HK1163173B - Method for culturing lactic acid bacterium and method for producing fermented milk - Google Patents

Description

Method for culturing lactic acid bacteria and method for producing fermented milk

Technical Field

The present invention relates to a method for culturing a bacteriocin-producing lactic acid bacterium, and a method for producing fermented milk containing a bacteriocin-producing lactic acid bacterium.

Background

Fermented milk such as yogurt (yogurt) was prepared by the following method: a starter is added to raw material milk (yogurt mix) in which raw milk (raw milk), skim milk, whey protein, and the like are mixed, and the yogurt mix is fermented. Lactic acid bacteria such as Lactobacillus bulgaricus (Lactobacillus bulgaricus) and Streptococcus thermophilus (Streptococcus thermophilus) are used as the starter.

After the yogurt mix is prepared, it is cooled to a temperature of 0-10 ℃ and shipped in a refrigerated state. Since lactic acid bacteria are alive in refrigerated yogurt, yogurt is gradually fermented during distribution and during the preservation period of the home, etc., and its acidity increases. As a result, the taste of yogurt changes even during the shelf life.

In addition, it is well known that some kinds of lactic acid bacteria produce antibacterial proteins or peptides called bacteriocins. As shown in the following patent documents 1 to 3, a bacteriocin-producing lactic acid bacterium can be used to improve the storage quality of food.

In the invention of patent document 1, Lactococcus (Lactococcus) which produces bacteriocin is used as a starter for yogurt. Lactococcus produces bacteriocins accompanied by fermentation of the yoghurt mix. The bacteriocin produced by lactococcus inhibits the increase in acidity of yogurt, and therefore, the storage quality of yogurt can be improved.

In the invention of patent document 2, Bifidobacterium (Bifidobacterium) and lactococcus are co-cultured using a liquid medium whose main components are milk and milk components. By adding the culture solution after co-culture as a food preservative to foods (bread, udon, etc.), the preservation quality of the foods can be improved and the foods can be imparted with excellent taste.

Patent document 3 discloses a flavor improving agent obtained by culturing Lactococcus lactis (Lactococcus lactis) using a whey medium to which a yeast extract or the like is added and removing Lactococcus lactis from the cultured whey medium. The flavor modifier can eliminate fishy smell of fish and seafood, and can bring good taste to food.

Patent document 1: japanese patent application laid-open publication No. 4-211360 "

Patent document 2: japanese patent application laid-open publication No. 8-187071 "

Patent document 3: japanese patent application laid-open No. 2004-283109 "

As described above, in the invention of patent document 1, a bacteriocin-producing lactococcus bacterium is used as a starter. Therefore, it is difficult to suppress an increase in acidity of yogurt in which lactococcus is not used as a starter.

In addition, in order to suppress an increase in acidity of yogurt during distribution and storage, a method of adding a food preservative used in patent document 2 or a flavor improving agent used in patent document 3 as an additive to a yogurt mix has been proposed. However, when an additive is added to a yogurt mix at a certain ratio, the original taste of yogurt is spoiled by yeast extract or the like as a raw material of the additive. Therefore, it is preferable to minimize the amount of the culture to be added when adding the culture of the bacteriocin-producing lactic acid bacteria to the yogurt mix.

Disclosure of Invention

The method for culturing lactic acid bacteria of the present invention comprises: a culture solution preparation step of preparing a culture solution containing whey degraded by proteolytic enzymes; and a culture step of inoculating a bacteriocin-producing lactic acid bacterium into the culture solution, and culturing the lactic acid bacterium while maintaining the pH of the culture solution inoculated with the lactic acid bacterium at not less than 5 but not more than 6.

The method of culturing lactic acid bacteria according to the present invention can obtain a culture solution having an antibacterial activity about ten times higher than that of a culture solution obtained by a conventional culture method. That is, the method of culturing lactic acid bacteria of the present invention can efficiently produce bacteriocins.

The method for preparing fermented milk of the present invention comprises: a raw milk producing step of producing a yogurt mix; a concentrated bacterial solution producing step of culturing a bacteriocin-producing lactic acid bacterium which is a bacteriocin producer, thereby producing a concentrated bacterial solution in which the bacteriocin-producing bacterium is concentrated; an addition step of adding not less than 0.01wt% but not more than 0.1wt% of the concentrated cell suspension to the yogurt mix, based on the total weight of the yogurt mix; and a fermentation step of fermenting the yogurt mix to which the concentrated bacterial liquid is added; the concentrated bacterial liquid generation process comprises the following steps: a culture solution preparation step of preparing a culture solution containing whey degraded by proteolytic enzymes; a culture step of inoculating the bacteriocin-producing bacteria into the culture solution, and culturing the bacteriocin-producing bacteria while maintaining the pH of the culture solution inoculated with the bacteriocin-producing bacteria at not less than 5 but not more than 6; and a separation step of separating the concentrated bacterial liquid from the culture liquid inoculated with the bacteriocin-producing strain.

