JP6340513B2 - Method for producing dairy lactic acid bacteria beverage - Google Patents

Description

  The present invention relates to a method for producing a dairy lactic acid bacteria beverage in which the number of living bacteria of lactic acid bacteria is 10 million / ml or more, and in particular, the number of living bacteria of lactic acid bacteria is maintained at 10 million / ml or more over 4 weeks. And a method for producing a dairy lactic acid bacteria beverage containing a peptide having ACE (angiotensin converting enzyme) inhibitory activity.

  Food containing live lactic acid bacteria is said to be able to improve the balance of the human digestive system and suppress the occurrence of illness by taking it, and is generally called probiotic food. Among such foods, dairy lactic acid bacteria beverages are prescribed that the number of live bacteria of lactic acid bacteria is 10 million / ml or more, and the number of live bacteria needs to be maintained within the expiration date. . In general, the shelf life of dairy lactic acid bacteria beverages is set to about 2 weeks, so this type of product needs to maintain the above viable count for about 3 weeks including the reserve rate. Furthermore, it is desirable to maintain the above viable count for about 4 weeks due to the manufacturing process of dairy lactic acid bacteria beverages. In conventional dairy lactic acid bacteria beverages, Lactobacillus casei having excellent acid resistance and lactic acid-producing ability has been mainly used as lactic acid bacteria.

  By the way, some lactic acid bacteria produce peptides having an ACE inhibitory activity exhibiting blood pressure lowering activity, and Lactobacillus helveticus is known as such a lactic acid bacterium. By using this Lactobacillus helveticus and culturing this lactic acid bacterium in an animal milk medium, a method for producing a fermented dairy product containing a peptide having an ACE inhibitory activity and a blood pressure lowering action has been proposed ( For example, see Patent Document 1.)

  In addition, two types of lactic acid bacteria are mixed and cultured to produce a lactic acid bacteria beverage. For example, a method for producing a lactic acid bacteria beverage by mixing and cultivating Bifidobacterium breve (a kind of heterolactic acid bacteria also called bifidobacteria) and lactobacillus casei in a medium such as skim milk containing milk as a main component Has been proposed (see, for example, Patent Document 2).

Japanese Patent No. 3028411 (page 2-3) JP-A-5-227946 (page 2-3)

  As disclosed in Patent Document 1, a functional fermented dairy product containing a peptide having ACE inhibitory activity can be obtained by culturing Lactobacillus helveticus in an animal milk medium. However, Lactobacillus helveticus produces a large amount of lactic acid at an early stage of culture, and the bacteria are weakened and killed by the lactic acid produced by itself. Therefore, it is difficult to obtain a dairy lactic acid bacteria beverage in which the number of living lactic acid bacteria is maintained at 10 million / ml or more over 4 weeks by the method of culturing alone using Lactobacillus helveticus. For this reason, lactic acid bacteria beverages cultured using Lactobacillus helveticus are generally commercialized as sterilized dairy lactic acid bacteria beverages (number of viable bacteria: 0). Such a dairy lactic acid bacteria beverage (bactericidal) is a functional food containing a peptide having ACE inhibitory activity, but is not a probiotic food in which lactic acid bacteria live and reach the intestine and have a beneficial function in the intestine.

  On the other hand, in the method of simply culturing two types of lactic acid bacteria, Lactobacillus casei and Lactobacillus helveticus, the bacteria are weak and the viable number of lactic acid bacteria is maintained at 10 million / ml or more over 4 weeks. No dairy lactic acid bacteria beverage is obtained (see experimental results below). In addition, in a simple mixed culture, the growth of Lactobacillus helveticus is suppressed and a sufficient amount of ACE inhibitory peptide is not produced.

  The present invention has been made in view of the above circumstances, and the number of living lactic acid bacteria is maintained at 10 million / ml or more for about 4 weeks, and is sufficient to exhibit a blood pressure lowering effect. It is an object of the present invention to provide a method for producing a dairy lactic acid bacteria beverage containing a peptide having an ACE inhibitory activity to an extent.

As a means for solving the above problems, dairy products having desired properties and characteristics by culturing two types of lactic acid bacteria, Lactobacillus casei and Lactobacillus helveticus, at different inoculation periods It has been found that a lactic acid bacteria beverage can be obtained. That is, the invention according to claim 1 is a method for producing a dairy lactic acid bacteria beverage in which the number of living lactic acid bacteria is 10 million / ml or more, wherein Lactobacillus casei is inoculated into the dairy material, After culturing Lactobacillus casei from the bacterium for 20 to 30 hours , the culture is inoculated with Lactobacillus helveticus and further cultured for 1 to 2 days.

