JP2014045743A - Method for improving survivability of microorganism to be used for food - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for creating a strain of high survivability by simply and efficiently improving resistance to oxygen stress of a microorganism, especially, of a lactobacillus.SOLUTION: Provided is a strain of a microorganism having improved oxygen-resistance and survivability in the late growth phase, selecting a streptonigrin-resistant strain by culturing a microorganism in a culture medium containing streptonigrin. The microorganism is one usable for food, preferably a probiotic, more preferably, lactobacillus (especially lactobacillus bacteria) or bifidus bacteria.

Description

  The present invention provides a method for producing oxygen-resistant microorganisms, microorganisms having improved survival, and methods for producing oxygen-sensitive microorganisms, particularly microorganisms used in foods such as probiotics. More particularly, the present invention relates to microorganisms used in foods such as probiotics, and more particularly to lactic acid bacteria and bifidobacteria.

  Microorganisms such as lactic acid bacteria and bifidobacteria are widely distributed in nature, including intestinal tracts of humans and animals, and are used for the production of various fermented foods. In recent years, the health effects and probiotics due to the intake of useful microorganisms such as lactic acid bacteria and bifidobacteria have attracted attention. Probiotics are defined as “viable cells that have a useful effect on the host when an appropriate amount is ingested”, and lactic acid bacteria and bifidobacteria are typical probiotics. In probiotics, it is important for living microorganisms to reach the intestinal tract of humans and animals, so that microorganisms used as probiotics must be kept alive in the product. In addition, in the production of fermented foods, lactic acid bacteria and bifidobacteria are often added as starters, but the viable cell count and metabolic activity of the microorganisms used as starters greatly affect the productivity and stability of production. Thus, it is industrially very important to improve the survival of microorganisms used as lactic acid bacteria and other probiotics.

  A plurality of factors are involved in reducing the survival of lactic acid bacteria and bifidobacteria, but it is known that oxygen stress is one of the main factors (Non-Patent Document 1). Oxygen stress means that cells are damaged by oxygen molecules themselves or reactive oxygen species (hydrogen peroxide, superoxide, hydroxy radicals, etc.) generated from oxygen molecules. There is a defense mechanism against. However, among useful lactic acid bacteria, there are bacteria that are weak to oxygen and sensitive to oxygen. In particular, many lactic acid bacteria used as probiotics have been isolated from an anaerobic environment such as the intestinal tract, and many are sensitive to oxygen. Bifidobacteria are obligately anaerobic bacteria, and the survival loss due to oxygen stress is more problematic than lactic acid bacteria.

  Up to now, several methods for improving survival, including reduction of oxygen stress, have been studied. For example, a peroxidase mix for decomposing peroxide (Patent Document 1), a method of adding live or dead cells of lactic acid bacteria with high protective effect (Patent Document 2, Patent Document 3), and oxygen permeation The use of low-container containers has been proposed. However, the use of additives leads to an increase in production cost and there is a concern about the influence on taste and flavor. On the other hand, since a container with low oxygen permeability once loses its effect and problems such as an increase in container cost, a fundamental problem has not been solved.

For the reasons described above, in order to solve the problem of survival, it is most desirable to improve the survival of the target microorganism itself or to select a strain having high survival. Bifidobacterium longum FERM BP-778 is an example of the isolation of a highly viable strain from nature. Compared with the same type of Bifidobacterium, fermentability of milk, in fermented milk It is reported that all of the survival properties are high (Patent Document 4). However, it takes a lot of time and labor to select such a highly viable strain from nature. Moreover, the obtained highly viable strain does not always have useful properties as probiotics. For this reason, it has been studied to produce mutant strains with improved survival from existing useful strains. So far, mutant strains with improved survival have been produced with Lactobacillus gasseri for lactic acid bacteria and Bifidobacterium bifidum for bifidobacteria (Patent Documents 5 and 6). In each case, the cultured bacteria are stored at a low temperature, and strains with improved survival are selected from the surviving strains. However, it takes a lot of time and labor to acquire a strain with improved survival by such a method. In the invention described in Patent Document 6, survivability is improved by repeating the work of culturing the surviving strain again when Bifidobacterium bifidum YIT4007 strain is stored at low temperature for 21 days under aerobic conditions and then storing it again at low temperature several times. Have acquired shares. On the other hand, in the invention described in Patent Document 5, by introducing mutation into DNA polymerase III of Lactobacillus gasseri OLL2716, which is the parent strain, a strain having increased DNA mutation efficiency was first created, and the viability was improved using that strain. The strains that have been screened are screened. In addition, it has been reported that the improvement in survival is due to the deficiency of the riboflavin transporter, and the Lactobacillus gasseri OLL2716 strain and Lactobacillus delbrüc Lactobacillus delbrueckii subsp. Bulgaricus T-11 strain has been successfully improved. However, there are many restrictions on the use of genetically modified microorganisms for food.

