HK1159688A1 - Freeze-dried microbial cell powder and method for producing same - Google Patents

Abstract

Provided is a freeze-dried microbial cell powder suitable for prolonged storage which can be obtained by a convenient and effective freeze-drying method. A method for producing a freeze-dried microbial cell powder comprising freeze-drying a suspension, in which cells of a lactic acid bacterium and/or a bifidobacterium are suspended in a solution of sugars, to give a freeze-dried microbial cell powder, wherein the sugars are trehalose and sucrose and the trehalose and sucrose concentrations in the suspension before the freeze-drying are each greater than or equal to 4.5 wt%. The weight ratio of trehalose to sucrose is preferably 5:1 to 1:5.

Description

Lyophilized powder of thallus and its preparation method

Technical Field

The present invention relates to a freeze-dried powdery microbial cell and a method for producing the same, and more particularly, to a freeze-dried powdery microbial cell which can be stored for a long period of time even under a specific high-temperature condition, and a method for producing the same.

Background

In recent years, strains having probiotic functions have attracted attention. Moreover, preventive medicine based on improvement/normalization of the digestive tract by taking these bacteria and an immune regulation effect accompanying this has been actively used. Among them, lactic acid bacteria, bifidus bacteria, and the like are also called probiotics, and have been ingested in the form of various foods since ancient times. Recently, development of functions has been more vigorous than ever before, and development of commercial products combining novel functionalities has also been carried out.

For the purpose of improving/normalizing the digestive tract, a large number of processed food groups containing these fungi have been developed. And work for taking up these fungi easily and efficiently is still under investigation. For example, yogurt is a common example of these food groups, but these fungi are also powdered for easier ingestion. The powdery cells can also be used as a novel raw material for conveniently producing processed foods, and have high utility value.

However, when these fungi are powdered, if these fungi do not have a necessary amount of viable bacteria remaining at the time of ingestion or food production, the effects of these fungi cannot be obtained. Therefore, it is desired to develop an effective method for preserving fungi and a method for easily preparing the fungi into powder. For example, it is desired to develop a method for improving survivability of freeze-dried cells.

In order to achieve the above object, various studies have been made so far.

For example, patent document 1 aims to improve the solubility in the intestine without dissolving in gastric juice by coating and granulating useful bacteria such as lactic acid bacteria and lactobacillus bifidus that are killed under acidic conditions or at high temperatures for storage. Specifically, a physiologically active substance is coated by 3 layers to prepare a granulated substance, and a saccharide is used in one of the layers.

Patent document 2 proposes a method for improving the viability of bacteria belonging to the genus bifidobacterium by adding a viability-improving agent containing a saccharide selected from the group consisting of glycerol, xylitol, ribitol, arabitol and mannitol to a culture medium. The method aims to maintain the viability and the number of bacteria when stored under aerobic conditions or low pH conditions. In particular, the main aim of this technique is to increase the survival rate in the culture medium system.

Further, patent document 3 provides an enteric lactic acid bacterium composition which is obtained by mixing a polyglycerin fatty acid ester into a lactic acid bacterium powder and has excellent long-term stability, acid resistance and enteric solubility. The above techniques are mainly focused on the storage stability and tolerance under specific conditions.

On the other hand, various techniques have been developed for improving the viability of fungi during cryopreservation and freeze-drying storage.

For example, patent document 4 discloses a method of freeze-drying in order to provide a production method with less damage or death of the bacterial cells and high survival rate in the freezing or freeze-drying step, the method including: adding polysaccharide partial hydrolysate derived from rhizoma Amorphophalli powder into lactobacillus liquid dispersion, and freeze drying. The polysaccharide partial hydrolysate is a substance having a specific molecular weight obtained by enzymatic decomposition, and a complicated process is required for producing the hydrolysate. The main object of this technique is to improve the survival rate of freeze-thawed cells, and there is no suggestion or mention at all about the survival rate of freeze-dried powder cells produced by this technique after storage.

