EA046292B1 - METHOD FOR OBTAINING HIGHLY VITAL DRIED MICROBIAL CELLS - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to a method for producing highly viable dried microbial cells having high viability after storage.

State of the art

There are many microorganisms having beneficial enzymatic activities, and such microorganisms are widely used to produce functional food materials such as carbohydrates, amino acids and phospholipids.

In particular, among such microorganisms, lactic acid bacteria have traditionally been widely used to produce dairy products such as yogurt and cheese, and in recent years there have been advances in the development of food products, beverages and the like. using dried lactic acid bacteria. In this case, to obtain the effect of lactic acid bacteria, it is desirable to use living cells, but in the process of obtaining dried cells, the cells are often damaged and killed, and it is difficult to obtain the required number of living cells.

As a method of reducing cell damage and death, for example, a method of adjusting the dispersion medium that is used when drying cells (PTL 1, PTL 2 and NPL 1) is known.

However, the only method known to reduce cell damage and death is one that is carried out before the cells are dried.

List of cited references

Patent documents.

PTL 1: Japanese Patent No. 3504365.

PTL 2: Publication of pending Japanese patent application JP-A-2010-4787.

Non-patent documents.

NPL 1: G.L. DE ANTONI et al., Trehalose, a Cryoprotectant for Lactobacillus bulgaricus, Cryobiology, 26, p. 149-153, 1989.

The essence of the invention

Technical challenge

Therefore, the purpose of the present invention is to develop a new method for reducing cell damage and death.

The solution of the problem

The inventors of the present invention have conducted extensive research to achieve the above object, and as a result, although heat stress usually causes a decrease in the viability of microorganisms, they have unexpectedly discovered that viability after storage is increased if the dried cells are subjected to a variable temperature treatment corresponding to heat stress, after obtaining the dried cells, thereby completing the present invention.

Thus, the present invention relates to a method for producing highly viable dried microbial cells, characterized in that the dried microbial cells are subjected to variable temperature treatment.

In addition, the present invention relates to a method for increasing the viability of dried microbial cells, characterized in that the dried microbial cells are subjected to variable temperature treatment.

Advantageous effects of the invention

According to the present invention, the post-storage viability of the previously obtained dried microbial cells can be increased using a simple variable temperature treatment method.

Description of Embodiments

The method for producing highly viable dried microbial cells of the present invention (hereinafter referred to as the production method of the present invention) is configured such that the dried microbial cells are subjected to variable temperature treatment.

The dried microbial cells that are used in the production method of the present invention are not particularly limited, but examples thereof include cells obtained by drying microorganisms such as lactic acid bacteria. Examples of lactic acid bacteria include lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus casei, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus cremoris, Lactobacillus helveticus, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus yoghurti, Lactobacillus delbruecki subsp. bulgarius, Lactobacillus delbrueckii subspecies delbrueckii, Lactobacillus johnsonii and Lactobacillus mali , bacteria of the genus Bifidobacterium, such as Bifidobacterium bifidum, Bifidobacterium breve and Bifidobacterium longum, bacteria of the genus Streptococcus, such as Streptococcus thermophilus and Streptococcus lactis, bacteria of Lactococcus, such as Lactococcus lactis subspecies lactis, Lactococcus lactis subspecies cremoris, Lactococcus plantarum and Lactococcus raffinolactis, a also bacteria of the genus Enterococcus, such as Enterococcus faecalis and Enterococcus faecium. You can combine one type or two or more types of lactic acid bacteria. Among microorganisms, lactic acid bacteria are preferred. Among lactic acid bacteria, the preferred ones are lactic acid bacteria of the genus Lactobacillus, and the species Lactobacillus casei

- 1 046292 is preferred, and Lactobacillus casei YIT 9029 (FERM BP-1366, deposit date: May 1, 1981, International Depository of Patent Organisms, National Institute of Technology and Evaluation (No. 120, 2-5-8 Kazusakamatari, Kizarazu- shi, Chiba-ken, 292-0818, Japan) is especially preferred.