The method for producing fermented milk of the present invention can reduce the amount of concentrated cell suspension added as an additive to a yogurt mix. Therefore, the acidity of yogurt can be prevented from increasing without deteriorating the original taste of yogurt.

In addition, the present invention aims to provide a method for culturing lactic acid bacteria, by which bacteriocins can be efficiently produced, and a method for producing fermented milk, by which an increase in the degree of lactic acid in fermentation can be suppressed.

The objects, features, forms and advantages of the present invention will become apparent from the following summary of the invention and the accompanying drawings.

Drawings

FIG. 1 is a schematic diagram illustrating the composition of whey degradation medium used in example 1;

FIG. 2 is a diagram showing the result of culturing Lactobacillus gasseri (Lactobacillus gasseri) in example 1;

FIG. 3 is a schematic diagram illustrating the composition of whey degradation medium used in example 2;

FIG. 4 is a diagram illustrating the culture results of Lactobacillus gasseri in example 2;

fig. 5 is a graph illustrating a corresponding relationship between the gravitational acceleration and the antibacterial activity of the concentrated bacterial liquid at the time of centrifugal separation;

fig. 6 is a schematic view illustrating respective compositions of yogurt mixes used in example 4;

FIG. 7 is a graph showing the change of acidity with time of yogurt of example 4 stored at 5 ℃;

FIG. 8 is a graph showing the change of acidity with time of yogurt of example 4 stored at 10 ℃;

fig. 9 is a schematic view illustrating respective compositions of yogurt mixes used in example 5; and

FIG. 10 is a graph showing the change of acidity with time of the yogurt of example 5 stored at 5 ℃.

Detailed Description

The following describes embodiments of the present invention. The method for culturing lactic acid bacteria in the present embodiment is a method of culturing lactic acid bacteria by adding an alkaline solution to a medium so as to maintain the pH of the medium within a certain range (pH of not less than 5 but not more than 6). Thus, a culture of lactic acid bacteria having very high antibacterial activity per viable bacteria count can be obtained.

In the method for culturing lactic acid bacteria according to the present embodiment, the lactic acid bacteria to be cultured are lactic acid bacteria capable of producing bacteriocins (hereinafter, simply referred to as "bacteriocin-producing bacteria"). By using the method for culturing lactic acid bacteria of the present embodiment, the following bacteria can be cultured: lactic acid bacteria belonging to the genus Lactobacillus, such as Lactobacillus gasseri; and lactic acid bacteria belonging to the genus lactococcus such as lactococcus lactis. Specifically, lactic acid bacteria include: such as Lactobacillus gasseri OLL2959(NITEBP-224, patent microorganism Collection), Lactococcus lactis subsp. OLS3311(Lactococcus lactis subsp. lactis OLS3311, FERM BP-10966, patent organism Collection), Lactococcus lactis subsp. OLS3312(Lactococcus lactis subsp. cremoris OLS3312, FERM BP-10967, patent organism Collection), etc.

The method for culturing lactic acid bacteria according to the present embodiment is specifically described. First, a proteolytic enzyme such as protease is added to a whey aqueous solution containing whey to degrade whey protein in the whey aqueous solution. Whey protein such as concentrated Whey Protein (WPC) and isolated Whey Protein (WPI) may be added to the aqueous whey solution before adding the proteolytic enzyme.

Then, a yeast extract such as a brewers yeast extract is added to the whey aqueous solution to prepare a whey degradation medium for culturing bacteriocin-producing bacteria. In addition to whey protein, a meat extract, a fish meat extract, or the like may be added to the whey degradation medium as a nitrogen source. In addition, inorganic nutrients such as iron sulfate and magnesium sulfate, and emulsifiers such as decaglycerol monooleate and sorbitol monooleate may be added to the whey degradation medium. Sodium ascorbate and the like may also be added to the whey degradation medium.

