WO2010119956A1 - Lactobacillus survival rate improvement agent and storage method using same - Google Patents
Lactobacillus survival rate improvement agent and storage method using same Download PDFInfo
- Publication number
- WO2010119956A1 WO2010119956A1 PCT/JP2010/056856 JP2010056856W WO2010119956A1 WO 2010119956 A1 WO2010119956 A1 WO 2010119956A1 JP 2010056856 W JP2010056856 W JP 2010056856W WO 2010119956 A1 WO2010119956 A1 WO 2010119956A1
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- WIPO (PCT)
- Prior art keywords
- lactic acid
- acid bacteria
- survival
- galactomannan
- molecular weight
- Prior art date
Links
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C3/00—Preservation of milk or milk preparations
- A23C3/08—Preservation of milk or milk preparations by addition of preservatives
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- 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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof
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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
- C12N1/04—Preserving or maintaining viable microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the present invention relates to an agent for improving the survival of lactic acid bacteria during storage such as refrigeration or freezing storage using galactomannan whose molecular weight is controlled by molecular weight reduction treatment, and a storage method using the same.
- a lactic acid bacterium refers to a bacterium that uses saccharides as a raw material and does not produce a spoilage substance by fermentation and uses lactic acid as a main product, and as described in Non-Patent Document 1, a homolactic lactic acid bacterium that produces only lactic acid as a final product And heterolactic acid bacteria that produce alcohol and acetic acid in parallel.
- lactic acid bacteria are known for industrial use. According to the definition using lactic acid production efficiency as an index, there is an interpretation that the genus Bifidobacterium is excluded from lactic acid bacteria, but since it produces lactic acid, it is generally considered as a lactic acid bacterium in a broad sense.
- Representative bacteria that are often referred to as lactic acid bacteria, including the genus include the genus Lactobacillus, the genus Enterococcus, the genus Lactococcus, and the genus Pediococcus.
- Leuconostoc There are six genera of Leuconostoc, and the former three are used for yogurt and intestinal regulating agents. In both cases, it is thought that by producing a large amount of lactic acid by fermentation and changing its own living environment to acidic, it inhibits the growth of other microorganisms and thereby suppresses contamination.
- lactic acid bacteria inhibit the growth of other microorganisms, but are resistant to lactic acid produced by themselves. Low. Therefore, the number of viable bacteria decreases with time in the storage process before ingestion of the product added with lactic acid bacteria. Therefore, by adding an excessive amount of lactic acid bacteria at the time of production, it is possible to secure a sufficient number of living lactic acid bacteria at the expiration date and surpass the decrease in the number of living bacteria.
- Patent Document 1 proposes a method for finding a strain of the genus Lactobacillus having a low killing rate even in a highly acidic environment at the end of the culture by a screening operation and improving survival.
- Patent Document 2 describes the survival of Bifidobacterium sp. By culturing propionic acid or naphthoquinone ring compounds that promote the growth of Bifidobacterium sp. A method for improving the performance has been proposed. Further, in Patent Document 3, when Lactobacillus helveticus is used, co-fermentation with Lactobacillus acidophilus suppresses an increase in lactic acid acidity during storage and improves survival. A method has been proposed.
- Patent Document 4 a food or drink containing Bifidobacterium is treated with a specific resin to selectively remove an acid that inhibits the survival of Bifidobacterium, Methods for improving the survival of bacteria belonging to the genus Bacteria have been proposed.
- Patent Documents 5 to 12 propose methods for improving survival by adding a physiologically active compound to a medium or culture of lactic acid bacteria.
- a physiologically active compound include sorbitol, bovine lactoferrin, bovine apolactoferrin, bovine lactoferrin iron, malic acid or a salt thereof, lactitol, phospholipid, dead lactic acid bacteria, manganese, etc.
- the method of improving the survival using the improved survival strain found by making full use of the screening operation sacrifices versatility in exchange for high specificity, and is widely used in industrial applications.
- the desired lactic acid bacteria intended by the practitioner cannot be survived.
- Techniques that secrete growth-promoting substances of the target lactic acid bacteria by co-fermentation or suppress the production of survival inhibitory substances are also effective for improving survival, but have high specificity for the combination of strains, Lactic acid bacteria intended by the practitioner cannot survive.
- Technology that improves survival by adsorbing / removing acidic substances with resin can achieve the purpose of improving survival, but the acid adsorption / removal process is complicated, and it is actually a high-value-added product.
- the application mode is extremely limited because it is not applicable to solid lactic acid bacteria foods.
- the method of improving the survival by adding a physiologically active compound to the culture medium or culture of lactic acid bacteria has proven its effectiveness, although there are unclear parts in its mechanism of action. While having versatility and simplicity, high concentrations of physiologically active compounds are required to improve the survival of lactic acid bacteria depending on the type of the compound. In this case, the food and drink containing the lactic acid bacteria has an influence on the taste.
- Orla-Jensen, S.M. The Lactic Acid Bacteria, Host and Son, Copenhagen (1919) Sir Erie Metnikov, “The Longevity of Life”, University of Japan Civilization Association Office, p236 (1912) Honmichi: History of bifidobacteria research, bifidobacteria research, Tomokazu Kitaoka, Japan bifidobacteria center, Tokyo p1 (1994) Japanese Patent Laid-Open No. 2003-219861 JP 7-227207 A Japanese Patent Laid-Open No. 10-099018 Japanese Patent Laid-Open No.
- the present invention has been made to solve the above-described problems, and is prepared from a medium or culture by adding an appropriate physiologically active compound to a medium or culture of lactic acid bacteria in a trace amount and at an optimum concentration.
- An object of the present invention is to provide an agent for improving the survival of lactic acid bacteria in storage of food and drink, and a method using the same.
- the lactic acid bacteria survival improver of the present invention which has been made to achieve the above object, has a molecular weight controlled to 5 to 310 kDa, a main chain of D-mannose units bonded with ⁇ -1,4, and ⁇ -1,6.
- a low molecular weight-treated galactomannan having a side chain of bound D-galactose units and having a composition ratio of D-mannose to D-galactose of 2.5: 1 to 15: 1 is contained as an active ingredient.
- Such a low molecular weight treatment galactomannan can be obtained by subjecting at least one kind of galactomannan selected from a group of different galactomannans to a low molecular weight treatment.
- the lactic acid bacteria survival improver is added to the culture medium or culture of the lactic acid bacteria, so that the culture medium or the culture and the food and drink containing it This improves the survival of the lactic acid bacteria during storage.
- the storage may be low-temperature storage selected from refrigerated storage and frozen storage.
- the addition amount of the low molecular weight-treated galactomannan is 0.01 to 1.0% by weight with respect to the weight of the medium or the culture.
- the lactic acid bacterium-containing food or drink of the present invention is a product to which a lactic acid bacterium survival improver is added.
- the lactic acid bacteria survival improver of the present invention and the preservation method for improving the survival of lactic acid bacteria using the same will be described in detail later, but the lactic acid bacteria containing a low molecular weight-treated galactomannan as an active ingredient
- a survival improver to the medium or culture of lactic acid bacteria, it is possible to improve the survival of lactic acid bacteria during storage such as distribution and storage of the culture medium / culture and foods and drinks produced therefrom. Has the function and effect.
- the storage method of the present invention when the culture medium / culture product or food and drink containing lactic acid bacteria are stored, especially refrigerated or frozen, the decrease in the number of surviving lactic acid bacteria is small even after storage for 3 weeks.
- the survivability during distribution storage is stable at a high value, and the original purpose of using lactic acid bacteria in a medium / culture or food / drink can be achieved.
- the storage method of the present invention can be carried out simply, inexpensively and safely by using an appropriate amount of the survival improver of lactic acid bacteria of the present invention in a small amount in a medium / culture or food / drink.
- a storage method for improving the survival of lactic acid bacteria using a lactic acid bacteria survival improver to which the present invention is applied and a method for storing lactic acid bacteria by the number of times of freezing when the lactic acid bacteria culture medium is frozen and stored in a storage method not applying the present invention It is a figure which shows the comparison of residual property.
- another storage method for improving the viability of lactic acid bacteria using the lactic acid bacteria survival improver to which the present invention is applied and in the refrigerated storage time when the lactic acid bacteria culture medium is refrigerated and stored in a storage method not applying the present invention. It is a figure which shows the comparison of lactic acid bacteria survivability of.
- the preservation method for improving the survival of lactic acid bacteria using the lactic acid bacteria survival improver of the present invention is a specific polysaccharide having a high water retention function as an active ingredient, that is, a low molecular weight reduction. Addition of lactic acid bacteria survival improver containing treated galactomannan (lower molecular weight-treated galactomannan) to 0.01 to 1.0% by weight with respect to the medium or culture of lactic acid bacteria To solve this problem.
- the food and drink to which the survival improver of lactic acid bacteria of the present invention is added solves the above technical problem by using a galactomannan subjected to a low molecular weight treatment as an effective and essential component. is there.
- the addition amount of the galactomannan subjected to the low molecular weight treatment is less than 0.01% by weight relative to the medium or culture of the lactic acid bacteria, and the survival of the lactic acid bacteria is almost the same as in the non-added group, The addition effect of the said galactomannan is hardly seen.
- the addition amount exceeds 1.0% by weight the survival of lactic acid bacteria loses the dependency on the addition amount and almost reaches a plateau, so that the survival improvement effect according to the addition amount cannot be obtained. .
