JPWO2010119956A1 - Lactic acid bacteria survival improver and storage method using the same - Google Patents

Abstract

In order to improve the survival of lactic acid bacteria during refrigeration or freezing storage, a substance exhibiting a significant effect in a small amount is provided. Lactic acid bacteria survival improver containing a specific polysaccharide having a high water retention function, that is, a low molecular weight-treated galactomannan (low molecular weight-treated galactomannan) as an active ingredient, a lactic acid bacteria culture medium or culture On the other hand, by adding so that the final concentration is in the range of 0.01 to 1.0% by weight, the survival of lactic acid bacteria during refrigeration or freezing storage can be improved. In addition, it is possible to provide foods and drinks to which a lactic acid bacteria survival improver containing the low molecular weight-treated galactomannan as an active ingredient is added.

Description

  The present invention relates to an agent for improving the survival of lactic acid bacteria during storage such as refrigeration or frozen storage using galactomannan whose molecular weight is controlled by a 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.

  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.

  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.

  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 noted in his book 1907, “Long Life Expectancy”, that Bulgaria has many long-lived people, and when lactic acid bacteria in Bulgaria were ingested, the amount of spoilage substances decreased, which could 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 intestine even when ingested for most of the early developed probiotic products, including yogurt found by Mechnikov Became. Therefore, in recent years, research has been developed with the aim of developing a technique for allowing viable bacteria to reach the intestines and development of a high survival strain.

  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.

  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.

  For example, Patent Document 1 proposes a method of 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.

  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.

  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 salts 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.

  However, the method of improving survival by using the survival improvement strains found by making full use of screening operations 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. Although technology that improves survival by adsorbing and removing acidic substances with resin can achieve the purpose of improving survival, the acid adsorption / removal process is complicated, and in fact it is 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, a high concentration of physiologically active compound is 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.

  Therefore, a substance that improves the survival of lactic acid bacteria, which is highly effective in a small amount, has been desired.

Orla-Jensen, S.M. : The Lactic Acid Bacteria, Host and Son, Copenhagen (1919) Elie Mechnikov “The Theory of Longevity” Dainippon Civilization Association Office, p236 (1912) Honmichi: History of bifidobacteria research, bifidobacteria research, Tomokazu Kitaoka, Japan bifidobacteria center, Tokyo p1 (1994) JP 2003-219861 A JP-A-7-227207 JP-A-10-099018 Japanese Patent Laid-Open No. 11-032724 Japanese Patent Publication No.57-004291 JP-A-6-253734 JP 7-184540 A JP-A-8-038044 JP-A-11-113484 JP 2007-097447 A JP 2008-005811 A JP 2008-199905 A

  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 of 5 to 310 kDa and a β-1,4 linked D-mannose unit main chain and α-1,6. A low molecular weight-treated galactomannan having a side chain of bound D-galactose units and having a constituent 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-treated galactomannan can be obtained by subjecting at least one kind of galactomannan selected from different galactomannan groups to a low molecular weight treatment.

  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.

  In this storage method, the storage may be low-temperature storage selected from refrigerated storage and frozen storage.

  Moreover, in this storage method, it is preferable that the addition amount of the low molecular weight treatment galactomannan is 0.01 to 1.0% by weight with respect to the weight of the culture medium or the culture.

  Moreover, the lactic acid bacteria containing food / beverage of this invention is a thing to which the survival improvement agent of lactic acid bacteria 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 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.

  In the storage method of the present invention, when the culture medium / culture or food / beverage products containing lactic acid bacteria are stored, especially refrigerated or frozen, the number of remaining lactic acid bacteria decreases little even after storage for 3 weeks.

  That is, the viability during distribution storage is stable at a high value, and the original purpose of using lactic acid bacteria in a culture 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 a suitable amount of the survival improver for 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. In 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. It is a figure which shows the comparison of lactic-acid-bacteria survival at the time of refrigeration preservation | save in the food / beverage products to which the lactic-acid-bacteria survival improver which applies this invention was added, and the food / beverage which does not apply this invention.

Preferred form for carrying out the invention

  Hereinafter, although the preferable form for implementing this invention is demonstrated in detail, the scope of the present invention is not limited to these forms.

  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.

  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.

  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. .

  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.

  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.

  Galactomannan, which is 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.

  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, and a main chain of β-1,4 linked D-mannose units and α-1,6 linked. 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, and the 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, and the genus Torulaspora can also be used. These can be cultured alone or in combination.

  The fermentation is carried out by a degrading enzyme that yeast inductively produces with galactomannan as a raw material. Degrading enzymes that are inducibly 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 a gene recombination technique. 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 1 mL of the reaction solution, and more preferably 0.001 unit per 1 mL of the 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.

  Yeast and enzyme groups that are used as a catalyst or catalyst for the production of the low molecular weight-treated 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.

  Below, the concrete implementation example is shown in detail about the preservation | save method which improves the survival property of lactic acid bacteria using the survival improvement agent of lactic acid bacteria to which this invention is applied.

(Preparation Example 1)
Candida utilis was inoculated into a liquid medium containing 0.5 wt% 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.

(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. After adding untreated galactomannan and the low molecular weight-treated galactomannan obtained in Example 1 to lactate dehydrogenase so as to have a final concentration of 0.005% by weight, it is 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%.

(Preparation Example 3)
To a 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.

(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 culture solution diluted as appropriate and any solution of pure water, erythritol, lactitol, xylitol, and 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.

  EXAMPLES Examples of the present invention will be shown below and the present invention will be described more specifically. However, the present invention is not limited to these examples.

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 track changes in the number of viable bacteria. The result is shown in FIG.

  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.

(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.

  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.

(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.

  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.

  From the results of Examples 1 to 3, the lactic acid bacteria are stable for a long period of time even when 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.

  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)

  The molecular weight is controlled to 5 to 310 kDa, the main chain of β-1,4-linked D-mannose units and the side chain of α-1,6-linked D-galactose units, and D-mannose and D-galactose The lactic acid bacteria survival improver characterized by including the low molecular weight process galactomannan whose composition ratio is 2.5: 1-15: 1 as an active ingredient.   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.   The storage method according to claim 2, wherein the addition amount of the low molecular weight-treated galactomannan is 0.01 to 1.0% by weight based on the weight of the medium or the culture. 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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