Patent document 5 proposes a preservation solution that has good bacterial survival rate and activity even after thawing frozen bacteria. The cryopreservation liquid for bacteria contains trehalose and/or polyethylene glycol as effective components. Thus, the preservation solution is characterized by being capable of suppressing damage during freezing and being immediately used after thawing. This technology is a system containing moisture, and particularly a technology for solving the problem in the process from freezing to thawing.

In patent document 6, lactobacillus fermentum is coated with a protective film having starch and sugar to prepare a spray-dried powder. This technique is a technique that focuses on improving the viability of the bacteria upon spray drying.

Further, in patent document 7, storage stability in a dry state is improved by allowing a dry microorganism such as a dry lactic acid bacterium to coexist with an L-arginine acidic amino acid salt. The main object of this technique is to improve the storage stability of microorganisms when they are formulated into a preparation. The results of storing the bifidus preparation produced by this technique at 40 ℃ for 2 weeks are shown, but the survival rate is only about 25%, and the preparation is not a technique that can withstand long-term storage for several months.

In addition, non-patent document 1 shows the following study results: from the analysis of the mechanism in this freeze-drying, it has been ascertained that the damage upon freeze-drying of microorganisms is mainly caused by the change in the physical state of phospholipids constituting cell membranes and the structural change of proteins; based on this finding, E.coli and the like were freeze-dried in the presence of trehalose or sucrose. However, there is no suggestion or mention at all of the use of trehalose in combination with sucrose. Further, no corresponding studies have been made on the long-term storage of a specific freeze-dried product and the storage under a specific high-temperature condition.

Further, in non-patent document 2, for the same purpose as described above, studies on freeze-drying and storage of lactobacillus salivarius have been made. In this document, 4% trehalose; 4% of sucrose; 18% skimmed milk powder; 4% trehalose and 18% skim milk powder; 4% sucrose and 18% skimmed milk powder; 4% trehalose and 4% sucrose; and 4% trehalose, 4% sucrose and 18% skimmed milk powder as cryoprotectant, and the lyophilized powder of thallus is stored at-85 deg.C for survival rate study. The cell suspension was lyophilized to have a sugar concentration of about 3.2% and a skim milk powder concentration of 14.4%. According to the results disclosed in this document, it is described that the storage stability at-85 ℃ of the freeze-dried powdery cells could not be sufficiently improved by using 4% sucrose, but the storage stability at-85 ℃ of the freeze-dried powdery cells could be improved by using 4% sucrose and 18% skim milk powder. Further, it was described that the storage stability at-85 ℃ of freeze-dried powdery microbial cells was improved by using 4% trehalose alone. Further, the results of storing powdered cells obtained by freeze-drying a mixed solution of 4% trehalose, 4% sucrose and 18% skim milk powder at room temperature (temperature not shown) for 7 weeks are shown. It is mentioned here that when stored in an environment under a specific humidity condition (humidity 2.8 to 5.6%, water activity 0.028 to 0.056), high storage stability can be maintained. However, it is not possible to maintain high preservability (survival rate of about 40% and 10%, respectively) in an environment with a humidity of 0% and a humidity of 8.8%, and it is not practical to limit the conditions under which the room temperature can be preserved to extremely low humidity and to narrow the humidity range. As described above, the technique of non-patent document 2 shows the result of a storage test under an environmental condition deviating from a normal living environment, and further, no suggestion or mention is made at all of storage under a high temperature condition, and it is difficult to apply the technique to normal food.

