WO2010101175A1 - Freeze-dried microbial cell powder and method for producing same - Google Patents

Freeze-dried microbial cell powder and method for producing same Download PDF

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Publication number
WO2010101175A1
WO2010101175A1 PCT/JP2010/053421 JP2010053421W WO2010101175A1 WO 2010101175 A1 WO2010101175 A1 WO 2010101175A1 JP 2010053421 W JP2010053421 W JP 2010053421W WO 2010101175 A1 WO2010101175 A1 WO 2010101175A1
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freeze
trehalose
sucrose
producing
dried powdered
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PCT/JP2010/053421
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French (fr)
Japanese (ja)
Inventor
政幸 上條
正樹 寺原
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明治乳業株式会社
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Priority to JP2011502775A priority Critical patent/JP5583114B2/en
Priority to CN201080004172.6A priority patent/CN102272287B/en
Priority to SG2011049061A priority patent/SG172859A1/en
Publication of WO2010101175A1 publication Critical patent/WO2010101175A1/en
Priority to HK12100228.0A priority patent/HK1159688A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

  • the present invention relates to a freeze-dried powdered cell and a method for producing the same, and particularly to a freeze-dried powdered cell that can be stored for a long time even under a predetermined high temperature condition and a method for producing the same.
  • a large number of processed food groups containing these fungi have been developed for the purpose of improving and normalizing the digestive tract. Moreover, the device for ingesting these fungi simply and effectively is also made
  • Patent Document 1 by coating and granulating useful bacteria such as lactic acid bacteria and bifidobacteria that die under acidic or high temperature, the solubility in the intestine is not improved in the gastric juice in the sense of preservation. want to be.
  • a physiologically active substance is granulated by three-layer coating, and sugar is used as one of them.
  • Patent Document 2 proposes a method of adding a survival improver containing a saccharide selected from glycerol, xylitol, adonitol, arabitol, and mannitol to improve the survival of Bifidobacterium. Yes.
  • the purpose of this method is to maintain the survival and the number of bacteria during storage under aerobic conditions and low pH conditions.
  • this technique aims to improve the survival rate of the system in the medium.
  • Patent Document 3 a polyglycerol fatty acid ester is mixed with lactic acid bacteria cell powder to provide an enteric lactic acid bacteria composition having excellent long-term stability, acid resistance and enteric properties.
  • the above technique focuses on storage and tolerance under specific conditions.
  • Patent Document 4 discloses that a lot of konjac powder derived from lactic acid bacteria is dispersed in a lactic acid bacteria dispersion for the purpose of providing a production method with little damage or death of the bacteria in the freezing or freeze-drying process and having a high survival rate.
  • a method of freeze-drying by adding a saccharide partial hydrolyzate is disclosed.
  • the polysaccharide partial hydrolyzate has a predetermined molecular weight obtained by enzymatic degradation, and requires a complicated process for producing it.
  • this technique is mainly intended to improve the survival rate in freeze-thawing, and there is no suggestion or reference regarding the survival rate after storage of freeze-dried powdered cells produced by this technique.
  • Patent Document 5 proposes a preservation solution having a good survival rate and activity even after thawing frozen bacteria.
  • This cryopreservation solution for fungi contains trehalose and / or polyethylene glycol as active ingredients. This is characterized by the fact that it is a preservative solution that can be used immediately after thawing, while preventing damage during freezing. It is a system that contains water, and is a technology that solves the problem in the process of freezing to thawing.
  • Lactobacillus fermentum is coated with a protective film containing starch and saccharide to form a spray-dried powder.
  • This technique is a technique that focuses on enhancing the survival of bacteria during spray drying.
  • Patent Document 7 improves the storage stability in a dry state by allowing a dry microorganism such as a dry lactic acid bacterium to coexist with an L-arginine acidic amino acid salt.
  • This technology is mainly intended to improve the storage stability of microorganisms when formulating microorganisms (fungi).
  • the results of bifidobacteria preparations produced by this technique were stored for 2 weeks at 40 ° C., the survival rate is only about 25%, and it cannot withstand long-term storage for several months. It was.
  • Non-Patent Document 1 from the mechanism analysis in lyophilization, it was found that the damage during lyophilization of microorganisms is largely due to changes in the physical state of phospholipids constituting the cell membrane and structural changes in proteins, In contrast, the results of lyophilization of Escherichia coli and the like in the presence of trehalose or sucrose are shown. However, there is no suggestion or mention about using a mixture of trehalose and sucrose. Furthermore, no investigation has been made on the long-term storage of specific freeze-dried products and storage at a predetermined high temperature condition.
  • Non-Patent Document 2 studies on lyophilization and storage of Lactobacillus salivarius have been conducted for the same purpose as described above.
  • sucrose and 18% nonfat dry milk as a freeze-drying protective agent
  • freeze-dried powdered cells are stored at -85 ° C, and the survival rate is examined.
  • the sugar concentration in the cell suspension at the time of freeze-drying is about 3.2%
  • the nonfat dry milk concentration is 14.4%.
  • Non-Patent Document 2 shows the results of a storage test under environmental conditions that deviate from the normal living environment, and there is no suggestion or reference regarding storage under high temperature conditions. It was still difficult to apply the technology to general food.
  • powdery microbial cell for use as a powdery microbial cell that can be applied to foods that can be stored at room temperature for a long period of time, it can be used under special conditions at high temperatures (30-40 ° C) within the range expected for normal living environment temperatures. It is necessary to have high survivability without limitation of storage conditions. A powdery cell having no such property cannot guarantee its quality and cannot be used as a food material.
  • an object of the present invention is to provide a lyophilized powdery microbial cell suitable for long-term storage and obtained by simple and effective lyophilization, and a method for producing the same.
  • lactic acid bacteria and bifidobacteria are useful even if they are stored for a long time after freeze-drying, or even if they are stored in a living temperature range (especially in summer) that is considered to have a significant impact on storage stability, not at very low temperatures.
  • a lyophilized powdery cell that can be stored in a state where the survival rate of the fungus is high, can be directly ingested as a powder after storage, or can be effectively used as a raw material in food production, and its production A method is provided.
  • the food composition containing the obtained freeze-dried powdered microbial cell is also provided.
  • the present invention provides a lyophilized powdery microbial cell suitable for long-term storage and obtained by simple and effective lyophilization.
  • a lyophilized powdery cell is obtained by freeze-drying a suspension in which lactic acid bacteria and / or bifidobacteria are suspended in a saccharide solution to obtain a lyophilized powdered cell.
  • the concentration of each of trehalose and sucrose is preferably 4.5 to 15% by weight, more preferably 8 to 12% by weight.
  • the weight ratio of trehalose to sucrose is preferably 5: 1 to 1: 5.
  • preferred examples of the lactic acid bacteria and / or bifidobacteria include fungi belonging to the genus Bifidobacterium.
  • fungi belonging to the genus Bifidobacterium include Bifidobacterium bifidum strains, Bifidobacterium longum strains, and the like.
  • Bifidobacterium bifidum strain include the Bifidobacterium bifidum OLB6378 strain.
  • a preferred example of the Bifidobacterium longum strain is a Bifidobacterium longum OLB6001 strain.
  • Another aspect of the present invention is a freeze-dried powdered cell produced by the method for producing a freeze-dried powdered cell as described above.
  • Still another embodiment of the present invention is a food composition comprising an effective amount of the above lyophilized powdered microbial cells.
  • the method for producing a freeze-dried powdered cell that can be stored for a long period of time according to the present invention is particularly effective when it is necessary to guarantee long-term storage, such as a solid or powdered food. That is, the powder freeze-dried powder produced by the production method of the present invention has a significantly improved survival rate during high-temperature storage, so that it is useful even after use after storage, such as lactic acid bacteria and / or bifidobacteria. The effect of can be obtained.
  • the production method of the present invention is a simple method, it does not require any special equipment or complicated steps, and there is no increase in costs associated therewith.
  • the lyophilized powdery cell produced by the production method of the present invention can be used as it is, it can be easily ingested directly after storage or effectively used as a raw material in food production. be able to.
  • the trehalose concentration and the sucrose concentration in the suspension before lyophilization were each 6.6% by weight (a sugar solution containing 20% by weight of trehalose and sucrose was used as a sugar solution before mixing with the cells). It is a graph which shows the result of the storage test in 20 degreeC, 30 degreeC, and each temperature of 40 degreeC of the freeze-dried powdery cell body of Bifidobacterium bifidum prepared on a certain condition.
  • the conditions in which the trehalose concentration and the sucrose concentration in the suspension before lyophilization are 10% by weight (a solution containing 30% by weight of trehalose and sucrose as a sugar solution before mixing with the cells) were used.
  • Examples of lactic acid bacteria and bifidobacteria used in the present invention include bacteria belonging to the genus Bifidobacterium, such as Bifidobacterium longum strains, Bifidobacterium infantitis (Bifidobacterium infantitis). ) Strains, Bifidobacterium breve strains, Bifidobacterium bifidum strains, Bifidobacterium adrescentis strains and the like. Still further, fungi belonging to the genus Lactobacillus and Streptococcus can be exemplified, for example, Lactobacillus gasseri strain, Lactobacillus bulgaricus strain, Etc. can be specifically mentioned. However, the present invention is not limited to these species, and these strains can be used alone or in combination of two or more.
  • preferable examples include a Bifidobacterium bifidum strain and a Bifidobacterium longum strain.
  • Bifidobacterium bifidum OLB6378 strain, Bifidobacterium longum OLB6001 strain and the like are mentioned as examples of deposited strains of these preferable examples.
  • (A) Deposit of Bifidobacterium bifidum OLB6378 strain The Bifidobacterium bifidum OLB6378 strain used in the present invention was deposited under the following conditions. (1) Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818 Phone number 0438-20-5580 (3) Accession number: NITE BP-31 (4) Display for identification: Bifidobacterium bifidum OLB6378 (5) Original deposit date: October 26, 2004 (6) Date of transfer to deposit under the Budapest Treaty: January 18, 2006 (B) Deposit of Bifidobacterium longum OLB6001 strain The Bifidobacterium longum OLB6001 strain used in the present invention was deposited under the following conditions.