- the medium used in the present invention includes milk such as MRS medium and Briggsliver broth medium in addition to a medium obtained by adding a growth promoting substance to whole milk, skim milk, or reduced milk from these milk powders. It is also possible to apply a medium that does not contain. In addition, fermentation in these culture media is performed under aerobic conditions.
- the obtained culture may be used for food as it is as a food containing lactic acid bacteria, or may be used for beverages as dairy lactic acid bacteria fermented dairy products after adjusting the taste and texture.
- Lactic acid bacteria that improve the viability used in the present invention are not particularly limited, and include, for example, the aforementioned Bifidobacterium genus, Lactobacillus genus, Enterococcus genus, Lactococcus genus, Pediococcus genus, Leuconostoc genus, and the like, more specifically Lactobacillus. . casei, L .; acidophilus, L. et al. Helveticus is a preferred example.
- the galactomannan used as a raw material for the low molecular weight treated galactomannan has a main chain of ⁇ -1,4-linked D-mannose units and a side chain of ⁇ -1,6-linked D-galactose units.
- the origin of the galactomannan is not limited to the raw material, i.e., the origin, and need not be highly purified.
- locust bean seed powder is preferable, but as other galactomannan, guar gum, cassia gum, soy bean full derived from soybean seed coat, tamson gum and the like can be mentioned. These are highly safe substances that are used as thickeners in the food industry and as tableting aids in the pharmaceutical industry.
- galactomannans of different origins differ in the composition ratio of mannose units and galactose units, they may be derived from a single source or a galactomannan group consisting of a mixture of different sources as raw materials for the low molecular weight processed galactomannan. It may also be an aqueous solution or suspension. Furthermore, there is no restriction
- the molecular structure obtained after the treatment has a molecular weight of 5 to 310 kDa, a main chain of D-1,4-linked D-mannose units and ⁇ -1,6 bonds. It must have a side chain of D-galactose units so that the constituent ratio of D-mannose and D-galactose is 2.5: 1 to 15: 1.
- a method for adjusting the ratio is as follows: Alternatively, a chemical method using an acid catalyst or the like, or a physical method such as a high-pressure hydrothermal reaction using heat or pressure may be used.
- Microorganisms used for fermentation may be naturally occurring wild strains or mutant strains obtained using genetic recombination techniques, but in terms of fermentation rate, ease of handling, safety, etc.
- Yeast is practical. Many food yeasts can be used in the present invention, but are preferably selected from the genus Candida and the genus Saccharomyces.
- the genus Phaffia the genus Phodotorula, the genus Zygosaccharomyces, the genus Kluyveromyces, the genus Torulaspora, and the like can also be used. These can be cultured alone or in combination.
- the above fermentation is performed by a degrading enzyme that yeast inductively produces with galactomannan as a raw material.
- Degrading enzymes that are inductively produced by yeast contain ⁇ -galactosidase, ⁇ -mannosidase, and ⁇ -mannanase, and the enzyme group catalyzes a low molecular weight reaction of galactomannan as a raw material. Therefore, for production of the low molecular weight-treated galactomannan, not only live yeasts but also the degrading enzyme group, hemicellulase showing substrate specificity for galactomannan and the like can be used.
- the enzyme may be a naturally occurring wild type or a mutant obtained by subcloning, glycosylation or amino acid modification using gene recombination techniques. Further, it may be a fusion protein with other protein or peptide, or an enzyme fragment.
- the amount of ⁇ -galactosidase, ⁇ -mannosidase, and ⁇ -mannanase used is not particularly limited, but is preferably 0.0001 unit or more and 1000 units or less per mL of the reaction solution, and more preferably 0.00 per unit of reaction solution. 001 unit or more and 100 unit or less.
- One unit of ⁇ -galactosidase and ⁇ -mannosidase is defined as the amount of enzyme that produces 1 mmol of p-nitrophenol per minute from a p-nitrophenolated substrate at 37 ° C. and pH 7.5.
- One unit of ⁇ -mannanase is defined as the amount of enzyme showing a reducing power equivalent to 1 mmol of mannose from mannan at 37 ° C. and pH 7.5.
- Yeast and enzyme groups that are used as a catalyst or a catalyst for the production of the low molecular weight treatment galactomannan can be subjected to a low molecular weight treatment in a free form or an immobilized form.
- the immobilized form refers to cross-linking to a support and inclusion / encapsulation in a semipermeable membrane.
- Candida utilis was inoculated in a liquid medium containing 0.5% by weight locust bean seed powder as a raw material galactomannan, ammonium sulfate and a trace amount of inorganic salts as a nitrogen source, and liquid culture was performed at 30 ° C. for 24 hours. After completion of the culture, the supernatant was concentrated and dialyzed to obtain a crude galactomannan extract with a reduced molecular weight. This was subjected to gel filtration chromatography to obtain a fraction having a molecular weight of about 270 kDa.
- Lactic acid dehydrogenase is an enzyme with extremely low resistance to freezing denaturation.
- the residual activity of the enzyme after freezing and thawing is an indicator of the effect of preventing freezing denaturation.
- Untreated galactomannan and the low molecular weight-treated galactomannan obtained in Example 1 were added to lactate dehydrogenase to a final concentration of 0.005% by weight, cooled at a rate of 1 ° C. per minute, ⁇ It was kept frozen at 20 ° C. for 24 hours.
- the residual activity in the untreated galactomannan addition group was 45%, whereas in the low-molecular-weight treated galactomannan addition group, 100% remaining Showed activity.
- the low molecular weight-treated galactomannan was found to have an excellent antifreezing effect at an extremely low concentration of 0.005%.
- Preparation Example 2 Dissolve 520 g of whole milk powder and 0.75 g of yeast extract in pure water to make 2.1 L. Dispense 300 mL into triangular Kolben and sterilize in a hot water bath at 95 ° C. for 30 minutes. A whole fat medium was used. Here, in the same whole fat medium, L. 1.0 mL of a culture solution obtained by growing caseiJCM1134 strain to 1.0 ⁇ 10 9 cfu / mL was inoculated and statically cultured at 30 ° C. for 18 hours.
- the appropriate diluted culture solution and a solution of pure water, erythritol, lactitol, xylitol, or low molecular weight-treated galactomannan are 1: 1 was followed, and the time-dependent change in the number of viable bacteria during freezing or refrigerated storage was followed.
- the number of viable bacteria in the storage test is 100 mL by dissolving 1.0 g glucose, 0.5 g milk casein hydrolyzate, 0.3 g yeast extract, 0.3 g malt extract, and 1.0 g agar in pure water. Of these, 10 mL was dispensed into a petri dish and used as a plate medium for viable cell count measurement. In this flat plate medium, 1.0 mL of the liquid medium after storage was seeded and left to stand overnight at 30 ° C., and the formed colonies were counted.
- Example 1 L After culturing casei in the above liquid medium, pure water (D.W.), erythritol (ERT), lactitol (LCT), low molecular weight-treated galactomannan (1.0% 10 9 cfu / mL) (SCLBG). The final concentration of each was set to 0.50%. This was frozen at ⁇ 18 ° C. and then thawed repeatedly at room temperature to monitor changes in the number of viable bacteria. The result is shown in FIG.
- Example 2 After culturing casei in the liquid medium, it was diluted with pure water, erythritol, lactitol, and low molecular weight-treated galactomannan so as to be 2.0 ⁇ 10 10 cfu / mL. At this time, final concentrations of erythritol, lactitol, and xylitol were set to 0.4 mol / L, and final concentrations of low molecular weight-treated galactomannan were set to 0.05 wt%, 0.1 wt%, and 0.5 wt%. . This was followed over 3 weeks for changes in the number of viable bacteria during storage at 4 ° C. The result is shown in FIG.
- Example 3 10 g of commercially available yogurt starter was cultivated in the above liquid medium in 500 g of skim milk sterilized at 80 ° C. for 10 minutes in a water bath. Casei was added and mixed with 5.0 mL of galactomannan having a reduced molecular weight as a lactic acid bacteria survival improver, and dispensed into a cup by 50 mL. The final concentration of the low molecular weight treatment galactomannan at this time was set to 0.20%. After the preparation, it was fermented at 40 ° C. for 5 hours to produce yogurt. As a control, yogurt was prepared with the same composition except that the low molecular weight-treated galactomannan was removed. Changes in the number of viable bacteria during storage of these prepared yogurts during storage at 4 ° C. were followed over 3 weeks. The results are shown in FIG.
- the lactic acid bacteria are stable for a long period of time even if the medium or culture of lactic acid bacteria and foods and drinks containing them are refrigerated and frozen using the lactic acid bacteria survival improver of the present invention. Therefore, it was shown that the effective period of foods and drinks containing lactic acid bacteria can be set as long as several days to several weeks.
- this invention contributes to the improvement of the storage technique in the product group which appealed for the utilization of live lactic acid bacteria, and also contributes to the improvement of the distribution system.
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Abstract
Provided is a substance, minute amounts of which are very effective in improving the survival rate of lactobacillus being stored refrigerated or frozen. The lactobacillus survival rate improvement agent contains, as an active ingredient, a specific strongly moisture-retaining polysaccharide, i.e. galactomannan processed to reduce the molecular weight thereof (low-molecular galactomannan). By adding the lactobacillus survival rate improvement agent to a lactobacillus culture or culture medium such that the final concentration is between 0.01% and 1.0% by weight, the survival rate of lactobacillus being stored refrigerated or frozen can be improved. This also allows the provision of foods and beverages to which a lactobacillus survival rate improvement agent, containing low-molecular galactomannan as an active ingredient, has been added.