Documents of the prior art

Patent document

Patent document 1: japanese laid-open patent publication No. 5-186337

Patent document 2: japanese laid-open patent publication No. 11-137172

Patent document 3: japanese patent laid-open No. 2001-64189

Patent document 4: japanese laid-open patent publication No. 7-313140

Patent document 5: japanese patent laid-open publication No. 2001-327280

Patent document 6: japanese patent laid-open publication No. 2005-52100

Patent document 7: international publication WO2006/106806

Non-patent document

Non-patent document 1: leslie et al, "protection of Both cell Membranes and proteins of non-invasive bacteria by Trehalose and Sucrose during Drying" (Trehalose and Sucrose Process Membranes and proteins in Integrated bacteria Drying), "Applied and environmental microorganisms (Applied and environmental Microbiology), USA, 10.1995, 3592. sup. 3597

Non-patent document 2: gaber Zayed et al, "Influence of trehalose and moisture content on the viability of Lactobacillus salivarius during lyophilization and storage (Influence of trehalase and moisture content on clearance of Lactobacillus salivarius)," Process Biochemistry, 2004, Vol.39, p.1081-1086

Disclosure of Invention

Problems to be solved by the invention

As described above, various studies have been made to preserve useful fungi and to find effective forms. In particular, various attempts have been made to maintain the functions of fungi and the survival rate after long-term storage of the fungi in the form of a powder, such as freeze-dried cells or dried cells, because of the ease of the form of the product. For example, in a specific attempt, there are significant limitations such as effective only for a specific fungus or effective for a plurality of fungi, but the storage conditions require ultra-low temperature and low humidity, and thus no industrially effective means has been developed. In particular, in order to use the powdery cells as powdery cells for foods that can be stored at room temperature for a long period of time, it is necessary to have high viability under high-temperature (30 to 40 ℃) conditions that are within a range of normal living environment temperatures, not limited to special storage conditions. The quality of powdery cells having no such properties cannot be guaranteed, and it is not certain that they can be used as a material for food.

Accordingly, an object of the present invention is to provide freeze-dried powdery microbial cells suitable for long-term storage and obtained by simple and effective freeze-drying, and a method for producing the same. In particular, it is intended to provide a freeze-dried powdery microbial cell which can be stored in a state of high survival rate of useful bacteria such as lactic acid bacteria and Lactobacillus bifidus even in a temperature range of living (particularly, summer) which is not at an extremely low temperature and is considered to have a great influence on storage stability even when stored for a long period of time after freeze-drying, can be easily taken as a powder after storage, or can be effectively used as a raw material in food production, and a method for producing the same. Further, a food composition containing the freeze-dried powder of the obtained microbial cell is provided.

Means for solving the problems

As a result of intensive studies in view of the above-mentioned conventional problems, the present inventors have found that the viability of powdery cells of useful bacteria (lactic acid bacteria and/or bifidus bacteria), particularly the survival rate during high-temperature storage, is remarkably improved by freeze-drying a suspension obtained by suspending lactic acid bacteria and/or bifidus bacteria in a sugar solution, and have completed the present invention.

The present invention provides a freeze-dried powdery microbial cell which is suitable for long-term storage and can be obtained by simple and effective freeze-drying.

That is, one aspect of the present invention is a method for producing freeze-dried powdered microbial cells, which comprises freeze-drying a suspension of lactic acid bacteria and/or lactobacillus bifidus suspended in a saccharide solution to obtain freeze-dried powdered microbial cells, wherein the saccharide is trehalose and sucrose, and the concentration of each of trehalose and sucrose in the suspension before freeze-drying is 4.5 wt% or more.

In the present invention, the concentration of each of trehalose and sucrose is preferably 4.5 to 15 wt%, more preferably 8 to 12 wt%.

In the invention, the weight ratio of the trehalose to the sucrose is preferably 5: 1-1: 5.

In the present invention, preferable examples of the lactic acid bacteria and/or lactobacillus bifidus include fungi belonging to the genus Bifidobacterium (Bifidobacterium).

Preferred examples of fungi belonging to the genus Bifidobacterium include Bifidobacterium bifidum and Bifidobacterium longum.

Preferred examples of the Bifidobacterium bifidum strain include Bifidobacterium bifidum (Bifidobacterium bifidum) OLB6378 strain and the like.