  • the Bifidobacterium bifidum OLB6378 strain and the Bifidobacterium longum OLB6001 strain used in the present invention have the following mycological properties.
  • Bifidobacterium bifidum OLB6378 is a Gram-positive obligate anaerobe derived from human infant feces. Lactobacilli MRS Broth (BD) is inoculated with this bacterium, and cultured in anaerobic state by using AnaeroPack Kenki (manufactured by Mitsubishi Gas Chemical Company) at 37 ° C. for 18 hours, a Y-shaped microbial form is observed.
  • BiBIF- a specific primer for Bifidobacterium bifidum (intestinal flora symposium 8, molecular biological detection / identification of intestinal flora, Tomohiro Mitsuoka, Takahiro Matsumoto), specifically, BiBIF-, which is a species-specific primer for 16S rDNA.
  • PCR products were observed by PCR using 1: CCA CAT GAT CGC ATG TGA TT and BiBIF-2: CCG AAG GCT TGC TCC CAA A.
  • the Bifidobacterium longum OLB6001 strain is a Gram-positive obligate anaerobic bacterium derived from human adult feces, and the shape of the fungus is a gonococcal or branched polymorph and does not have spore formation or motility.
  • This bacterium is applied on a BL agar medium (Eiken) plate and cultured by a steel wool method at 37 ° C. for 48 hours, colonies having an opaque circular hemispherical gloss are formed.
  • the saccharide as a protective agent used in the present invention is a mixture of trehalose and sucrose.
  • the concentration of trehalose and sucrose in the suspension before lyophilization is 4.5% by weight or more, preferably 8% by weight, from the viewpoint of obtaining lyophilized powdery cells suitable for long-term storage at high temperatures. That's it.
  • the upper limit of the concentration is preferably 15% by weight, more preferably 12% by weight from the viewpoint that the effect of long-term storage stability at high temperatures is equivalent even if the concentration is excessively increased.
  • the weight ratio of trehalose to sucrose is not particularly limited, but is preferably 5: 1 to 1: 5, more preferably 3: 1 from the viewpoint of obtaining lyophilized powder cells suitable for long-term storage at high temperatures.
  • the carbohydrates are preferably mixed with cultured bacteria in the form of a carbohydrate-containing aqueous solution, and the bacteria are resuspended.
  • the lyophilized powdery cell of the present invention can be obtained by freeze-drying the suspension thus obtained.
  • the saccharide-containing aqueous solution may contain, for example, milk protein, amino acids, ascorbic acid and the like in addition to water and saccharides.
  • the manufacturing method of the lyophilized powdery microbial cell of this invention is not specifically limited, For example, it consists of the following procedures. 1) A desired fungus is cultured according to a conventional method. 2) Use the culture solution containing the cultured bacteria as it is in the next 3), or concentrate or solid-liquid separate the culture solution containing the cultured bacteria by centrifugation or the like. Bacteria separated as a liquid (bacterial cell liquid) or solid content is obtained and used in the following 3). 3) The obtained bacterial cell liquid or bacterial cell and a solution containing a predetermined concentration of sugar (protective agent) are mixed to obtain a suspension, and then the suspension is freeze-dried to obtain the present invention. To obtain a freeze-dried powdered cell.
  • freeze-drying method examples include a method using a freeze-dryer. For example, after rapid pre-freezing at a low temperature (for example, ⁇ 30 to ⁇ 90 ° C.), the temperature is reduced at room temperature (for example, 0 to 20 ° C.). It may be dried under a low pressure (preferably 1000 Pa or less, more preferably 100 Pa or less), and the temperature of the freeze dryer is increased to, for example, 30 to 70 ° C. while maintaining the degree of vacuum. .
  • the lyophilized powdery cell of the present invention can be ingested as it is because it uses trehalose and sucrose which can also be used as food additives, and it can be added to various food compositions. Can also be used.
  • the freeze-dried powdered cell of the present invention shows a predetermined survival rate even after long-term storage, it can be used as a raw material for inoculum such as fermented milk.
  • the food composition examples include various foods and beverages (soft drinks, fermented milk, yogurt, adjusted powdered milk, etc.).
  • the food composition can be used as it is, or can be used according to a conventional method for ordinary food compositions such as mixing with other foods or food ingredients.
  • the food composition may be in the state of a commonly used food or drink, for example, solid (powder, granule, etc.), paste, liquid or suspension.
  • components in the food composition are not particularly limited, and examples include water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, and flavors. These components may be used individually by 1 type, or can also be used in combination of 2 or more type. In addition, as another component in a food composition, you may use the foodstuff which contains many synthetic products and / or synthetic products.
  • Example 1 According to the procedure shown below, freeze-dried powdered cells were produced based on the production method of the present invention. 1) The Bifidobacterium bifidum OLB6378 strain was subjected to neutralization culture in a casein decomposition medium (a medium containing enzymatically decomposed casein as a protein). 2) Centrifugation (320 ° C., 10000 G for 20 minutes) of 320 ml of the culture solution was performed to remove 307.2 ml of the supernatant to obtain a cell pellet fraction (12.8 ml).
  • casein decomposition medium a medium containing enzymatically decomposed casein as a protein
  • the protective agent liquid used in this experimental example is as shown in Table 1 below.
  • Table 2 shows the results of the Bifidobacterium bifidum preservation test for Samples 1 and 2 and Comparative Samples 1, 2 and 3 according to the present invention.
  • the survival rate was low about the comparative samples 1 and 2
  • saving for 7 days at 20 degreeC is described together for reference.
  • Comparative Examples 1 and 2 are tests on a trehalose-only system typified by Non-Patent Document 2, but it was found that the objective could not be sufficiently achieved with trehalose alone.
  • Non-Patent Document 2 describes the effect of storage under ultra-low temperature and humidity conditions, but it has been found that the desired effect cannot be obtained at the living environment temperature as in this experiment.
  • Example 2 Experiments were conducted on the effects of long-term storage (up to 6 months) of the freeze-dried microbial cells obtained by the production method of the present invention under high temperature conditions (20 ° C, 30 ° C, 40 ° C). .
  • the freeze-dried powdered cells used were produced according to the same production method as Samples 1 and 2, except that the amount charged was increased about 30 times. Therefore, it demonstrates as the samples 1 and 2 below.
  • the results obtained by the above experiment are shown in FIGS.
  • sample 1 (see FIG. 1), containing 6.6% by weight of each of trehalose and sucrose obtained by mixing a solution containing 20% by weight of trehalose and a solution containing 20% by weight of sucrose.
  • Protectant solution and sample 2 (see FIG. 2), each containing 10% by weight of trehalose and sucrose obtained by mixing a solution containing 30% by weight trehalose and a solution containing 30% by weight sucrose.
  • Both the protective agent solution maintained a sufficient viable count even after 6 months even under storage temperature conditions of 20 ° C. and 30 ° C., and showed very high survival. Although it is a considerably extreme living environment temperature condition, with respect to storage at 40 ° C., the sample 1 shows 93.9% survival after 1 month storage. Therefore, it was found that the concentration used in Sample 1 was more preferable as the concentration of the protective agent solution under these conditions.
  • Example 3 Experiments were performed in the same manner as in Example 1 except that the samples shown in Table 3 below were used and the samples were stored at 40 ° C. for 3 months. The results are shown in Table 4. From Table 4, it was found that there was no difference between the mass ratio of trehalose and sucrose in the range of 3: 1 to 1: 3 and the survival rate was 11 to 17%.
  • Example 4 According to the procedure shown below, freeze-dried powdered cells were produced based on the production method according to the present invention.
  • Bifidobacterium longum OLB6001 strain was neutralized and cultured in a casein decomposition medium (a medium containing enzymatically decomposed casein as a protein).
  • a casein decomposition medium a medium containing enzymatically decomposed casein as a protein.
  • Centrifugation 5 ° C., 10000 G for 20 minutes
  • 5400 ml of the culture broth removed 5200 ml of the supernatant to obtain a cell pellet fraction (200 ml).
  • 3) 100 ml of a protective agent solution containing a predetermined concentration of sugar was added to the cell pellet fraction (200 ml), the cells were suspended, frozen at ⁇ 80 ° C.
  • freeze-dried To 1 g of freeze-dried powdered cells immediately after lyophilization, physiological saline was added to cover the cells, and the number of viable bacteria in the water-covered solution was measured using a BL agar plate medium. 5) Furthermore, freeze-dried after 2 g of freeze-dried powdered cells were placed in lamizip (registered trademark; trade name of plastic bag) and stored at 40 ° C. for 8 days, 30 days, 82 days, and 124 days. Similarly, the number of viable cells was measured on a BL agar plate medium for powdered cells.
  • lamizip registered trademark; trade name of plastic bag
  • Example 8 A sugar solution (hereinafter referred to as Sample 6) containing 20% by weight of trehalose and sucrose was used. The contents of trehalose and sucrose in the suspension before lyophilization formed by mixing the sugar solution and the cell pellet fraction were 6.7% by weight, respectively.
  • Comparative Example 5 A solution containing 6% by weight of skim milk powder, 1.7% by weight of lactose, 0.4% by weight of amino acid (lysine, etc.) and 4% by weight of other components (dextrin, etc.) (hereinafter referred to as Comparative Sample 4) is used. It was. The results are shown in Table 3 and FIG.
  • Table 5 shows that the freeze-dried microbial cell of Comparative Example 5 (circled number 1 in FIG. 3) shows only 0.002% survival when stored at 40 ° C. for about 4 months.
  • the freeze-dried microbial cell of Example 8 (circled number 2 in FIG. 3) shows a high survival rate of 40.0% when stored at 40 ° C. for about 4 months.
  • the method for producing lyophilized powdered cells that can be stored for a long period of time according to the present invention, lactic acid bacteria and / or useful after storage, especially when it is necessary to guarantee long-term storage, such as solids and powdered foods
  • the effect of bifidobacteria can be obtained.
