Method for producing dried microbial cells with high viability

Abstract

A new technology is provided that reduces damage and death of bacterial cells through a method for producing highly viable dried microbial cells, which is characterized by subjecting dried microbial cells to a temperature-changing treatment.

Description

[Technical field]

[0001] The present invention relates to a method for producing dried microbial cells that have high survival rates after storage. [Background technology]

[0002] Many microorganisms have useful enzyme activities and are widely used in the production of functional food materials such as carbohydrates, amino acids, and phospholipids.

[0003] In particular, among microorganisms, lactic acid bacteria have been widely used for the production of dairy products such as yogurt and cheese, and in recent years, the development of foods and beverages using dried lactic acid bacteria has progressed. In order to obtain the effects of lactic acid bacteria, it is desirable to use live bacterial cells, but in the process of obtaining dried bacterial cells, the bacterial cells are often damaged and killed, making it difficult to obtain the required amount of live bacterial cells.

[0004] Known techniques for reducing damage and death of bacterial cells include, for example, a technique for adjusting the dispersion medium used when drying bacterial cells (Patent Documents 1 and 2, Non-Patent Document 1).

[0005] However, the only techniques known to reduce damage and death of bacteria are those that are carried out before the bacteria are dried. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 3504365 [Patent Document 2] JP 2010-4787 A [Non-patent literature]

[0007] [Non-Patent Document 1] GLDE ANTONI et al., "Trehalose, a Cryoprotectant for Lactobacillus bulgaricus", Cryobiology 26, p.149-153, 1989. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, an object of the present invention is to provide a new technology for reducing damage and death of bacterial cells. [Means for solving the problem]

[0009] As a result of intensive research to solve the above problems, the inventors have unexpectedly found that, although heat stress is usually a factor that reduces the viability of microorganisms, by subjecting the dried cells to a temperature change treatment that corresponds to heat stress after obtaining the cells, the survival rate after storage is improved, and thus completed the present invention.

[0010] That is, the present invention relates to a method for producing dried microbial cells with high viability, which comprises subjecting the dried microbial cells to a temperature change treatment.

[0011] The present invention also relates to a method for improving the viability of dried microbial cells, which comprises subjecting the dried microbial cells to a temperature change treatment. Effect of the Invention

[0012] According to the present invention, the survival rate of dried microbial cells once produced can be improved after storage by a simple method of alternating temperature treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The method for producing dried microbial cells with high viability according to the present invention (hereinafter referred to as the "production method of the present invention") comprises subjecting the dried microbial cells to a temperature-changing treatment.

[0014] The dried microbial cells used in the production method of the present invention are not particularly limited, but examples thereof include dried cells of microorganisms such as lactic acid bacteria. Examples of lactic acid bacteria include Lactobacillus casei, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus cremoris, Lactobacillus helveticus, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus yoghurti, Lactobacillus delbrueckii subsp. bulgaricus ... Lactobacillus bacteria such as Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus johnsonii, and Lactobacillus mali; Bifidobacterium bacteria such as Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium longum; Streptococcus bacteria such as Streptococcus thermophilus and Streptococcus lactis; Lactococcus lactis subsp. lactis), Lactococcus lactis subsp. cremoris (Lactococcus lactis subsp.Examples of the lactic acid bacteria include Lactococcus bacteria such as Lactococcus cremoris, Lactococcus plantarum, and Lactococcus raffinolactis, and Enterococcus bacteria such as Enterococcus faecalis and Enterococcus faecium. These lactic acid bacteria may be used alone or in combination of two or more kinds. Among these microorganisms, lactic acid bacteria are preferred. Among these lactic acid bacteria, Lactobacillus lactic acid bacteria are preferred, Lactobacillus casei is more preferred, and Lactobacillus casei YIT 9029 (FERM BP-1366, date of deposit: May 1, 1981, National Institute of Technology and Evaluation, Patent Organism Deposit Center (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan)) is particularly preferred.

[0015] The method for drying the microorganism to obtain dried cells is not particularly limited, and spray drying, freeze drying, etc. well known to those skilled in the art may be used. Specific methods include, for example, those described in Japanese Patent No. 3504365, JP 2010-4787 A, GLDE ANTONI et al., "Trehalose, a Cryoprotectant for Lactobacillus bulgaricus", Cryobiology 26, p.149-153, 1989, International Publication WO2017 / 073752, etc. Among these methods, the method described in International Publication WO2017 / 073752 is preferred.

[0016] More specifically, the method described in International Publication WO2017 / 073752 is carried out as follows.

[0017] First, the microorganisms are cultured according to a conventional method, and then the cells are harvested according to a conventional method. Washing may be carried out between, before, or after the culturing and harvesting of the microorganisms, if necessary.

[0018] The collected microbial cells are suspended in an aqueous solution containing a protective agent, an antioxidant, and a chelating agent, preferably in a dispersion medium of an aqueous solution consisting of a protective agent, an antioxidant, and a chelating agent (hereinafter, this may also be simply referred to as the "dispersion medium"), and the suspension is dried to obtain dried microbial cells.