Preferred examples of the Bifidobacterium longum (Bifidobacterium longum) strain include Bifidobacterium longum (Bifidobacterium longum) OLB6001 strain and the like.

Further, another aspect of the present invention relates to a freeze-dried powder-form bacterial cell produced by the method for producing a freeze-dried powder-form bacterial cell as described above.

Still another aspect of the present invention relates to a food composition containing an effective amount of the freeze-dried powder of microbial cells described above.

Effects of the invention

The method for producing lyophilized powder-form bacterial cells which can be stored for a long period of time according to the present invention can exhibit its effect particularly when long-term storage of solid materials, powder-form foods, and the like is required. That is, the survival rate of the powdery freeze-dried powdery microbial cells produced by the production method of the present invention is significantly improved when stored at high temperature, and therefore, the effect of useful lactic acid bacteria and/or Lactobacillus bifidus can be obtained even when used after storage.

Further, since the production method of the present invention is a simple method, a special apparatus and a complicated process are not required, and the cost increase associated therewith is not caused.

In addition, since the freeze-dried powdery microbial cells produced by the production method of the present invention can be used as they are, they can be easily taken as powders after storage or can be effectively used as raw materials for food production.

Drawings

FIG. 1 is a graph showing the results of storage tests of freeze-dried powdered cells of Bifidobacterium bifidum at temperatures of 20 ℃, 30 ℃ and 40 ℃ under conditions in which the trehalose concentration and the sucrose concentration in the suspension before freeze-drying were 6.6 wt%, respectively (as sugar solutions before mixing with the cells, sugar solutions containing 20 wt% of trehalose and sucrose, respectively, were used).

FIG. 2 is a graph showing the results of storage tests of freeze-dried powdered cells of Bifidobacterium bifidum at temperatures of 20 ℃, 30 ℃ and 40 ℃ under conditions in which the trehalose concentration and sucrose concentration in the suspension before freeze-drying were 10 wt%, respectively (as sugar solutions before mixing with the cells, sugar solutions containing 30 wt% of trehalose and sucrose, respectively, were used).

FIG. 3 is a graph showing the results of a storage test at a temperature of 40 ℃ for freeze-dried powdered cells of Bifidobacterium longum (circle number 2 in the figure) and freeze-dried powdered cells for comparison (circle number 1 in the figure) prepared under the condition that the trehalose concentration and sucrose concentration in the suspension before freeze-drying were 6.7 wt%, respectively (as a sugar solution before mixing with the cells, a sugar solution containing 20 wt% of trehalose and sucrose, respectively, was used).

Detailed Description

The present invention will be described in detail below, but the present invention is not limited to the various embodiments described below.

Examples of the lactic acid bacteria and bifidus bacteria used in the present invention include those belonging to the genus Bifidobacterium (Bifidobacterium), and specific examples thereof include Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium bifidum (Bifidobacterium bifidum), and Bifidobacterium adolescentis (Bifidobacterium adolescentis). Further, there can be exemplified fungi belonging to the genus Lactobacillus (Lactobacillus) and the genus Streptococcus (Streptococcus), and specific examples thereof include Lactobacillus gasseri (Lactobacillus gasseri) strain, Lactobacillus bulgaricus (Lactobacillus bulgaricus) strain, and Streptococcus thermophilus (Streptococcus thermophilus) strain. However, the present invention is not limited to these strains, and for these strains, it may be used alone or in combination of 2 or more.

Preferred examples of the above-mentioned strains include Bifidobacterium bifidum (Bifidobacterium bifidum) and Bifidobacterium longum (Bifidobacterium longum).

Examples of the deposited strain of these preferred embodiments include Bifidobacterium bifidum (OLB 6378 strain) and Bifidobacterium longum (OLB 6001 strain).

(A) Deposit of Bifidobacterium bifidum OLB6378 Strain

The bifidobacterium bifidum OLB6378 strain used in the present invention was deposited under the following conditions.

