JP2020120630A - Cultured lactic acid bacteria with high content of reduced glutathione and method of production thereof - Google Patents

Abstract

To provide cultured lactic acid bacteria that can maintain high content of intracellular reduced glutathione (GSH).SOLUTION: Cultured lactic acid bacteria with high content of reduced glutathione have a modified pH by acid addition to cultured Streptococcus bacteria producing reduced glutathione in cystine-containing milk medium.SELECTED DRAWING: Figure 2

Description

TECHNICAL FIELD The present invention relates to a lactic acid bacterium culture containing high content of reduced glutathione.

Glutathione, which is known to have an antioxidant action as well as a liver function improving action, an immunostimulating action, and the like, is a tripeptide consisting of glutamic acid, cysteine, and glycine, and exists in an oxidized form and a reduced form. The oxidized form is a molecule in which two molecules of reduced glutathione are linked by a disulfide bond. Reduced glutathione (GSH) has a thiol group and reduces active oxygen species such as free radicals and peroxides. Can be erased.

Regarding this reduced glutathione, a method for producing a culture containing reduced glutathione at a high concentration by culturing Streptococcus thermophilus in a milk medium supplemented with a predetermined amount of cystine has been proposed. (For example, refer to Patent Document 1).

In this proposal, the solid content of non-fat milk (SNF; Solids Not Fat) is 10 to 20%, the conditions of the culture temperature of 30 to 34° C. and the culture time of 12 to 24 hours are described, and 0.002 to 0.004% of cystine is added. It is described that the amount of reduced glutathione produced in the culture medium is large, and more specifically, the amount of reduced glutathione is reduced only by about 5 to 32% even when stored for 21 days (Patent Document 1, Table 1) is disclosed.

Further, a method for producing fermented milk has been proposed in which an auxiliary material adjusted to pH 3 to 4 with lactic acid is added to fermented milk having pH 3 to 4 (see, for example, Patent Document 2). Thereby, the concentration of the polysaccharide produced by the microorganism is high, and the fermented dairy food product having a good flavor can be obtained.

JP, 2013-188177, A JP, 2014-027943, A

By the way, the present applicant has conducted various studies on new products based on the technique of Patent Document 1, and found that reduced glutathione-producing bacteria were cultured in a milk medium containing high nonfat milk solids (SNF). However, during storage of the lactic acid bacterium culture, reduced glutathione (hereinafter also referred to as “GSH”) accumulated in the cells was released outside the cells and changed to oxidized glutathione (hereinafter also referred to as “GSSG”). Then, a new problem that the GSH amount in the obtained lactic acid bacterium culture cannot be maintained was confirmed.

This phenomenon is observed not only in the culture of only the reduced glutathione-producing bacteria alone but also in the mixed culture of the reduced glutathione-producing bacteria and other bacteria, as long as the milk medium contains high nonfat milk solids (SNF). Similarly, it was confirmed that reduced glutathione (GSH) accumulated in the cells was released outside the cells and changed to oxidized glutathione.

An object of the present invention is to obtain a lactic acid bacterium culture capable of maintaining high intracellular reduced glutathione (GSH) and a method for producing the same.

The reduced-glutathione-rich lactic acid bacterium culture according to the present invention is characterized by being a pH-adjusted product by acid addition of a Streptococcus genus reduced glutathione-producing bacterial culture in a cystine-containing milk medium.

The reduced-glutathione-rich lactic acid bacterium culture according to the present invention is characterized by being a mixed culture of the above-mentioned Streptococcus genus reduced glutathione-producing bacterium and Lactobacillus bacterium.

The lactic acid bacterium culture containing high content of reduced glutathione according to the present invention is characterized in that the pH-adjusted product is adjusted to pH 4.1 to 4.5.

The reduced glutathione-rich lactic acid bacterial culture according to the present invention is produced by culturing Streptococcus genus reduced glutathione-producing bacteria in a cystine-containing milk medium, and before the growth curve reaches the logarithmic phase to the stationary phase, the culture was carried out. It is characterized in that the pH is adjusted by adding an acid to the culture.

The method for producing a lactic acid bacterium culture having a high content of reduced glutathione according to the present invention is characterized in that the cystine-containing milk medium contains 20% or more of non-fat milk solids (SNF).