Inoculating bacteriocin-producing bacteria to the whey degradation medium, and culturing the bacteriocin-producing bacteria. Preferably, the bacteriocin-producing bacteria are cultured until the pH of the whey degradation medium becomes not higher than 6, and then the bacteriocin-producing bacteria are cultured while the pH of the whey degradation medium in which the bacteriocin-producing bacteria are cultured is adjusted to be in the range of not less than 5 but not higher than 6. More preferably, the bacteriocin-producing bacteria are cultured until the pH of the whey degradation medium becomes not higher than 5.8, and then the bacteriocin-producing bacteria are cultured while the pH of the whey degradation medium is adjusted to be in a range of not lower than 5.2 but not higher than 5.8. Still preferably, the bacteriocin-producing bacteria are cultured while adjusting the pH of the whey degradation medium to a range of not less than 5.5 but not more than 5.8. The pH may be adjusted by adding an alkaline solution to the whey degradation medium. As the alkaline solution, an aqueous potassium carbonate solution, an aqueous sodium bicarbonate solution, or the like can be used.

After culturing the bacteriocin-producing bacteria, a concentrated bacterial solution containing concentrated bacteriocin-producing bacteria is separated from a whey degradation medium (culture solution) in which the bacteriocin-producing bacteria are cultured. The concentrated bacterial suspension can be separated by centrifugation or membrane separation. When the concentrated bacterial liquid is separated by centrifugation, the gravitational acceleration is preferably not lower than 6000 (G).

The antibacterial activity of the thus obtained concentrated bacterial liquid of a bacteriocin-producing bacterium is several tens times or more higher than that of a concentrated bacterial liquid of a bacteriocin-producing bacterium cultured without adjusting the pH of the whey degradation medium during the culturing. That is, by culturing the bacteriocin-producing bacteria while maintaining the pH of the whey-degrading medium in the range of 5 to 6, the antibacterial activity of the concentrated bacterial liquid can be controlled and a concentrated bacterial liquid having a high antibacterial activity can be efficiently obtained.

When the concentrated bacterial suspension of the present embodiment is added to food as a food preservative, the amount of the concentrated bacterial suspension of the present embodiment to be added to food is smaller than the amount of a conventional food preservative that is generally required. That is, the concentrated bacterial liquid of the present embodiment added to food as a food preservative can suppress the change in the original taste of food and can also improve the storage quality of food.

Next, a method for producing the fermented milk (yogurt) of the present embodiment will be specifically described. First, a yogurt mix as a raw milk is prepared. The yogurt mix is prepared by mixing skim milk powder, whey protein, water, and the like into raw milk. In addition, granulated sugar, fruit pulp, fruit juice, etc. can be added into the yogurt mix.

After homogenizing and sterilizing the yogurt mix in the same manner as in the conventional manner, the yogurt mix is inoculated with a starter, the bacteriocin-producing bacteria obtained by the above-described method for culturing lactic acid bacteria, or a concentrated bacterial solution thereof. The inoculum size of the concentrated bacterial liquid of the bacteriocin-producing bacteria is preferably not less than 0.01wt% but not more than 0.1wt% based on the total weight of the yogurt mix. The amount of the inoculum of the concentrated bacterial liquid of the bacteriocin-producing strain is more preferably not less than 0.01wt% but not more than 0.05 wt%.

The lactic acid bacterium used as a starter may be the same lactic acid bacterium as the bacteriocin-producing bacterium or may be a different lactic acid bacterium. In addition, concentrated cell suspension may be added to the yogurt mix before the step of homogenizing the yogurt mix or the step of sterilizing the yogurt mix.

Yogurt is produced by fermenting a yogurt mix inoculated with bacteriocin-producing bacteria or a concentrate thereof which is a cell suspension. The fermentation conditions may be the same as conventional conditions. In the yogurt produced by the method discussed in this embodiment, the increase in acidity immediately after the production is suppressed, as compared with the yogurt to which the concentrated cell suspension is not added. This is because the activity of lactobacillus bulgaricus in yogurt is inhibited by bacteriocin contained in a high concentration in the concentrated cell suspension. Therefore, the yogurt produced by the method discussed in this embodiment can stably maintain the beautiful taste immediately after preparation over the entire shelf life (about 2 weeks) as compared with the existing yogurt.

In the method for producing fermented milk according to the present embodiment, the amount of the concentrated bacterial suspension used as a food preservative can be reduced to about 1/10, which is the amount of a food preservative generally used. Thus, the original taste of yogurt can be prevented from being spoiled by the addition of concentrated cell suspension.