In addition, in the lactic acid bacterium Lactococcus lactis , a hydrogen peroxide resistant strain has been reported as a reactive oxygen resistant strain (Non-patent Document 2). However, in this method, since hydrogen peroxide itself is an unstable substance, it is difficult to obtain a resistant strain. Moreover, no strains with improved survival were obtained with other types of lactic acid bacteria.

  As described above, the production of oxygen stress-resistant strains from existing useful strains is an effective means, but it takes a lot of time and labor to produce strains with improved survival, excluding genetic engineering techniques. Needed.

JP-A-10-262550 JP 2009-232716 A JP 2008-5811 A Japanese Patent No. 3875237 JP 2011-135804 A Japanese Patent No. 4881304

Yuji Yamamoto, “Factors Affecting Behavior and Survivability of Microorganisms Used as Probiotics at Low Temperature Storage”, Journal of the Japan Society of Refrigerating and Air Conditioning, Japan Society of Refrigerating and Air Conditioning, April 15, 2011, No. 1 86, p. 317-322 Rochat T. et al., Appl. Envioron. Microbiol., 2005, 71, p. 2782-2788

  As described above, it takes a lot of time and labor to produce an oxygen stress resistant strain. In addition, when gene recombination technology is used, there are many restrictions on the use of the resulting microorganisms in food. An object of the present invention is to provide a method for producing a mutant strain having high survival by improving the oxygen stress resistance of microorganisms easily and efficiently.

  In order to solve the above-mentioned problems, the present inventors have intensively studied on a method for producing a strain having improved oxygen stress resistance in microorganisms having improved oxygen stress resistance, particularly lactic acid bacteria and bifidobacteria used as probiotics. Went. In oxygen stress, active oxygen generated from oxygen rather than oxygen molecules themselves often damage cells and cause fatal damage. In view of this, the present inventors have focused on selecting mutants that are resistant to active oxygen in order to produce strains with improved oxygen stress resistance. And, by using streptonigrin (Streptonigrin) as an active oxygen generator, by selecting a strain resistant to this, it has been found that oxygen stress resistance performance is improved, and a strain with high survival can be easily obtained. The present invention has been completed.

That is, the present invention is as follows.
1. A method for producing an oxygen-resistant microorganism comprising culturing a microorganism in a streptonigrin-containing medium and selecting a streptonigrin-resistant strain.
2. 2. The method according to 1 above, wherein the microorganism is a microorganism that can be used in food.
3. 3. The method according to 2 above, wherein the microorganism that can be used in the food is probiotic.
4). 4. The method according to 3 above, wherein the probiotic is a lactic acid bacterium.
5. 4. The method according to 3 above, wherein the probiotic is Bifidobacterium.
6). 5. The method according to 4 above, wherein the lactic acid bacterium is a genus Lactobacillus.
7). 7. The method according to 6 above, wherein the Lactobacillus bacterium is Lactobacillus acidophilus or Lactobacillus gasseri .
8). A method for producing a microorganism having improved survival under an aerobic condition by the method according to claim 1.
9. A method for improving the oxygen resistance performance of a microorganism, comprising culturing the microorganism in a streptonigrin-containing medium to cause streptonigrin resistance.
10. A method for improving survival of microorganisms under aerobic conditions by the method according to claim 9.
11. An oxygen-resistant microorganism obtained by the method according to claim 1.
12 The microorganism according to claim 11, which has improved survival under aerobic conditions.
13. 13. Lactobacillus acidophilus SH138 strain (deposited as deposit number NITE P-1380) or Lactobacillus gasseri SH208 strain (deposited as deposit number NITE P-1381) Microorganisms.