  • no special equipment or complicated processes are required, and there is no cost increase associated therewith, which is economically advantageous.
  • the lyophilized powdery cell of the present invention can be used as it is, it can be easily ingested directly as a powder even after storage, or can be effectively used as a raw material in food production. Value is high.

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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

凍結乾燥粉末状菌体及びその製造方法Freeze-dried powdered cell and method for producing the same
　本発明は、凍結乾燥粉末状菌体及びその製造方法に関し、特に所定の高温条件下においても長期保存が可能である凍結乾燥粉末状菌体及びその製造方法に関する。 The present invention relates to a freeze-dried powdered cell and a method for producing the same, and particularly to a freeze-dried powdered cell that can be stored for a long time even under a predetermined high temperature condition and a method for producing the same.
　近年、プロバイオティクス機能を有する菌株に注目が集まっている。そして、これらの菌の摂取による消化管内の改善・健常化とそれに伴う免疫調整作用による予防医学が盛んになってきている。その中でも特に乳酸菌、ビフィズス菌などはプロバイオティクス菌とも称され、古来より様々な食品として摂取されている。最近では、以前よりもその機能開発がさらに盛んになり、新規な機能性を盛り込んだ商品としての開発も進んでいる。 In recent years, attention has been focused on strains having probiotic functions. In addition, improvement and normalization of the gastrointestinal tract by ingestion of these bacteria and preventive medicine based on the accompanying immunomodulating action have become popular. Among them, lactic acid bacteria, bifidobacteria and the like are also called probiotic bacteria and have been ingested as various foods since ancient times. Recently, the development of functions has become more popular than before, and the development of products incorporating new functionality is also progressing.
　前記の消化管内の改善・健常化等の目的の為に、これら菌類を含む加工食品群が多数開発されている。また、これら菌類を簡便かつ効果的に摂取するための工夫もなされている。例えば、これら食品群の一般的な一例としてヨーグルトが挙げられるが、さらに摂取しやすいようにこれらの菌類を粉末状とすることが行われている。この粉末状菌体はまた、簡便に応用加工食品を製造するための新規な原料として用いることもでき、その利用価値は高い。 A large number of processed food groups containing these fungi have been developed for the purpose of improving and normalizing the digestive tract. Moreover, the device for ingesting these fungi simply and effectively is also made | formed. For example, yogurt is given as a general example of these food groups, and these fungi are made into a powder form so that they can be easily ingested. This powdery microbial cell can also be used as a novel raw material for easily producing an applied processed food, and its utility value is high.
　しかし、これらの菌類を粉末状とした場合、摂取時や食品製造時にこれらの菌類が必要量以上生残していなければ、これらの菌類の効果を得ることができない。従って、菌類の有効な保存方法、及び、粉末状に簡便に製造するための方法が望まれている。例えば、凍結乾燥した場合における生残性を向上させる方法が望まれている。 However, when these fungi are powdered, the effects of these fungi cannot be obtained unless they are surviving more than the required amount at the time of ingestion or food production. Therefore, an effective method for preserving fungi and a method for easily producing powders are desired. For example, a method for improving the survivability when freeze-dried is desired.
　上記目的のために、これまでも様々な検討が行われている。
　例えば、特許文献１には、酸性下又は高温下で死滅する乳酸菌、ビフィズス菌等の有用菌を被覆造粒することで、保存という意味で胃液中において溶解せず腸内での溶性の向上を目指している。具体的には生理活性物質を３層被覆により造粒物とし、その一つに糖類を用いている。 Various studies have been conducted for the above purpose.
For example, in Patent Document 1, by coating and granulating useful bacteria such as lactic acid bacteria and bifidobacteria that die under acidic or high temperature, the solubility in the intestine is not improved in the gastric juice in the sense of preservation. want to be. Specifically, a physiologically active substance is granulated by three-layer coating, and sugar is used as one of them.
　また、特許文献２ではビフィドバクテリウム属細菌の生残性改善に、グリセロール、キシリトール、アドニトール、アラビトール、マンニトールから選ばれる糖類を含有する生残性改善剤を培地に添加する方法を提案している。この方法においては、好気条件や低ｐＨ条件下で保存時の生残性、菌数維持を目的としている。特にこの技術においては、培地における系での生残率向上をその主旨とする。 Patent Document 2 proposes a method of adding a survival improver containing a saccharide selected from glycerol, xylitol, adonitol, arabitol, and mannitol to improve the survival of Bifidobacterium. Yes. The purpose of this method is to maintain the survival and the number of bacteria during storage under aerobic conditions and low pH conditions. In particular, this technique aims to improve the survival rate of the system in the medium.
　また、特許文献３においては、乳酸菌菌体粉末にポリグリセリン脂肪酸エステルを混ぜ、長期安定性が優れ且つ耐酸性、腸溶性に優れた腸溶性乳酸菌組成物を提供する。以上の技術は、特定条件下における保存や耐性に主眼をおいているものである。 In Patent Document 3, a polyglycerol fatty acid ester is mixed with lactic acid bacteria cell powder to provide an enteric lactic acid bacteria composition having excellent long-term stability, acid resistance and enteric properties. The above technique focuses on storage and tolerance under specific conditions.
　一方、菌類の凍結保存、凍結乾燥保存についての生残性改良についても種々の技術が開発されている。
　例えば、特許文献４には、凍結又は凍結乾燥工程において菌体の損傷又は死滅が少なく、かつ生残率の高い製造方法を提供する目的で、乳酸菌類の菌体分散液にコンニャク粉由来の多糖類部分加水分解物を添加して凍結乾燥する方法が開示されている。多糖類部分加水分解物は酵素分解することで得られる所定の分子量を有するものであり、これを製造する繁雑な工程が必要である。また、この技術は凍結融解における生残率向上を主旨としており、この技術で製造された凍結乾燥粉末状菌体の保存後の生残率については全く示唆も言及もされていない。 On the other hand, various techniques have also been developed for improving the survival of fungi in frozen storage and freeze-dried storage.
For example, Patent Document 4 discloses that a lot of konjac powder derived from lactic acid bacteria is dispersed in a lactic acid bacteria dispersion for the purpose of providing a production method with little damage or death of the bacteria in the freezing or freeze-drying process and having a high survival rate. A method of freeze-drying by adding a saccharide partial hydrolyzate is disclosed. The polysaccharide partial hydrolyzate has a predetermined molecular weight obtained by enzymatic degradation, and requires a complicated process for producing it. Further, this technique is mainly intended to improve the survival rate in freeze-thawing, and there is no suggestion or reference regarding the survival rate after storage of freeze-dried powdered cells produced by this technique.
　特許文献５においては、凍結菌を融解後であっても菌生存率及び活性が良好な保存液が提案されている。この菌用凍結保存液はトレハロース及び／又はポリエチレングリコールを有効成分として含有する。これにより凍結時の障害を抑制し、融解後に即使用可能な保存液である点を特徴とする。水分を含んだ系であり、特に凍結から融解という過程における問題を解決する技術である。 Patent Document 5 proposes a preservation solution having a good survival rate and activity even after thawing frozen bacteria. This cryopreservation solution for fungi contains trehalose and / or polyethylene glycol as active ingredients. This is characterized by the fact that it is a preservative solution that can be used immediately after thawing, while preventing damage during freezing. It is a system that contains water, and is a technology that solves the problem in the process of freezing to thawing.
　また、特許文献６では、ラクトバチルスファーメンタムを澱粉質、糖質を有する保護膜で被覆して噴霧乾燥粉体とする。この技術では、噴霧乾燥時の菌の生残性を高めることに重点を置いた技術である。 In Patent Document 6, Lactobacillus fermentum is coated with a protective film containing starch and saccharide to form a spray-dried powder. This technique is a technique that focuses on enhancing the survival of bacteria during spray drying.
　さらに、特許文献７では、乾燥乳酸菌等の乾燥微生物とＬ－アルギニン酸性アミノ酸塩とを共存させることで乾燥状態での保存安定性を向上させている。この技術は微生物（菌）を製剤化する際の微生物の保存安定性向上を主旨としている。尚、この技術で製造したビフィズス菌製剤を４０℃で２週間保存した結果が示されているが、その生残率は僅か２５％程度であり、数ヶ月に及ぶ長期保存に耐えられるものではなかった。 Furthermore, Patent Document 7 improves the storage stability in a dry state by allowing a dry microorganism such as a dry lactic acid bacterium to coexist with an L-arginine acidic amino acid salt. This technology is mainly intended to improve the storage stability of microorganisms when formulating microorganisms (fungi). Although the results of bifidobacteria preparations produced by this technique were stored for 2 weeks at 40 ° C., the survival rate is only about 25%, and it cannot withstand long-term storage for several months. It was.
　また、非特許文献１では、その凍結乾燥における機序解析から、微生物の凍結乾燥時における損傷が、細胞膜を構成するリン脂質の物理状態の変化とタンパク質の構造変化とによるところが大きいことを突き止め、これに対して大腸菌などについてトレハロースまたはスクロース存在下にて凍結乾燥を行う研究結果を示している。しかし、トレハロースとスクロースを混合して用いることについては全く示唆も言及もされていない。さらに、具体的な凍結乾燥物の長期保存について、また所定の高温条件における保存についてはなんら対応検討がなされていない。 Further, in Non-Patent Document 1, from the mechanism analysis in lyophilization, it was found that the damage during lyophilization of microorganisms is largely due to changes in the physical state of phospholipids constituting the cell membrane and structural changes in proteins, In contrast, the results of lyophilization of Escherichia coli and the like in the presence of trehalose or sucrose are shown. However, there is no suggestion or mention about using a mixture of trehalose and sucrose. Furthermore, no investigation has been made on the long-term storage of specific freeze-dried products and storage at a predetermined high temperature condition.