In the above-described method for producing a dairy lactic acid bacteria beverage, the time from inoculating Lactobacillus casei to inoculating Lactobacillus helveticus, that is , the time for culturing Lactobacillus casei alone is 20 hours. More preferably, it is set to ˜24 hours.

According to the manufacturing method of the invention according to claim 1, first, Lactobacillus casei is inoculated into the dairy raw material (medium), and the Lactobacillus casei is cultured alone for 20 to 30 hours from the inoculation , thereby Lactobacillus casei grows in the medium. Lactobacillus casei is inoculated after Lactobacillus casei grows to some extent, but Lactobacillus casei has a relatively small amount of lactic acid produced at the beginning of the culture. Does not adversely affect growth. And the peptide which has ACE inhibitory activity is produced with the proliferation of Lactobacillus helveticus. Moreover, since a large amount of lactic acid is produced with the growth of Lactobacillus helveticus, the lactic acid in Lactobacillus helveticus weakens the bacteria and is likely to die. On the other hand, Lactobacillus casei is acid-resistant, and the culture is completed in 1 to 2 days from the inoculation time of Lactobacillus helveticus. Most of the fungi are thought to be Lactobacillus casei) and ACE-inhibiting peptides.
Therefore, according to the production method of the invention according to claim 1, the number of viable bacteria of lactic acid bacteria is maintained at 10 million / ml or more for about 4 weeks, and ACE inhibition is sufficient to show a blood pressure lowering effect. A dairy lactic acid bacteria beverage containing a peptide having activity can be obtained.

Further, with the 2 0 24 hours the time until the inoculated Lactobacillus helveticus after inoculated Lactobacillus casei, Lactobacillus helveticus after Lactobacillus casei has been properly grown Since the inoculation is performed, both lactic acid bacteria of Lactobacillus casei and Lactobacillus helveticus grow well. Therefore, the effect of the invention according to claim 1 can be obtained more reliably.

The storability of the dairy lactic acid bacteria drink obtained using the production method according to the present invention is compared with the storability of the dairy lactic acid bacteria drink obtained by simultaneously inoculating Lactobacillus casei and Lactobacillus helveticus. It is a graph. It is a graph which shows the relationship between the preservation | save property of the dairy product lactic acid bacteria drink when changing the number of days which mixed culture culture of Lactobacillus casei and Lactobacillus helveticus after cultivating Lactobacillus casei alone. Changes in systolic blood pressure when lactic acid bacteria culture solution obtained using the production method according to the present invention was administered to spontaneously hypertensive rats, changes in systolic blood pressure when water for injection was administered to spontaneously hypertensive rats, and It is a graph shown by contrast. Changes in diastolic blood pressure when lactic acid bacterium culture solution obtained using the production method according to the present invention is administered to spontaneously hypertensive rats, changes in diastolic blood pressure when water for injection is administered to spontaneously hypertensive rats, and It is a graph shown by contrast.

Hereinafter, preferred embodiments of the present invention will be described.
In the method according to the present invention, Lactobacillus casei and Lactobacillus casei and lactobacillus Helveticas is mixed and cultured. By combining single culture and mixed culture in this way, the number of viable lactic acid bacteria can be maintained at 10 million / ml or more for about 4 weeks, and ACE produced as Lactobacillus helveticus grows. A dairy lactic acid bacteria beverage containing an inhibitory peptide is obtained.

The single culture operation is performed by inoculating Lactobacillus casei in a solution (medium) in which a dairy material, for example, skim milk is dissolved in water. The temperature at the time of culture shall be 37 degreeC, for example. The time of single culture (the time from inoculating Lactobacillus casei to inoculating Lactobacillus helveticus) is 20 hours or more, and if shorter than that, the growth of Lactobacillus casei becomes insufficient, A dairy lactic acid bacteria beverage having a live lactic acid bacteria count of 10 million / ml or more cannot be obtained. On the other hand, if the single culture time becomes too long, Lactobacillus casei grows too much and Lactobacillus helveticus in the mixed culture phase becomes difficult to grow. As a result, the ACE inhibitory active peptide contained in the dairy lactic acid bacteria beverage The amount of is insufficient. For this reason, the time for single culture is 30 hours or less, more preferably 24 hours or less. Therefore, only culture time is 2 0 30 hours, more preferably from 20 hours to 24 hours.