Examples

Next, an example of the method for culturing lactic acid bacteria according to the present invention will be described with reference to the drawings.

Example 1

Fig. 1 is a schematic diagram illustrating the composition of the whey degradation medium used in example 1. First, the whey degradation medium used in example 1 was prepared. Specifically, an aqueous whey solution was prepared by mixing 8.70 wt% of whey powder (manufactured by mingzhi dairy), 1.50 wt% of concentrated whey protein (WPC80, manufactured by NZMP) and 88.80 wt% of water, based on the total weight of the whey degradation medium. Then, the whey protein in the aqueous whey solution was degraded by adding 0.10 wt% of proteolytic ENZYME (protease a "AMANO" G, manufactured by AMANO ENZYME corporation) to the aqueous whey solution.

Then, to the whey aqueous solution in which whey protein was decomposed, 0.20 wt% of brewer' S yeast extract (manufactured by Korea beer Co.), 0.50 wt% of fish meat extract (manufactured by Maruhanarich food Co.), 0.10 wt% of sodium ascorbate, and 0.05wt% of iron sulfate (FeSO4), 0.05wt% of emulsifier (SUN SOFT Q-17S (Decarol monooleate, manufactured by Sun chemical Co.) were added to prepare a whey degradation medium.

Then, Lactobacillus gasseri OLL2959(NITE BP-224, patent microorganism Collection) was inoculated into the whey degradation medium so that the viable cell count was 2-4X 10⁷cfu/ml. After the lactobacillus gasseri OLL2959 was cultured until the pH value of the whey degradation medium became 5.5, the lactobacillus gasseri OLL2959 was cultured by neutralization. Specifically, while adding an aqueous potassium carbonate solution (40 wt%) to the whey degradation medium and stirring so that the pH of the whey degradation medium is not lower than 5.5 from time to time, lactobacillus gasseri OLL2959 was cultured at 34 ℃ for 22 hours (neutralization culture). Wherein the neutralization culture is performed by blowingUnder anoxic conditions of carbon dioxide. After the neutralization culture, the number of viable bacteria of lactobacillus gasseri OLL2959 in the whey degradation medium (culture solution) was measured according to the infusion culture method using BCP medium. The viable bacteria count of Lactobacillus gasseri OLL2959 is 1.81X 10¹⁰cfu/ml。

The whey degradation medium (culture solution) after the culture was neutralized by centrifugation (acceleration of gravity: 6000G), to obtain a concentrated solution of Lactobacillus gasseri OLL2959 of example 1. The antibacterial activity of the concentrated bacterial suspension of example 1 was measured by using a method which will be described later. The antibacterial activity of the concentrated bacterial suspension of example 1 per 1ml was 72400AU (Arbitrary Unit). In addition, the concentrated bacterial liquid of example 1 was used at a ratio of 1X 10⁹The antibacterial activity of cfu was about 4000 AU.

In addition, using the whey degradation medium of example 1, lactobacillus gasseri OLL2959 was subjected to neutralization culture while changing the pH conditions of the whey degradation medium. As a result, the antibacterial activity of the concentrated bacterial suspension obtained by the neutralization culture at a pH of 5.2 to 5.8 was the same as that of the concentrated bacterial suspension of example 1. The antimicrobial activity of the concentrated bacterial suspension obtained by the neutralization culture at pH 5 to 6 was slightly lower than that of the concentrated bacterial suspension of example 1.

To confirm the effect of the neutralization culture, the whey degradation medium inoculated with lactobacillus gasseri OLL2959 in the same manner as in example 1 was allowed to stand at 37 ℃ for 20 hours to statically culture lactobacillus gasseri OLL2959 (comparative example 1). The whey degradation medium (culture solution) statically cultured was centrifuged (acceleration of gravity: 6000G), thereby obtaining the concentrated bacterial suspension of comparative example 1.

In the whey degradation medium (culture solution) after static culture, the viable bacteria count of lactobacillus gasseri OLL2959 was 2.63 × 10⁹cfu/ml. In addition, the antibacterial activity per 1ml of the concentrated bacterial suspension of comparative example 1 was less than 100 AU. Concentrated bacterial suspension of comparative example 1 at a ratio of 1X 10⁹The antibacterial activity of cfu is less than about 40 AU.