(1) The name of the depository institution: independent administrative law human product evaluation technology substrate organization patent microorganism collection center

(2) The contact way is as follows: zi-code 292-

Telephone number 0438-20-5580

(3) The preservation number is: NITE BP-31

(4) Representation for recognition: bifidobacterium bifidum OLB6378

(5) The original preservation date: average 16 years (2004) 10 months and 26 days

(6) Deposit transfer date based on budapest treaty: 2006, 1 month and 18 days

(B) Deposit of Bifidobacterium longum strain OLB6001

The bifidobacterium longum strain OLB6001 used in the present invention was deposited under the following conditions.

(1) The name of the depository institution: patent biological collection center of institute for Integrated Industrial and technology research of independent administrative Law

(2) The contact way is as follows: zip code 305 plus 8566 arrowhead county bubo city east 1-1-1 central 6 th

Telephone number 029-

(3) The preservation number is: FERM P-13610

(4) Representation for recognition: bifidobacterium longum No.7

(5) The preservation date is as follows: 4 months and 20 days in 5 years (1993)

(6) Budapest treaty based deposit transfer

Day of acceptance: equal to 22 years (2010) and 3 months and 2 days

Acceptance number: FERM ABP-11242

The bifidobacterium bifidum OLB6378 strain and bifidobacterium longum OLB6001 strain used in the present invention have the following bacteriological properties.

Bifidobacterium bifidum OLB6378 strain is gram-positive obligate anaerobacter from human infant feces. When this strain was inoculated into lactobacillus MRS Broth (BD) and cultured at 37 ℃ for 18 hours in an anaerobic state using naeropack KENKI (manufactured by mitsubishi gas chemical corporation), a Y-shaped bacterial form was observed. Further, the method comprises the steps of using a specific primer for Bifidobacterium bifidum (Bifidobacterium bifidum) (in-solution フロ - ラシンポジウム 8 (in-intestinal flora culture collection 8), molecular biological detection/identification of intestinal flora; Rakakipedia, Songbu, Longbai), specifically using a species-specific primer BiBIF-1 for 16S rDNA: CCA CAT GAT CGC ATG TGA TT and BiBIF-2: CCG AAG GCT TGC TCC CAAA PCR products were observed by PCR.

Bifidobacterium longum strain OLB6001 is a gram-positive obligate anaerobic bacillus derived from adult feces, and has a bacterium form of bacillus or branched polymorphic form, and is sporulation-free and motility-free. When this strain was spread on a BL agar medium (Rongyan) plate and cultured at 37 ℃ for 48 hours by the stainless steel wool method, an opaque, round, hemispherical, and glossy colony was formed. The strain has fermentability for arabinose, xylose, ribose, glucose, fructose, galactose, sucrose, maltose, lactose, melibiose, raffinose and melezitose.

The sugar used as the protective agent in the invention is a mixture of trehalose and sucrose. The concentration of each of trehalose and sucrose in the suspension before lyophilization is 4.5% by weight or more, preferably 8% by weight or more, from the viewpoint of obtaining lyophilized powder cells suitable for long-term storage at high temperature. The upper limit of the concentration is preferably 15% by weight, more preferably 12% by weight, from the viewpoint of the same effect even when the concentration is excessively increased for long-term storage at high temperatures. The weight ratio of trehalose to sucrose is not particularly limited, but is preferably 5: 1 to 1: 5, more preferably 3: 1 to 1: 3, and particularly preferably 1: 1 to 1: 1, from the viewpoint of obtaining freeze-dried powdery cells suitable for long-term storage at high temperature.

Preferably, these sugars are mixed with the cultured bacteria in the form of an aqueous solution containing sugars, and the bacteria are resuspended. The suspension thus obtained is freeze-dried to obtain the freeze-dried powdery microbial cells of the present invention.

The aqueous solution containing a saccharide may contain, for example, milk protein, amino acid, ascorbic acid, and the like in addition to water and the saccharide.

