fermented soy milk having excellent storage stability of live bacteria, it is possible to use the obtained live bacteria preparation as a starter.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention does not receive a restriction | limiting at all by these Examples. In addition, the operation method of the process (1) to the process (5) used by the Example and the comparative example, the manufacturing method of a viable preparation, and the measurement method of the survival rate of a viable preparation are shown below.

Step (1) Step of culturing cells The cells are 1% by weight of tryptone, 0.5% by weight of yeast extract, 0.5% by weight of glucose, 0.5% by weight of lactose, and 0.1% by weight of Tween 80. %, L-cysteine hydrochloride was inoculated into 50 ml of a sterilized liquid medium containing 0.02% by weight, and static culture was performed at 37 ° C. for 20 hours to obtain a culture solution.

Step (2) Collecting the cells The cells obtained by centrifuging the culture solution obtained in the above step (1) at 5000 rpm for 10 minutes at 4 ° C. were washed twice with 25 ml of sterilized water and centrifuged. After separation, 5 ml of a bacterial cell concentrate was obtained.

Step (3) Drying Step Transfer the entire amount of the bacterial cell concentrate obtained in the above step (2) to a 10 mL glass vial and use a freeze dryer (VirTis Advantage Plus) for 20 hours at −25 ° C. After freezing, it was freeze-dried for 48 hours at a vacuum degree of 5 Pa and a shelf temperature of 25 ° C. to obtain a dried product.

Step (4) Step of culturing dried cells in a solid medium Obtained in Step (3) above on a sterilized solid medium (a plate obtained by adding 20 ml of a medium to a 90Φ × 20 mm plate) to the liquid medium. A bacterial cell dispersion obtained by dispersing a certain amount of the lyophilized product in physiological saline was applied so that the number of colonies was 100 ± 10, and cultured at 37 ° C. for 72 hours.

Step (5) Step of selecting large colonies One bacterial cell having a colony size of 3 mm or more was selected from the agar plate obtained in the step (4).

Measurement of the survival rate when lyophilized in the absence of a dispersion medium The number of viable cells in the concentrated cell culture liquid obtained by culturing and collecting the cells in the above liquid medium was measured by a conventional method. The total viable count in the concentrate was determined. In addition, the number of viable cells in a lyophilized product obtained by lyophilizing the bacterial cell concentrate in the absence of a dispersion medium was measured by a conventional method, and the total number of viable cells in the lyophilized material was determined and dried. The survival rate at that time was calculated from the following equation.

  Survival rate (%) when freeze-dried in the absence of a dispersion medium = (total viable count of freeze-dried product / total viable count of cell concentrate) × 100.

Production method of viable cell preparation The bacterial cells were inoculated into 1 L of the above liquid medium, followed by stationary culture at 37 ° C for 20 hours. After culturing, the cells obtained by centrifuging the culture solution at 5000 rpm for 10 minutes at 4 ° C. were washed twice with 500 ml of sterilized water, and after centrifugation, 20 ml of microbial cell concentrate was obtained. The bacterial cell concentrate contains 0.4% by weight of sucrose, 0.2% by weight of trehalose, 0.2% by weight of sodium glutamate, 0.2% by weight of histidine, and 0.2% by weight of malic acid. 2 ml of the resulting dispersion medium was added and freeze-dried to obtain a freeze-dried product. The lyophilized product was dispersed in dextrin so that the viable cell count was 1.0 × 10 ¹⁰ cfu / g to obtain a viable cell preparation.

Measurement of survival rate during production of viable cell preparations Obtain the total viable cell count in the bacterial cell concentrate and the total cell count in the lyophilized product by a conventional method. Calculated from the formula.
Survival rate (%) at the time of production of a viable cell preparation = (total number of bacteria in lyophilized product / total number of cells in cell concentrate) × 100.

Storage stability of live bacterial preparations The storage stability of live bacterial preparations was determined by the survival rate after storage.
Aluminum pouch (product name: Lamidip) containing 1 g of silica gel (manufactured by Fuji Silysia Chemical Co., Ltd.) dispensed with 10 g of live bacteria preparation in a polyethylene bag with a chuck (product name Unipack B-8, manufactured by Seinichi Co., Ltd.) , Manufactured by Seinichi Co., Ltd.) and sealed by heat sealing was used as a storage sample. The number of viable bacteria per 1 g of viable bacterial preparation after storage at 25 ° C., 1 month, 3 months and 6 months was measured by a conventional method, and the survival rate was determined from the following formula.
Survival rate (%) = (Number of viable bacteria per 1 g of viable bacterial preparation after storage / 1.0 × ^{10 10} ) × 100.

Example 1
Using the lactic acid bacteria Pediococcus acidilactici R037 strain, the steps (1) to (5) were repeated three times to obtain selected cells derived from the R037 strain. Using the selected cells, a viable cell preparation having a viable cell count of 1.0 × 10 ¹⁰ cfu / g was obtained.

(Comparative Example 1)
Using the lactic acid bacterium Pediococcus acidilactici R037 strain, the steps (1) to (3) were repeated three times to obtain unselected cells derived from the R037 strain. Using the unselected cells, a viable cell preparation having a viable cell count of 1.0 × 10 ¹⁰ cfu / g was obtained.

(Example 2)
Using the lactic acid bacterium Lactobacillus brevis kaneka-01 strain, the steps (1) to (5) were repeated three times to obtain selected cells derived from kaneka-01. Using the selected cells, a viable cell preparation having a viable cell count of 1.0 × 10 ¹⁰ cfu / g was obtained.