Description
本発明は、低分子化処理により分子量が制御されたガラクトマンナンを利用して冷蔵又は冷凍保蔵のような保蔵中における乳酸菌の生残性を向上させる剤、及びそれを用いた保蔵方法に関する。
The present invention relates to an agent for improving the survival of lactic acid bacteria during storage such as refrigeration or freezing storage using galactomannan whose molecular weight is controlled by molecular weight reduction treatment, and a storage method using the same.
乳酸菌は、糖類を原料として、発酵により腐敗物質を生産せずに乳酸を主な生産物とする菌を指しており、非特許文献1に記載のように、乳酸のみを最終産物として作り出すホモ乳酸菌と、アルコールや酢酸なども並行して生産するヘテロ乳酸菌とに分類されている。
A lactic acid bacterium refers to a bacterium that uses saccharides as a raw material and does not produce a spoilage substance by fermentation and uses lactic acid as a main product, and as described in Non-Patent Document 1, a homolactic lactic acid bacterium that produces only lactic acid as a final product And heterolactic acid bacteria that produce alcohol and acetic acid in parallel.
これら乳酸菌が腸内に常在し、ヘテロ乳酸菌の一種であるビフィズス菌が優勢にあると、摂取した栄養成分の吸収が良好に行われ、その機作には乳酸菌生菌が関与しているという報告がある。現在、このビフィズス菌を関与成分とした特定保健用食品が多数認可され、市販されている。
When these lactic acid bacteria exist in the intestine and bifidobacteria, a kind of heterolactic acid bacteria, prevail, the ingested nutrients are absorbed well, and the mechanism involves the live lactic acid bacteria. There is a report. At present, many foods for specified health use containing this bifidobacteria are approved and are commercially available.
工業利用されている乳酸菌として、様々なものが知られている。乳酸の生産効率を指標とする定義によると、ビフィドバクテリウム(Bifidobacterium)属は乳酸菌から外れるという解釈もあるが、乳酸を産生することから、一般に広義の乳酸菌として考えられている。当該属を含め、一般に乳酸菌と呼ばれて利用されることが多い代表的な細菌には、ラクトバシラス(Lactobacillus)属、エンテロコッカス(Enterococcus)属、ラクトコッカス(Lactococcus)属、ペディオコッカス(Pediococcus)属、リューコノストック(Leuconostoc)属の6属が挙げられ、特に前3者は、ヨーグルトや整腸剤に利用されている。いずれも発酵によって多量の乳酸を生産し、自らの生活環境を酸性に変えることで、他の微生物の増殖を阻害しそれによる汚染を抑制しているものと考えられている。
Various lactic acid bacteria are known for industrial use. According to the definition using lactic acid production efficiency as an index, there is an interpretation that the genus Bifidobacterium is excluded from lactic acid bacteria, but since it produces lactic acid, it is generally considered as a lactic acid bacterium in a broad sense. Representative bacteria that are often referred to as lactic acid bacteria, including the genus, include the genus Lactobacillus, the genus Enterococcus, the genus Lactococcus, and the genus Pediococcus. There are six genera of Leuconostoc, and the former three are used for yogurt and intestinal regulating agents. In both cases, it is thought that by producing a large amount of lactic acid by fermentation and changing its own living environment to acidic, it inhibits the growth of other microorganisms and thereby suppresses contamination.
人体に有益な乳酸菌を摂取するという考えは、パスツール研究所に所属していたロシアの科学者であるイリヤ・メチニコフの発案だとされる。メチニコフは1907年出版の著書『不老長寿論』の中で、ブルガリアに長寿者が多いことに着目し、ブルガリアの乳酸菌を摂取させたところ、腐敗物質が減少したので、人の自家中毒を防止できて長寿になると報告した(非特許文献2)。しかしその後の研究で、メチニコフが見出したヨーグルトをはじめ、初期に開発されたほとんどのプロバイオティクス製品については、摂取してもほとんどの乳酸菌が胃で死滅してしまい、腸に到達しないことが明らかになった。そこで、近年、生菌を腸に到達させるための技術や高生残性株の開発を目指して研究が展開されている。
The idea of ingesting lactic acid bacteria beneficial to the human body is said to be the idea of Ilya Mechnikov, a Russian scientist who belonged to the Pasteur Institute. Mechnikov noticed that there are many long-lived people in Bulgaria in his book 1907, published in 1907, and when lactic acid bacteria in Bulgaria were ingested, the amount of spoilage substances decreased, so it was possible to prevent human self-poisoning. Reported longevity (Non-patent Document 2). However, in subsequent studies, it was clear that most lactic acid bacteria were killed in the stomach and did not reach the intestines even when ingested for most early-stage probiotic products, including yogurt found by Mechnikov Became. Therefore, in recent years, research has been conducted with the aim of developing techniques for allowing viable bacteria to reach the intestines and developing high survival strains.
乳酸菌の工業利用には、前述の通り、生菌を腸へ届け、生理機能を発現させることが肝要であるが、乳酸菌は、他の微生物の増殖を阻害する半面、自ら生産する乳酸に対する耐性が低い。そのため、乳酸菌を添加した製品の摂取前の保蔵の過程で、生菌数が経日的に低下してしまう。そこで、製造時に過剰量の乳酸菌を添加することで、賞味期限における乳酸菌の十分な生菌数を確保して、生菌数の低下を凌いでいるのが現状である。
As mentioned above, it is important for industrial use of lactic acid bacteria to deliver viable bacteria to the intestines and to express physiological functions. However, lactic acid bacteria inhibit the growth of other microorganisms, but are resistant to lactic acid produced by themselves. Low. Therefore, the number of viable bacteria decreases with time in the storage process before ingestion of the product added with lactic acid bacteria. Therefore, by adding an excessive amount of lactic acid bacteria at the time of production, it is possible to secure a sufficient number of living lactic acid bacteria at the expiration date and surpass the decrease in the number of living bacteria.
製品の流通・保蔵中における乳酸菌の生残性を向上させる方法に関し、(1)乳酸菌の品種改良、(2)共発酵、(3)生残性を低下させる酸の除去、(4)生残性向上物質の添加について、多くの研究成果に基づく技術開発が成され、提案されている。
Concerning methods for improving the survival of lactic acid bacteria during product distribution and storage, (1) breeding of lactic acid bacteria, (2) co-fermentation, (3) removal of acids that reduce survival, (4) survival Technological development based on many research results has been made and proposed for the addition of property-improving substances.
例えば、特許文献1には、培養終期の高酸性環境下においても死滅速度の低いLactobacillus属の菌株をスクリーニング操作により見出し、生残性の向上を図る方法が提案されている。
For example, Patent Document 1 proposes a method for finding a strain of the genus Lactobacillus having a low killing rate even in a highly acidic environment at the end of the culture by a screening operation and improving survival.
特許文献2には、Bifidobacterium属菌の増殖を促進させるプロピオン酸またはナフトキノン環化合物を代謝産物として菌体外に生産する異種の微生物と共に同一系内にて培養することで、Bifidobacterium属菌の生残性を向上させる方法が提案されている。また、特許文献3には、ラクトバシラス・ヘルベティカス(Lactobacillus helveticus)の利用に際し、ラクトバシラス・アシドフィルス(Lactobacillus acidophilus)と共発酵させることで、保蔵中の乳酸酸性度の上昇を抑え、生残性を向上させる方法が提案されている。
Patent Document 2 describes the survival of Bifidobacterium sp. By culturing propionic acid or naphthoquinone ring compounds that promote the growth of Bifidobacterium sp. A method for improving the performance has been proposed. Further, in Patent Document 3, when Lactobacillus helveticus is used, co-fermentation with Lactobacillus acidophilus suppresses an increase in lactic acid acidity during storage and improves survival. A method has been proposed.
特許文献4には、ビフィドバクテリウム属菌を含有する飲食物を特定樹脂で処理することで、ビフィドバクテリウム属菌の生残性を阻害する酸を選択的に除去して、ビフィドバクテリウム属菌の生残性を向上させる方法が提案されている。
In Patent Document 4, a food or drink containing Bifidobacterium is treated with a specific resin to selectively remove an acid that inhibits the survival of Bifidobacterium, Methods for improving the survival of bacteria belonging to the genus Bacteria have been proposed.
さらに、特許文献5~12には、乳酸菌の培地または培養物に、生理活性化合物を添加することで、生残性を向上させる方法が提案されている。そのような化合物は、ソルビトール、牛ラクトフェリン、牛アポラクトフェリン、牛ラクトフェリン鉄、リンゴ酸またはその塩、ラクチトール、リン脂質、乳酸菌の死菌体、マンガン等多岐に亙るが、いずれの方法も、乳酸菌の問題点、即ち(1)耐酸性が低く、低pH領域における長期間生存が困難であること、(2)栄養要求性が複雑且つ厳密で、純粋な牛乳培地では増殖しないこと、(3)酸素が存在する状態では生育が遅い偏性嫌気性生菌であることのいずれかを解決し、乳酸菌の生残性を改善させた技術である。
Furthermore, Patent Documents 5 to 12 propose methods for improving survival by adding a physiologically active compound to a medium or culture of lactic acid bacteria. Such compounds include sorbitol, bovine lactoferrin, bovine apolactoferrin, bovine lactoferrin iron, malic acid or a salt thereof, lactitol, phospholipid, dead lactic acid bacteria, manganese, etc. Problems: (1) Low acid resistance and difficulty in long-term survival in low pH range, (2) Complex and strict auxotrophy and no growth on pure milk medium, (3) Oxygen This is a technique that solves any of the obligately anaerobic viable bacteria that grow slowly in the presence of lactic acid bacteria and improves the survival of lactic acid bacteria.