The method for producing the freeze-dried powdery microbial cells of the present invention is not particularly limited, and includes, for example, the following steps.

1) The desired fungi are cultured according to a conventional method.

2) The culture solution containing the cultured bacteria is used as it is in the following 3), or the culture solution containing the cultured bacteria is concentrated by centrifugation or the like or subjected to solid-liquid separation to obtain a concentrated culture solution (bacterial fluid) or a cell separated as a solid component, and used in the following 3).

3) The resulting microbial cell liquid or microbial cell is mixed with a solution containing a sugar (protective agent) at a predetermined concentration to obtain a suspension, and the suspension is freeze-dried to obtain a freeze-dried powdery microbial cell of the present invention.

The freeze-drying method may be, for example, a method using a freeze-dryer, and the like, and for example, the freeze-drying method may be a method of rapidly pre-freezing at a low temperature (for example, -30 to-90 ℃), drying at room temperature (for example, 0 to 20 ℃) and under reduced pressure (preferably, a degree of vacuum of 1000Pa or less, and more preferably, 100Pa or less), raising the temperature of the freeze-dryer to, for example, 30 to 70 ℃ under the condition of maintaining the degree of vacuum, and further drying.

The freeze-dried powdery cells of the present invention can be directly ingested because they use trehalose and sucrose which are also useful as food additives; and may also be added to various food compositions for use. The freeze-dried powdery microbial cells of the present invention exhibit a specific survival rate even after long-term storage, and therefore, can be used as a raw material for an inoculum such as fermented milk.

The food composition may be any of various foods and beverages (e.g., cold drinks, fermented milk, yogurt, modified milk powder, etc.). The food composition may be used as it is, or may be used by mixing with other foods or food ingredients according to a conventional method in a usual food composition. The food composition may be in the form of a food or drink that is generally used, and may be in any form such as solid (powder, granule, etc.), paste, liquid, or suspension.

The other ingredients in the food composition are not particularly limited, and examples thereof include water, proteins, sugars, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, seasonings, and the like. These components may be used alone in 1 kind, or may be used in combination of 2 or more kinds. Further, as other components in the food composition, synthetic products and/or foods containing a large amount of synthetic products may be used.

Examples

The present invention will be described below with reference to examples using anaerobic bacteria (Lactobacillus bifidus) which are generally difficult to store, but the present invention is not limited thereto. In the present specification, the expression of% means weight% unless otherwise specified.

[ Experimental example 1]

Lyophilized powder cells were produced by the production method of the present invention according to the following steps.

1) Bifidobacterium bifidum (strain OLB 6378) was subjected to neutralization culture in a casein decomposing medium (medium containing enzymatically decomposed casein as a protein).

2) The culture medium (320 ml) was centrifuged (at 4 ℃ C. for 20 minutes at 10000G) to remove 307.2ml of the supernatant, thereby obtaining a cell pellet fraction (12.8 ml).

3) A protective agent solution (2ml) containing a sugar at a specific concentration was added to the cell pellet fraction (4ml), and the cells were suspended, frozen at-80 ℃ and freeze-dried.

4) Immediately after the freeze-drying, 0.5g of freeze-dried powdery cells were rehydrated with physiological saline, and the number of viable bacteria in the rehydrated liquid was measured using BL agar plate medium.

5) Further, 0.5g of freeze-dried powdery fungus was added to the suspension of Lami-Zip (registered trademark; trade name of plastic bag), viable cell count was measured by using BL agar plate medium in the same manner as for freeze-dried powdery cells after storage at 20 ℃ or 30 ℃ for 53 days.

The protective agent solutions used in the present experimental examples are shown in table 1 below.

[ Table 1]

The results of the above-described preservation test of Bifidobacterium bifidum in the inventive samples 1 and 2 and the comparative samples 1, 2 and 3 are shown in Table 2. In addition, since the survival rates in comparative samples 1 and 2 were low, the results of the case of storing at 20 ℃ for 7 days are shown only with reference.