  According to the present invention, a microorganism having improved oxygen resistance can be produced efficiently by a simple method. In particular, oxygen resistance can be improved in useful microorganisms used in foods, such as probiotics such as lactic acid bacteria and bifidobacteria, and survival under aerobic conditions can be improved. In foods that require microorganisms to reach the intestinal tract in a living state, the viability of the microorganisms during production and storage is an important requirement. By using the microorganisms produced according to the present invention, it is not necessary to use additives for maintaining survival and expensive containers with low oxygen permeability as in the prior art, so that the original flavor and palatability of foods can be obtained. The manufacturing cost can be reduced without any influence. Moreover, when using the microorganism obtained by this invention as a starter of fermented food, productivity and stability of manufacture can be improved.

  Furthermore, the method of the present invention is expected to provide oxygen resistance by applying to not only lactic acid bacteria and bifidobacteria sensitive to oxygen used in foods but also a wide variety of microorganisms. For example, it is possible to produce a microorganism having a high survivability among microorganisms used for production of useful substances and use this microorganism to increase the productivity.

  Streptonigrin used in the method of the present invention is a relatively stable reagent and is easier to handle and use than hydrogen peroxide used in the conventional method. And since streptonigrin is an active oxygen generator and an effective mutagenic agent, it can be mutated by simply culturing it in a streptonigrin-containing medium without performing mutation treatment on microorganisms in advance. And selection of oxygen-tolerant strains.

It is a graph which shows the low-temperature survival property on the aerobic condition of a parent strain and a streptonigrin resistant strain. A shows the result of measuring the number of viable bacteria every week after storing the parent strain of Lactobacillus acidophilus (JCM1132 strain) and the streptonigrin resistant strain (SH138 and SH141) at 4 ° C. B shows the results of measuring the number of viable bacteria every week after storing the parent strain of Lactobacillus gasseri (JCM1131 strain) and the streptonigrin resistant strains (SH195, SH196, SH208, SH211 and SH212) at 4 ° C.

  The method for producing the oxygen-resistant microorganism of the present invention includes culturing the microorganism in a streptonigrin-containing medium and selecting a streptonigrin-resistant strain.

  The streptonigrin used is a known antitumor agent containing aminoquinone, and the substance name is 3-methyl-4- (2-hydroxy-3,4-dimethoxyphenyl) -5-amino-6- [5,8. -Dioxo-6-methoxy-7-aminoquinolin-2-yl] pyridine-2-carboxylic acid. Streptonigrin forms a complex with intracellular iron, and decomposes DNA and RNA by generating active oxygen with an iron catalyst. The DNA degradation activity of streptonigrin is suppressed by SOD and catalase, which are active oxygen degrading enzymes, or EDTA, which is an iron chelator, so at least three types of active oxygen (superoxide, hydrogen peroxide, hydroxy radical) (Bolxan AD. Et al., Mutant Res., 2001, 488: 25-37). That is, when the cell's ability to react with active oxygen is improved, or when iron ions that catalyze the generation of active oxygen are reduced, it is presumed to be resistant to streptonigrin. It is considered that a strain showing resistance to active oxygen can be obtained. This is demonstrated in the examples below. That is, in the examples, lactic acid bacteria generally used as probiotics, and by isolating a streptonigrin resistant strain in a Lactobacillus bacterium characterized by being susceptible to oxygen stress, oxygen tolerance and survival It was possible to easily obtain a strain with improved

  Since the mechanism of generation of active oxygen in cells by streptonigrin is common to microorganisms in general, a method for obtaining a strain having improved oxygen tolerance and survival by selecting a streptonigrin-resistant strain is described above. It can be applied to other microorganisms and is expected to bring about similar effects.