　さらに、非特許文献２では、上記同様の目的からラクトバチルス・サリバリウスの凍結乾燥および保存についての検討が行われている。この文献では、４％トレハロース、４％スクロース、１８％脱脂粉乳、４％トレハロースと１８％脱脂粉乳、４％スクロースと１８％脱脂粉乳、４％トレハロースと４％スクロース、および４％トレハロースと４％スクロースと１８％脱脂粉乳を凍結乾燥保護剤に用い凍結乾燥粉末状菌体を－８５℃で保存し、その生残率を検討している。尚、凍結乾燥時の菌体懸濁液中の糖濃度は約３．２％、脱脂粉乳濃度は１４．４％となる。当該文献に開示された結果によれば、４％スクロースは凍結乾燥粉末状菌体の－８５℃保存性を十分に高めることはできないが、４％スクロースと１８％脱脂粉乳では凍結乾燥粉末状菌体の－８５℃における保存性を高めることができたと報告されている。さらに、４％トレハロース単独で凍結乾燥粉末状菌体の－８５℃保存性を高める結果が報告されている。加えて、４％トレハロースと４％スクロースと１８％脱脂粉乳の混合溶液で凍結乾燥して得た粉末状菌体を室温（温度未記載）で７週間保存した結果が示されている。ここでは特定の湿度条件（湿度２．８～５．６％、水分活性で０．０２８～０．０５６）の環境下で保存すれば、高い保存性が維持されると述べられている。しかし、湿度０％および湿度８．８％の環境下では高い保存性を維持できず（それぞれの生残率は約４０％および１０％）、室温保存可能な条件は極めて低い湿度で、しかも非常に狭い湿度範囲に限られているものであり実用的ではない。このように、非特許文献２の技術は、通常の生活環境から乖離した環境条件での保存試験結果を示しており、さらに高温条件での保存については全く示唆も言及もされておらず、この技術を一般的な食品に応用することは未だ困難なものであった。 Furthermore, in Non-Patent Document 2, studies on lyophilization and storage of Lactobacillus salivarius have been conducted for the same purpose as described above. In this document, 4% trehalose, 4% sucrose, 18% nonfat dry milk, 4% trehalose and 18% nonfat dry milk, 4% sucrose and 18% nonfat dry milk, 4% trehalose and 4% sucrose, and 4% trehalose and 4% Using sucrose and 18% nonfat dry milk as a freeze-drying protective agent, freeze-dried powdered cells are stored at -85 ° C, and the survival rate is examined. In addition, the sugar concentration in the cell suspension at the time of freeze-drying is about 3.2%, and the nonfat dry milk concentration is 14.4%. According to the results disclosed in this document, 4% sucrose cannot sufficiently enhance the storage stability of freeze-dried powdered cells at −85 ° C., but 4% sucrose and 18% nonfat dry milk can freeze-dried powdered fungi. It has been reported that the preservability of the body at -85 ° C could be improved. Furthermore, it has been reported that 4% trehalose alone enhances the storage stability of freeze-dried powdered cells at −85 ° C. In addition, the results are shown in which powdered cells obtained by lyophilization with a mixed solution of 4% trehalose, 4% sucrose and 18% nonfat dry milk were stored at room temperature (temperature not described) for 7 weeks. Here, it is stated that high storage stability is maintained when stored in an environment of specific humidity conditions (humidity 2.8 to 5.6%, water activity 0.028 to 0.056). However, high storage stability cannot be maintained in an environment of 0% humidity and 8.8% humidity (survival rates of about 40% and 10%, respectively), and room temperature storage conditions are extremely low humidity and very high However, it is limited to a narrow humidity range and is not practical. Thus, the technology of Non-Patent Document 2 shows the results of a storage test under environmental conditions that deviate from the normal living environment, and there is no suggestion or reference regarding storage under high temperature conditions. It was still difficult to apply the technology to general food.
特開平５－１８６３３７号公報Japanese Patent Laid-Open No. 5-186337 特開平１１－１３７１７２号公報Japanese Patent Laid-Open No. 11-137172 特開２００１－６４１８９号公報JP 2001-64189 A 特開平７－３１３１４０号公報JP-A-7-313140 特開２００１－３２７２８０号公報JP 2001-327280 A 特開２００５－５２１００号公報JP 2005-52100 A 国際公開ＷＯ２００６／１０６８０６号公報International Publication WO2006 / 106806
　上記のように、これまでも有用菌類の保存に関して、また有効な形態に関しての検討は種々行われてきた。特に、粉末状の凍結乾燥菌体もしくは乾燥菌体などについては、その製品形態の簡便性から様々なアプローチがなされているが、いまだ菌類の機能維持、長期保存後の生残率については検討の余地が大きい。例えば、特定のアプローチが、特定の菌類のみにしか効果がなかったり、あるいは多くの菌類に効果があったとしても保存条件として超低温かつ低湿を必要とするなどの著しい制限があったりするため、工業的には未だ有効な手段は開発されていないと言える。特に、常温で長期保存可能な食品への応用が可能な粉末状菌体として用いるためには、通常の生活環境温度で想定される範囲内における高温（３０～４０℃）条件下で、特殊な保存条件の制限無く、高い生残性を持つことが必要である。このような性質を有しない粉末状菌体は、その品質を保証できず、食品の材料として用い得るものとはなり得ない。 As described above, various studies have been made on the preservation of useful fungi and on the effective form. In particular, for powdered freeze-dried cells or dry cells, various approaches have been made due to the simplicity of the product form, but the maintenance of fungal functions and the survival rate after long-term storage are still being investigated. There is a lot of room. For example, because certain approaches are effective only for certain fungi, or even if they are effective for many fungi, there are significant limitations such as requiring ultra-low temperature and low humidity as storage conditions. It can be said that effective means have not been developed yet. In particular, for use as a powdery microbial cell that can be applied to foods that can be stored at room temperature for a long period of time, it can be used under special conditions at high temperatures (30-40 ° C) within the range expected for normal living environment temperatures. It is necessary to have high survivability without limitation of storage conditions. A powdery cell having no such property cannot guarantee its quality and cannot be used as a food material.
　したがって、本発明の目的は、長期保存に適し、簡便且つ有効な凍結乾燥によって得られる凍結乾燥粉末状菌体、及びその製造方法を提供することである。特に、凍結乾燥後に長期保存をしたとしても、またその長期保存が超低温でなく保存性に影響が大きいと考えられる生活温度帯（特に夏期）で行なわれたとしても、乳酸菌やビフィズス菌などの有用菌の生残率が高い状態で保存することができ、保存後においても簡便に粉末として直接摂取したり、食品製造において原料として有効に活用することができる凍結乾燥粉末状菌体、及びその製造方法を提供するものである。さらには、得られた凍結乾燥粉末状菌体を含有する食品組成物をも提供するものである。 Therefore, an object of the present invention is to provide a lyophilized powdery microbial cell suitable for long-term storage and obtained by simple and effective lyophilization, and a method for producing the same. In particular, lactic acid bacteria and bifidobacteria are useful even if they are stored for a long time after freeze-drying, or even if they are stored in a living temperature range (especially in summer) that is considered to have a significant impact on storage stability, not at very low temperatures. A lyophilized powdery cell that can be stored in a state where the survival rate of the fungus is high, can be directly ingested as a powder after storage, or can be effectively used as a raw material in food production, and its production A method is provided. Furthermore, the food composition containing the obtained freeze-dried powdered microbial cell is also provided.
　上記従来の問題点に鑑み本発明者らは鋭意研究を進めたところ、糖質の溶液に乳酸菌及び／又はビフィズス菌が懸濁している懸濁液を、凍結乾燥させることによって、有用菌（乳酸菌及び／又はビフィズス菌）の粉末状菌体の生残性、特に高温保存時の生残率が著しく向上することが判り、本発明を完成するに至った。 In light of the above-described conventional problems, the present inventors have conducted extensive research. As a result, a suspension in which lactic acid bacteria and / or bifidobacteria are suspended in a saccharide solution is freeze-dried to obtain useful bacteria (lactic acid bacteria). And / or the viability of the powdered cells of Bifidobacteria), in particular, the survival rate during high-temperature storage was remarkably improved, and the present invention was completed.
　本発明によって、長期保存に適し、簡便且つ有効な凍結乾燥によって得られる凍結乾燥粉末状菌体が提供される。 The present invention provides a lyophilized powdery microbial cell suitable for long-term storage and obtained by simple and effective lyophilization.
　即ち、本発明の一態様は、糖質の溶液に乳酸菌及び／又はビフィズス菌が懸濁している懸濁液を凍結乾燥させて、凍結乾燥粉末状菌体を得る凍結乾燥粉末状菌体の製造方法であって、前記糖質がトレハロース及びスクロース（ショ糖）であり、かつ、凍結乾燥前の前記懸濁液中のトレハロース及びスクロースの各々の濃度が４．５重量％以上である凍結乾燥粉末状菌体の製造方法である。 That is, in one embodiment of the present invention, a lyophilized powdery cell is obtained by freeze-drying a suspension in which lactic acid bacteria and / or bifidobacteria are suspended in a saccharide solution to obtain a lyophilized powdered cell. A freeze-dried powder, wherein the carbohydrates are trehalose and sucrose (sucrose), and each concentration of trehalose and sucrose in the suspension before freeze-drying is 4.5% by weight or more This is a method for producing fungal cells.
　本発明において、トレハロース及びスクロースの各々の濃度は、好ましくは４．５～１５重量％、より好ましくは８～１２重量％である。 In the present invention, the concentration of each of trehalose and sucrose is preferably 4.5 to 15% by weight, more preferably 8 to 12% by weight.
　本発明において、トレハロースとスクロースとの重量比は、好ましくは５：１～１：５である。 In the present invention, the weight ratio of trehalose to sucrose is preferably 5: 1 to 1: 5.
