JP6018771B2 - Reduced glutathione-rich fermented lactic acid bacteria and method for producing the same - Google Patents

Description

  The present invention relates to a fermented lactic acid bacterium containing a reduced glutathione at a high concentration, having excellent physiological functions such as antioxidant activity, and having a good flavor, and a method for producing the same.

  Amino acids or peptides have various functions, and are added to food and drink for the purpose of improving product quality or imparting physiological functions. In dairy products, for example, it has been reported that by adding cysteine to fermented dairy products, the survival of lactic acid bacteria can be maintained even under severe light irradiation conditions, and deterioration of flavor can be suppressed (Patent Document 1). . In addition, it is known that by adding cystine to a milk beverage, it is possible to suppress deterioration odors and precipitation of milk components caused by high-temperature sterilization and heat storage (Patent Document 2).

  Patent Document 3 discloses a cysteine-enriched fermented milk food blended with a peptide containing a high content of cysteine, which promotes biosynthesis of glutathione in vivo and increases the concentration of glutathione in body cells. It is intended. Glutathione is a tripeptide composed of glutamic acid, cysteine, and glycine, and is known to have an antioxidative effect in addition to a liver function improving action, an immunostimulating action, and the like. That is, glutathione has an oxidized form and a reduced form, but reduced glutathione (GSH) has a thiol group and can reduce and eliminate active oxygen species such as free radicals and peroxides. . Oxidized bimolecular glutathione becomes oxidized glutathione (GS-SG) bonded to each other (SS bond).

  Among the lactic acid bacteria used in fermented milk products, some have the ability to produce such reduced glutathione. For this reason, if the content of reduced glutathione in lactic acid bacteria can be increased, a fermented milk product having excellent physiological functions such as antioxidant activity can be obtained. For example, arteriosclerosis is considered to be one of the main causes of oxidation of low density lipoprotein (LDL) in blood vessels. That is, macrophages hardly take in native LDL, but oxidized LDL is taken up actively, which changes into foam cells containing a large amount of lipid droplets and accumulates in the subendothelium, leading to arteriosclerosis. Therefore, if a component having high antioxidant activity can be ingested daily as a food or drink, it is considered that LDL oxidation can be suppressed and arteriosclerosis can be effectively prevented.

  In yeast, a method of adding cysteine to a medium is known as a method for increasing the content of reduced glutathione (Non-patent Document 1). On the other hand, Non-Patent Document 1 describes that the content of reduced glutathione did not increase even when cystine, an amino acid related to cysteine, was added to the medium (page 539, right column, line 17). -19th line). As a method for increasing the content of reduced glutathione in lactic acid bacteria, a mixture of cysteine, glycine, and glutamic acid, which are constituent amino acids of reduced glutathione, has been prepared so far that the final concentration of each amino acid in the medium is about 0.003% or 0. A method of adding and culturing so as to be 006% has been reported (Non-patent Document 2). However, this method has a problem that an unpleasant flavor peculiar to cysteine is strongly generated and is not suitable as a food or drink.

JP 2002-17254 A JP 2009-55802 A JP 2006-197834 A

Catalino G. Alfafara, Akihisa Kanda, Toru Shioi, Hiroshi Shimizu, Suteaki Shioya and Ken-ichi Suga.Effect of amino acids on glutathione production by Saccharomyces cerevisiae.Applied Microbiology and Biotechnology Volume 36, Number 4, 538-540 Leonides Fernandes and James L. Steele.Glutathione Content of Lactic Acid Bacteria.Journal of Dairy Science 76 1233-1242 (1993)

  Accordingly, an object of the present invention is to provide a fermented lactic acid bacterium containing reduced glutathione at a high concentration and having a good flavor.

  As a result of diligent research to solve the above-mentioned problems, the present inventors added cystine, which had not been increased in reduced glutathione content in yeast, to the medium in the culture of lactic acid bacteria. Conversely, the inventors found that a fermented product containing a large amount of reduced glutathione in the lactic acid bacteria and having an excellent flavor was obtained, and the present invention was completed.