(Comparative Example 2)
Using the lactic acid bacterium Lactobacillus brevis kaneka-01, the steps (1) to (3) were repeated three times to obtain an unselected cell derived from kaneka-01. Using the unselected cells, a viable cell preparation having a viable cell count of 1.0 × 10 ¹⁰ cfu / g was obtained.

Example 3
Using the lactic acid bacterium Lactobacillus delbrueckii KLAB-4 strain, the steps (1) to (5) were repeated three times to obtain KLAB-4-derived selected cells. Using the selected cells, a viable cell preparation having a viable cell count of 1.0 × 10 ¹⁰ cfu / g was obtained.

(Comparative Example 3)
Using the lactic acid bacterium Lactobacillus delbrueckii KLAB-4 strain, the steps (1) to (3) were repeated three times to obtain an unselected microbial cell derived from KLAB-4. Using the unselected cells, a viable cell preparation having a viable cell count of 1.0 × 10 ¹⁰ cfu / g was obtained.

  For each of the selected strains of Examples 1 to 3 and the non-selected strains of Comparative Examples 1 to 3, the survival rate when freeze-dried in the absence of a dispersion medium and the survival rate at the time of manufacturing a viable preparation were determined. The results are shown in Tables 1 and 2. In addition, the storage stability of each viable bacterial preparation after 1 month, 3 months and 6 months at 25 ° C. was measured and shown in Table 3. In addition, operation of Examples 1-3 and Comparative Examples 1-3 was performed 3 times, and Table 1-Table 3 were shown as an average value of the result of three selection stocks and a non-selection stock, respectively.

As shown in Table 1, the selected cells obtained by repeating the series of steps (1) to (5) three times were lyophilized under the same conditions as in step (3) in the absence of a dispersion medium. The survival rate was 40% or more. On the other hand, the survival rate when the unselected cells obtained by repeating the steps (1) to (3) three times is lyophilized in the absence of a dispersion medium varies depending on the strain, but is 10%. To 20%. In addition, as shown in Table 2, the selected cells were superior to the unselected cells in the survival rate when freeze-dried at the time of producing the viable cell preparation.

  From the above results, the selected strain obtained by the selection method of the present invention has an excellent survival rate in the drying step during the production of the viable bacterial preparation, and increases the production efficiency of the viable bacterial preparation by using the microbial cells. be able to.

  In addition, from the results shown in Table 3, the survival rate of the viable preparation prepared using the selected cells obtained by repeating the series of steps (1) to (5) at 25 ° C. is At 6 months, it was over 90%. On the other hand, the survival rate of the viable preparation prepared by using the unselected cells obtained by repeating the steps (1) to (3) three times at 25 ° C. is 6 months. 60% to 70%. From the above results, it can be seen that the viable bacterial preparation of the present invention has excellent storage stability.

Claims (15)

(1) Step of culturing microbial cells, (2) Step of collecting bacteria, (3) Step of drying, (4) Step of culturing dried cells in solid medium, (5) Step of selecting large colonies A viable preparation excellent in storage stability, prepared using a microorganism obtained by repeating a series of steps including the steps (1) to (5) at least once. The viable preparation according to claim 1, wherein the drying in (3) is freeze-drying. The viable preparation according to claim 1 or 2, wherein the drying of (3) is performed in the absence of a dispersion medium. A viable cell preparation using cells showing a survival rate of 40% or more when lyophilized in the absence of a dispersant. In the freeze-drying, 5 mL of the bacterial cell concentrate is placed in a 10 mL vial, frozen at −25 ° C. for 20 hours using a freeze dryer, and then freeze-dried at a vacuum degree of 5 Pa and a shelf temperature of 25 ° C. for 48 hours. The viable microbe preparation according to claim 4, which is performed under conditions. The live microorganism preparation according to any one of claims 1 to 5, wherein the microorganism is a lactic acid bacterium. The viable preparation according to claim 6, wherein the lactic acid bacterium is a lactic acid bacterium belonging to the genus Pediococcus or Lactobacillus. The live bacterium preparation according to claim 6, wherein the lactic acid bacterium is Pediococcus acidilactici, Lactobacillus brevis, or Lactobacillus delbrueckii. A method for producing a microbial cell preparation prepared by drying microbial cells and adding an excipient as necessary, wherein (1) a step of culturing the microbial cells, and (2) a step of collecting the cells A microorganism obtained by repeating at least once a series of steps including (3) a step of drying, (4) a step of culturing dried cells in a solid medium, and (5) a step of selecting large colonies. A method characterized by that. A method for screening microbial cells having excellent storage stability and / or survival rate in the drying step when preparing the microbial cells, wherein (1) culturing microbial cells , (2) a step of collecting bacteria, (3) a step of drying, (4) a step of culturing dried cells in a solid medium, and (5) a series of steps including a step of selecting large colonies at least once. A method characterized by repeating the above. The method according to claim 9 or 10, wherein the drying in (3) is lyophilization. The method according to any one of claims 9 to 11, wherein the drying of (3) is performed in the absence of a dispersion medium. The method according to any one of claims 9 to 12, wherein the microorganism is a lactic acid bacterium. The method according to claim 13, wherein the lactic acid bacterium is a lactic acid bacterium belonging to the genus Pediococcus or Lactobacillus. The method according to claim 14, wherein the lactic acid bacterium is Pediococcus acidilactici, Lactobacillus brevis, or Lactobacillus delbrueckii.