The microorganism that is the target of the production method of the present invention is preferably a microorganism that can be used in food, and more preferably a probiotic. The probiotic lactobacilli, Lactococcus (Lactococcus) bacteria, Streptococcus (Streptococcus) bacteria, Enterococcus (Enterococcus) genus bacteria, Weissella (Weissella) bacteria, Pediococcus (Pediococcus) bacteria, leuco Examples thereof include lactic acid bacteria and bifidobacteria such as Leuconostoc bacteria. The Lactobacillus bacteria, Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus brevis (Lactobacillus brevis ), Lactobacillus casei ( Lactobacillus paracasei ), Lactobacillus plantarum , Lactobacillus paraplantarum , Lactobacillus paraplantarum , Lactobacillus helvetics (lvactobacillus helvetics ) And Lactobacillus delbrueckii and the like. Bifidobacterium longum , Bifidobacterium breve , Bifidobacterium adolescentis , Bifidobacterium bifidum ( Bifidobacterium bifidum ) , Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium shoe Delon gum (Bifidobacterium pseudolongum), and the like.

  Examples of microorganisms to which the method of the present invention can be applied other than lactic acid bacteria and bifidobacteria include Clostridium bacteria that are sensitive to oxygen.

Specific examples of microorganisms obtained by the method of the present invention are as follows. Lactobacillus acidophilus SH138 strain (Independent administrative agency, Product Evaluation Technology Foundation, Patent Microorganism Deposit Center (Kazusa Kamashizu, Kisarazu City, Chiba Prefecture) -5-8) deposited on June 22, 2012 under the accession number NITE P-1380) and SH141 strain, and Lactobacillus gasseri SH208 strain (Patented by National Institute of Technology and Evaluation) (Deposit number NITE P-1381 dated June 22, 2012) at the Microorganism Deposit Center (2-5-8 Kazusa Kamashizu, Kisarazu City, Chiba)), SH195, SH211, SH212 and SH196 Can be mentioned.

  In the method of selecting a strain having higher survival than the natural world, not only does it require a lot of labor and time as described above, but the selected strain does not necessarily have a purposeful property, for example, a property useful as a probiotic. It does not have. By using the method of the present invention, the viability can be increased in a strain already used as a useful strain, and a useful strain with improved survival can be easily produced.

  In the streptonigrin-containing medium used in the production method of the present invention, streptonigrin was added to a medium suitable for culturing the strain used as the parent strain at a concentration higher than the minimum growth inhibitory concentration of the strain against streptonigrin. Medium. When Lactobacillus bacteria are used, it is preferable to add streptonigrin to a GYP medium, MRS medium, skim milk medium, or the like.

  In the production method of the present invention, first, the parent strain is inoculated into a medium containing the above streptonigrin at a concentration higher than the minimum growth inhibitory concentration and cultured. The culture may be carried out by a culture method and culture conditions suitable for the strain to be used. In the case of Lactobacillus bacteria, it is preferable to perform stationary culture or shaking culture at 37 ° C. for 24 to 72 hours. Since streptonigrin is also a mutagenic agent, by culturing the parent strain in the above medium, mutations are induced in the parent strain to produce various mutant strains, of which the streptonigrin resistant strain survives and grows. In this way, a strain that grows in a medium containing streptonigrin at a concentration higher than the minimum growth inhibitory concentration is obtained as a streptonigrin resistant strain.

  Oxygen tolerance of the obtained streptonigrin-resistant strain, that is, survival under aerobic conditions, is determined by measuring the number of viable bacteria after storing the culture solution under aerobic conditions (in the atmosphere) for a certain period of time. It can be measured. In this way, a strain with improved oxygen tolerance and survival can be obtained.

  Microorganisms with improved oxygen tolerance and survival produced by the method of the present invention can be added to foods as probiotics or used as starters for fermented foods. Alternatively, it can be used as a preparation of various dosage forms by blending known carriers and excipients and adding food additives as necessary. Moreover, it can be used for substance manufacture as a strain | stump | stock which can produce a useful substance.

  Hereinafter, the present invention will be described by way of examples. The following examples are given for the purpose of illustrating the present invention and are not intended to limit the present invention.

Is a lactic acid bacteria are commonly used as probiotics, have the characteristics of being weak in oxidative stress, and the mutant strain having excellent oxygen resistance and viability using the Lactobacillus acidophilus and Lactobacillus gasseri as follows Production did.