The method for producing a reduced-glutathione-rich lactic acid bacterium culture according to the present invention is characterized in that the Streptococcus genus reduced glutathione-producing bacterium and Lactobacillus bacterium are mixed and cultured in the cystine-containing milk medium. ..

The method for producing a culture of lactic acid bacteria having a high content of reduced glutathione according to the present invention is characterized in that the acid to be added is an organic acid.

INDUSTRIAL APPLICABILITY The present invention has an effect that a lactic acid bacterium culture product containing a high amount of intracellular reduced glutathione (GSH) and having good storage stability can be obtained.

It is a diagram which shows the change of the intracellular GSH amount of Streptococcus bacterium (ST-1) single bacterium fermented milk and the mixed culture fermented milk of Lactobacillus bacterium (LcS). It is explanatory drawing which shows the transition of the amount of GSH and the amount of GSSG inside and outside a microbial cell. It is explanatory drawing which shows the difference in the preservability of the amount of GSH and GSSG of the extracellular body by the presence or absence of addition of lactic acid. It is explanatory drawing which shows the difference in the intracellular GSH amount by the addition of lactic acid, and its preservation.

The lactic acid bacterium culture containing reduced glutathione at a high content of the present invention is a pH-adjusted product obtained by adding an acid to a reduced glutathione-producing bacterial culture of Streptococcus in a cystine-containing milk medium. The addition of acid inhibits the transfer of reduced glutathione (GSH) accumulated in the bacterial cells to the outside of the bacterial cells, so that a high intracellular GSH amount can be maintained.

The Streptococcus genus reduced glutathione-producing bacterium of the present invention is not particularly limited as long as it is a Streptococcus genus bacterium having reduced glutathione-producing ability, for example, Streptococcus thermophlus (Streptococcus thermophlus), Streptococcus agalactiae ( Streptococcus agalactiae) and other bacteria belonging to the genus Streptococcus can be used, and these can be used alone or in combination of two or more.

Among these, Streptococcus thermophilus is particularly preferable. As Streptococcus thermophilus, Streptococcus thermophilus YIT 2001, Streptococcus thermophilus ATCC 19258 and the like can be used, but among these, the reduced glutathione content is high, so Streptococcus thermophilus YIT 2001 (FERM BP- 7538, deposit date: January 31, 2001) is preferably used.

Streptococcus genus reduced glutathione-producing bacteria may be cultivated with a single or a plurality of bacteria of the same genus, but when commercialized, mixed culture with lactic acid bacteria of other genera to give the product a preferable performance other than reduced glutathione production. Good. Examples thereof include Streptococcus bacteria, Lactococcus bacteria, Lactobacillus microorganisms, and the like. Particularly, good yogurt can be obtained by co-culturing Lactobacillus casei YIT9029 (FERM BP-1366, deposit date: May 18, 1987) with Streptococcus genus reduced glutathione-producing bacteria. It is preferable that a beverage can be obtained.

When culturing a reduced glutathione-producing lactic acid bacterium, a milk medium is preferably used because the fermented material that has been cultured is highly adaptable to foods. As this milk medium, liquid milk derived from animals and plants such as milk, goat milk, sheep milk, soy milk, skim milk powder, milk powder such as whole milk powder, milk reduced from concentrated milk, etc., or diluted with water Can be used.

Cysteine is added to the milk medium of the present invention in order to produce reduced glutathione. The cystine used in the present invention includes L-cystine, D-cystine and a mixture thereof, and L-cystine is preferably used. The amount of cystine added to the milk medium is usually 0.001 to 0.01% (w/w), preferably 0.001 to 0.006% (w/w).

In addition, non-fat milk solids (SNF) in the milk medium is contained in a high concentration (20% (w/w) or more, or 20% to 25% (w/w)) during storage of the lactic acid bacterium culture. In addition, intracellular reduced glutathione (GSH) was released outside the cells and changed to oxidized glutathione (GSSG), which caused a new problem that an effective amount of intracellular GSH could not be maintained. Even if the culture was simply stopped in this state and stored in a refrigerator at 10° C., the release of GSH outside the bacterial cells could not be stopped and the intracellular GSH amount could not be maintained.