Fig. 2 illustrates respective measurement results of viable bacteria count and antibacterial activity of concentrated bacterial liquid of lactobacillus gasseri OLL2959 after cultivation in example 1 and comparative example 1. As described above, the concentrated bacterial suspension of example 1 was used at a ratio of 1X 10⁹The antibacterial activity of cfu was 4000AU, while the concentrated bacterial solution of comparative example 1 was 1X 10 per 1X 10⁹cfu has an antibacterial activity of less than about 40 AU. That is, the antibacterial activity per viable bacteria count of the concentrated cell suspension obtained by the neutralization culture (example 1) was about 100 times that of the concentrated cell suspension obtained by the static culture (comparative example 1).

In addition, the viable bacteria count of lactobacillus gasseri OLL2959 in the whey degradation medium (culture solution) after the neutralization culture is about one order of magnitude higher than that in the whey degradation medium (culture solution) after the static culture. The following conclusions are inferred from this: since lactobacillus gasseri OLL2959 is made active by maintaining the pH of the whey degradation medium at not less than 5.5 at the time of neutralization culture, bacteriocins are efficiently produced.

In this manner, the pH of the whey degradation medium is adjusted to a range of 5 to 6 during the culture and the bacteriocin-producing bacteria are subjected to neutralization culture, whereby the antibacterial activity of the concentrated bacterial liquid can be controlled to a very high level.

Example 2

Fig. 3 is a schematic diagram illustrating the composition of the whey degradation medium used in example 2. To prepare the whey degradation medium used in example 2, a whey aqueous solution in which whey protein was decomposed was prepared in the same manner as in example 1.

To the whey aqueous solution in which whey protein was decomposed, 0.20 wt% of brewers yeast extract (manufactured by Korea beer Co.), 0.50 wt% of fish meat extract (manufactured by MARUHA NICHRO food Co., Ltd.), 0.10 wt% of sodium ascorbate, 0.05wt% of iron sulfate (FeSO4) and 0.05wt% of emulsifier (SUN SOFT 81S (sorbitol monooleate), manufactured by Sun chemical Co., Ltd.) were added to prepare a whey degradation culture. The emulsifier used in example 2 was different from that used in example 1.

Then, the whey degradation medium was inoculated with Lactobacillus gasseri OLL2959 so that the viable bacteria count was 2-4X 10⁷cfu/ml. After culturing the lactobacillus gasseri OLL2959 until the pH value of the whey degradation medium becomes 5.5, the lactobacillus gasseri OLL2959 is cultured in a neutralized manner, as in example 1. Specifically, while adding an aqueous potassium carbonate solution (40 wt%) to the whey degradation medium so that the pH of the whey degradation medium is frequently not lower than 5.5, the Lactobacillus gasseri OLL2959 was neutralized and cultured at 34 ℃ for 22 hours. After the neutralization culture, the number of viable bacteria of lactobacillus gasseri OLL2959 in the whey degradation medium (culture solution) was measured using the same method as in example 1. The viable bacteria count of the strain Lactobacillus gasseri OLL2959 is 1.84X 10¹⁰cfu/ml。

The concentrated bacterial liquid of example 2 was separated by centrifuging (acceleration of gravity: 6000G) to neutralize the whey degradation medium (culture liquid) after the culture. The antibacterial activity per 1ml of the concentrated bacterial suspension of example 2 was 51200 AU. Example 2 concentrated cell suspension per 1X 10⁹The antibacterial activity of cfu was about 2800 AU. The antibacterial activity was measured by the same method as in example 1 (described later).

In addition, the whey degradation medium of example 2 was used while changing the pH conditions of the whey degradation medium, and lactobacillus gasseri OLL2959 was cultured in a neutralized manner. As a result, the antibacterial activity of the concentrated bacterial suspension obtained by the neutralization culture at a pH of 5.2 to 5.8 was the same as that of the concentrated bacterial suspension of example 2. The antimicrobial activity of the concentrated bacterial suspension obtained by the neutralization culture at a pH of 5 to 6 was slightly lower than that of the concentrated bacterial suspension of example 2.

To confirm the effect of the neutralization culture, the whey degradation medium inoculated with lactobacillus gasseri OLL2959 in the same manner as in example 2 was statically cultured at 37 ℃ for 20 hours (comparative example 2). The statically cultured whey degradation medium (culture solution) was centrifuged (acceleration of gravity: 6000G) to separate the concentrated bacterial solution of comparative example 2.

In the whey degradation medium (culture solution) after static culture, the viable bacteria count of lactobacillus gasseri OLL2959 was 2.63 × 10⁹cfu/ml. Further, the antibacterial activity per 1ml of the concentrated bacterial suspension of comparative example 2 was less than 100AU, and the antibacterial activity per 1X 10 of the concentrated bacterial suspension of comparative example 2 was 1X 10⁹The antibacterial activity of cfu is less than about 40 AU.