しかし、スクリーニング操作を駆使して漸く見出した生残性改良株を用いた生残性を向上させる方法は、高い特異性と引き換えに汎用性が犠牲となっており、工業利用の際に汎用されその実施者が意図する所望の乳酸菌を生残させることができない。共発酵により対象乳酸菌の増殖促進物質を分泌、または生残阻害物質の生成を抑制する技術も生残性を改善させるためには有効であるが、菌株の組合せに特異性が高く、同様に、実施者が意図する乳酸菌を生残させることができない。酸性物質を樹脂により吸着・除去することで生残性の改善を図る技術は、生残性向上させる目的を達成できるものの、酸の吸着・除去工程が複雑で、実際には高付加価値製品にのみ適用可能であること、また、固形の乳酸菌食品には適用不可であることなどから、利用態様が極めて限定される。一方、乳酸菌の培地または培養物に生理活性化合物を添加することで、生残性を向上させる方法は、その作用機作に未解明の部分もあるものの、その有効性も実証されており、高い汎用性と簡便性を備えている反面、その化合物の種類によっては、乳酸菌の生残性を改善するために高い濃度の生理活性化合物を要求される。この場合、当該乳酸菌を含む飲食物に、味質面で影響が出る。
However, the method of improving the survival using the improved survival strain found by making full use of the screening operation sacrifices versatility in exchange for high specificity, and is widely used in industrial applications. The desired lactic acid bacteria intended by the practitioner cannot be survived. Techniques that secrete growth-promoting substances of the target lactic acid bacteria by co-fermentation or suppress the production of survival inhibitory substances are also effective for improving survival, but have high specificity for the combination of strains, Lactic acid bacteria intended by the practitioner cannot survive. Technology that improves survival by adsorbing / removing acidic substances with resin can achieve the purpose of improving survival, but the acid adsorption / removal process is complicated, and it is actually a high-value-added product. The application mode is extremely limited because it is not applicable to solid lactic acid bacteria foods. On the other hand, the method of improving the survival by adding a physiologically active compound to the culture medium or culture of lactic acid bacteria has proven its effectiveness, although there are unclear parts in its mechanism of action. While having versatility and simplicity, high concentrations of physiologically active compounds are required to improve the survival of lactic acid bacteria depending on the type of the compound. In this case, the food and drink containing the lactic acid bacteria has an influence on the taste.
そのため、微量で著効を示す乳酸菌の生残性を向上させる物質が嘱望されていた。
Therefore, a substance that improves the survival of lactic acid bacteria, which is highly effective in a small amount, has been desired.
本発明は、上述の課題を解決するためになされたもので、適切な生理活性化合物を乳酸菌の培地または培養物に、微量かつ最適な濃度で添加することにより、その培地や培養物から調製された飲食物の保蔵における乳酸菌の生残性を向上させる剤、及びそれを用いた方法を提供することを目的とする。
The present invention has been made to solve the above-described problems, and is prepared from a medium or culture by adding an appropriate physiologically active compound to a medium or culture of lactic acid bacteria in a trace amount and at an optimum concentration. An object of the present invention is to provide an agent for improving the survival of lactic acid bacteria in storage of food and drink, and a method using the same.
前記の目的を達成するためになされた本発明の乳酸菌生残性向上剤は、分子量が5~310kDaに制御され、β-1,4結合したD-マンノース単位の主鎖およびα-1,6結合したD-ガラクトース単位の側鎖を有し、D-マンノースとD-ガラクトースとの構成比が2.5:1~15:1である低分子化処理ガラクトマンナンを、有効成分として含むものである。
The lactic acid bacteria survival improver of the present invention, which has been made to achieve the above object, has a molecular weight controlled to 5 to 310 kDa, a main chain of D-mannose units bonded with β-1,4, and α-1,6. A low molecular weight-treated galactomannan having a side chain of bound D-galactose units and having a composition ratio of D-mannose to D-galactose of 2.5: 1 to 15: 1 is contained as an active ingredient.
このような低分子化処理ガラクトマンナンは、由来の異なるガラクトマンナン群から選ばれる少なくとも1種類のガラクトマンナンに、低分子化処理を施して、得ることができる。
Such a low molecular weight treatment galactomannan can be obtained by subjecting at least one kind of galactomannan selected from a group of different galactomannans to a low molecular weight treatment.
本発明の乳酸菌の生残性を向上させる保蔵方法は、乳酸菌生残性向上剤を、乳酸菌の培地又は培養物に添加することによって、前記培地又は前記培養物とそれを含ませた飲食物との何れかの保蔵中における前記乳酸菌の生残性を向上させるというものである。
In the preservation method for improving the survival of lactic acid bacteria according to the present invention, the lactic acid bacteria survival improver is added to the culture medium or culture of the lactic acid bacteria, so that the culture medium or the culture and the food and drink containing it This improves the survival of the lactic acid bacteria during storage.
この保蔵方法は、前記保蔵が、冷蔵保蔵及び冷凍保蔵から選ばれる低温保蔵であってもよい。
In this storage method, the storage may be low-temperature storage selected from refrigerated storage and frozen storage.
また、この保蔵方法は、前記低分子化処理ガラクトマンナンの添加量が、前記培地又は前記培養物の重量に対し、0.01~1.0重量%であることが好ましい。
Further, in this storage method, it is preferable that the addition amount of the low molecular weight-treated galactomannan is 0.01 to 1.0% by weight with respect to the weight of the medium or the culture.
また本発明の乳酸菌含有飲食物は、乳酸菌の生残性向上剤が添加されているものである。
Also, the lactic acid bacterium-containing food or drink of the present invention is a product to which a lactic acid bacterium survival improver is added.
本発明の乳酸菌生残性向上剤、及びそれを用いて、乳酸菌の生残性を向上させる保蔵方法は、より詳細には後述するが、低分子化処理ガラクトマンナンが有効成分として含まれた乳酸菌生残性向上剤を乳酸菌の培地又は培養物へ添加することにより、培地・培養物やそれから製造した飲食品を流通・保管のような保蔵中に、乳酸菌の生残性を向上させるような優れた作用・効果を奏する。
The lactic acid bacteria survival improver of the present invention and the preservation method for improving the survival of lactic acid bacteria using the same will be described in detail later, but the lactic acid bacteria containing a low molecular weight-treated galactomannan as an active ingredient By adding a survival improver to the medium or culture of lactic acid bacteria, it is possible to improve the survival of lactic acid bacteria during storage such as distribution and storage of the culture medium / culture and foods and drinks produced therefrom. Has the function and effect.
本発明の保蔵方法で、乳酸菌が含まれた培地・培養物や飲食品を、保蔵、特に冷蔵または冷凍保蔵した場合、3週間保蔵後でも、乳酸菌の生残数の低下は少ない。
In the storage method of the present invention, when the culture medium / culture product or food and drink containing lactic acid bacteria are stored, especially refrigerated or frozen, the decrease in the number of surviving lactic acid bacteria is small even after storage for 3 weeks.
即ち、流通保蔵中における生残性は高い数値で安定し、培地・培養物や飲食品中で乳酸菌を用いる本来の目的が達成可能となる。
That is, the survivability during distribution storage is stable at a high value, and the original purpose of using lactic acid bacteria in a medium / culture or food / drink can be achieved.
本発明の保蔵方法は、本発明の乳酸菌の生残性向上剤を、培地・培養物や飲食品中、微量で適量用いることによって、簡便かつ安価で、安全に行うことができる。
The storage method of the present invention can be carried out simply, inexpensively and safely by using an appropriate amount of the survival improver of lactic acid bacteria of the present invention in a small amount in a medium / culture or food / drink.
以下、本発明を実施するための好ましい形態について、詳細に説明するが、本発明の範囲はこれらの形態に限定されるものではない。
Hereinafter, preferred modes for carrying out the present invention will be described in detail, but the scope of the present invention is not limited to these modes.
前記の技術課題に対し、本発明の乳酸菌生残性向上剤を用いて、乳酸菌の生残性を向上させる保蔵方法は、有効成分として水分保持機能の高い特定の多糖類、即ち、低分子化処理を施したガラクトマンナン(低分子化処理ガラクトマンナン)を含有する乳酸菌の生残性向上剤を、乳酸菌の培地又は培養物に対して、0.01~1.0重量%となるように添加することで解決するものである。
In response to the above technical problem, the preservation method for improving the survival of lactic acid bacteria using the lactic acid bacteria survival improver of the present invention is a specific polysaccharide having a high water retention function as an active ingredient, that is, a low molecular weight reduction. Addition of lactic acid bacteria survival improver containing treated galactomannan (lower molecular weight-treated galactomannan) to 0.01 to 1.0% by weight with respect to the medium or culture of lactic acid bacteria To solve this problem.
また、本発明の乳酸菌の生残性向上剤が添加された飲食物は、低分子化処理を施されたガラクトマンナンが有効且つ必須な成分とする構成により、前記の技術課題を解決するものである。
In addition, the food and drink to which the survival improver of lactic acid bacteria of the present invention is added solves the above technical problem by using a galactomannan subjected to a low molecular weight treatment as an effective and essential component. is there.