  The mixed culture operation is performed by inoculating Lactobacillus helveticus into a culture solution of Lactobacillus casei and maintaining the temperature of the single culture period as it is. This mixed culture is performed for 1 to 2 days. If the period of the mixed culture is shorter than 1 day, Lactobacillus helveticus does not grow sufficiently, and the amount of the ACE inhibitory active peptide contained in the dairy lactic acid bacteria beverage becomes insufficient. On the other hand, when the period of the mixed culture becomes 3 days, not only Lactobacillus helveticus but also Lactobacillus casei will weaken and die, and the number of living lactic acid bacteria will be less than 10 million / ml within 2 weeks. .

  A lactic acid bacteria beverage (product) obtained by adding a syrup such as liquid sugar, fructose, and glucose to a lactic acid bacteria culture solution obtained by mixed culture after single culture as described above and homogenizing the mixture. To do.

Next, examples of the present invention will be described in comparison with comparative examples.
A 10% solution of skim milk powder dissolved in water was used as a liquid medium, and Lactobacillus casei was inoculated into the medium, and Lactobacillus casei was cultured at a temperature of 37 ° C. for 1 day. One day after inoculating Lactobacillus casei, Lactobacillus helveticus was inoculated into the culture solution, and Lactobacillus casei and Lactobacillus helveticus were further cultured for one day. For comparison, Lactobacillus casei and Lactobacillus helveticus were inoculated simultaneously in the same medium as described above, and Lactobacillus casei and Lactobacillus helveticus were mixed and cultured at a temperature of 37 ° C. for 2 days.

  The lactic acid bacteria culture solution obtained by the mixed culture after the single culture by the above-described operation and the lactic acid bacteria culture solution obtained by the mixed culture from the beginning are each stored for 0 to 28 days, and the live bacteria of the lactic acid bacteria within the storage period The number change was examined. The number of viable bacteria was measured by a dilution plate culture method using a GYP agar medium. The result is shown in FIG. A in the graph of FIG. 1 shows a change in the number of viable bacteria in a lactic acid bacteria culture solution obtained by single culture-mixed culture, and B shows a change in the number of viable bacteria in a lactic acid bacteria culture solution obtained by mixed culture.

  As can be clearly seen from the results shown in the graph of FIG. 1, in the lactic acid bacterium culture solution obtained by single culture-mixed culture, the number of viable bacteria decreased very little. The number is maintained above 10 million / ml. Therefore, the dairy lactic acid bacteria drink commercialized using this lactic acid bacteria culture solution has sufficient preservability. On the other hand, in the lactic acid bacteria culture solution obtained by the mixed culture, the viable count is markedly reduced, and the viable count becomes 10 million / ml or less after storage for about 2 weeks.

  Next, in the case of preparing a lactic acid bacteria culture solution by mixing and culturing after single culture by the same operation as described above, one day after inoculating Lactobacillus casei, Lactobacillus helveticus is inoculated, and then Lactobacillus When the period of mixed culture of casei and Lactobacillus helveticus was changed to 1 day, 2 days and 3 days, the storage stability of the obtained lactic acid bacteria culture solution was examined. The result is shown in FIG. C in the graph of FIG. 2 shows the change in the number of viable bacteria in the lactic acid bacteria culture solution obtained with the mixed culture period as 1 day, and D is the number of viable bacteria in the lactic acid bacteria culture solution obtained with the mixed culture period as 2 days E shows the change in the number of viable bacteria in the lactic acid bacteria culture solution obtained by setting the mixed culture period to 3 days.

  As can be seen from the results shown in the graph of FIG. 2, in the lactic acid bacteria culture solution obtained by single culture-mixed culture when the mixed culture period was 1 day and 2 days, the decrease in the number of viable bacteria was observed in both cases. The number of viable bacteria is maintained at 10 million / ml or more even if stored for about 4 weeks. On the other hand, when the mixed culture period is 3 days, in the lactic acid bacteria culture solution obtained by single culture-mixed culture, the number of viable bacteria is significantly reduced, and the number of viable bacteria is 1,000 after storage for about 10 days. It becomes 10,000 / ml or less. Therefore, from the viewpoint of storage stability of lactic acid bacteria beverages, it is appropriate to set the mixed culture period to 1 day to 2 days.

  In addition, in the case of preparing a lactic acid bacteria culture solution by performing mixed culture after single culture by the same operation as described above, inoculating Lactobacillus helveticus one day after inoculating Lactobacillus casei, When the period of mixed culture of casei and Lactobacillus helveticus was changed to 1, 2 and 3 days, the ACE inhibitory activity in the obtained lactic acid bacteria culture solution was examined. The ACE inhibitory activity was measured using a measurement kit (manufactured by Dojindo Laboratories, Inc., ACE Kit-WST). The results are shown in Table 1. “IC50 (μl / ml)” in Table 1 represents the concentration necessary to inhibit ACE activity by 50%, and the smaller the value, the higher the ACE inhibitory activity.