Fig. 4 illustrates respective measurement results of viable bacteria count and antibacterial activity of concentrated bacterial liquid of lactobacillus gasseri OLL2959 after culture in example 2 and comparative example 2. As described above, the concentrated bacterial suspension of example 2 was used at a ratio of 1X 10⁹The antibacterial activity of cfu was 2800 AU. Concentrated bacterial suspension of comparative example 2 at a ratio of 1X 10⁹The antibacterial activity of cfu is less than about 40 AU. That is, the antibacterial activity per viable bacteria count of the concentrated cell suspension obtained by the neutralization culture (example 2) was 70 times or more the antibacterial activity per viable bacteria count of the concentrated cell suspension obtained by the static culture (comparative example 2).

In addition, the viable bacterial count of the lactobacillus gasseri OLL2959 after the neutralization culture is about one order of magnitude higher than that of the lactobacillus gasseri OLL2959 after the static culture. The following can also be deduced from example 2: since lactobacillus gasseri OLL2959 is made active, bacteriocins are produced efficiently.

Thus, by culturing lactobacillus gasseri OLL2959 under the conditions of example 2, the antibacterial activity of the concentrated bacterial suspension can be controlled to a very high level. Further, since the antibacterial activity of the concentrated bacterial suspension of example 1 or example 2 is several tens to several hundreds times that of the concentrated bacterial suspension of comparative example 1 or comparative example 2, it can be seen that the emulsifier that can be used in the whey degradation medium is not particularly limited.

Method for measuring antibacterial activity

Then, a method for measuring the antibacterial activity of the concentrated bacterial suspension of lactobacillus gasseri OLL2959 will be discussed by taking the concentrated bacterial suspension of example 1 as an example. The antibacterial activity of the concentrated bacterial solutions of example 2, comparative example 1 and comparative example 2 was also measured by the same method.

MRS medium (BECTON, manufactured by DICKINSON) is used. Test media were prepared by adding 0.1% (v/v) indicator bacteria based on MRS medium. Lactobacillus delbrueckii subsp. bulgaricus ATCC11842 (model strain) was used as an indicator.

The cryopreserved concentrated bacterial suspension of example 1 was kept in a hot water bath for 5 minutes, and then a 1% (v/v) aqueous solution of the concentrated bacterial suspension of example 1 was prepared. The aqueous solution of the concentrated bacterial suspension was gradually diluted at a rate of 2 times each time to obtain a plurality of diluted solutions of the concentrated bacterial suspension, and the dilution rate levels were different. Dilution rate levels ranged from 8-12. Thus, dilution 2 of concentrated bacterial suspension was prepared⁸-2¹²Double solution. The diluted solutions were added to the test media, respectively, using anaeropack⁸-2¹²The test medium in double solution was incubated at 37 ℃ for 24 hours under anaerobic conditions.

After the hypoxic culture, the highest level of dilution rate (N) indicating that the bacteria did not grow was confirmed. Then, the antibacterial Activity (AU) of the concentrated bacterial suspension of example 1 was obtained based on the maximum level (N) of the dilution ratio and the concentration (0.01: 1%) of the aqueous solution of the concentrated bacterial suspension. The antibacterial activity can be obtained based on the following calculation formula.

The antibacterial Activity (AU) is the highest level of dilution (N)/concentration of the aqueous solution of the concentrated bacterial suspension (0.01).

Example 3

In example 3, the conditions for centrifugation of the concentrated bacterial liquid of lactobacillus gasseri being neutralized and cultured are discussed. The whey degradation medium (culture solution) after the neutralization culture of example 1 was centrifuged with various values of the acceleration of gravity set. Then, the antibacterial activity of each concentrated bacterial liquid under different centrifugal separation conditions was obtained.

Fig. 5 is a graph illustrating a relationship between the acceleration of gravity and the antibacterial activity of the concentrated bacterial liquid. As shown in FIG. 5, when the centrifugal separation is performed, the antibacterial activity of the concentrated bacterial suspension increases as the acceleration of gravity increases. Also, when the acceleration of gravity was not less than 6000G at the time of centrifugal separation, the antibacterial activity became constant (approximately 72400 AU). It was thus clear that a concentrated bacterial liquid having a high antibacterial activity can be efficiently obtained by centrifuging the concentrated bacterial liquid at a gravitational acceleration of not less than 6000G.