  That is, the present invention is a fermented lactic acid bacterium obtained by cultivating reduced glutathione-producing lactic acid bacteria in a medium containing cystine.

  Moreover, this invention is fermented dairy products containing the said lactic-acid-bacteria fermented material.

  Furthermore, the present invention is a method for producing a fermented lactic acid bacterium characterized by culturing reduced glutathione-producing lactic acid bacteria in a medium containing cystine.

  Since the fermented lactic acid bacterium of the present invention contains a large amount of reduced glutathione in the microbial cells, an excellent physiological function such as an antioxidant effect can be obtained by ingesting this. That is, even if lactic acid bacteria produce reduced glutathione, if it is released and is present outside the cells, it will be immediately oxidized, and its physiological function is not fully expressed even if ingested. However, since the fermented lactic acid bacterium of the present invention has a large amount of reduced glutathione protected in the microbial cells and released after reaching the intestine, it has excellent physiological functions in vivo such as LDL oxidation inhibitory activity. It is obtained. Furthermore, this fermented lactic acid bacterium has good flavor, excellent palatability, and high safety, so it can be taken on a daily basis, reducing the risk of various diseases such as arteriosclerosis and inflammation, and preventing aging. Useful. Further, although cystine has been conventionally considered not to increase the content of reduced glutathione, in the present invention, by using a medium supplemented with cystine, the fermentation of lactic acid bacteria has a high content of reduced glutathione and a good flavor. It is possible to obtain a product, and such an effect is not suggested by any prior literature.

In Example 1, it is a figure which shows the reduced glutathione density | concentration in the microbial cell of the lactic acid bacteria culture | cultivated in the culture medium which added cystine or 0.01%. In Example 3, it is the figure which showed the relationship of the amount of cystine addition to a culture medium, the oxidation lag time extension rate of the obtained fermented dairy product, and reduced glutathione content.

The fermented lactic acid bacteria of the present invention are obtained by cultivating reduced glutathione-producing lactic acid bacteria in a medium containing cystine. The reduced glutathione-producing lactic acid bacterium is not particularly limited as long as it is a lactic acid bacterium having a reduced glutathione-producing ability. Examples include Lactobacillus microorganisms such as Lactococcus microorganisms, Lactobacillus fermentum, Lactobacillus helveticus, etc., and these can be used alone or in combination of two or more. Among these, Streptococcus microorganisms are preferable in that reduced glutathione-producing ability is high, and Streptococcus thermophilus is particularly preferable. As Streptococcus thermophilus, Streptococcus thermophilus YIT 2001 (FERM BP-7538 ; National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center on January 31, 2001 ( East Tsukuba City, Ibaraki, Japan 305-8586) 1-chome 1-address 1 center 6) was deposited as deposit number FERM P-18189, transferred to an international deposit based on the Budapest Treaty on April 5, 2001, and deposited under a deposit number of FERM BP-7538 ), Streptococcus thermophilus ATCC 19258 and the like can be used. Among them, the reduced glutathione content is high, and Streptococcus thermophilus YIT 2001 is preferably used.

  When cultivating the above-mentioned reduced glutathione-producing lactic acid bacteria, various media on which lactic acid bacteria grow, such as milk medium and MRS medium, can be adapted, but medium adjustment is simple, and adaptation of the cultured fermented product to foods A milk medium is preferably used in that it has high properties. As milk medium, liquid milk derived from animals and plants such as cow's milk, goat's milk, sheep milk and soy milk, milk powder such as skim milk powder, whole milk powder, milk reduced from concentrated milk, etc., diluted as it is or with water Can be used. Moreover, the non-fat milk solid content (SNF) of a milk culture medium is 10-20 w / w% normally, Preferably it is 12-16 w / w%. Such a range is preferable because the reduced glutathione content becomes high. To these media, components used in normal lactic acid bacteria media may be added, and for example, yeast extract, surfactant and the like can be added.