Lactobacillus acidophilus JCM1132 (preserved in RIKEN BioResource Center, Microbial Materials Development Office (2-1 Hirosawa, Wako, Saitama)), Lactobacillus gasseri JCM1131 (RIKEN BioResource Center Microorganisms) The material development room (stored in 2-1 Hirosawa, Wako City, Saitama Prefecture) was used as the parent stock. The minimum growth inhibitory concentration of each strain against streptonigrin (Sigma) was measured. As the medium, GYP medium (glucose 1%, yeast extract 1%, proteose peptone 0.5%, sodium acetate 0.2%, beef extract 0.2%, magnesium sulfide heptahydrate 0.02%, manganese sulfide four Hydrate 0.001%, iron sulfide heptahydrate 0.001%, sodium chloride 0.001%, Tween 80 0.05%, adjusted to pH 6.8). The minimum growth inhibitory concentration was 0.625 μg / ml for Lactobacillus acidophilus JCM1132, and 2.5 μg / ml for Lactobacillus gasseri JCM1131.

Subsequently, streptonigrin having a stepwise higher concentration than the determined minimum growth inhibitory concentration (0.625, 1.25, 2.5, 5.0, 10.0 μg / ml for Lactobacillus acidophilus JCM1132, 2.5, 5.0, 10.0, 20.0 μg / ml for Lactobacillus gasseri JCM1131) ml) was added to the GYP medium, and left or shake cultured at 37 ° C. for 24-72 hours, and the strain grown in the presence of a streptonigrin concentration higher than the minimum growth inhibitory concentration was purified as a streptonigrin resistant strain. did. The SH138 and SH141 strains were obtained as the streptonigrin resistant strains of Lactobacillus acidophilus , and the SH195, SH196, SH208, SH211 and SH212 were obtained as the streptonigrin resistant strains of Lactobacillus gasseri .

In order to measure the survival of the obtained streptonigrin resistant strain, a culture solution of Lactobacillus acidophilus or Lactobacillus gasseri that had been left standing at 37 ° C. in an MRS liquid medium (Difco) until the absorbance reached A600 = 1.0 was used. Using. This culture solution was stored at 4 ° C. for 28 days under aerobic conditions, and the culture solution diluted appropriately every 7 days was seeded on an MRS agar medium and cultured to determine the viable cell count. The results are shown in FIG.

As is clear from FIG. 1, the number of viable bacteria in the parent strain, Lactobacillus acidophilus JCM1132, decreased significantly with the passage of days, whereas in the Lactobacillus acidophilus streptonigrin-resistant strains, both SH138 and SH141 strains, Survival improved, and after 28 days, the number of viable bacteria was about 500 to 1000 times higher than that of the wild type. On the other hand, the streptonigrin resistant strains of Lactobacillus gasseri (SH195, SH196, SH208, SH211 and SH212) also showed viable cell counts about 30 to 200 times higher than the parent strain at the 14th day after the start of storage. After 21 days, the number of viable bacteria decreased, but the improvement of survival was confirmed in 2 weeks, which is a general expiration date for yogurt and the like. Thus, by isolating a streptonigrin resistant strain, a strain with improved survival could be easily obtained.

Claims (13)

  A method for producing an oxygen-resistant microorganism comprising culturing a microorganism in a streptonigrin-containing medium and selecting a streptonigrin-resistant strain.   The method according to claim 1, wherein the microorganism is a microorganism that can be used in food.   The method according to claim 2, wherein the microorganism that can be used in the food is probiotic.   4. The method of claim 3, wherein the probiotic is a lactic acid bacterium.   4. The method of claim 3, wherein the probiotic is Bifidobacterium.   The method according to claim 4, wherein the lactic acid bacterium is a Lactobacillus bacterium. The method according to claim 6, wherein the Lactobacillus genus bacterium is Lactobacillus acidophilus or Lactobacillus gasseri .   A method for producing a microorganism having improved survival under an aerobic condition by the method according to claim 1.   A method for improving the oxygen resistance performance of a microorganism, comprising culturing the microorganism in a streptonigrin-containing medium to cause streptonigrin resistance.   A method for improving survival of microorganisms under aerobic conditions by the method according to claim 9.   An oxygen-resistant microorganism obtained by the method according to claim 1.   The microorganism according to claim 11, which has improved survival under aerobic conditions. 13. Lactobacillus acidophilus SH138 strain (deposited as deposit number NITE P-1380) or Lactobacillus gasseri SH208 strain (deposited as deposit number NITE P-1381) Microorganisms.

Images