低分子化処理を施されたガラクトマンナンの添加量は、乳酸菌の培地又は培養物に対して、0.01重量%未満では、乳酸菌の生残性は無添加群と比してほぼ同等で、当該ガラクトマンナンの添加効果はほとんど見られない。また、添加量が1.0重量%を超える範囲では、乳酸菌の生残性は、添加量依存性を逸し、ほぼプラトーに達するため、添加量に応じた生残性向上効果を得ることができない。
The addition amount of the galactomannan subjected to the low molecular weight treatment is less than 0.01% by weight relative to the medium or culture of the lactic acid bacteria, and the survival of the lactic acid bacteria is almost the same as in the non-added group, The addition effect of the said galactomannan is hardly seen. In addition, in the range where the addition amount exceeds 1.0% by weight, the survival of lactic acid bacteria loses the dependency on the addition amount and almost reaches a plateau, so that the survival improvement effect according to the addition amount cannot be obtained. .
本発明に用いられる培地には、全乳、脱脂乳、またはこれらの粉乳からの還元乳等に生育促進物質等を添加した培地の他、MRS培地、ブリッグスリバーブロス(Briggsliver broth)培地といった、乳を含まない培地も、適用することが可能である。なお、これら培地における発酵は、好気性条件下にて行われる。
The medium used in the present invention includes milk such as MRS medium and Briggsliver broth medium in addition to a medium obtained by adding a growth promoting substance to whole milk, skim milk, or reduced milk from these milk powders. It is also possible to apply a medium that does not contain. In addition, fermentation in these culture media is performed under aerobic conditions.
得られた培養物は、そのまま乳酸菌を含有食品として食用に供してもよく、また、味質や食感を調整し、酪農乳酸菌の発酵乳製品として飲料に供してもよい。
The obtained culture may be used for food as it is as a food containing lactic acid bacteria, or may be used for beverages as dairy lactic acid bacteria fermented dairy products after adjusting the taste and texture.
本発明に用いられる生残性を向上させる乳酸菌は、特に限定されず、例えば前述したBifidobacterium属、Lactobacillus属、Enterococcus属、Lactococcus属、Pediococcus属、Leuconostoc属等が挙げられ、より具体的にはLactobacillus.casei、L.acidophilus、L.helveticusが好ましい例として挙げられる。
Lactic acid bacteria that improve the viability used in the present invention are not particularly limited, and include, for example, the aforementioned Bifidobacterium genus, Lactobacillus genus, Enterococcus genus, Lactococcus genus, Pediococcus genus, Leuconostoc genus, and the like, more specifically Lactobacillus. . casei, L .; acidophilus, L. et al. Helveticus is a preferred example.
低分子化処理を施すことにより特定の分子構造を有する低分子化処理ガラクトマンナンに、タンパク質の冷凍変性防止効果があることはこれまでの研究で、既に見出されていた(特開2008-143986号公報)。しかし、このような低分子化処理ガラクトマンナンが乳酸菌に対して作用し、その生残性を向上させる効果を有していることは、知られておらず、タンパク質の冷凍変性防止効果とは全く作用効果が相違する優れた乳酸菌生残性向上効果を奏する。
It has already been found in previous studies that a low molecular weight-treated galactomannan having a specific molecular structure by performing a low molecular weight treatment has an effect of preventing freezing and denaturation of proteins (JP 2008-143986 A). Issue gazette). However, it is not known that such a low molecular weight-treated galactomannan acts on lactic acid bacteria and has an effect of improving its survival, and it is completely free from the effect of preventing freezing and denaturation of proteins. There is an excellent effect of improving the survival of lactic acid bacteria with different effects.
低分子化処理ガラクトマンナンの原料となるガラクトマンナンは、β-1,4結合したD-マンノース単位の主鎖およびα-1,6結合したD-ガラクトース単位の側鎖を有しているものであれば、そのガラクトマンナンの由来は、原素材即ち起源が限定されるものではなく、また、高度に精製された状態のものである必要もない。ガラクトマンナンとしては、イナゴ豆の種子粉末が好ましいが、他のガラクトマンナンとして、グアーガム、カシアガム、大豆種皮由来のソイビーンフル、タムソンガムなどが挙げられる。これらは、食品産業においては増粘剤として、また医薬品産業においては製錠補助剤として使用されている安全性の高い物質である。由来の異なるガラクトマンナンはマンノース単位とガラクトース単位の構成比率が異なるが、当該低分子化処理ガラクトマンナンの原料として、単一起源からの由来物でも、異起源からの混合物からなるガラクトマンナン群でもよく、また、水溶液または懸濁液でもよい。さらに、原料となるガラクトマンナンの濃度には、特に制限はない。
The galactomannan used as a raw material for the low molecular weight treated galactomannan has a main chain of β-1,4-linked D-mannose units and a side chain of α-1,6-linked D-galactose units. If present, the origin of the galactomannan is not limited to the raw material, i.e., the origin, and need not be highly purified. As the galactomannan, locust bean seed powder is preferable, but as other galactomannan, guar gum, cassia gum, soy bean full derived from soybean seed coat, tamson gum and the like can be mentioned. These are highly safe substances that are used as thickeners in the food industry and as tableting aids in the pharmaceutical industry. Although galactomannans of different origins differ in the composition ratio of mannose units and galactose units, they may be derived from a single source or a galactomannan group consisting of a mixture of different sources as raw materials for the low molecular weight processed galactomannan. It may also be an aqueous solution or suspension. Furthermore, there is no restriction | limiting in particular in the density | concentration of the galactomannan used as a raw material.
当該ガラクトマンナンの低分子化処理は、処理後得られる分子の構造が、分子量が5~310kDaであって、β-1,4結合したD-マンノース単位の主鎖およびα-1,6結合したD-ガラクトース単位の側鎖を有し、D-マンノースとD-ガラクトースの構成比が2.5:1~15:1となるようにするものでなければならず、その比率を調整する方法は、酸触媒等を用いた化学的手法でもよく、熱や圧力などを利用した高圧水熱反応などの物理的手法でもよい。しかし、反応の選択性や効率性、環境負荷などを考慮した場合、微生物発酵や酵素反応等の生化学的手法により、当該低分子化処理を施すことがより好ましい。発酵に使用される微生物は天然に存在する野生株であっても、遺伝子組換え技術を利用して得られた変異株でもよいが、発酵速度、ハンドリングの良さ、安全性等の面から、食品酵母が実用的である。食品酵母として多くのものが本発明に使用可能であるが、キャンディダ(Candida)属、サッカロミセス(Saccharomyces)属から選択されるのが好ましい。また、ファフィア(Phaffia)属、フォドトルーラ(Phodotorula)属、チゴサッカロミセス(Zygosaccharomyces)属、クルイフェロミセス(Kluyveromyces)属、トルラスポラ(Torulaspora)属なども利用可能である。これらを単独で、または混合して培養することができる。
In the molecular weight reduction treatment of the galactomannan, the molecular structure obtained after the treatment has a molecular weight of 5 to 310 kDa, a main chain of D-1,4-linked D-mannose units and α-1,6 bonds. It must have a side chain of D-galactose units so that the constituent ratio of D-mannose and D-galactose is 2.5: 1 to 15: 1. A method for adjusting the ratio is as follows: Alternatively, a chemical method using an acid catalyst or the like, or a physical method such as a high-pressure hydrothermal reaction using heat or pressure may be used. However, when the selectivity and efficiency of the reaction, the environmental load, and the like are taken into consideration, it is more preferable to perform the molecular weight reduction treatment by a biochemical method such as microbial fermentation or enzymatic reaction. Microorganisms used for fermentation may be naturally occurring wild strains or mutant strains obtained using genetic recombination techniques, but in terms of fermentation rate, ease of handling, safety, etc. Yeast is practical. Many food yeasts can be used in the present invention, but are preferably selected from the genus Candida and the genus Saccharomyces. In addition, the genus Phaffia, the genus Phodotorula, the genus Zygosaccharomyces, the genus Kluyveromyces, the genus Torulaspora, and the like can also be used. These can be cultured alone or in combination.