Further, even in mixed culture of reduced glutathione-producing bacteria and other bacteria, reduced glutathione (GSH) accumulated in the cells is similarly produced in a milk medium containing high nonfat milk solids (SNF). It was confirmed that it was released outside the cells and changed to oxidized glutathione (GSSG).

In the pH-adjusted product obtained by adding an acid to the culture of the present invention to adjust the pH, even if the non-fat milk solid content (SNF) is 20% (w/w) or more, the lactic acid bacteria culture is stored in the cells during storage. Accumulated reduced glutathione is difficult to be released to the outside of the bacterial cell, and it inhibits transfer of reduced glutathione (GSH) accumulated in the bacterial cell to the outside of the bacterial cell, so that high intracellular GSH can be maintained. .. Specifically, the intracellular GSH amount is 2000 ng/mL or more, preferably 2500 ng/mL or more, and more preferably 3000 ng/mL or more. Further, the ratio of intracellular GSH to the total amount of intracellular GSH, intracellular GSSG, extracellular GSH, and extracellular GSSG is 30% or more, preferably 40% or more, and more preferably 55% or more. is there. Furthermore, the ratio of the extracellular GSH amount to the total amount of the intracellular GSH, the intracellular GSSG, the extracellular GSH, and the extracellular GSSG is 40% or less, preferably 30% or less, and more preferably 20%. It is the following.

Although the pH of the medium rapidly shifts to the acidic side by the addition of acid, a preferable pH-adjusted product may be adjusted to a pH that would normally be reached after 24 hours. Specifically, the pH may be adjusted to 4.1 to 4.5. Further, the acid may be added so that the lactic acid acidity according to the neutralization titration method becomes 19.0±2.0.

Regarding the acid for pH adjustment, in order to prevent the reduced glutathione accumulated in the cells of Streptococcus reduced glutathione-producing bacteria from being released to the outside of the cells, it is added to make it acidic. Good. Regarding the acid, it is possible to add various acids, but organic acids are preferable because many are recognized as additives in foods, and lactic acid is the same as the acid produced by the reduced glutathione-producing bacteria itself, More preferable.

Regarding the pH adjustment of the present invention, streptococcus reduced glutathione-producing bacteria are sufficiently reduced glutathione accumulated in the cells, and yet, the accumulated reduced glutathione is carried out before the extracellular transfer. Optimal. In many cases, the reduced glutathione is sufficiently accumulated in the cells of the reduced glutathione-producing bacterium at about the peak, that is, before the growth curve reaches the logarithmic phase to the stationary phase.

More specifically, Streptococcus reduced glutathione-producing bacteria are cultured in a cystine-containing milk medium for 12 to 20 hours, and an acid is added to the cultured culture to adjust the pH before the growth curve reaches from the logarithmic phase to the stationary phase. To adjust.

As for the pH-adjusted product whose pH was drastically lowered using acid, as a result of unspecified large number of people who confirmed the flavor, those whose pH was drastically lowered by adding lactic acid within 12 hours of culture were It has been found that it has a good aftertaste and a refreshing feeling as compared with the one for 24 hours, and the rapid pH adjustment by the addition of lactic acid does not deteriorate the flavor.

The pH adjustment of the present invention inhibits the release of reduced glutathione accumulated outside the cells outside the cells, but the release of the reduced glutathione outside the cells is stored in a refrigerated state, The inhibition can be continued for 7 days after the addition of the acid, and the accumulation of reduced glutathione in the cells can be sufficiently maintained.

The reduced glutathione high-content lactic acid bacterium culture shown in the present invention, since reduced glutathione is accumulated in the bacterial cells, the culture itself can be used as a yogurt food, a yogurt beverage, or the like, or the culture itself or the bacterial cells can be collected. It may be provided as a food, a supplement agent or the like by sterilizing it and drying it. In this case, the storage stability is also much higher than the storage of the culture itself in a liquid.

FIG. 1 is a diagram showing changes in the intracellular GSH amount between Streptococcus bacterium (ST-1) single bacterium fermented milk and mixed culture fermented milk of Lactobacillus bacterium (LcS). FIG. 2 is an explanatory diagram showing changes in the amount of GSH inside and outside the cells and the amount of GSSG. FIG. 3 is an explanatory diagram showing the difference in the preservability of the intracellular and extracellular GSH amount and GSSG amount depending on the presence or absence of lactic acid addition. FIG. 4 is an explanatory diagram showing the difference between the intracellular GSH amount and the preservability due to the addition of lactic acid.