[0019] The water used as the dispersion medium is not particularly limited, but for example, drinkable water such as purified water or deionized water can be used.

[0020] The protective agent used in the dispersion medium is not particularly limited, but examples thereof include glutamic acid or its salts such as sodium glutamate and potassium glutamate, disaccharides such as trehalose, sucrose, lactose, and maltose, glycerol, maltodextrin, cyclodextrin, and skim milk powder. Among these protective agents, it is preferable to use glutamic acid or its salts and / or disaccharides, and more preferably sodium glutamate and / or trehalose. The content of the protective agent in the dispersion medium is not particularly limited, but is preferably 1 to 40% by mass, and more preferably 5 to 30% by mass.

[0021] The antioxidant used in the dispersion medium is not particularly limited, but may be, for example, ascorbic acid or a salt thereof such as sodium ascorbate or calcium ascorbate, vitamin E, catechin, glutathione, astaxanthin, etc., and among these antioxidants, sodium ascorbate is preferred. The content of the antioxidant in the dispersion medium is not particularly limited, but may be, for example, preferably 0.01 to 10% by mass, more preferably 0.05 to 5% by mass.

[0022] Furthermore, the chelating agent used in the dispersion medium is not particularly limited, but examples of the chelating agent that can be used include ethylenediaminetetraacetic acid (EDTA), citric acid or a salt thereof such as sodium citrate, phytic acid, etc. The content of the chelating agent in the dispersion medium is not particularly limited, but is preferably, for example, 0.1 to 10 mass%, more preferably 0.5 to 5 mass%.

[0023] A preferred embodiment of the dispersion medium is an aqueous solution containing sodium glutamate, trehalose, sodium ascorbate and sodium citrate.

[0024] The amount of microbial cells suspended in the dispersion medium is not particularly limited, but for example, the number of microbial cells in the suspension is 1.0 × 10 5 ~4.0×10 14 cfu / mL, approximately 1.0×10 7 ~4.0×10 13 A level of cfu / mL is more preferable.

[0025] The method for drying the suspension is not particularly limited, and known drying methods such as freeze-drying and spray-drying can be used, but freeze-drying is preferred to increase the survival rate of microorganisms in the drying process. Drying conditions in the freeze-drying method include, for example, a freezing treatment at -35°C to -45°C for 6 to 12 hours, followed by a drying treatment at 12°C to 32°C for 40 to 90 hours.

[0026] The dried microbial cells obtained as described above are then subjected to a temperature-changing treatment. Prior to the temperature-changing treatment, the dried microbial cells may be crushed in a mill, packed in capsules made of hydroxypropylmethylcellulose or the like in amounts of 0.2 g each without degassing under normal atmospheric composition, and placed in a bag made of aluminum or the like together with an oxygen scavenger, and then subjected to the temperature-changing treatment in that state.

[0027] Variable temperature treatment is a process where the dried microbial cells are usually preserved at a temperature different from room temperature for a certain period of time. Specifically, the following (a) and (b) are performed. (a) Heating to 30°C or higher for one day or more (b) Cooling to below 10°C for one day or more The treatment (a) and (b) may be carried out alone, in combination, or repeatedly.

[0028] Preferred examples of the above treatment (a) include the following. (a1) Heating treatment at 30° C. or higher, preferably 35 to 37° C., for one day or more, preferably two days (a2) Heating to 30° C. or higher, preferably 35 to 37° C., for one day or more, preferably one day

[0029] Preferred examples of the treatment (b) above include the following. (b1) Cooling to 10°C or lower, preferably 2 to 10°C, for one day or more, preferably one day

[0030] As the variable temperature treatment, for example, it is preferable to carry out the above-mentioned (a) or to carry out the above-mentioned (b) and then carry out (a); it is more preferable to carry out the above-mentioned (a1) or to carry out the above-mentioned (b1) and then carry out (a1) or to carry out the above-mentioned (a2); it is particularly preferable to carry out the above-mentioned (a1).

[0031] In the case of heating, such a temperature-changing treatment can be carried out using a heating device such as a dryer or an autoclave, and in the case of cooling, such as a cooling device such as a refrigerator or a freezer. In addition, in these temperature-changing treatments, the pressure, etc. are not particularly limited.

[0032] The thus obtained dried microbial cells have a survival rate of 110% or more after 6 months of storage compared to when the dried microbial cells are not subjected to temperature change treatment, and have a survival rate of 120% or more after 12 months of storage compared to when the dried microbial cells are not subjected to temperature change treatment, and preferably have a survival rate of 130 to 140%.

[0033] This highly viable microbial dried cells can be used for the same purposes as conventional microbial dried cells. Specifically, it can be used in foods and beverages as it is or by mixing with other food materials that are usually added to foods. Examples of foods include processed meat foods such as ham and sausage, processed seafood foods such as kamaboko and chikuwa, bread, confectionery, butter, yogurt, fermented milk, etc. Examples of beverages include soft drinks, dairy lactic acid bacteria beverages, lactic acid bacteria beverages, etc. Furthermore, examples of the form of the food and beverage include the commonly used forms of food and beverage, such as solid forms such as powder and granules, paste forms, liquid forms, etc. Furthermore, the microbial dried cells may be formulated into, for example, tablets, powders, chewable agents, hard capsules, soft capsules, pills, etc. EXAMPLES

[0034] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, the viable cell count of Lactobacillus casei was measured by the following method.