上記発酵は、原料であるガラクトマンナンにより酵母が誘導的に生産する分解酵素によって行われる。酵母が誘導的に産生する分解酵素には、α-ガラクトシダーゼ、β-マンノシダーゼ、β-マンナナーゼが含有されており、当該酵素群が原料であるガラクトマンナンの低分子化反応を触媒している。従って、当該低分子化処理ガラクトマンナンの製造には、酵母生菌のみならず、当該分解酵素群の他、ガラクトマンナンにも基質特異性を示すヘミセルラーゼ等も利用可能である。酵素は天然に存在する野生型であっても、遺伝子組換え技術を利用してサブクローニング、配糖化やアミノ酸修飾により得られた変異型でもよい。また、他のタンパク質またはペプチドとの融合タンパク質、または酵素断片でもよい。α-ガラクトシダーゼ、β-マンノシダーゼ、β-マンナナーゼの使用量は特に限定されるものではないが、好ましくは反応液1mLあたり0.0001単位以上1000単位以下であり、より好ましくは反応液1mLあたり0.001単位以上100単位以下である。なお、α-ガラクトシダーゼおよびβ-マンノシダーゼの1単位は、37℃、pH7.5において、p-ニトロフェノール化した基質から毎分1mmolのp-ニトロフェノールを生成する酵素量と定義する。また、β-マンナナーゼの1単位は、37℃、pH7.5において、マンナンから毎分1mmolのマンノース相当の還元力を示す酵素量と定義する。
The above fermentation is performed by a degrading enzyme that yeast inductively produces with galactomannan as a raw material. Degrading enzymes that are inductively produced by yeast contain α-galactosidase, β-mannosidase, and β-mannanase, and the enzyme group catalyzes a low molecular weight reaction of galactomannan as a raw material. Therefore, for production of the low molecular weight-treated galactomannan, not only live yeasts but also the degrading enzyme group, hemicellulase showing substrate specificity for galactomannan and the like can be used. The enzyme may be a naturally occurring wild type or a mutant obtained by subcloning, glycosylation or amino acid modification using gene recombination techniques. Further, it may be a fusion protein with other protein or peptide, or an enzyme fragment. The amount of α-galactosidase, β-mannosidase, and β-mannanase used is not particularly limited, but is preferably 0.0001 unit or more and 1000 units or less per mL of the reaction solution, and more preferably 0.00 per unit of reaction solution. 001 unit or more and 100 unit or less. One unit of α-galactosidase and β-mannosidase is defined as the amount of enzyme that produces 1 mmol of p-nitrophenol per minute from a p-nitrophenolated substrate at 37 ° C. and pH 7.5. One unit of β-mannanase is defined as the amount of enzyme showing a reducing power equivalent to 1 mmol of mannose from mannan at 37 ° C. and pH 7.5.
当該低分子化処理ガラクトマンナンの製造に触媒的または触媒として利用される酵母や酵素群は、遊離形態または固定化形態で低分子化処理に供する事が可能である。固定化形態とは支持体への架橋、半透過性膜への包含・封入を指す。
Yeast and enzyme groups that are used as a catalyst or a catalyst for the production of the low molecular weight treatment galactomannan can be subjected to a low molecular weight treatment in a free form or an immobilized form. The immobilized form refers to cross-linking to a support and inclusion / encapsulation in a semipermeable membrane.
以下に、本発明を適用する乳酸菌の生残性向上剤を用いた乳酸菌の生残性を向上させる保蔵方法について、具体的に実施した例を、詳細に示す。
Hereinafter, an example in which the preservation method for improving the survival of lactic acid bacteria using the survival improver of lactic acid bacteria to which the present invention is applied will be described in detail.
(調製実施例1)
原料のガラクトマンナンとして0.5重量%のイナゴ豆の種子粉末、窒素源として硫酸アンモニウムおよび微量の無機塩類を含む液体培地にCandida utilisを植菌し、30℃,24時間液体培養を行った。培養終了後、上清の濃縮・透析を行い、低分子化処理ガラクトマンナン粗抽出液を得た。これをゲルろ過クロマトグラフィーに供し、分子量約270kDaの画分を得た。当該分子量画分の構成単糖の比率および結合様式に関し、β-1,4結合したD-マンノース単位の主鎖およびα-1,6結合したD-ガラクトース単位の側鎖を有し、その構成比が10.5:1であるガラクトマンナンであることが判明した。 (Preparation Example 1)
Candida utilis was inoculated in a liquid medium containing 0.5% by weight locust bean seed powder as a raw material galactomannan, ammonium sulfate and a trace amount of inorganic salts as a nitrogen source, and liquid culture was performed at 30 ° C. for 24 hours. After completion of the culture, the supernatant was concentrated and dialyzed to obtain a crude galactomannan extract with a reduced molecular weight. This was subjected to gel filtration chromatography to obtain a fraction having a molecular weight of about 270 kDa. Concerning the ratio of monosaccharides constituting the molecular weight fraction and the binding mode, it has a main chain of β-1,4-linked D-mannose units and a side chain of α-1,6-linked D-galactose units. It was found to be galactomannan with a ratio of 10.5: 1.
原料のガラクトマンナンとして0.5重量%のイナゴ豆の種子粉末、窒素源として硫酸アンモニウムおよび微量の無機塩類を含む液体培地にCandida utilisを植菌し、30℃,24時間液体培養を行った。培養終了後、上清の濃縮・透析を行い、低分子化処理ガラクトマンナン粗抽出液を得た。これをゲルろ過クロマトグラフィーに供し、分子量約270kDaの画分を得た。当該分子量画分の構成単糖の比率および結合様式に関し、β-1,4結合したD-マンノース単位の主鎖およびα-1,6結合したD-ガラクトース単位の側鎖を有し、その構成比が10.5:1であるガラクトマンナンであることが判明した。 (Preparation Example 1)
Candida utilis was inoculated in a liquid medium containing 0.5% by weight locust bean seed powder as a raw material galactomannan, ammonium sulfate and a trace amount of inorganic salts as a nitrogen source, and liquid culture was performed at 30 ° C. for 24 hours. After completion of the culture, the supernatant was concentrated and dialyzed to obtain a crude galactomannan extract with a reduced molecular weight. This was subjected to gel filtration chromatography to obtain a fraction having a molecular weight of about 270 kDa. Concerning the ratio of monosaccharides constituting the molecular weight fraction and the binding mode, it has a main chain of β-1,4-linked D-mannose units and a side chain of α-1,6-linked D-galactose units. It was found to be galactomannan with a ratio of 10.5: 1.
(調製実施例2)
乳酸脱水素酵素は冷凍変性に対し、著しく耐性の低い酵素であり,換言すれば冷凍解凍後の当該酵素の残存活性は、冷凍変性防止効果の指標となる。未処理のガラクトマンナン及び実施例1で得られた低分子化処理ガラクトマンナンを終濃度0.005重量%となるように乳酸脱水素酵素へ添加後、毎分1℃の割合で冷却し、-20℃で24時間凍結保持した。解凍後、当該乳酸脱水素酵素の残存活性を測定した結果、未処理のガラクトマンナン添加区の残存活性は45%であったのに対し、低分子化処理ガラクトマンナン添加区では,100%の残存活性を示した。当該低分子化処理ガラクトマンナンは、0.005%という極めて低濃度の添加において優れた冷凍変性防止効果を有していることが判明した。 (Preparation Example 2)
Lactic acid dehydrogenase is an enzyme with extremely low resistance to freezing denaturation. In other words, the residual activity of the enzyme after freezing and thawing is an indicator of the effect of preventing freezing denaturation. Untreated galactomannan and the low molecular weight-treated galactomannan obtained in Example 1 were added to lactate dehydrogenase to a final concentration of 0.005% by weight, cooled at a rate of 1 ° C. per minute, − It was kept frozen at 20 ° C. for 24 hours. As a result of measuring the residual activity of the lactate dehydrogenase after thawing, the residual activity in the untreated galactomannan addition group was 45%, whereas in the low-molecular-weight treated galactomannan addition group, 100% remaining Showed activity. The low molecular weight-treated galactomannan was found to have an excellent antifreezing effect at an extremely low concentration of 0.005%.
乳酸脱水素酵素は冷凍変性に対し、著しく耐性の低い酵素であり,換言すれば冷凍解凍後の当該酵素の残存活性は、冷凍変性防止効果の指標となる。未処理のガラクトマンナン及び実施例1で得られた低分子化処理ガラクトマンナンを終濃度0.005重量%となるように乳酸脱水素酵素へ添加後、毎分1℃の割合で冷却し、-20℃で24時間凍結保持した。解凍後、当該乳酸脱水素酵素の残存活性を測定した結果、未処理のガラクトマンナン添加区の残存活性は45%であったのに対し、低分子化処理ガラクトマンナン添加区では,100%の残存活性を示した。当該低分子化処理ガラクトマンナンは、0.005%という極めて低濃度の添加において優れた冷凍変性防止効果を有していることが判明した。 (Preparation Example 2)
Lactic acid dehydrogenase is an enzyme with extremely low resistance to freezing denaturation. In other words, the residual activity of the enzyme after freezing and thawing is an indicator of the effect of preventing freezing denaturation. Untreated galactomannan and the low molecular weight-treated galactomannan obtained in Example 1 were added to lactate dehydrogenase to a final concentration of 0.005% by weight, cooled at a rate of 1 ° C. per minute, − It was kept frozen at 20 ° C. for 24 hours. As a result of measuring the residual activity of the lactate dehydrogenase after thawing, the residual activity in the untreated galactomannan addition group was 45%, whereas in the low-molecular-weight treated galactomannan addition group, 100% remaining Showed activity. The low molecular weight-treated galactomannan was found to have an excellent antifreezing effect at an extremely low concentration of 0.005%.
(調製実施例3)
0.5重量%のイナゴ豆の種子粉末溶液に,α-ガラクトシダーゼ(Aspergillus niger由来)30U/mL,β-マンノシダーゼ(Aspergillus oryzae由来)10U/mL、β-マンナナーゼ(Bacillus subtilis由来)10U/mLを40℃にて保温しながら、48時間作用させた。なお、反応系は20mM MOPS緩衝液(pH7.5)にて緩衝化させた。反応終了後、常法により脱塩・脱タンパク質を行い、ゲルろ過クロマトグラフィーに供し、実施例1と同様の分子量および主鎖と側鎖との構成比を有するガラクトマンナンを得た。得られたガラクトマンナンには実施例2と同様の高い冷凍変性防止効果が確認できた。 (Preparation Example 3)
To 0.5% by weight locust bean seed powder solution, α-galactosidase (derived from Aspergillus niger) 30 U / mL, β-mannosidase (derived from Aspergillus oryzae) 10 U / mL, β-mannanase (derived from Bacillus subtilis) 10 U / mL The mixture was allowed to act for 48 hours while being kept at 40 ° C. The reaction system was buffered with 20 mM MOPS buffer (pH 7.5). After completion of the reaction, desalting and deproteinization were performed by a conventional method and subjected to gel filtration chromatography to obtain a galactomannan having the same molecular weight as in Example 1 and a constitutional ratio between the main chain and the side chain. The obtained galactomannan was confirmed to have the same effect of preventing freezing denaturation as in Example 2.