  Cystine is added to the medium. The cystine used in the present invention includes L-cystine, D-cystine, DL-cystine and a mixture thereof, and L-cystine is preferably used. The amount of cystine added in the medium is usually 0.001 to 0.01 w / w%, preferably 0.001 to 0.004 w / w%, more preferably 0.001 to 0.003 w / w%, particularly preferably. Is 0.001 to 0.002 w / w%. If the amount of cystine added is too large, the hydrogen sulfide odor may be felt immediately after production, which may cause a problem in flavor. When the content is in the range of 0.001 to 0.003 w / w%, a reduced glutathione content and a good flavor are obtained.

  The culture conditions of the lactic acid bacteria for obtaining the fermented lactic acid bacteria of the present invention are not particularly limited, but the culture temperature is 30 to 42 ° C, preferably 30 to 34 ° C. When the temperature is high, powdery feeling may be felt, and the content of reduced glutathione may be low. When the temperature is in the range of 30 to 34 ° C., a product having a good flavor and a high content of reduced glutathione is obtained. The culture is usually performed at such a temperature for about 12 to 24 hours. Moreover, as culture conditions at this time, a method suitable for culturing lactic acid bacteria used from standing, stirring, shaking, aeration and the like may be appropriately selected.

The fermented lactic acid bacterium thus obtained contains reduced glutathione at a high concentration in a protected state in the microbial cells. For example, the reduced glutathione content in the microbial cells is preferably 1200 ng / mL. More preferably, it contains 1800 ng / mL or more. Further, reduced glutathione-producing lactic acid bacteria are contained in an amount of 1.0 × 10 ³ cfu / mL to 1.0 × 10 ¹⁵ cfu / mL, preferably 1.0 × 10 ⁵ cfu / mL to 1.0 × 10 ¹³ cfu / mL. To do. Foods and drinks can be prepared by mixing the fermented cells as they are or by collecting the cells recovered from the fermented products by centrifugation or the like with other sub-materials that are permitted to be added to foods. As a fermented product using a milk medium, the fermented milk product can be used as it is or by mixing with other auxiliary materials. The fermented milk product obtained in this way contains reduced glutathione at a high concentration in a protected state in the fungus body. For example, the fermented milk product preferably has a reduced glutathione content in the fungus of 600 ng / mL, more preferably 1000 ng / mL or more. Further, the reduced glutathione-producing lactic acid bacterium is contained in 5.0 × 10 ² cfu / mL to 5.0 × 10 ¹⁴ cfu / mL, preferably 5.0 × 10 ⁴ cfu / mL to 5.0 × 10 ¹² cfu / mL. To do.

  Here, fermented dairy products include fermented soy milk or beverages such as fermented milk, dairy lactic acid bacteria beverages, hard yogurt, soft yogurt, plain yogurt, kefir, cheese, etc. Is. In addition, the fermented milk product of the present invention includes foods and drinks using various lactic acid bacteria, for example, plain type, flavored type, fruit type, soft type, drink type, hard type, frozen type, etc. It is.

  These fermented dairy products include, in addition to sweeteners such as syrup, other various food materials such as various sugars, thickeners, emulsifiers, various vitamins, etc. You may mix | blend an arbitrary component. Specific examples of these food materials include sugars such as sucrose, glucose, fructose, palatinose, trehalose, lactose, xylose, maltose, sorbitol, xylitol, erythritol, lactitol, palatinit, reduced starch syrup, reduced maltose starch syrup, etc. High-sweetness sweeteners such as sugar alcohol, aspartame, thaumatin, sucralose, acesulfame K, stevia, agar, gelatin, carrageenan, guar gum, xanthan gum, pectin, locust bean gum, gellan gum, carboxymethylcellulose, soy polysaccharide, propylene glycol alginate, etc. Various thickeners (stable), sucrose fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, lecithin emulsifier, etc. Milk fat such as cream, butter, sour cream, acidulants such as citric acid, lactic acid, acetic acid, malic acid, tartaric acid, gluconic acid, various vitamins such as vitamin A, vitamin B, vitamin C, vitamin E, calcium , Magnesium, zinc, iron, manganese and other minerals, yogurt, berry, orange, pear, perilla, citrus, apple, mint, grape, apricot, pair, custard cream, peach, Flavors such as melon, banana, tropical, herbal, tea and coffee can be listed.