　本発明において、前記乳酸菌及び／又はビフィズス菌の好ましい例として、ビフィドバクテリウム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）属に属する菌類などが挙げられる。
　ビフィドバクテリウム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）属に属する菌類の好ましい例として、ビフィドバクテリウム・ビフィダム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ）株、ビフィドバクテリウム・ロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ）株などが挙げられる。
　ビフィドバクテリウム・ビフィダム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ）株の好ましい例として、ビフィドバクテリウム・ビフィダム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ）ＯＬＢ６３７８株などが挙げられる。
　ビフィドバクテリウム・ロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ）株の好ましい例として、ビフィドバクテリウム・ロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ）ＯＬＢ６００１株などが挙げられる。 In the present invention, preferred examples of the lactic acid bacteria and / or bifidobacteria include fungi belonging to the genus Bifidobacterium.
Preferable examples of fungi belonging to the genus Bifidobacterium include Bifidobacterium bifidum strains, Bifidobacterium longum strains, and the like.
Preferable examples of the Bifidobacterium bifidum strain include the Bifidobacterium bifidum OLB6378 strain.
A preferred example of the Bifidobacterium longum strain is a Bifidobacterium longum OLB6001 strain.
　また本発明の別の態様は、上記いずれかに記載の凍結乾燥粉末状菌体の製造方法によって製造された凍結乾燥粉末状菌体である。 Another aspect of the present invention is a freeze-dried powdered cell produced by the method for producing a freeze-dried powdered cell as described above.
　本発明のさらに別の態様は、有効量の上記凍結乾燥粉末状菌体を含んでなる食品組成物である。 Still another embodiment of the present invention is a food composition comprising an effective amount of the above lyophilized powdered microbial cells.
　本発明による長期保存可能な凍結乾燥粉末状菌体の製造方法は、特に固形物・粉末状食品など、長期保存を保証する必要がある場合などに特にその効果が得られる。すなわち、本発明の製造方法により製造された粉凍結乾燥末状菌体は、高温保存時の生残率が著しく向上しているために、保存後に使用しても有用な乳酸菌及び／又はビフィズス菌の効果を得ることができる。
　また、本発明の製造方法は簡便な方法であるために、特別な装置や複雑な工程を必要とせず、それに伴うコスト増加もない。
　また本発明の製造方法により製造された凍結乾燥粉末状菌体は、そのままで使用することができるために、保存後においても簡便に粉末として直接摂取したり、食品製造において原料として有効に活用することができる。 The method for producing a freeze-dried powdered cell that can be stored for a long period of time according to the present invention is particularly effective when it is necessary to guarantee long-term storage, such as a solid or powdered food. That is, the powder freeze-dried powder produced by the production method of the present invention has a significantly improved survival rate during high-temperature storage, so that it is useful even after use after storage, such as lactic acid bacteria and / or bifidobacteria. The effect of can be obtained.
In addition, since the production method of the present invention is a simple method, it does not require any special equipment or complicated steps, and there is no increase in costs associated therewith.
In addition, since the lyophilized powdery cell produced by the production method of the present invention can be used as it is, it can be easily ingested directly after storage or effectively used as a raw material in food production. be able to.
凍結乾燥前の懸濁液中のトレハロース濃度とスクロース濃度がそれぞれ６．６重量％（菌体との混合前の糖溶液として、トレハロースとスクロースをそれぞれ２０重量％含有するものを使用した。）である条件下で調製したビフィドバクテリウム・ビフィダムの凍結乾燥粉末状菌体の、２０℃、３０℃、４０℃の各温度下での保存試験の結果を示すグラフである。The trehalose concentration and the sucrose concentration in the suspension before lyophilization were each 6.6% by weight (a sugar solution containing 20% by weight of trehalose and sucrose was used as a sugar solution before mixing with the cells). It is a graph which shows the result of the storage test in 20 degreeC, 30 degreeC, and each temperature of 40 degreeC of the freeze-dried powdery cell body of Bifidobacterium bifidum prepared on a certain condition. 凍結乾燥前の懸濁液中のトレハロース濃度とスクロース濃度がそれぞれ１０重量％（菌体との混合前の糖溶液として、トレハロースとスクロースをそれぞれ３０重量％含有するものを使用した。）である条件下で調製したビフィドバクテリウム・ビフィダムの凍結乾燥粉末状菌体の、２０℃、３０℃、４０℃の各温度下での保存試験の結果を示すグラフである。The conditions in which the trehalose concentration and the sucrose concentration in the suspension before lyophilization are 10% by weight (a solution containing 30% by weight of trehalose and sucrose as a sugar solution before mixing with the cells) were used. It is a graph which shows the result of the storage test in each temperature of 20 degreeC, 30 degreeC, and 40 degreeC of the freeze-dried powdery cell body of Bifidobacterium bifidum prepared below. 凍結乾燥前の懸濁液中のトレハロース濃度とスクロース濃度がそれぞれ６．７重量％（菌体との混合前の糖溶液として、トレハロースとスクロースをそれぞれ２０重量％含有するものを使用した。）である条件下で調製したビフィドバクテリウム・ロンガムの凍結乾燥粉末状菌体（図中の丸数字２）、及び、比較用の凍結乾燥粉末状菌体（図中の丸数字１）の、４０℃の温度下での保存試験の結果を示すグラフである。The trehalose concentration and sucrose concentration in the suspension before lyophilization were each 6.7% by weight (the sugar solution before mixing with the bacterial cells was one containing 20% by weight of trehalose and sucrose). 40 of the lyophilized powdery cell of Bifidobacterium longum prepared under certain conditions (circle number 2 in the figure) and the lyophilized powdery cell body for comparison (circle number 1 in the figure) It is a graph which shows the result of the storage test under the temperature of ° C.
　以下、本発明を詳細に説明するが、本発明は以下に述べる個々の形態には限定されない。 Hereinafter, the present invention will be described in detail, but the present invention is not limited to the individual forms described below.
　本発明で用いる乳酸菌やビフィズス菌としては、ビフィドバクテリウム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）属に属する菌類が例示でき、例えばビフィドバクテリウム・ロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ）株、ビフィドバクテリウム・インファンチス（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｉｎｆａｎｔｉｓ）株、ビフィドバクテリウム・ブレーベ（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｒｅｖｅ）株、ビフィドバクテリウム・ビフィダム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ）株、ビフィドバクテリウム・アドレッセンティス（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ａｄｏｌｅｓｃｅｎｔｉｓ）株等を具体的に挙げることができる。またさらには、ラクトバシルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ）属、ストレプトコッカス（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ）属に属する菌類が例示でき、例えばラクトバシルス・ガセリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｇａｓｓｅｒｉ）株、ラクトバシルス・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｂｕｌｇａｒｉｃｕｓ）株、ストレプトコッカス・サーモフィラス（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｔｈｅｒｍｏｐｈｉｌｕｓ）株等を具体的に挙げることができる。しかし、本発明はこれらの種に限定されるものではなく、またこれらの菌株については、単独あるいは２以上を組み合わせて使用することができる。 Examples of lactic acid bacteria and bifidobacteria used in the present invention include bacteria belonging to the genus Bifidobacterium, such as Bifidobacterium longum strains, Bifidobacterium infantitis (Bifidobacterium infantitis). ) Strains, Bifidobacterium breve strains, Bifidobacterium bifidum strains, Bifidobacterium adrescentis strains and the like. Still further, fungi belonging to the genus Lactobacillus and Streptococcus can be exemplified, for example, Lactobacillus gasseri strain, Lactobacillus bulgaricus strain, Etc. can be specifically mentioned. However, the present invention is not limited to these species, and these strains can be used alone or in combination of two or more.
　上記の中でも好ましい例として、ビフィドバクテリウム・ビフィダム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ）株、ビフィドバクテリウム・ロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ）株などが挙げられる。
　これら好ましい例の、寄託された菌株の例として、ビフィドバクテリウム・ビフィダム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ）ＯＬＢ６３７８株、ビフィドバクテリウム・ロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ）ＯＬＢ６００１株などが挙げられる。 Among these, preferable examples include a Bifidobacterium bifidum strain and a Bifidobacterium longum strain.
Bifidobacterium bifidum OLB6378 strain, Bifidobacterium longum OLB6001 strain and the like are mentioned as examples of deposited strains of these preferable examples.
（Ａ）ビフィドバクテリウム・ビフィダムＯＬＢ６３７８株の寄託
　本発明で用いるビフィドバクテリウム・ビフィダムＯＬＢ６３７８株は、下記の条件で寄託した。
（１）　寄託機関名：独立行政法人製品評価技術基盤機構　特許微生物寄託センター
（２）　連絡先：〒２９２－０８１８　千葉県木更津市かずさ鎌足２－５－８
　　　　　　　　電話番号０４３８－２０－５５８０
（３）　受託番号：ＮＩＴＥ　ＢＰ－３１
（４）　識別のための表示：Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ　ＯＬＢ６３７８
（５）　原寄託日：　平成１６年（２００４年）１０月２６日
（６）　ブタペスト条約に基づく寄託への移管日：２００６年１月１８日
（Ｂ）ビフィドバクテリウム・ロンガムＯＬＢ６００１株の寄託
　本発明で用いるビフィドバクテリウム・ロンガムＯＬＢ６００１株は、下記の条件で寄託した。
（１）　寄託機関名：独立行政法人産業技術総合研究所
特許生物寄託センター
（２）　連絡先：〒３０５－８５６６　
茨城県つくば市東１－１－１ 中央第６
　　　　　　　　　電話番号０２９－８６１－６０２９
（３）　受託番号：ＦＥＲＭ　Ｐ－１３６１０
（４）　識別のための表示：Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ　Ｎｏ．７
（５）　寄託日： 平成５年（１９９３年）４月２０日
（６）　ブタペスト条約に基づく寄託への移管
　　　　受領日：平成２２年（２０１０年）３月２日
　　　　受領番号：ＦＥＲＭ　ＡＢＰ－１１２４２ (A) Deposit of Bifidobacterium bifidum OLB6378 strain The Bifidobacterium bifidum OLB6378 strain used in the present invention was deposited under the following conditions.
(1) Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-31
(4) Display for identification: Bifidobacterium bifidum OLB6378
(5) Original deposit date: October 26, 2004 (6) Date of transfer to deposit under the Budapest Treaty: January 18, 2006 (B) Deposit of Bifidobacterium longum OLB6001 strain The Bifidobacterium longum OLB6001 strain used in the present invention was deposited under the following conditions.