The method of obtaining dried cells by drying microorganisms is not particularly limited, and spray drying, lyophilization or the like, well known to those skilled in the art, can be used. Specific examples of the method include the methods described in Japanese Patent No. 3504365, Japanese Unexamined Patent Application Publication JP-A-2010-4787, G.L. Publication DE ANTONI et al., Trehalose, a Cryoprotectant for Lactobacillus bulgaricus, Cryobiology, 26, p. 149-153, 1989, WO 2017/073752, etc. Among these methods, the method described in international application WO 2017/073752 is preferred.

More specifically, the method described in WO 2017/073752 is carried out as follows.

The microorganisms are first cultured in a conventional manner and then the cells are collected in a conventional manner. It should be noted that, if necessary, washing can be performed between, before, or after culturing microorganisms and collecting cells.

The microbial cells thus collected are introduced into a dispersion medium consisting of an aqueous solution containing a protector, an antioxidant and a chelating agent, preferably an aqueous solution consisting of a protector, an antioxidant and a chelating agent (hereinafter also simply called a dispersion medium) and the resulting suspension is suspended therein dried to thereby obtain dried microbial cells.

The water used in the dispersion medium is not particularly limited, but, for example, drinking water such as purified water or deionized water can be used.

The protector used in the dispersion medium is not particularly limited, but examples include glutamic acid or a salt thereof such as monosodium glutamate or monopotassium glutamate, a disaccharide such as trehalose, sucrose, lactose or maltose, glycerol, maltodextrin, cyclodextrin , skimmed milk powder, etc. Among the protectors, it is preferable to use glutamic acid or its salt and/or disaccharide, and more preferably monosodium glutamate and/or trehalose. The content of the protector in the dispersion medium is not particularly limited, but is preferably, for example, 1 to 40 mass%, more preferably 5 to 30 mass%.

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

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

As a preferred type of dispersion medium, an aqueous solution containing monosodium glutamate, trehalose, sodium ascorbate and sodium citrate can be exemplified.

The number of microbial cells that are suspended in the dispersion medium is not particularly limited, however, for example, the number of microbial cells in the suspension is from about 1.0 x 10 ⁵ to 4.0 x 10 ¹⁴ CFU/ml, more preferably from about 1.0 x 10 ⁷ to 4 .0x10 ¹³ CFU/ml.

The method of drying the suspension is not particularly limited, and for example, a known drying method such as lyophilization or spray drying can be used, but in order to increase the viability level of microorganisms in the drying step, lyophilization is preferable. Examples of drying conditions in the lyophilization method include conditions in which a freezing treatment is carried out at a temperature of -35 to -45°C for 6 to 12 hours and then a drying treatment at a temperature of 12 to 32°C for 40 to 90 hours.

The dried microbial cells obtained as described above are then subjected to variable temperature treatment. It should be noted that the dried microbial cells before treatment with alternating temperatures, for example, are crushed using a grinder, and the crushed cells are placed in hydroxypropyl methylcellulose capsules in an amount of 0.2 g per capsule with a regular air composition without degassing, then the capsules are placed in a bag or similar container , made of aluminum or similar material, together with an oxygen scavenger, and in this state the dried microbial cells can be subjected to variable temperature treatment.

Variable temperature treatment is a treatment in which the temperature load is different from the room temperature at which dried microbial cells are typically stored and is applied for a specified period or longer. In particular, they perform one, two

- 2,046,292 or more treatments selected from the group consisting of the following treatments (a) and (b):

(a) treatment by heating to 30°C or higher for 1 day or more;

(b) cooling to 10°C or lower for 1 day or more.

Treatments (a) and (b) can be carried out separately or in combination, or can be carried out repeatedly.

Preferred examples of processing (a) include the following types of processing:

(a1) treatment by heating to 30°C or higher, preferably 35 to 37°C for 1 day or more, preferably 2 days; and (a2) treatment by heating to 30°C or higher, preferably 35 to 37°C for 1 day or more, preferably 1 day.