[ Table 2]

^*1: days of storage: 7 days

As shown in Table 2, in examples 1 to 4 based on the method of the present invention, the number of viable cells was not reduced even when the freeze-dried powdery microbial cells were stored under a predetermined high-temperature condition for 53 days. In particular, it was found that the survival rate was dramatically improved even at a high temperature rather close to the temperature of the living environment as in examples 2 and 4.

As described above, the production method of the present invention is a technique sufficiently applicable to solid/powder foods having a relatively long shelf life.

In addition, comparative examples 1 and 2 are tests on a system using only trehalose as represented by non-patent document 2, but it is found that the object cannot be sufficiently achieved by using only trehalose. In particular, non-patent document 2 describes the effect when the storage is performed under ultra-low temperature and humidity conditions, but it is found that the desired effect cannot be obtained at the temperature of the living environment as in the present experiment.

In comparative examples 3 and 4, a trehalose/sucrose combination system at the same concentration as that in non-patent document 2 was investigated, but it was found that the number of viable bacteria was halved under temperature conditions assumed to be living environments.

It is thus clear that the freeze-dried powder cells obtained by the methods of these comparative examples are not suitable for solid/powder foods having a long shelf life, and are not suitable for practical use in such foods.

[ Experimental example 2]

The lyophilized powder-form bacterial cells obtained by the production method of the present invention were further subjected to an experiment for their effects when stored at high temperature (20 ℃, 30 ℃, 40 ℃) for a long period of time (up to 6 months).

The preparation was carried out in the same manner as in samples 1 and 2, except that the amount of lyophilized powder of microbial cells used was increased to about 30 times. Therefore, the following description will be made of samples 1 and 2.

The results obtained by the above experiments are shown in fig. 1 and 2.

As is apparent from the figure, even when sample 1 (see fig. 1. protectant solutions containing 6.6 wt% of trehalose and sucrose, respectively, obtained by mixing a solution containing 20 wt% of trehalose and a solution containing 20 wt% of sucrose) and sample 2 (see fig. 2. protectant solutions containing 10 wt% of trehalose and sucrose, respectively, obtained by mixing a solution containing 30 wt% of trehalose and a solution containing 30 wt% of sucrose) were both kept at storage temperatures of 20 ℃ and 30 ℃, a sufficient viable cell count was maintained after 6 months, and very high viability was exhibited.

For storage at 40 ℃, sample 1 showed 93.9% viability after 1 month of storage, despite storage under rather extreme ambient temperature conditions. Therefore, under these conditions, the concentration of the protecting agent solution is more preferably the concentration used in sample 1.

[ Experimental example 3]

An experiment was performed in the same manner as in example 1, except that the samples shown in table 3 below were used and stored at 40 ℃ for 3 months. The results are shown in Table 4. As is clear from Table 4, when the mass ratio of trehalose to sucrose was 3: 1 to 1: 3, the survivability was 11 to 17%, and the effect was comparable to each other without any difference.

[ Table 3]

[ Table 4]

[ Experimental example 4]

Lyophilized powder cells were produced by the production method of the present invention according to the following steps.

1) Bifidobacterium longum (Bifidobacterium longum) OLB6001 strain was subjected to neutralization culture in a casein degradation medium (medium containing enzymatically degraded casein as a protein).

2) 5400ml of the culture solution was centrifuged (at 4 ℃ C. for 20 minutes at 10000G) to remove 5200ml of the supernatant, and a cell pellet fraction (200ml) was obtained.

3) 100ml of a protective agent solution containing a sugar at a predetermined concentration was added to the cell pellet fraction (200ml), and the cells were suspended, frozen at-80 ℃ and freeze-dried.

4) Immediately after the freeze-drying, 1g of the freeze-dried powder of the cells was rehydrated with physiological saline, and the number of viable cells in the rehydrated liquid was measured using BL agar plate medium.