Example 1
First, it was verified that when the SNF concentration was high, the amount of intracellular glutathione in the fermented product of lactic acid bacteria was smaller than that when the SNF concentration was low. Specifically, in this Example 1, Streptococcus thermophilus YIT2001 (hereinafter also referred to as "ST-1") and Lactobacillus casei YIT9029 (hereinafter also referred to as "LcS") were used as the test strains. .. Inoculate 10% (w/w) non-fat dry milk aqueous solution (0.01% (v/v) for ST-1 and 0.5% (v/v) for LcS) with cryopreserved bacterial solution of only ST-1 or both bacteria, The cells were precultured at 37°C for 22 hours.

First, a milk culture medium (non-fat milk solid content (SNF) 16% (w/w) and 22.7% ( w/w), L-cystine 0-0.006%) 10 L, and culturing with stirring at 34.5±0.5° C. until the pH reached less than 4.4 was obtained to obtain each fermented lactic acid bacterium. The content of reduced glutathione in the cells of the lactic acid bacterium fermentation product was measured. The results are shown in Table 1 below. From Table 1, it was found that when the SNF was 20% or more, the amount of intracellular reduced glutathione was lower than when the SNF was 16%.

The method for measuring reduced glutathione in the cells was performed as follows. First, 1 mL of the sample was diluted with 9 mL of 10 mM ethylenediaminetetraacetic acid (EDTA). After centrifuging 600 μL of this solution (20,000 g, 4° C., 10 min), the supernatant was removed to collect bacterial cells. Φ0.1 mm glass beads (1,000 mg) and borate buffer solution (2 mM EDTA, 100 mM borate buffer solution, pH 8.0) 800 μL were added, and the cells were shaken (6.5/ms, 60 s) to disrupt the cells. The supernatant was recovered by centrifugation (20,000 g, 4° C., 10 min).

To 200 μL of this supernatant, 300 μL of 100 mM borate buffer (pH 8.0) containing 1 mM 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) was added, and the temperature was adjusted to 60°C. The derivatization reaction was carried out for 15 minutes. After ice-cooling for 10 minutes, 200 μL of 0.1N hydrochloric acid was added, and centrifugal filtration (10,000 g, 4° C., 2 min) with a filter having a pore size of 0.45 μm was analyzed and quantified by HPLC/fluorescence detection method.

Next, FIG. 1 shows the results of verifying changes in the intracellular GSH amount with respect to the culture time. That is, using a ST-1 single bacterium and a mixed bacterium of ST-1 and LcS, L-cystine was added to 0.002% in a milk medium (non-fat milk solids (SNF) 16% (w/w) and 22.7%. (w/w)) 10 L was inoculated, stirred and cultured, and the fermented product of lactic acid bacteria was ingested with time, and the content of reduced glutathione in the cells of the fermented product of lactic acid bacteria was measured. As shown in FIG. 1, it was found that the intracellular GSH amount peaked at 12 hours of culture and gradually decreased.

Example 2
In this example, the time course of glutathione is shown. Specifically, as test strains, as in Example 1, ST-1 single bacteria and mixed bacteria of ST-1 and LcS were used. ST-1 alone or a cryopreserved bacterial solution of both bacteria was inoculated into a 10% (w/w) non-fat dry milk solution (0.01% (v/v) for ST-1 and 0.5% (v/v) for LcS), It precultured at 37 degreeC for 24 hours.

5 mL of the obtained preculture liquid was added to L-cystine in a 3 L jar fermenter at a concentration of 0.002% (w/w) in a milk medium (non-fat milk solids (SNF) 22.7% (w/w)). w)) 2 L was inoculated with ST-1 single bacterium or a mixed bacterium of ST-1 and LcS, and cultured with stirring at 34° C. for 26 hours to obtain a fermented lactic acid bacterium.