0.5重量%のイナゴ豆の種子粉末溶液に,α-ガラクトシダーゼ(Aspergillus niger由来)30U/mL,β-マンノシダーゼ(Aspergillus oryzae由来)10U/mL、β-マンナナーゼ(Bacillus subtilis由来)10U/mLを40℃にて保温しながら、48時間作用させた。なお、反応系は20mM MOPS緩衝液(pH7.5)にて緩衝化させた。反応終了後、常法により脱塩・脱タンパク質を行い、ゲルろ過クロマトグラフィーに供し、実施例1と同様の分子量および主鎖と側鎖との構成比を有するガラクトマンナンを得た。得られたガラクトマンナンには実施例2と同様の高い冷凍変性防止効果が確認できた。 (Preparation Example 3)
To 0.5% by weight locust bean seed powder solution, α-galactosidase (derived from Aspergillus niger) 30 U / mL, β-mannosidase (derived from Aspergillus oryzae) 10 U / mL, β-mannanase (derived from Bacillus subtilis) 10 U / mL The mixture was allowed to act for 48 hours while being kept at 40 ° C. The reaction system was buffered with 20 mM MOPS buffer (pH 7.5). After completion of the reaction, desalting and deproteinization were performed by a conventional method and subjected to gel filtration chromatography to obtain a galactomannan having the same molecular weight as in Example 1 and a constitutional ratio between the main chain and the side chain. The obtained galactomannan was confirmed to have the same effect of preventing freezing denaturation as in Example 2.
(調製実施例2)
全脂乳粉520g、酵母抽出物0.75gを純水にて溶解して、2.1Lとしたもののうち、300mLを三角コルベンに分注し、湯せんにて95℃、30分間滅菌したものを全脂培地とした。ここに同全脂培地にて事前に、L.caseiJCM1134株を1.0×109cfu/mLにまで増殖させた培養液を1.0mL植菌し、30℃にて18時間静置培養した。菌体数が4.0×1010cfu/mLとなるまで培養後、適宜希釈済みの当該培養液及び純水、エリスリトール、ラクチトール、キシリトール、低分子化処理ガラクトマンナンの何れかの溶液を1:1で混合し、冷凍又は冷蔵保蔵中における生菌数の経時変化を追跡した。 (Preparation Example 2)
Dissolve 520 g of whole milk powder and 0.75 g of yeast extract in pure water to make 2.1 L. Dispense 300 mL into triangular Kolben and sterilize in a hot water bath at 95 ° C. for 30 minutes. A whole fat medium was used. Here, in the same whole fat medium, L. 1.0 mL of a culture solution obtained by growing caseiJCM1134 strain to 1.0 × 10 9 cfu / mL was inoculated and statically cultured at 30 ° C. for 18 hours. After culturing until the number of cells reaches 4.0 × 10 10 cfu / mL, the appropriate diluted culture solution and a solution of pure water, erythritol, lactitol, xylitol, or low molecular weight-treated galactomannan are 1: 1 was followed, and the time-dependent change in the number of viable bacteria during freezing or refrigerated storage was followed.
全脂乳粉520g、酵母抽出物0.75gを純水にて溶解して、2.1Lとしたもののうち、300mLを三角コルベンに分注し、湯せんにて95℃、30分間滅菌したものを全脂培地とした。ここに同全脂培地にて事前に、L.caseiJCM1134株を1.0×109cfu/mLにまで増殖させた培養液を1.0mL植菌し、30℃にて18時間静置培養した。菌体数が4.0×1010cfu/mLとなるまで培養後、適宜希釈済みの当該培養液及び純水、エリスリトール、ラクチトール、キシリトール、低分子化処理ガラクトマンナンの何れかの溶液を1:1で混合し、冷凍又は冷蔵保蔵中における生菌数の経時変化を追跡した。 (Preparation Example 2)
Dissolve 520 g of whole milk powder and 0.75 g of yeast extract in pure water to make 2.1 L. Dispense 300 mL into triangular Kolben and sterilize in a hot water bath at 95 ° C. for 30 minutes. A whole fat medium was used. Here, in the same whole fat medium, L. 1.0 mL of a culture solution obtained by growing caseiJCM1134 strain to 1.0 × 10 9 cfu / mL was inoculated and statically cultured at 30 ° C. for 18 hours. After culturing until the number of cells reaches 4.0 × 10 10 cfu / mL, the appropriate diluted culture solution and a solution of pure water, erythritol, lactitol, xylitol, or low molecular weight-treated galactomannan are 1: 1 was followed, and the time-dependent change in the number of viable bacteria during freezing or refrigerated storage was followed.
保蔵試験における生菌数は、グルコース1.0g、ミルクカゼイン加水分解物0.5g、酵母抽出物0.3g、麦芽抽出物0.3g、寒天1.0gを純水にて溶解して、100mLとしたもののうち、10mLをシャーレに分注したものを生菌数測定用平板培地とした。本平板培地に保蔵後の液体培地1.0mLを蒔き、30℃にて一昼夜静置培養し、形成されたコロニーをカウントした。
The number of viable bacteria in the storage test is 100 mL by dissolving 1.0 g glucose, 0.5 g milk casein hydrolyzate, 0.3 g yeast extract, 0.3 g malt extract, and 1.0 g agar in pure water. Of these, 10 mL was dispensed into a petri dish and used as a plate medium for viable cell count measurement. In this flat plate medium, 1.0 mL of the liquid medium after storage was seeded and left to stand overnight at 30 ° C., and the formed colonies were counted.
以下に、本発明の実施例を示し、本発明を更に具体的に説明するが、本発明はこれらの実施例に限定されるものではない。
Hereinafter, examples of the present invention will be shown and the present invention will be described more specifically. However, the present invention is not limited to these examples.
(実施例1)
L.caseiを前記液体培地にて培養後、1.0×109cfu/mLとなるように、純水(D.W.)、エリスリトール(ERT)、ラクチトール(LCT)、低分子化処理ガラクトマンナン(SCLBG)で希釈した。この際のそれぞれの終濃度を0.50%に設定した。これを-18℃にて凍結した後、常温で解凍を繰り返して生菌数の変化を追跡した。その結果を図1に示す。 Example 1
L. After culturing casei in the above liquid medium, pure water (D.W.), erythritol (ERT), lactitol (LCT), low molecular weight-treated galactomannan (1.0% 10 9 cfu / mL) (SCLBG). The final concentration of each was set to 0.50%. This was frozen at −18 ° C. and then thawed repeatedly at room temperature to monitor changes in the number of viable bacteria. The result is shown in FIG.
L.caseiを前記液体培地にて培養後、1.0×109cfu/mLとなるように、純水(D.W.)、エリスリトール(ERT)、ラクチトール(LCT)、低分子化処理ガラクトマンナン(SCLBG)で希釈した。この際のそれぞれの終濃度を0.50%に設定した。これを-18℃にて凍結した後、常温で解凍を繰り返して生菌数の変化を追跡した。その結果を図1に示す。 Example 1
L. After culturing casei in the above liquid medium, pure water (D.W.), erythritol (ERT), lactitol (LCT), low molecular weight-treated galactomannan (1.0% 10 9 cfu / mL) (SCLBG). The final concentration of each was set to 0.50%. This was frozen at −18 ° C. and then thawed repeatedly at room temperature to monitor changes in the number of viable bacteria. The result is shown in FIG.
図1から明らかな通り、低分子化処理ガラクトマンナンを用いた場合、凍結回数が増加しても乳酸菌の生残菌数がほぼ維持されていたが、純水、エリスリトール、ラクチトールを用いた場合、凍結回数の増加に応じて、乳酸菌の生残菌数が減少した。
As is clear from FIG. 1, when the low molecular weight-treated galactomannan was used, the survival number of lactic acid bacteria was almost maintained even when the number of freezing cycles was increased, but when pure water, erythritol, or lactitol was used, As the number of freezes increased, the number of surviving lactic acid bacteria decreased.
(実施例2)
L.caseiを前記液体培地にて培養後、2.0×1010cfu/mLとなるように、純水、エリスリトール、ラクチトール、低分子化処理ガラクトマンナンで希釈した。この際、エリスリトール、ラクチトール、キシリトールの各終濃度を各0.4mol/L、低分子化処理ガラクトマンナンの終濃度を0.05重量%、0.1重量%、0.5重量%に設定した。これを4℃にて保蔵中における生菌数の経時変化を3週間に亙って追跡した。その結果を図2に示す。 (Example 2)
L. After culturing casei in the liquid medium, it was diluted with pure water, erythritol, lactitol, and low molecular weight-treated galactomannan so as to be 2.0 × 10 10 cfu / mL. At this time, final concentrations of erythritol, lactitol, and xylitol were set to 0.4 mol / L, and final concentrations of low molecular weight-treated galactomannan were set to 0.05 wt%, 0.1 wt%, and 0.5 wt%. . This was followed over 3 weeks for changes in the number of viable bacteria during storage at 4 ° C. The result is shown in FIG.