  The fermented dairy product of the present invention has a good flavor and contains a large amount of reduced glutathione in the microbial cells, and by taking this, an excellent antioxidant effect and the like can be obtained. Since it is excellent in the effect of inhibiting lipid oxidation in vivo such as LDL, it is useful as a fermented milk food for inhibiting lipid oxidation, inhibiting LDL oxidation, or preventing or improving arteriosclerosis.

  Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to these examples.

Example 1
Effect of cystine addition on the reduced glutathione content in cells:
Streptococcus thermophilus YIT 2001 was used as a test strain. A modified GAM medium (4 ml) was inoculated with 0.5% (w / w) of the YIT 2001 cryopreserved bacterial solution and cultured at 37 ° C. for 16 hours. This was inoculated with 0.2% (v / v) of 10% (w / v) nonfat dry milk aqueous solution and statically cultured at 37 ° C. for 20 hours. 0.06 ml of this culture solution was added to a milk medium (skim milk powder 16.8% (w / w) (non-fat milk solids (non-fat milk solids)) in 50 ml Kolben to which L-cystine was added to a concentration of 0.01% (w / w). SNF) 16% (w / w))) was inoculated into 30 ml, and left to stand at 34 ° C. for 24 hours to obtain a lactic acid bacteria fermentation product. After the culture, the content of reduced glutathione in the cells was measured by the following method. The results are shown in FIG.

(Measurement method of reduced glutathione: HPLC method)
1 mL of the sample was diluted 5-fold with 4 mL of 10 mM ethylenediaminetetraacetic acid (EDTA) solution. After 600 μL of this diluted solution was centrifuged (20,000 g, 4 ° C., 10 min), the supernatant (whey) was removed to obtain bacterial cells. Add φ 0.1 mm glass beads (1,000 mg) and 2 mM EDTA-added 100 mM borate buffer (pH = 8.0) 800 μL to the cells and shake with FastPrep-24 (6.5 ms ^-1 , 60 s And the cells were crushed. After crushing the crushed sample (20,000 g, 4 ° C., 10 min), 200 μL of the supernatant was recovered. To this was added 300 μL of 100 mM borate buffer (pH = 8.0) containing 1 mM 4- (aminosulfonyl) -7-fluoro-2,1,3-benzooxadiazole (ABD-F), and 60 ^o Derivatization reaction was carried out with C for 15 minutes. After ice-cooling for 15 minutes, 200 μL of 0.1 N hydrochloric acid was added and analyzed and quantified by HPLC / fluorescence detection.

(HPLC conditions)
Mobile phase: 0.1% trifluoroacetic acid (TFA): acetonitrile = 88: 12
Wavelength: Excitation wavelength 380 nm, fluorescence wavelength 510 nm
Temperature: Column 40 ° C, sample 10 ° C
Flow rate: 1.0 mL / min
Injection volume: 10 μL
Column used: Mightysil RP-18 GP Aqua inner diameter 4.6 mm, length 150 mm (Kanto Chemical)
)

  From FIG. 1 and Table 2, it was shown that the addition of cystine to the medium significantly increased the reduced glutathione in the microbial cells.