Each of these fermented lactic acid bacteria was recovered every 2 hours from the 6th hour of culture, and the amount of reduced/oxidized glutathione inside and outside the cells was measured. The results are shown in FIG. 2 and Table 2.

The method for measuring reduced and oxidized glutathione in the cells is as follows. That is, 1 mL of the sample was diluted with 9 mL of 10 mM ethylenediaminetetraacetic acid (EDTA). After centrifuging 600 μL of this solution (20,000 g, 4° C., 10 min), the supernatant was removed to collect bacterial cells. To this, add 800 μL of φ0.1 mm glass beads (1,000 mg) and 2 mM EDTA-added 100 mM borate buffer (pH 8.0), and shake (6.5/ms, 60 s) with FastPrep-24 (MP Biomedicals) to microbial cells. After crushing, the mixture was centrifuged (20,000 g, 4° C., 10 min) and the supernatant was collected.

Purified water or reducing agent (5 mM TCEP, Nacalai Tesque) 10 μL was added to 200 μL of the supernatant, and 2 mM EDTA added 100 mM borate buffer (pH 8.0) 290 μL and lmM 4-(aminosulfonyl)-7-fluoro- 300 μL of 100 mM borate buffer (pH 8.0) containing 2,1,3-benzoxadiazole (ABD-F) was added, and a derivatization reaction was performed at 60° C. for 15 minutes. After ice-cooling for 10 minutes, 200 μL of 0.1N hydrochloric acid was added, and centrifugal filtration (10,000 g, 4° C., 2 min) with a filter having a pore size of 0.45 μm was analyzed and quantified by HPLC/fluorescence detection method.

The method for measuring reduced and oxidized glutathione outside the cells is as follows. That is, 600 μL of a solution prepared by diluting 1 mL of a sample with 9 mL of 10 mM ethylenediaminetetraacetic acid (EDTA) was centrifuged (20,000 g, 4° C., 10 min), and then the supernatant was recovered. Purified water or reducing agent (5 mM TCEP, Nacalai Tesque) 10 μL was added to 200 μL of the supernatant, and 0.5 mM 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) was added to this. ) Addition 590 μL of 400 mM borate buffer (pH 8.0) was added, and a derivatization reaction was performed at 60° C. for 20 minutes. After ice-cooling for 15 minutes, 200 μL of 0.4N hydrochloric acid was added, and the mixture was analyzed and quantified by HPLC/fluorescence detection method.

As shown in FIG. 2 and Table 2, with respect to the change in the intracellular GSH amount for each culture time, the intracellular GSH amount increased until 18 hours, but after that, the intracellular GSH amount decreased. I knew that.

Example 3
In this example, it is shown that a shortened culturing time can provide a bacterial solution having a high content of intracellular GSH, and that it can be maintained at a high level by adding lactic acid after culturing. Specifically, a mixed bacterium of ST-1 and LcS was used as the test strain. 0.01% (v/v) of ST-1 and 0.5% (v/v) of LcS were inoculated into a 10% (w/w) skim milk powder aqueous solution, and precultured at 37° C. for 24 hours.

0.25 mL of the obtained preculture liquid was added to 300 mL Kolben so that L-cystine was added at a concentration of 0.002% (w/w) in a milk medium (non-fat milk solids (SNF) 22.7% (w/w). )) 100 mL was inoculated and statically cultured at 34° C. for 12 hours to obtain a fermented lactic acid bacterium. After the culture was completed, lactic acid was added to adjust the pH to 4.3±0.1 and stored at 4° C. for up to 7 days. The amount of reduced glutathione in the cells of the fermented lactic acid bacterium before and after storage was measured by the same method as in Example 1, and the residual rate after storage was calculated.

The number of viable LcS bacteria in the fermented lactic acid bacterium was measured in a cephalothin-added BCP medium (final concentration 2 μg/mL). The viable cell count of ST-1 was calculated by subtracting the viable cell count of LcS from the total viable cell count of ST-1·LcS. The results are shown in Table 3. As shown in Table 3, the residual ratio of intracellular reduced glutathione amount in the case of adding lactic acid was overwhelmingly higher than that in the case of not adding lactic acid.