(1) Depositary institution name: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (2) Contact: 305-8566, Japan
Ibaraki Prefecture Tsukuba City East 1-1-1 Central 6th
Phone number 029-861-6029
(3) Accession number: FERM P-13610
(4) Display for identification: Bifidobacterium longum No. 7
(5) Deposit date: April 20, 1993 (6) Transfer to deposit under the Budapest Treaty Receipt date: March 2, 2010 Receipt number: FERM ABP-11242
　本発明で用いるビフィドバクテリウム・ビフィダムＯＬＢ６３７８株、及び、ビフィドバクテリウム・ロンガムＯＬＢ６００１株は、以下の菌学的性質を有するものである。 The Bifidobacterium bifidum OLB6378 strain and the Bifidobacterium longum OLB6001 strain used in the present invention have the following mycological properties.
　ビフィドバクテリウム・ビフィダムＯＬＢ６３７８株は、ヒト乳幼児糞便由来のグラム陽性偏性嫌気性桿菌である。Ｌａｃｔｏｂａｃｉｌｌｉ　ＭＲＳ　Ｂｒｏｔｈ（ＢＤ）に本菌を接種し、ＡｎａｅｒｏＰａｃｋ・ケンキ（三菱ガス化学社製）の使用による嫌気状態にて３７℃１８時間培養すると、Ｙ字型の菌形態が観察される。また、Ｂｉｆｏｄｏｂａｃｔｅｉｒｕｍ　ｂｉｆｉｄｕｍの特異的プライマー（腸内フローラシンポジウム８、腸内フローラーの分子生物学的検出・同定、光岡知足、松本隆広）、具体的には、１６Ｓ　ｒＤＮＡの種特異的プライマーであるＢｉＢＩＦ－１：ＣＣＡ　ＣＡＴ　ＧＡＴ　ＣＧＣ　ＡＴＧ　ＴＧＡ　ＴＴ、およびＢｉＢＩＦ－２：ＣＣＧ　ＡＡＧ　ＧＣＴ　ＴＧＣ　ＴＣＣ　ＣＡＡ　Ａを用いたＰＣＲでＰＣＲ産物が認められた。
　ビフィドバクテリウム・ロンガムＯＬＢ６００１株は、ヒト成人糞便由来のグラム陽性偏性嫌気性菌であり、菌形状は桿菌または分岐状の多形であり、芽胞の形成、運動性はない。ＢＬ寒天培地（栄研）平板上で本菌を塗布し、スチールウール法にて３７℃４８時間培養すると、不透明な円形半球状の光沢を有するコロニーを形成する。アラビノース、キシロース、リボース、グルコース、フラクトース、ガラクトース、シュークロース、マルトース、ラクトース、メリビオース、ラフィノース、メレチトースに対する発酵性を有する。 Bifidobacterium bifidum OLB6378 is a Gram-positive obligate anaerobe derived from human infant feces. Lactobacilli MRS Broth (BD) is inoculated with this bacterium, and cultured in anaerobic state by using AnaeroPack Kenki (manufactured by Mitsubishi Gas Chemical Company) at 37 ° C. for 18 hours, a Y-shaped microbial form is observed. In addition, a specific primer for Bifidobacterium bifidum (intestinal flora symposium 8, molecular biological detection / identification of intestinal flora, Tomohiro Mitsuoka, Takahiro Matsumoto), specifically, BiBIF-, which is a species-specific primer for 16S rDNA. PCR products were observed by PCR using 1: CCA CAT GAT CGC ATG TGA TT and BiBIF-2: CCG AAG GCT TGC TCC CAA A.
The Bifidobacterium longum OLB6001 strain is a Gram-positive obligate anaerobic bacterium derived from human adult feces, and the shape of the fungus is a gonococcal or branched polymorph and does not have spore formation or motility. When this bacterium is applied on a BL agar medium (Eiken) plate and cultured by a steel wool method at 37 ° C. for 48 hours, colonies having an opaque circular hemispherical gloss are formed. It has fermentability to arabinose, xylose, ribose, glucose, fructose, galactose, sucrose, maltose, lactose, melibiose, raffinose, and meletitose.
　本発明で用いる保護剤としての糖質は、トレハロースとスクロースとの混合物である。凍結乾燥前の懸濁液中のトレハロースとスクロースの各々の濃度は、高温下での長期保存に好適な凍結乾燥粉末状菌体を得る観点から、４．５重量％以上、好ましくは８重量％以上である。該濃度の上限は、濃度を過度に増大させても高温下での長期保存性の効果が同等になる観点から、好ましくは１５重量％、より好ましくは１２重量％である。トレハロースとスクロースとの重量比は、特に限定されないが、高温下での長期保存に好適な凍結乾燥粉末状菌体を得る観点から、好ましくは５：１～１：５、より好ましくは３：１～１：３、特に好ましくは１：１～１：１である。
　これら糖質は、糖質含有水溶液の形態で、培養した菌と混合し、この菌を再懸濁させることが好ましい。こうして得られた懸濁液を凍結乾燥させることによって、本発明の凍結乾燥粉末状菌体を得ることができる。
　なお、糖質含有水溶液中には、水及び糖質に加えて、例えば、乳たん白質、アミノ酸、アスコルビン酸等を含有させてもよい。 The saccharide as a protective agent used in the present invention is a mixture of trehalose and sucrose. The concentration of trehalose and sucrose in the suspension before lyophilization is 4.5% by weight or more, preferably 8% by weight, from the viewpoint of obtaining lyophilized powdery cells suitable for long-term storage at high temperatures. That's it. The upper limit of the concentration is preferably 15% by weight, more preferably 12% by weight from the viewpoint that the effect of long-term storage stability at high temperatures is equivalent even if the concentration is excessively increased. The weight ratio of trehalose to sucrose is not particularly limited, but is preferably 5: 1 to 1: 5, more preferably 3: 1 from the viewpoint of obtaining lyophilized powder cells suitable for long-term storage at high temperatures. ˜1: 3, particularly preferably 1: 1 to 1: 1.
These carbohydrates are preferably mixed with cultured bacteria in the form of a carbohydrate-containing aqueous solution, and the bacteria are resuspended. The lyophilized powdery cell of the present invention can be obtained by freeze-drying the suspension thus obtained.
The saccharide-containing aqueous solution may contain, for example, milk protein, amino acids, ascorbic acid and the like in addition to water and saccharides.
　本発明の凍結乾燥粉末状菌体の製造方法は、特に限定されないが、例えば以下の手順からなる。
１）所望の菌類を常法に従い培養する。
２）培養された菌を含む培養液をそのまま、次の３）で使用するか、あるいは、培養された菌を含む培養液を、遠心分離等により濃縮もしくは固液分離して、濃縮された培養液（菌体液）もしくは固形分として分離した菌体を得て、これを、次の３）で使用する。
３）得られた菌体液または菌体と、所定濃度の糖質（保護剤）を含有する溶液を混合して、懸濁液を得た後、この懸濁液を凍結乾燥させて、本発明の凍結乾燥粉末状菌体を得る。
　凍結乾燥の方法としては、凍結乾燥機を用いる方法等を挙げることができ、例えば、低温（例えば－３０～－９０℃）で急速に予備凍結した後、室温（例えば０～２０℃）かつ減圧下（好ましくは１０００Ｐａ以下、より好ましくは１００Ｐａ以下の真空度）で乾燥させ、真空度をこのまま維持したまま、凍結乾燥機の温度を例えば３０～７０℃に上昇させて、更に乾燥させればよい。 Although the manufacturing method of the lyophilized powdery microbial cell of this invention is not specifically limited, For example, it consists of the following procedures.
1) A desired fungus is cultured according to a conventional method.
2) Use the culture solution containing the cultured bacteria as it is in the next 3), or concentrate or solid-liquid separate the culture solution containing the cultured bacteria by centrifugation or the like. Bacteria separated as a liquid (bacterial cell liquid) or solid content is obtained and used in the following 3).
3) The obtained bacterial cell liquid or bacterial cell and a solution containing a predetermined concentration of sugar (protective agent) are mixed to obtain a suspension, and then the suspension is freeze-dried to obtain the present invention. To obtain a freeze-dried powdered cell.
Examples of the freeze-drying method include a method using a freeze-dryer. For example, after rapid pre-freezing at a low temperature (for example, −30 to −90 ° C.), the temperature is reduced at room temperature (for example, 0 to 20 ° C.). It may be dried under a low pressure (preferably 1000 Pa or less, more preferably 100 Pa or less), and the temperature of the freeze dryer is increased to, for example, 30 to 70 ° C. while maintaining the degree of vacuum. .
　本発明の凍結乾燥粉末状菌体は、食品添加物としても使用しうるトレハロースとスクロースとを使用しているためにそのまま摂取することも可能であり、また様々な食品組成物にこれを添加して用いることも可能である。また、本発明の凍結乾燥粉末状菌体は長期保存後であっても所定の菌生残率を示すため、これを発酵乳などの種菌等の原料として用いることも可能である。 The lyophilized powdery cell of the present invention can be ingested as it is because it uses trehalose and sucrose which can also be used as food additives, and it can be added to various food compositions. Can also be used. In addition, since the freeze-dried powdered cell of the present invention shows a predetermined survival rate even after long-term storage, it can be used as a raw material for inoculum such as fermented milk.
　食品組成物としては各種飲食品（清涼飲料、発酵乳、ヨーグルト、調整粉乳等）が挙げられる。食品組成物はそのまま使用したり、他の食品ないし食品成分と混合するなど、通常の食品組成物における常法にしたがって使用することができる。また、食品組成物の性状は、通常用いられる飲食品の状態、例えば、固体状（粉末、顆粒状その他）、ペースト状、液状ないし懸濁状のいずれでもよい。 Examples of the food composition include various foods and beverages (soft drinks, fermented milk, yogurt, adjusted powdered milk, etc.). The food composition can be used as it is, or can be used according to a conventional method for ordinary food compositions such as mixing with other foods or food ingredients. In addition, the food composition may be in the state of a commonly used food or drink, for example, solid (powder, granule, etc.), paste, liquid or suspension.