Example 2
Effect of cystine addition on reduced glutathione content and flavor:
Streptococcus thermophilus YIT 2001 was used as a test strain. A 10% (w / v) non-fat dry milk aqueous solution was inoculated with 0.1% (v / v) of YIT2001 bacterial solution, followed by static culture at 37 ° C. for 24 hours. UHT pasteurized milk (skimmed milk powder 16.8% (w / w) (SNF16% (w / w))) to which 20 mL of this culture solution (viable cell count: 9.5 × 10 ⁸ cfu / ml) was added with varying L-cystine concentration, L-cystine 0-0.004% (w / w)) was inoculated into 10 L, and stirred and cultured at 34 ° C. for 24 hours to obtain a lactic acid bacteria fermentation product. After homogenizing 515 g of the lactic acid bacteria fermentation product, syrup (8.75% (w / w) per glucose fructose liquid sugar product, 0.41% (w / w) per stabilizer (pectin) product) and flavor (0.11% (v / w)) to obtain 1000 g of fermented milk product. The reduced glutathione content of the fermented lactic acid bacteria and the fermented dairy product was measured in the same manner as in Example 1. Lactic acid bacteria fermented products were measured at the end of culture (initial), and fermented dairy products were measured on the day of preparation (initial) and after storage at 10 ° C. for 21 days. Moreover, the flavor of the obtained fermented dairy product on the day of preparation (initial) and after storage at 10 ° C. for 21 days was evaluated according to the following criteria. Furthermore, the viable count of YIT2001 in fermented lactic acid bacteria and fermented dairy products was measured by the pour method using a BCP-added plate count agar medium. The results are shown in Table 1.

(Flavor evaluation standard)
○: Hydrogen sulfide-like odor is not observed △: Hydrogen sulfide-like odor is slightly recognized ×: Hydrogen sulfide-like odor is recognized

  As the amount of cystine added increases, the content of reduced glutathione increases. However, at 0.003% or more, it hardly increases. In addition, when the cystine addition amount was 0.004%, a hydrogen sulfide-like odor was strongly observed at the initial stage. In addition, since the number of viable bacteria was almost the same in all cases, it was found that the increase in the content of reduced glutathione was not due to the increase in the number of viable bacteria.

Example 3
Antioxidant activity of fermented dairy products:
The fermented milk product obtained in Example 2 and containing cystine added at 0% (no addition), 0.001%, 0.002%, 0.003%, and 0.004% per charged milk was used as a test sample. 1 ml of the sample was diluted 5-fold with 4 ml of 10 mM EDTA, and centrifuged (8000 rpm, 4 ° C., 10 min) to recover the cells and further washed with 10 mM EDTA. The washed cells were suspended in 1 ml of water, 4 times the amount of ethanol was mixed with this suspension, and the mixture was extracted by shaking at room temperature for 2 hours. After extraction, the mixture was centrifuged (8000 rpm, 10 minutes) to collect the entire supernatant and dried under reduced pressure to obtain an ethanol extract. The ethanol extract was sample loaded on Sep-pak plus tC18 and eluted with 10% acetonitrile. The collected eluate was dried under reduced pressure and dissolved in 0.4 ml of water, and then LDL oxidation inhibitory activity was measured by the following method. Further, the reduced glutathione content in the test sample (fermented milk product) was measured by the same method as in Example 1. The results are shown in FIG.

(Measurement method of LDL oxidation inhibitory activity)
An LDL fraction was prepared from human serum by ultracentrifugation and dialyzed overnight against PBS under anaerobic conditions. After dialysis, the concentration was adjusted to 0.1 mg protein / mL. After adding 50 μl of sample to this LDL solution, 2,2′-azobis-4-dimethylvaleronitrile (V-70; oxidation initiator) is added to a final concentration of 0.25 mM and kept at 37 ° C. The time course of lipid peroxide concentration was measured by the conjugated diene method (absorbance at 234 nm). The time from the addition of V-70 to the start of the increase in lipid peroxide concentration (oxidation lag time) was determined. The extension rate (%) of the oxidation lag time of the LDL solution when the sample was added was calculated from the following formula and used as an index of LDL oxidation inhibition activity.