Example 4
In this example, changes over time of glutathione with and without the addition of lactic acid are shown. Specifically, as in Example 1, a single ST-1 bacterium and a mixed bacterium of ST-1 and LcS were used. ST-1 alone or a cryopreserved bacterial solution of both bacteria was inoculated into a 10% (w/w) non-fat dry milk solution (0.01% (v/v) for ST-1 and 0.5% (v/v) for LcS), It precultured at 37 degreeC for 24 hours.

0.25 mL of the obtained preculture liquid was added to 300 mL Kolben so that L-cystine was added at a concentration of 0.002% (w/w) in a milk medium (non-fat milk solids (SNF) 22.7% (w/w). )) 100 mL was inoculated and statically cultured at 34° C. for 12 hours to obtain a fermented lactic acid bacterium. One of the obtained fermentation products of lactic acid bacteria was sampled and stored at 10°C. At the time of sampling, the amount of reduced/oxidized glutathione inside and outside the cells on the 2nd day of storage and the 7th day of storage was measured in the same manner as in Example 2.

Further, other lactic acid fermentation products were prepared by adding lactic acid to pH 4.3±0.1 after completion of the culture, sampling and storing at 10° C. as in the case of the first sample. At the time of sampling, the amount of reduced/oxidized glutathione inside and outside the cells on the second and seventh storage days was measured in the same manner as in Example 2. Results are shown in FIG.

As shown in Fig. 3, when the pH was rapidly reduced by adding lactic acid, the amount of reduced glutathione in the cells was overwhelming on both the 2nd day of storage and the 7th day of storage compared to the case where lactic acid was not added. It was shown that it was preserved. In addition, on the 7th day of storage at 10° C., when lactic acid was not added, the ratio of intracellular GSH to the total amount of intracellular GSH, intracellular GSSG, extracellular GSH, and extracellular GSSG was 20.2%. The ratio of in vitro GSH was 48.1%. On the other hand, on the 7th day of storage at 10° C., when lactic acid was added, the ratio of intracellular GSH to the total amount of intracellular GSH, intracellular GSSG, extracellular GSH, and extracellular GSSG was 60.6%. The ratio of outer GSH was 13.6%, showing a significant difference between the two.

Example 5
In this example, it is shown that acids other than lactic acid also have an effect of maintaining intracellular GSH. Specifically, ST-1 was used as the test strain. A 10% (w/w) skim milk powder aqueous solution was inoculated with 0.01% (v/v) of each, and precultured at 37° C. for 24 hours. 0.25 mL of this preculture liquid was added to L-cystine in 300 mL Kolben at a concentration of 0.004% (w/w) in a milk medium (non-fat milk solids (SNF) 22.7% (w/w)). 100 mL was inoculated and statically cultured at 34° C. for 12 hours to obtain a lactic acid bacterium fermented product.

After completion of the culture, an inorganic acid (hydrochloric acid, phosphoric acid) or an organic acid (lactic acid, citric acid, succinic acid) was added, and the mixture was stored at 10°C for up to 7 days. The amount of reduced glutathione (GSH) in the cells of the fermented lactic acid bacterium before and after storage was measured by the same method as in Example 1, and the residual rate after storage was calculated. The results are shown in Table 4. From the results of Table 4, it was found that the organic acid has a higher residual ratio of intracellular reduced glutathione by adding the acid than the inorganic acid. It was also found that the effect is particularly high when lactic acid is added.

Example 6
In this Example, it is shown that the fermented product of lactic acid bacterium that has been cultured for 18 hours and the fermented milk product obtained by using the lactic acid bacterium have a high GSH content in cells and a high storage stability by post-addition of lactic acid. Specifically, ST-1 and LcS were used as the test strains.

Cryopreserved bacterial liquor of both bacteria was inoculated into 10% (w/w) non-fat dry milk solution (0.01% (v/v) for ST-1 and 0.5% (v/v) for LcS) and incubated at 37°C for 21 hours. Precultured. 25 mL of this preculture liquid was inoculated into 10 L of a milk medium (non-fat milk solids (SNF) 22.7% (w/w), L-cystine 0.004%) supplemented with L-cystine, and cultured with stirring at 34°C for 18 hours. After that, lactic acid was added to pH 4.3±0.1 to obtain a fermented lactic acid bacterium.