5) Further, 2g of freeze-dried powdery fungus was added to Lami-Zip (registered trademark; trade name of plastic bag), viable cell count was measured using BL agar plate medium similarly for freeze-dried powdery cells after storage at 40 ℃ for each of 8 days, 30 days, 82 days, and 124 days.

The following protective solutions were used in the present experimental examples.

(1) Example 8

Sugar solutions each containing 20 wt% of trehalose and sucrose were used (hereinafter referred to as sample 6). The trehalose and sucrose contents of the suspension before freeze-drying, which was obtained by mixing the sugar solution with the cell pellet fraction, were 6.7% by weight, respectively.

(2) Comparative example 5

A solution containing 6 wt% of skim milk powder, 1.7 wt% of lactose, 0.4 wt% of amino acid (lysine and the like), 4 wt% of other components (dextrin and the like) was used (hereinafter referred to as comparative sample 4.).

The results are shown in table 5 and fig. 3. As is clear from Table 5, the lyophilized powder form of the microbial cells of comparative example 5 (circle number 1 in FIG. 3) showed viability of only 0.002% when stored at 40 ℃ for about 4 months; on the other hand, the lyophilized powder of the bacterial cells of example 8 (circled number 2 in FIG. 3) showed a high viability of 40.0% when stored at 40 ℃ for about 4 months.

[ Table 5]

Industrial applicability

The method for producing lyophilized powder-form bacterial cells which can be stored for a long period of time according to the present invention can provide the effect of useful lactic acid bacteria and/or Lactobacillus bifidus even after storage, particularly in the case where long-term storage of solid materials, powder-form foods, and the like is required. Further, it is economically advantageous because it does not require a special apparatus and a complicated process, and does not increase the cost associated therewith. Further, since the freeze-dried powdery microbial cells of the present invention can be used as they are, they can be directly taken in the form of powder after storage, and can be effectively used as a raw material for food production, and therefore, they have high utility values.

Claims (8)

1. A method for producing a freeze-dried powdery microbial cell, comprising freeze-drying a suspension obtained by suspending lactic acid bacteria and/or Lactobacillus bifidus in a sugar solution to obtain a freeze-dried powdery microbial cell, wherein the sugar is trehalose and sucrose, and the concentration of each of trehalose and sucrose in the suspension before freeze-drying is 6.6 to 12 wt%.

2. The method for producing a freeze-dried powder of microbial cells according to claim 1, wherein the concentration of each of trehalose and sucrose in the suspension before freeze-drying is 6.6 to 10% by weight.

3. The method for producing a freeze-dried powder of microbial cells according to claim 1 or 2, wherein the concentration of each of trehalose and sucrose in the suspension before freeze-drying is 8 to 10% by weight.

4. The method for producing a lyophilized powder bacterial cell according to claim 1 or 2, wherein the lactic acid bacterium and/or Lactobacillus bifidus is a fungus belonging to the genus Bifidobacterium (Bifidobacterium).

5. The method for producing a lyophilized powder bacterial cell according to claim 4, wherein the lactic acid bacterium and/or Lactobacillus bifidus is a Bifidobacterium bifidum (Bifidobacterium bifidum) strain.

6. The method for producing a lyophilized powder bacterial cell according to claim 5, wherein the lactic acid bacterium and/or Lactobacillus bifidus is Bifidobacterium bifidum (Bifidobacterium bifidum) OLB6378 strain.

7. The method for producing a lyophilized powder form of microbial cell according to claim 4, wherein the lactic acid bacterium and/or Lactobacillus bifidus is a Bifidobacterium longum (Bifidobacterium longum) strain.

8. The method for producing a lyophilized powder microbial cell according to claim 7, wherein the lactic acid bacterium and/or Lactobacillus bifidus is Bifidobacterium longum (Bifidobacterium longum) OLB6001 strain.