　食品組成物中のその他の成分は、特に限定されないが、水、タンパク質、糖質、脂質、ビタミン類、ミネラル類、有機酸、有機塩基、果汁、フレーバー類等が挙げられる。これらの成分は、１種単独で用いてもよいし、あるいは２種以上を組み合わせて使用することもできる。なお、食品組成物中のその他の成分としては、合成品及び／又は合成品を多く含む食品を用いてもよい。 Other components in the food composition are not particularly limited, and examples include water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, and flavors. These components may be used individually by 1 type, or can also be used in combination of 2 or more type. In addition, as another component in a food composition, you may use the foodstuff which contains many synthetic products and / or synthetic products.
　以下、一般的に保存が難しい嫌気性菌（ビフィズス菌）を用いた実施例に基づいて本発明を説明するが、本発明はこれにより限定されるものではない。なお、本明細書において、％表示は、明示しない場合には重量％を示す。 Hereinafter, the present invention will be described based on examples using anaerobic bacteria (Bifidobacteria) that are generally difficult to preserve, but the present invention is not limited thereto. In addition, in this specification,% display shows weight%, when not showing clearly.
［実験例１］
　以下に示す手順に従い、本発明の製造方法に基づき凍結乾燥粉末状菌体を製造した。
１）ビフィドバクテリウム・ビフィダム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ）ＯＬＢ６３７８株をカゼイン分解培地（酵素分解したカゼインをタンパクとする培地）で中和培養を行った。
２）培養液３２０ｍｌを遠心分離（４℃、１００００Ｇで２０分間）して上清３０７．２ｍｌを除去し、菌体ペレット画分（１２．８ｍｌ）を得た。
３）菌体ペレット画分（４ｍｌ）に所定濃度の糖質を含む保護剤液２ｍｌを添加し、菌体を懸濁させ、－８０℃にて凍結して凍結乾燥を行った。
４）凍結乾燥終了直後の凍結乾燥粉末状菌体０．５ｇに生理食塩水を添加して覆水し、覆水液中の生菌数をＢＬ寒天平板培地で測定した。
５）さらに、凍結乾燥粉末状菌体０．５ｇをラミジップ（登録商標；プラスチック製の袋の商品名）に入れて２０℃又は３０℃で５３日間保存後の凍結乾燥粉末状菌体についても同様に生菌数をＢＬ寒天平板培地で用いて測定した。 [Experimental Example 1]
According to the procedure shown below, freeze-dried powdered cells were produced based on the production method of the present invention.
1) The Bifidobacterium bifidum OLB6378 strain was subjected to neutralization culture in a casein decomposition medium (a medium containing enzymatically decomposed casein as a protein).
2) Centrifugation (320 ° C., 10000 G for 20 minutes) of 320 ml of the culture solution was performed to remove 307.2 ml of the supernatant to obtain a cell pellet fraction (12.8 ml).
3) To the cell pellet fraction (4 ml), 2 ml of a protective agent solution containing a predetermined concentration of sugar was added, the cells were suspended, frozen at −80 ° C. and lyophilized.
4) A physiological saline solution was added to 0.5 g of freeze-dried powdered cells immediately after completion of freeze-drying to cover the cells, and the number of viable bacteria in the solution was measured with a BL agar plate medium.
5) Furthermore, the same applies to freeze-dried powdered cells after 0.5 g of lyophilized powdered cells are stored in Lamizip (registered trademark; trade name of plastic bag) at 20 ° C. or 30 ° C. for 53 days. The viable cell count was measured using BL agar plate medium.
　本実験例において用いた保護剤液は以下に示す表１の通りである。 The protective agent liquid used in this experimental example is as shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　以上の本発明による試料１，２及び比較試料１，２，３についてのビフィドバクテリウム・ビフィダムの保存試験の結果を表２に示す。なお、比較試料１，２については生残率が低かったため、２０℃で７日間保存した場合の結果のみを参考として併記する。 Table 2 shows the results of the Bifidobacterium bifidum preservation test for Samples 1 and 2 and Comparative Samples 1, 2 and 3 according to the present invention. In addition, since the survival rate was low about the comparative samples 1 and 2, only the result at the time of preserve | saving for 7 days at 20 degreeC is described together for reference.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２より明らかなように、本発明の方法による実施例１～４では、凍結乾燥粉末状菌体を所定の高温条件下で５３日間保存しても、生菌数の減少が見られなかった。特に、実施例２、４のようにかなり生活環境温度に近い高温下であっても、その生残率を飛躍的に高めることができることが分かった。これにより本発明の製造方法は、品質保証期間が比較的長い固形状・粉末状食品に十分適用しうる技術であることが分かった。 As is clear from Table 2, in Examples 1 to 4 according to the method of the present invention, no decrease in the number of viable cells was observed even when the freeze-dried powdered cells were stored for 53 days under a predetermined high temperature condition. . In particular, it was found that the survival rate can be drastically increased even at a high temperature close to the living environment temperature as in Examples 2 and 4. As a result, it was found that the production method of the present invention is a technique that can be sufficiently applied to solid and powdered foods having a relatively long quality assurance period.
　一方、比較例１、２は非特許文献２に代表されるトレハロースのみの系についての試験であるが、トレハロースのみでは目的を十分に達成することができないことが分かった。特に、非特許文献２では超低温下、湿度条件下での保存時の効果が述べられているが、本実験のような生活環境温度では所望の効果を得ることができないことが分かった。 On the other hand, Comparative Examples 1 and 2 are tests on a trehalose-only system typified by Non-Patent Document 2, but it was found that the objective could not be sufficiently achieved with trehalose alone. In particular, Non-Patent Document 2 describes the effect of storage under ultra-low temperature and humidity conditions, but it has been found that the desired effect cannot be obtained at the living environment temperature as in this experiment.
　また、非特許文献２と同様の濃度のトレハロース・スクロース併用系について比較例３、４において検討を行ったが、生活環境を想定した温度条件下では生菌数は半減してしまうことが分かった。
　したがってこれら比較例の方法によって得られた凍結乾燥粉末状菌体は、品質保証期間が長い固形・粉末状食品に適用することが難しく、これら食品への実用に適する技術ではないことがわかった。 In addition, although the trehalose / sucrose combined system having the same concentration as in Non-Patent Document 2 was studied in Comparative Examples 3 and 4, it was found that the number of viable bacteria was halved under temperature conditions assuming a living environment. .
Therefore, it was found that the freeze-dried powdered cells obtained by the methods of these comparative examples are difficult to apply to solid / powdered foods with a long quality assurance period and are not suitable for practical use in these foods.
［実験例２］
　本発明の製造方法によって得られた凍結乾燥粉末状菌体について、さらに高温条件下（２０℃、３０℃、４０℃）で、長期保存（～６ヶ月）した場合の効果についての実験を行った。
　なお、用いた凍結乾燥粉末状菌体は、仕込み量を約３０倍に増やした以外は、試料１及び２と同じ製造方法に従い製造したものである。従って以下、試料１、２として説明する。
　以上の実験により得られた結果を図１及び図２に示す。 [Experiment 2]
Experiments were conducted on the effects of long-term storage (up to 6 months) of the freeze-dried microbial cells obtained by the production method of the present invention under high temperature conditions (20 ° C, 30 ° C, 40 ° C). .
The freeze-dried powdered cells used were produced according to the same production method as Samples 1 and 2, except that the amount charged was increased about 30 times. Therefore, it demonstrates as the samples 1 and 2 below.
The results obtained by the above experiment are shown in FIGS.
　図から明らかなように、試料１（図１参照。トレハロースを２０重量％含む溶液と、スクロースを２０重量％含む溶液を混合して得られた、トレハロースとスクロースを各６．６重量％ずつ含有する保護剤溶液）、及び、試料２（図２参照。トレハロースを３０重量％含む溶液と、スクロースを３０重量％含む溶液を混合して得られた、トレハロースとスクロースを各１０重量％ずつ含有する保護剤溶液）は共に、２０℃及び３０℃の保存温度条件下であっても６ヶ月後にも十分な生菌数を維持しており、非常に高い生残性を示した。
　かなり極端な生活環境温度条件ではあるが、４０℃保存については、試料１は１ヶ月保存後において９３．９％の生残性を示している。従ってこの条件においては、保護剤溶液の濃度として試料１で用いた濃度がより好ましいことが分かった。 As is apparent from the figure, sample 1 (see FIG. 1), containing 6.6% by weight of each of trehalose and sucrose obtained by mixing a solution containing 20% by weight of trehalose and a solution containing 20% by weight of sucrose. Protectant solution) and sample 2 (see FIG. 2), each containing 10% by weight of trehalose and sucrose obtained by mixing a solution containing 30% by weight trehalose and a solution containing 30% by weight sucrose. Both the protective agent solution) maintained a sufficient viable count even after 6 months even under storage temperature conditions of 20 ° C. and 30 ° C., and showed very high survival.
Although it is a considerably extreme living environment temperature condition, with respect to storage at 40 ° C., the sample 1 shows 93.9% survival after 1 month storage. Therefore, it was found that the concentration used in Sample 1 was more preferable as the concentration of the protective agent solution under these conditions.