Next, as an example of the method for producing fermented milk of the present invention, a method for producing yogurt by using the concentrated cell suspension of example 1 will be described.

Example 4

Fig. 6 is a schematic diagram illustrating the compositions of three yogurt mixes used in example 3. First, yogurt mixes of mixes a to C were prepared by mixing 83.90 wt% of cow's milk, 1.51 wt% of skim milk (both manufactured by mingzhi dairy), 0.80 wt% of concentrated whey protein (WPC34, manufactured by DOMO corporation) and water, based on the total weight of the yogurt mix. As shown in fig. 6, the mixing ratio of water is different in the mixtures a-C.

The yogurt mixes of mixes a-C were homogenized and sterilized in the same manner as conventionally done, and then the yogurt mixes of mixes a-C were cooled to about 40 ℃. After cooling, the yogurt mix of mixes a-C was inoculated with 2.00 wt% lactic acid bacteria starter. Lactic acid bacteria isolated from Mingzhi Bulgaria yogurt (manufactured by Mingzhi Dairy Co.) were used as lactic acid bacteria starter.

The yogurt mix of mixture B was inoculated with 0.05wt% of the concentrated bacterial suspension of example 1. The yogurt mix of mixture C was inoculated with 0.10 wt% of the concentrated bacterial suspension of example 1. The yogurt mix of mix a was not inoculated with the concentrated inoculum of example 1.

The yogurt mixes of the respective mixes a-C were fermented at a temperature of 40 ℃ until the lactic acid concentration became about 0.75% to prepare yogurt. Then, the yogurt of the mixtures a-C was refrigerated at temperatures of 5 ℃ and 10 ℃, respectively, and the lactic acid concentration in the yogurt of the mixtures a-C was measured within the usual shelf life (about 2 weeks from the preparation day).

FIG. 7 is a graph showing the change in lactic acid concentration with time when yogurt of the mixtures A to C were stored at 5 ℃. FIG. 8 is a graph showing the change with time in the lactic acid concentration when yogurt of the mixtures A to C were stored at 10 ℃.

As shown in fig. 7 and 8, the lactic acid concentrations of the yoghurts of the mixtures B and C were lower than that of the mixture a regardless of the storage temperature. That is, it was found that the yogurt of the mixture B and the mixture C inoculated with the concentrated cell suspension of example 1 was suppressed in fermentation in a refrigerated state, and the original taste and quality thereof could be maintained.

From the fact that the lactic acid concentration of the yogurt of the mixture C was slightly lower than that of the yogurt of the mixture B, it was found that the increase in the lactic acid concentration tended to be more suppressed as the inoculation amount of the concentrated cell suspension increased. However, as the amount of the concentrated cell suspension to be inoculated increases, the original taste of yogurt may be deteriorated. Since the difference in lactic acid concentrations of the yogurt of the mixture B and the mixture C is small, the inoculum amount of the concentrated cell suspension may be in the range of 0.01wt% to 0.05wt% if only the increase in lactic acid concentration of the yogurt is intended to be suppressed.

In the yogurt mixes of mixes B and C, there was no difference in the effect of suppressing the increase in the lactic acid content of the yogurt between the above case and the case of inoculating the concentrated cell suspension of example 1 before homogenizing and sterilizing the yogurt mixes. It is thus apparent that the bacteriocin-producing bacteria inoculated with the concentrated bacterial liquid may be dead or live, and that neither of the bacteriocin-producing bacteria nor the antibacterial activity of the concentrated bacterial liquid is affected.

Example 5

Fig. 9 is a schematic diagram illustrating the compositions of two yogurt mixes used in example 5. Whey Protein Isolate (WPI) was added to the yogurt mix of example 5 in place of Whey Protein Concentrate (WPC), and granulated sugar was also added.

First, yogurt mixes of each of mixes D and E were prepared by mixing 84.20 wt% of cow's MILK, 1.76 wt% of skim MILK (all manufactured by mingzhi dairy), 0.20 wt% of whey protein isolate (WPI, NEW ZEALAND MILK PRODUCTS), 4.50 wt% of granulated sugar, and water, based on the total weight of the yogurt mix. As shown in fig. 9, the mixing ratio of water is different in the mixtures D and E.