After homogenizing 530 g of the fermented lactic acid bacterium, syrup (fine liquor (FL): 7.36% (w/w) per product, calcium lactate: 0.055% (w/w) per product, cream: 1.5% per product ( w/w)) to obtain 1000 g of fermented milk product. Fermented lactic acid bacteria were stored at 5°C for 7 days, and fermented dairy products were stored at 10°C for 21 days.

With respect to the fermented lactic acid bacteria and the fermented milk product before and after storage, the amount of reduced glutathione in the cells was measured by the same method as in Example 1, and the residual rate was calculated. As a result, it was found that by adding lactic acid, the intracellular GSH amount remained in the fermented milk beverage mixed with syrup.

Example 7
In this example, the timing of pH adjustment was examined. Specifically, the same adjustment as in Example 4 was performed. That is, a cryopreserved bacterial solution of ST-1 and LcS was inoculated into a 10% (w/w) skim milk powder aqueous solution (0.01% (v/v) for ST-1 and 0.5% (v/v) for LcS), It was precultured at 37° C. for 21 hours.

25 mL of the preculture liquid was inoculated into 10 L of a milk medium (non-fat milk solid content (SNF) 22.7% (w/w)) containing L-cystine at a concentration of 0.004% (w/w), and the temperature was 34°C. After stirring and culturing for 18 hours, a lactic acid bacterium fermented product was obtained. After culturing, lactic acid was added to pH 4.3±0.1 and the degree of lactic acid by neutralization titration was 19.0±2.0, then sampled and stored at 10℃ for 7 days. The amount was measured (post-lactic acid addition).

As a comparative example, 10 L of a milk medium (non-fat milk solids (SNF) 22.7% (w/w)) added with L-cystine at a concentration of 0.004% (w/w) before adding a preculture liquid After adjusting the pH of the mixture to 6.0 by adding lactic acid, inoculate 25 mL of a mixed bacterial solution of ST-1 and LcS, and incubate at 34° C. for 32 hours (lactic acid acidity by neutralization titration becomes 19.0±2.0. Lactic acid bacteria fermented product (after addition of lactic acid) that has been stirred and cultured, and a milk medium (non-fat milk solids (SNF) 22.7% (w/w) added with L-cystine at a concentration of 0.004% (w/w). )) 10 L was inoculated with a mixed bacterial solution of ST-1 and LcS, and stirred and cultured at 34° C. for 32 hours (until the lactic acid acidity by the neutralization titration method was 19.0±2.0), the fermentation product of lactic acid bacteria (current formulation) Was prepared, sampled after completion of the culture, and stored at 10° C. for 7 days, and the amount of reduced/oxidized glutathione in the cells was measured.

The results are shown in Fig. 4. As shown in FIG. 4, it was confirmed that the pH adjustment by the addition of lactic acid was performed after 18 hours of culturing, so that the amount of reduced glutathione produced was large and that the amount of accumulated reduced glutathione was large even after storage for 7 days. It was

Claims (7)

A lactic acid bacterium culture containing a high amount of reduced glutathione, which is a pH-adjusted product of a culture of a reduced glutathione-producing bacterium belonging to the genus Streptococcus in a cystine-containing milk medium. The reduced-glutathione-rich lactic acid bacterium culture according to claim 1, which is a mixed culture of the Streptococcus genus reduced glutathione-producing bacterium and Lactobacillus genus. The reduced-glutathione-rich lactic acid bacterium culture according to claim 1 or 2, wherein the pH-adjusted product is adjusted to pH 4.1 to 4.5. Streptococcus reduced glutathione-producing bacteria are cultivated in a cystine-containing milk medium, and before the growth curve goes from the logarithmic phase to the stationary phase, an acid is added to the cultivated culture to adjust the pH. A method for producing a lactic acid bacterium culture. The method for producing a lactic acid bacterium culture according to claim 4, wherein the cystine-containing milk medium contains 20% or more non-fat milk solids (SNF). The method for producing a lactic acid bacterium culture according to claim 4 or 5, wherein the Streptococcus genus reduced glutathione-producing bacterium and the Lactobacillus genus are mixed and cultured in the cystine-containing milk medium. The method for producing a lactic acid bacterium culture according to any one of claims 4 to 6, wherein the acid to be added is an organic acid.

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