［実験例３］
　下記の表３に示す試料を用いかつ４０℃で３ケ月間保存した以外は実施例１と同様にして、実験した。結果を表４に示す。表４から、トレハロースとスクロースの質量比が３：１～１：３の間で生残性が１１～１７％とそれぞれ差はみられず、同等の効果を有していることがわかった。
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
 [Experiment 3]
Experiments were performed in the same manner as in Example 1 except that the samples shown in Table 3 below were used and the samples were stored at 40 ° C. for 3 months. The results are shown in Table 4. From Table 4, it was found that there was no difference between the mass ratio of trehalose and sucrose in the range of 3: 1 to 1: 3 and the survival rate was 11 to 17%.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
［実験例４］
　以下に示す手順に従い、本発明による製造方法に基づき凍結乾燥粉末状菌体を製造した。
１）　ビフィドバクテリウム・ロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ）ＯＬＢ６００１株を、カゼイン分解培地（酵素分解したカゼインをタンパクとする培地）で中和培養した。
２）　培養液５４００ｍｌを遠心分離（４℃、１００００Ｇで２０分間）して上清５２００ｍｌを除去し、菌体ペレット画分（２００ｍｌ）を得た。
３）　菌体ペレット画分（２００ｍｌ）に所定濃度の糖質を含む保護剤液１００ｍｌを添加し、菌体を懸濁させ、－８０℃にて凍結して凍結乾燥を行った。
４）　凍結乾燥終了直後の凍結乾燥粉末状菌体１ｇに生理食塩水を添加して覆水し、覆水液中の生菌数をＢＬ寒天平板培地で測定した。
５）　さらに、凍結乾燥粉末状菌体２ｇをラミジップ（登録商標；プラスチック製の袋の商品名）に入れて４０℃で８日間、３０日間、８２日間、１２４日間の各期間保存後の凍結乾燥粉末状菌体についても同様に、生菌数をＢＬ寒天平板培地で測定した。 [Experimental Example 4]
According to the procedure shown below, freeze-dried powdered cells were produced based on the production method according to the present invention.
1) Bifidobacterium longum OLB6001 strain was neutralized and cultured in a casein decomposition medium (a medium containing enzymatically decomposed casein as a protein).
2) Centrifugation (5 ° C., 10000 G for 20 minutes) of 5400 ml of the culture broth removed 5200 ml of the supernatant to obtain a cell pellet fraction (200 ml).
3) 100 ml of a protective agent solution containing a predetermined concentration of sugar was added to the cell pellet fraction (200 ml), the cells were suspended, frozen at −80 ° C. and freeze-dried.
4) To 1 g of freeze-dried powdered cells immediately after lyophilization, physiological saline was added to cover the cells, and the number of viable bacteria in the water-covered solution was measured using a BL agar plate medium.
5) Furthermore, freeze-dried after 2 g of freeze-dried powdered cells were placed in lamizip (registered trademark; trade name of plastic bag) and stored at 40 ° C. for 8 days, 30 days, 82 days, and 124 days. Similarly, the number of viable cells was measured on a BL agar plate medium for powdered cells.
　本実験例において用いた保護剤液は以下の通りである。
（１）実施例８
　トレハロースとスクロースをそれぞれ２０重量％含有する糖溶液（以下、試料６という。）を用いた。この糖溶液と菌体ペレット画分を混合してなる凍結乾燥前の懸濁液中のトレハロースとスクロースの含有率は、それぞれ６．７重量％であった。
（２）比較例５
　脱脂粉乳６重量％、乳糖１．７重量％、アミノ酸（リジン等）を０．４重量％、その他の成分（デキストリン等）を４重量％含有する溶液（以下、比較試料４という。）を用いた。
　結果を表３及び図３に示す。表５から、比較例５の凍結乾燥粉末状菌体（図３中の丸数字の１）が、４０℃で約４カ月間保存した場合に０．００２％の生残性しか示さないのに対し、実施例８の凍結乾燥粉末状菌体（図３中の丸数字の２）は、４０℃で約４カ月間保存した場合に４０．０％の高い生残性を示すことがわかる。 The protective agent solution used in this experimental example is as follows.
(1) Example 8
A sugar solution (hereinafter referred to as Sample 6) containing 20% by weight of trehalose and sucrose was used. The contents of trehalose and sucrose in the suspension before lyophilization formed by mixing the sugar solution and the cell pellet fraction were 6.7% by weight, respectively.
(2) Comparative Example 5
A solution containing 6% by weight of skim milk powder, 1.7% by weight of lactose, 0.4% by weight of amino acid (lysine, etc.) and 4% by weight of other components (dextrin, etc.) (hereinafter referred to as Comparative Sample 4) is used. It was.
The results are shown in Table 3 and FIG. Table 5 shows that the freeze-dried microbial cell of Comparative Example 5 (circled number 1 in FIG. 3) shows only 0.002% survival when stored at 40 ° C. for about 4 months. On the other hand, it can be seen that the freeze-dried microbial cell of Example 8 (circled number 2 in FIG. 3) shows a high survival rate of 40.0% when stored at 40 ° C. for about 4 months.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　本発明による長期保存可能な凍結乾燥粉末状菌体の製造方法によれば、特に固形物・粉末状食品など、長期保存を保証する必要がある場合などに、保存後にも有用な乳酸菌及び／又はビフィズス菌の効果を得ることができる。また、特別な装置や複雑な工程を必要とせず、それに伴うコスト増加もなく経済的にも有利である。さらに、本発明の凍結乾燥粉末状菌体はそのままで使用することができるために、保存後においても簡便に粉末として直接摂取したり、食品製造において原料として有効に活用することができ、その利用価値は高い。 According to the method for producing lyophilized powdered cells that can be stored for a long period of time according to the present invention, lactic acid bacteria and / or useful after storage, especially when it is necessary to guarantee long-term storage, such as solids and powdered foods The effect of bifidobacteria can be obtained. In addition, no special equipment or complicated processes are required, and there is no cost increase associated therewith, which is economically advantageous. Furthermore, since the lyophilized powdery cell of the present invention can be used as it is, it can be easily ingested directly as a powder even after storage, or can be effectively used as a raw material in food production. Value is high.

Claims (11)

  1. 　糖質の溶液に乳酸菌及び／又はビフィズス菌が懸濁している懸濁液を凍結乾燥させて、凍結乾燥粉末状菌体を得る凍結乾燥粉末状菌体の製造方法であって、前記糖質がトレハロース及びスクロースであり、かつ、凍結乾燥前の前記懸濁液中のトレハロース及びスクロースの各々の濃度が４．５重量％以上である凍結乾燥粉末状菌体の製造方法。 A method for producing a freeze-dried powdered cell by freeze-drying a suspension in which lactic acid bacteria and / or bifidobacteria are suspended in a carbohydrate solution to obtain a freeze-dried powdered cell, wherein the carbohydrate is A method for producing a freeze-dried powdered microbial cell that is trehalose and sucrose, and each of the concentrations of trehalose and sucrose in the suspension before lyophilization is 4.5% by weight or more.
  2. 　凍結乾燥前の前記懸濁液中のトレハロース及びスクロースの各々の濃度が４．５～１５重量％である、請求項１に記載の凍結乾燥粉末状菌体の製造方法。 The method for producing a freeze-dried powdered cell according to claim 1, wherein the concentration of trehalose and sucrose in the suspension before freeze-drying is 4.5 to 15% by weight.
  3. 　凍結乾燥前の前記懸濁液中のトレハロース及びスクロースの各々の濃度が８～１２重量％である、請求項１又は２に記載の凍結乾燥粉末状菌体の製造方法。 The method for producing a freeze-dried powdered cell according to claim 1 or 2, wherein the concentration of trehalose and sucrose in the suspension before lyophilization is 8 to 12% by weight.
  4. 　トレハロースとスクロースとの重量比が５：１～１：５である、請求項１～３のいずれか１項に記載の凍結乾燥粉末状菌体の製造方法。 The method for producing a freeze-dried powdered cell according to any one of claims 1 to 3, wherein the weight ratio of trehalose to sucrose is 5: 1 to 1: 5.
  5. 　前記乳酸菌及び／又はビフィズス菌が、ビフィドバクテリウム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）属に属する菌類である、請求項１～４のいずれか１項に記載の凍結乾燥粉末状菌体の製造方法。 The method for producing a freeze-dried powdered cell according to any one of claims 1 to 4, wherein the lactic acid bacterium and / or bifidobacteria are fungi belonging to the genus Bifidobacterium.
  6. 　前記乳酸菌及び／又はビフィズス菌が、ビフィドバクテリウム・ビフィダム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ）株である、請求項５に記載の凍結乾燥粉末状菌体の製造方法。 The method for producing a freeze-dried powdered microbial cell according to claim 5, wherein the lactic acid bacterium and / or bifidobacteria is a Bifidobacterium bifidum strain.
  7. 　前記乳酸菌及び／又はビフィズス菌が、ビフィドバクテリウム・ビフィダム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ）ＯＬＢ６３７８株である、請求項６に記載の凍結乾燥粉末状菌体の製造方法。 The method for producing a lyophilized powdery microbial cell according to claim 6, wherein the lactic acid bacterium and / or bifidobacteria is Bifidobacterium bifidum OLB6378 strain.
  8. 　前記乳酸菌及び／又はビフィズス菌が、ビフィドバクテリウム・ロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ）株である、請求項５に記載の凍結乾燥粉末状菌体の製造方法。 The method for producing a freeze-dried powdered cell according to claim 5, wherein the lactic acid bacterium and / or bifidobacteria is a Bifidobacterium longum strain.
  9. 　前記乳酸菌及び／又はビフィズス菌が、ビフィドバクテリウム・ロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ）ＯＬＢ６００１株である、請求項８に記載の凍結乾燥粉末状菌体の製造方法。 The method for producing a lyophilized powdery microbial cell according to claim 8, wherein the lactic acid bacterium and / or bifidobacteria is Bifidobacterium longum OLB6001 strain.
  10. 　請求項１～９のいずれか１項に記載の凍結乾燥粉末状菌体の製造方法によって製造された凍結乾燥粉末状菌体。 A freeze-dried powdered cell produced by the method for producing a freeze-dried powdered cell according to any one of claims 1 to 9.
  11. 　有効量の請求項１０に記載の凍結乾燥粉末状菌体を含んでなる食品組成物。 A food composition comprising an effective amount of the lyophilized powdery cell according to claim 10.
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JP2014068545A (en) * 2012-09-27 2014-04-21 Meiji Co Ltd Method for detecting bifidobacterium
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