L.caseiを前記液体培地にて培養後、2.0×1010cfu/mLとなるように、純水、エリスリトール、ラクチトール、低分子化処理ガラクトマンナンで希釈した。この際、エリスリトール、ラクチトール、キシリトールの各終濃度を各0.4mol/L、低分子化処理ガラクトマンナンの終濃度を0.05重量%、0.1重量%、0.5重量%に設定した。これを4℃にて保蔵中における生菌数の経時変化を3週間に亙って追跡した。その結果を図2に示す。 (Example 2)
L. After culturing casei in the liquid medium, it was diluted with pure water, erythritol, lactitol, and low molecular weight-treated galactomannan so as to be 2.0 × 10 10 cfu / mL. At this time, final concentrations of erythritol, lactitol, and xylitol were set to 0.4 mol / L, and final concentrations of low molecular weight-treated galactomannan were set to 0.05 wt%, 0.1 wt%, and 0.5 wt%. . This was followed over 3 weeks for changes in the number of viable bacteria during storage at 4 ° C. The result is shown in FIG.
図2から明らかな通り、低分子化処理ガラクトマンナンを用いた場合、冷蔵保存期間が3週間まで乳酸菌の生残菌数がほぼ維持されていたが、純水、エリスリトール、ラクチトールを用いた場合、冷蔵保存日数の増加に応じて、乳酸菌の生残菌数が減少した。
As is clear from FIG. 2, when the low molecular weight-treated galactomannan was used, the survival number of lactic acid bacteria was almost maintained until the refrigerated storage period was 3 weeks, but when pure water, erythritol, lactitol was used, As the refrigerated storage days increased, the number of surviving lactic acid bacteria decreased.
(実施例3)
湯せんにて80℃、10分間滅菌した脱脂乳500gに市販ヨーグルトスターターを10g、前記液体培地にて培養したL.caseiを5.0mLおよび乳酸菌生残性向上剤として低分子化処理ガラクトマンナンを添加混合し、50mLずつカップに分注した。この際の低分子化処理ガラクトマンナンの終濃度を0.20%に設定した。調製後、40℃、5時間発酵させてヨーグルトを製造した。対照として、低分子化処理ガラクトマンナンを除いた以外は同組成にてヨーグルトを調製した。これら調製したヨーグルトの4℃にて保蔵中における生菌数の経時変化を3週間に亙って追跡した。結果を図3に示す。 (Example 3)
10 g of commercially available yogurt starter was cultivated in the above liquid medium in 500 g of skim milk sterilized at 80 ° C. for 10 minutes in a water bath. Casei was added and mixed with 5.0 mL of galactomannan having a reduced molecular weight as a lactic acid bacteria survival improver, and dispensed into a cup by 50 mL. The final concentration of the low molecular weight treatment galactomannan at this time was set to 0.20%. After the preparation, it was fermented at 40 ° C. for 5 hours to produce yogurt. As a control, yogurt was prepared with the same composition except that the low molecular weight-treated galactomannan was removed. Changes in the number of viable bacteria during storage of these prepared yogurts during storage at 4 ° C. were followed over 3 weeks. The results are shown in FIG.
湯せんにて80℃、10分間滅菌した脱脂乳500gに市販ヨーグルトスターターを10g、前記液体培地にて培養したL.caseiを5.0mLおよび乳酸菌生残性向上剤として低分子化処理ガラクトマンナンを添加混合し、50mLずつカップに分注した。この際の低分子化処理ガラクトマンナンの終濃度を0.20%に設定した。調製後、40℃、5時間発酵させてヨーグルトを製造した。対照として、低分子化処理ガラクトマンナンを除いた以外は同組成にてヨーグルトを調製した。これら調製したヨーグルトの4℃にて保蔵中における生菌数の経時変化を3週間に亙って追跡した。結果を図3に示す。 (Example 3)
10 g of commercially available yogurt starter was cultivated in the above liquid medium in 500 g of skim milk sterilized at 80 ° C. for 10 minutes in a water bath. Casei was added and mixed with 5.0 mL of galactomannan having a reduced molecular weight as a lactic acid bacteria survival improver, and dispensed into a cup by 50 mL. The final concentration of the low molecular weight treatment galactomannan at this time was set to 0.20%. After the preparation, it was fermented at 40 ° C. for 5 hours to produce yogurt. As a control, yogurt was prepared with the same composition except that the low molecular weight-treated galactomannan was removed. Changes in the number of viable bacteria during storage of these prepared yogurts during storage at 4 ° C. were followed over 3 weeks. The results are shown in FIG.
図3から明らかな通り、低分子化処理ガラクトマンナンを用いた場合、冷蔵保存期間が3週間まで乳酸菌の生残菌数がほぼ維持されていたが、純水を用いた場合、冷蔵保存日数の増加に応じて、乳酸菌の生残菌数が減少した。
As is clear from FIG. 3, when the low molecular weight-treated galactomannan was used, the survival number of lactic acid bacteria was almost maintained until the refrigerated storage period was 3 weeks, but when pure water was used, The number of surviving bacteria of lactic acid bacteria decreased with the increase.
実施例1~3の結果から、本発明の乳酸菌の生残性向上剤を用いて、乳酸菌の培地・培養物やそれを含む飲食物を冷蔵・冷凍保蔵しても、乳酸菌は、長期間安定して生存するので、乳酸菌含有飲食物の有効期間を数日~数週間という長期に設定することが可能であることが示された。
From the results of Examples 1 to 3, the lactic acid bacteria are stable for a long period of time even if the medium or culture of lactic acid bacteria and foods and drinks containing them are refrigerated and frozen using the lactic acid bacteria survival improver of the present invention. Therefore, it was shown that the effective period of foods and drinks containing lactic acid bacteria can be set as long as several days to several weeks.
本発明の乳酸菌の生残性向上剤を用いた保蔵方法により、味質に影響を与えること無く、簡便に、培地・培養物や飲食品の保蔵中の乳酸菌の生残性を向上させることが可能となった。そのため本発明は、乳酸菌の生菌利用を訴求した製品群における保蔵技術の向上に貢献すると共に、物流システムの改善にも資するものである。
By the storage method using the survival improver of lactic acid bacteria of the present invention, it is possible to easily improve the survival of lactic acid bacteria during storage of culture media / cultures and foods and drinks without affecting the taste quality. It has become possible. Therefore, this invention contributes to the improvement of the storage technique in the product group which appealed for the utilization of live lactic acid bacteria, and also contributes to the improvement of the distribution system.
Claims (4)
- 分子量が5~310kDaに制御され、β-1,4結合したD-マンノース単位の主鎖およびα-1,6結合したD-ガラクトース単位の側鎖を有し、D-マンノースとD-ガラクトースとの構成比が2.5:1~15:1である低分子化処理ガラクトマンナンを、有効成分として含むことを特徴とする乳酸菌生残性向上剤。 Having a molecular weight of 5 to 310 kDa, a main chain of β-1,4 linked D-mannose units and a side chain of α-1,6 linked D-galactose units, and D-mannose and D-galactose A viability improver for lactic acid bacteria, comprising as an active ingredient low molecular weight-treated galactomannan having a composition ratio of 2.5: 1 to 15: 1.
- 請求項1に記載の乳酸菌生残性向上剤を、乳酸菌の培地又は培養物に添加することによって、前記培地又は前記培養物とそれを含ませた飲食物との何れかの保蔵中における前記乳酸菌の生残性を向上させる保蔵方法。 The lactic acid bacterium during storage of either the medium or the culture and a food or drink containing the lactic acid bacterium by adding the lactic acid bacteria survival improver according to claim 1 to the medium or culture of the lactic acid bacterium Storage method to improve the survival of food.
- 前記低分子化処理ガラクトマンナンの添加量が、前記培地又は前記培養物の重量に対し、0.01~1.0重量%であることを特徴とする請求項2に記載の保蔵方法。 The preservation method according to claim 2, wherein the addition amount of the low molecular weight-treated galactomannan is 0.01 to 1.0% by weight with respect to the weight of the medium or the culture.
- 請求項1に記載の乳酸菌生残性向上剤が添加されていることを特徴とする乳酸菌含有飲食物。
A lactic acid bacteria-containing food or drink comprising the lactic acid bacteria survival improver according to claim 1 added thereto.
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WO2005110107A1 (en) * | 2004-05-19 | 2005-11-24 | Morinaga Milk Industry Co., Ltd. | Fermented milk |
JP2008143986A (en) * | 2006-12-07 | 2008-06-26 | Nikken Kasei Kk | Galactomannan undergone low-molecularization treatment, its manufacturing method and its use method |
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WO2005110107A1 (en) * | 2004-05-19 | 2005-11-24 | Morinaga Milk Industry Co., Ltd. | Fermented milk |
JP2008143986A (en) * | 2006-12-07 | 2008-06-26 | Nikken Kasei Kk | Galactomannan undergone low-molecularization treatment, its manufacturing method and its use method |
Non-Patent Citations (1)
Title |
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HIROMI MANABE: "Suiyosei Shokumotsu Sen'i 'San Fiber' no Kino to Yoghurt eno Oyo", NEW FOOD INDUSTRY, vol. 40, no. 10, 1998, pages 27 - 32 * |
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