[0035] <Method for measuring viable Lactobacillus casei cell count> Dry Lactobacillus casei cells were serially diluted with physiological saline (0.85% by mass sodium chloride). 1 mL of the diluted solution was mixed with a plate count agar medium containing BCP and cultured at 37°C for 72 hours. The number of colonies that formed was counted and multiplied by the dilution rate to obtain the viable Lactobacillus casei cell count.

[0036] Example 1 Preparation of Lactobacillus casei dried cells and temperature treatment: Lactobacillus casei YIT 9029 was anaerobically cultured at 37°C for 20 hours in a medium (pH 7) containing 1% yeast extract by mass, 0.1% monopotassium phosphate by mass, 0.2% dipotassium phosphate by mass, and 2% lactose by mass. After the culture was completed, the liquid was cooled to a temperature of 20°C or less, and the pH was adjusted to 7.0 with 5N sodium hydroxide solution. The culture liquid was centrifuged (14,000G, 4°C, 30 minutes) to recover the resulting bacterial cells, which were then added to a dispersion medium containing 10% sodium glutamate by mass, 10% trehalose by mass, 1% sodium ascorbate by mass, and 1% sodium citrate by mass, at a concentration of 2.0 x 10 11 The cells were suspended at a concentration of cfu / mL. The cell suspension was dispensed into trays, and dried cells were prepared by freeze-drying. The freeze-drying was carried out using a freeze-dryer (TAKARA FREEZE-DRYER TF20-80TANNS, manufactured by Takara AT M Co., Ltd.) at a shelf temperature of -40°C for 9 hours, followed by 80 hours at 20°C. The obtained dried cells were pulverized in a mill, and without degassing under normal atmospheric composition, 0.2 g of each were packed into capsules (made of hydroxypropylmethylcellulose), and placed in an aluminum pouch together with an oxygen scavenger (manufactured by Mitsubishi Gas Chemical Co., Ltd.).

[0037] The aluminum pouch obtained above immediately after production was heated at 35°C for 2 days, and then stored at 22°C for 6 months (Method 1). The aluminum pouch obtained above immediately after production was cooled at 2°C for 1 day, heated at 35°C for 2 days, and then stored at 22°C for 6 months (Method 2). The aluminum pouch obtained above immediately after production was heated at 37°C for 1 day, and then stored at 22°C for 6 months (Method 3). The aluminum pouch obtained above was stored at 22°C for 6 months for comparison. The results of measuring the viable cell count before and after storage are shown in Table 1. The survival rate was calculated from these viable cell counts using the following formula, and the extent to which each method improved survival compared to the comparison method (without temperature change treatment) was calculated using the following formula. These results are also shown in Table 1.

[0038] [Table 1]

[0039] [Number 1] Survival rate (%) = (number of viable bacteria after storage / number of viable bacteria before storage) x 100

[0040] [Number 2] Survival improvement rate (%) = (survival rate of the implementation method / survival rate of the comparison method) x 100

[0041] These results showed that the survival rate after 6 months of storage using the implementation method was 25% or more, and that the implementation method had a survival rate that was at least 110% higher than that when no temperature change treatment was applied (comparative method).

[0042] Example 2 Long-term storage of dried Lactobacillus casei cells: The dried cells obtained in Example 1 after storage for 6 months were stored at 22° C. for an additional 6 months, and the viable cell counts before and after storage were measured in the same manner as in Example 1. The results are shown in Table 2. The survival rate and survival rate improvement rate were calculated in the same manner as in Example 1. These results are also shown in Table 2.

[0043] [Table 2]

[0044] These results showed that the survival rate after 12 months of storage using the implementation method was 21% or more, and that the implementation method had a survival rate of at least 130% or more than that when no temperature change treatment was applied (comparative method). [Industrial Applicability]

[0045] The present invention can be used to produce dried microbial cells. That's all

Claims

1. (a) Temperature-changing treatment of dried microbial cells of Lactobacillus lactic acid bacteria (a) Heating to 30-37°C for 1-2 days (a) is not included in the alternating temperature treatment.

2. A dried microbial cell of Lactobacillus lactic acid bacteria subjected to temperature change treatment (b) (b) Cooling to 2-10°C for 1 day and then subjected to temperature change treatment (a). (a) Heating to 30-37°C for 1-2 days (However, no temperature-changing treatment other than (a) and (b) is performed.)

3. (a) Temperature-changing treatment of dried microbial cells of Lactobacillus lactic acid bacteria (a) Heating to 30-37°C for 1-2 days (a) is not to be carried out as the temperature-changing treatment.

4. A dried microbial cell of Lactobacillus lactic acid bacteria subjected to temperature change treatment (b). (b) Cooling to 2-10°C for 1 day and then subjected to temperature change treatment (a). (a) Heating to 30-37°C for 1-2 days (a) and (b) are not used as temperature-changing treatments.

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