(Calculation formula for extension rate of oxidation lag time)
Rate of extension of oxidation lag time (%) = [(oxidation lag time T−oxidation lag time C) / oxidation lag time C] × 100
Oxidation lag time T: Oxidation lag time when sample is added to LDL solution Oxidation lag time C: Oxidation lag time when sample is not added to LDL solution

  From FIG. 2, all of the media added with cystine showed higher LDL oxidation-inhibiting activity than the non-added, and the amount of reduced glutathione was also high. The activity increased in the order of no addition, 0.001%, and 0.002%, but no significant difference was observed in the activity at 0.002% or more.

Example 4
Effect of incubation temperature on reduced glutathione content and powderiness:
Streptococcus thermophilus YIT 2001 was used as a test strain. A 10% (w / v) nonfat dry milk aqueous solution was inoculated with 0.1% (v / v) of YIT2001 bacterial solution and cultured at 37 ° C. for 24 hours. 20 ml of this culture solution (viable cell count: 8.6 × 10 ⁸ cfu / ml), UHT pasteurized milk with L-cystine concentration of 0.002% (w / w) (16.8% skim milk powder (SNF 16% (w / w)), L-cystine (0.002% (w / w)) was inoculated into 10 L, and stirred and cultured at 30 ° C., 34 ° C., and 37 ° C. for 24 hours to obtain a lactic acid bacteria fermentation product. After homogenizing 515 g of the lactic acid bacteria fermentation product, syrup (8.75% (w / w) per glucose fructose liquid sugar product, 0.41% (w / w) per stabilizer (pectin) product) and flavor (0.11% (v / w)) to obtain 1000 g of fermented milk product. The reduced glutathione content of the fermented lactic acid bacteria and the fermented dairy product was measured in the same manner as in Example 1. The lactic acid bacteria fermented product was measured at the end of the culture (initial), and the fermented milk product was measured on the day of preparation (initial). Furthermore, the viable count of YIT2001 in fermented lactic acid bacteria and fermented dairy products was measured by the pour method using a BCP-added plate count agar medium. Moreover, the flavor (powderiness) after storing the obtained fermented milk product on the day of preparation (initial) and at 21C for 21 days was evaluated according to the following evaluation criteria. The results are shown in Table 2.

(Flavor evaluation standard)
○: No powdery ×: Powdery

  From Table 2, the reduced glutathione content was higher in the 34 ° C. culture than in the 37 ° C. culture, and more in the 30 ° C. culture than the 34 ° C. culture. In 37 ° C. culture, a powdery feeling was felt, but in 34 ° C. culture and 30 ° C. culture, the powderiness could be improved. It was confirmed that the use of a bacterial solution cultured under low temperature conditions was favorable in terms of reduced glutathione content and flavor.

The fermented lactic acid bacterium of the present invention contains reduced glutathione at a high concentration in the microbial cell and has excellent antioxidant activity and the like. Therefore, it is useful as a food or drink effective for prevention of various diseases such as arteriosclerosis and inflammation, or a raw material thereof.
that's all

Claims (5)

A method for producing a fermented lactic acid bacterium comprising culturing Streptococcus thermophilus in a milk medium supplemented with 0.001 to 0.004 w / w% cystine.   The method for producing a fermented lactic acid bacterium according to claim 1, wherein the content of intracellular reduced glutathione in the fermented product is 1200 ng / mL or more.   The method for producing a fermented lactic acid bacterium according to claim 1 or 2, wherein the culture temperature at the time of culturing is 30 to 34 ° C.   The method for producing a fermented lactic acid bacterium according to any one of claims 1 to 3, wherein the Streptococcus thermophilus is Streptococcus thermophilus YIT 2001 (FERM BP-7538). A method for producing a fermented dairy product, comprising mixing the lactic acid bacteria fermented product obtained by the method for producing a fermented lactic acid bacterium according to any one of claims 1 to 4 and an auxiliary material.

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