The yogurt mixes of mix D, mix E were homogenized and sterilized in the same manner as conventionally performed, and the yogurt mixes of mix D, mix E were cooled to about 40 ℃. After cooling, the yogurt mix of mix D, mix E was inoculated with 3.00 wt% lactic acid bacteria starter. Lactic acid bacteria isolated from Mingzhi sour milk (manufactured by Mingzhi Dairy Co.) were used as lactic acid bacteria starter. The yogurt mix of mixture E was inoculated with 0.05wt% of the concentrated cell suspension of example 1. The yogurt mix of blend D was not inoculated with the concentrated cell suspension of example 1.

In example 5, a yogurt mix inoculated with 0.10 wt% of the concentrated cell suspension was not used. This is because, in example 4, it is obvious that even if the inoculum size of the concentrated bacterial suspension is 0.05wt%, the acidity-suppressing effect can be sufficiently obtained.

Until the lactic acid concentration became about 0.75%, the respective yogurt mixes of mix D, mix E were fermented at a temperature of 40 ℃ to prepare yogurt. The yogurt of mixture D, E was refrigerated at a temperature of 5 ℃, and the lactic acid concentration in the yogurt of mixture D, E was measured within the usual shelf life of the yogurt (approximately 2 weeks from the preparation day).

Fig. 10 is a graph showing changes over time in the lactic acid concentrations of the yogurts of the mixes D and E. As shown in fig. 10, the lactic acid concentration of the yogurt of blend E was lower than that of the yogurt of blend D. From this, it is apparent that even when the concentrated cell suspension of example 1 is used for preparing a yogurt with sugar, the increase in lactic acid concentration of the yogurt with sugar can be suppressed.

In the yogurt mix of mix E, there was no difference in the effect of suppressing the increase in the lactic acid concentration of yogurt between the above case and the case of inoculating the concentrated cell suspension of example 1 before homogenizing and sterilizing the yogurt mix.

In examples 4 and 5, the amount of the inoculated concentrated bacterial suspension of example 1 was 1/10 or less, which is the amount of a conventionally used food preservative, as a food preservative. That is, the fermented milk production methods described in examples 4 and 5 can suppress the change in the original taste of yogurt due to inoculation of the concentrated cell suspension of example 1, and can maintain the original taste of yogurt for a normal shelf life. The same tests as in examples 4 and 5 were carried out by using the concentrated bacterial suspension of example 2. As a result, the yogurt produced using the concentrated cell suspension of example 2 can suppress the change in the acidity of the yogurt, as in the case of the concentrated cell suspension of example 1.

The present invention has been described with reference to the embodiments shown in the drawings, but the present invention is not limited to the described forms, and it should be understood that the scope of the present invention is defined by the scope of the claims.

Claims (4)

1. A method of producing fermented milk comprising:

a raw milk producing step of producing a yogurt mix;

a concentrated cell suspension producing step of culturing a bacteriocin-producing bacterium which is a bacteriocin-producing lactic acid bacterium to produce a concentrated cell suspension containing the bacteriocin-producing bacterium, wherein the bacteriocin-producing lactic acid bacterium is Lactobacillus gasseri;

an addition step of adding a starter and not less than 0.01wt% but not more than 0.1wt% of the concentrated cell suspension to the yogurt mix, based on the total weight of the yogurt mix; and

a fermentation step of fermenting the yogurt mix to which the concentrated bacterial liquid is added;

wherein the concentrated bacterial liquid generating process comprises:

a culture solution preparation step of preparing a culture solution containing whey decomposed by a proteolytic enzyme;

a culture step of inoculating the bacteriocin-producing bacteria into the culture solution, and culturing the bacteriocin-producing bacteria while maintaining the pH of the culture solution inoculated with the bacteriocin-producing bacteria at not less than 5.2 but not more than 5.8; and

a separation step of separating the concentrated bacterial liquid from the culture liquid in which the bacteriocin-producing bacteria have been cultured.

2. The method for producing fermented milk according to claim 1,

the amount of the concentrated bacterial liquid added to the yogurt mix is not less than 0.01wt% but not more than 0.05wt% based on the total weight of the yogurt mix.

3. The method for producing fermented milk according to claim 1,

in the culture step, an alkaline solution is added to the culture solution inoculated with the bacteriocin-producing bacterium to adjust the pH of the culture solution inoculated with the bacteriocin-producing bacterium.

4. The method for producing fermented milk according to claim 1,

in the separation step, the concentrated bacterial liquid is centrifuged from the culture liquid in which the bacteriocin-producing bacteria are cultured at a gravitational acceleration of 6000G or more.