Preferred examples of processing (b) include the following processing:

(b1) cooling to 10°C or lower, preferably 2 to 10°C for 1 day or more, preferably 1 day.

In a preferred alternating temperature treatment, for example, treatment (a) is carried out, or after treatment (b) is then treated (a), and in a more preferred alternating temperature treatment, treatment (a1) is carried out, or after treatment (b1) is then carried out treatment (a1), or then treatment (a2) is carried out, and in a particularly preferred variable temperature treatment treatment (a1) is carried out.

Such variable temperature treatment in the case of heating can be carried out using a heating device such as a dryer or an autoclave, and in the case of cooling the treatment can be carried out using a cooling device such as a refrigerator or freezer. It should be noted that when carrying out such treatment with variable temperatures, pressure, etc. is not particularly limited.

The dried microbial cells thus obtained have a viability of 110% or higher compared to the case where the dried microbial cells are not subjected to variable temperature treatment when stored for 6 months, and have a viability of 120% or higher, preferably a viability of from 130 to 140% compared to the option when dried microbial cells are not subjected to variable temperature treatment when stored for 12 months.

Highly viable dried microbial cells can be used for the same purposes as conventional dried microbial cells. In particular, the highly viable dried microbial cells can be used in foods and beverages directly or by mixing with another nutrient that is commonly added to foods. Examples of food products include processed meat products such as ham and sausages, processed fish products such as kamaboko (made from surimi, finely minced white meat fish fillets) and chikuwa (a fish paste product like long sausages), bread, pastries products, butter and fermented milk such as yogurt. Further, examples of beverages include soft drinks, milk drinks with lactic acid bacteria and drinks with lactic acid bacteria. Moreover, examples of food and beverage forms include commonly used food and beverage forms such as solids such as powder and granules, paste, liquid and the like. In addition, the dried microbial cells can be formulated into, for example, a tablet, powder, chewable tablet, hard capsule, soft capsule, pill, and the like.

Examples

The present invention will now be described in detail with the help of examples, but the present invention is in no way limited to these examples. It should be noted that in the following examples, the number of viable cells of Lactobacillus casei was measured by the following method.

Method for measuring the number of viable cells of Lactobacillus casei.

Serial dilutions of dried Lactobacillus casei cells were prepared in physiological solution (0.85 wt.% sodium chloride). The diluted solution (1 ml) was mixed and titrated on BCP agar plates to count the number of cells and the cells were cultured at 37°C for 72 hours. The resulting colonies were then counted, the resulting number was multiplied by the dilution factor and the resulting value was used as an indicator of the number of viable Lactobacillus casei cells.

Example 1.

Preparation of dried Lactobacillus casei cells and treatment with variable temperatures.

Lactobacillus casei YIT 9029 was cultivated under anaerobic conditions at 37°C for 20 hours in a medium (pH 7) containing yeast extract (1 wt.%), potassium monophosphate (0.1 wt.%), dipotassium phosphate (0.2 wt. .%) and lactose (2 wt.%). After completion of cultivation, the culture solution was cooled to a liquid temperature of 20°C or lower and the pH of the solution was adjusted to 7.0 using 5N. sodium hydroxide solution. Cells obtained by centrifugation of the culture solution (14000xg, 4°C, 30 min),

- 3 046292 were collected and the cells were suspended with a titer of 2.0x1011 CFU/ml in a dispersion medium prepared to bring the total volume to 1000 ml in accordance with the composition containing monosodium glutamate (10 wt.%), trehalose (10 wt.%), sodium ascorbate (1 wt.%) and sodium citrate (1 wt.%). The cell suspension was placed in a tray and the dried cells were obtained by lyophilization. It should be noted that lyophilization was carried out using a lyophilizer (TAKARA FREEZE-DRYER TF20-80 TANNS, manufactured by TAKARA ATM Co., Ltd.) at a shelf temperature of -40°C for 9 hours and then at a shelf temperature of 20°C for 80 h. The resulting dried cells were crushed using a grinder and the crushed cells were placed into capsules (made from hydroxypropyl methylcellulose) in an amount of 0.2 g per capsule with a regular air composition without degassing, then the capsules were placed in an aluminum bag along with an oxygen absorber (manufactured by Mitsubishi Gas Chemical Company, Inc.).

The aluminum bag obtained above was immediately heated at 35°C for 2 days after preparation and then stored at 22°C for 6 months (implementation method 1). In addition, the aluminum bag obtained above was immediately cooled at 2°C for 1 day after preparation, then heated at 35°C for 2 days, and then stored at 22°C for 6 months (embodiment method 2). In addition, the aluminum bag obtained above was immediately heated at 37°C for 1 day after preparation and thereafter stored at 22°C for 6 months (implementation method 3). It should be noted that the aluminum bag obtained above was stored at 22°C for 6 months, and the result obtained was used for comparison. The results of measuring the number of viable cells before and after storage are given in table. 1. The viability level was also calculated based on the viable cell count using the formula below, and the viability improvement level of the respective embodiments was also calculated compared to the comparative method (without variable temperature treatment) using the formula below. The results are also shown in table. 1.

Table 1

Before storage After storage for 6 months Vitality level Vitality Enhancement Level Comparative method 2730x10 ^s cells/g 618x10 ^s cells/g 22.6% - Method 1 2730x10 ^s cells/g 745x10 ^s cells/g 27.3% 121% Method 2 2730x10 ^s cells/g 700x10 ^s cells/g 25.6% 113% Method 3 2730x10 ^s cells/g 720x10 ^s cells/g 26.4% 117%

Formula 1:

Viability level (%) = (number of viable cells after storage/number of viable cells before storage) x 100.

Formula 2:

Vitality improvement rate (%) = (viability level of implementation method/vitality level of comparative method) x 100.

According to the results obtained, it was found that the viability level after 6 months of storage in each of the embodiments was 25% or higher, and therefore, each of the embodiments provided a viability of at least 110% or higher compared to the variant where treatment with variable temperatures were not carried out (comparative method).

Example 2.

Long-term storage of dried Lactobacillus casei cells.

The dried cells after 6 months of storage obtained in Example 1 were then stored at 25°C for 6 months, and the results of measuring the number of viable cells before and after storage, measured in the same way as described in Example 1, are shown in Table. 2. The level of viability and the level of increase in viability were also calculated in the same way as described in example 1. The results are also shown in table. 2.

- 4 046292

table 2

Before storage After storage for 12 months Vitality level Vitality Enhancement Level Comparative method 2730x10 ^s cells/g 440x10 ^s cells/g 16.1% - Method 1 2730x10 ^s cells/g 590x10 ^s cells/g 21.6% 134% Method 2 2730x10 ^s cells/g 575x10 ^s cells/g 21.1% 131% Method 3 2730x10 ^s cells/g 580x10 ^s cells/g 21.2% 132%

According to the results obtained, it was found that the viability level after 12 months of storage in each of the embodiments was 21% or higher, and therefore, each of the embodiments provided a viability of at least 130% or higher compared to the variant when treatment with variable temperatures were not carried out (comparative method).

Industrial applicability

The present invention can be used to obtain dried microbial cells.

Claims (2)

CLAIM 1. A method for increasing the viability of dried microbial cells, in which the dried microbial cells are dried cells of lactic acid bacteria of the genus Lactobacillus, characterized in that the dried microbial cells are subjected to a variable temperature treatment, with one treatment (a) or a combination of (a) + ( b); wherein treatment (a) is heating at 30-37°C for 1 to 2 days and treatment (b) is cooling at 2-10°C for 1 to 2 days. 2. A method for increasing the viability of dried microbial cells according to claim 1, wherein treatment (a) is carried out after treatment (b).