  As can be seen from the results shown in Table 1, even when the mixed culture period is 1 day, the obtained lactic acid bacteria culture solution shows a somewhat higher ACE inhibitory activity, but the culture solution with a mixed culture period of 2 days is better. When the ACE inhibitory activity is higher and the mixed culture period is 3 days, the ACE inhibitory activity of the culture solution is further increased. From this result, from the viewpoint of the functionality (ACE inhibitory activity) of the lactic acid bacteria beverage, a mixed culture period of 1 day is sufficient, but it is more preferable to set it to 2 to 3 days. However, considering the storage stability and production efficiency of the lactic acid bacteria beverage described above, it is appropriate to set the mixed culture time to 1 to 2 days. In addition, the pH of the lactic acid bacteria culture solution after two days of mixed culture reached 3.6, which is a pH necessary for protein stabilization during the production of a dairy lactic acid bacteria beverage.

  Next, in the case of preparing a lactic acid bacteria culture by mixing and culturing after single culture by the same operation as described above, the time from inoculating Lactobacillus casei to inoculating Lactobacillus helveticus (Lactobacillus The number of viable bacteria of Lactobacillus casei and Lactobacillus helveticus in the obtained lactic acid bacteria culture solution was measured when the casei single culture time was changed from 10 hours to 40 hours. The mixed culture time for Lactobacillus casei and Lactobacillus helveticus was 48 hours. The results are shown in Table 2.

  From the results shown in Table 2, when the single culture time is 20 hours to 40 hours, the number of live bacteria of Lactobacillus casei and Lactobacillus helveticus in the lactic acid bacteria culture solution is 10 million to 100 million / ml or more, respectively. Thus, when considering the degree of decrease in the number of viable bacteria accompanying the storage days shown in FIG. In particular, when Lactobacillus casei and Lactobacillus helveticus are mixed and cultured after culturing Lactobacillus casei alone for 20 to 30 hours, the viable count of Lactobacillus casei is 10 million / ml or more. The viable count of Helveticas is over 100 million / ml. Furthermore, when Lactobacillus casei and Lactobacillus helveticus are mixed and cultured after culturing Lactobacillus casei alone for 20 to 24 hours, the viable counts of Lactobacillus casei and Lactobacillus helveticus are both 100 million / ml. That's it. In consideration of production efficiency, a single culture time of 20 to 30 hours is sufficient. Furthermore, in order to improve the production efficiency and to increase the number of viable bacteria in the lactic acid bacteria beverage, the single culture time is 20 hours to 24 hours is preferred. In addition, it is thought that Lactobacillus helveticus will mostly die over time.

  A 10% solution of skim milk powder dissolved in water is used as a liquid medium. Lactobacillus casei is inoculated into the medium and cultured at 37 ° C. for 1 day. Helveticas was inoculated, and Lactobacillus casei and Lactobacillus helveticus were mixed and cultured for 2 days to prepare a lactic acid bacteria culture solution.

  The obtained lactic acid bacteria culture solution was administered to spontaneously hypertensive rats, and the blood pressure lowering effect of this lactic acid bacteria culture solution was tested. In each of the test group and the control group, 6 12-week-old spontaneously hypertensive rats (SHR / Izm strain) were preliminarily raised for about 1 week and fasted for about 17 hours. For the test group, the lactic acid bacterium culture solution obtained above was forcibly administered orally in a volume of 4 ml / kg (body weight), and systolic blood pressure and dilatation after 4 hours, 8 hours and 24 hours had elapsed. Period blood pressure was measured. In the control group, water for injection was forcibly administered once in a volume of 4 ml / kg (body weight), and systolic blood pressure and diastolic blood pressure were measured after 4 hours, 8 hours and 24 hours from the administration. did. FIG. 3 shows changes in systolic blood pressure, and FIG. 4 shows changes in diastolic blood pressure. F in the graph of FIG. 3 shows the blood pressure change for the test group, and G shows the blood pressure change for the control group. Moreover, H in the graph of FIG. 4 shows the blood pressure change about a test group, and I shows the blood pressure change about a control group.

  As can be seen from the test results shown in FIGS. 3 and 4, the test group administered with the lactic acid bacteria culture solution produced by the method according to the present invention had systolic blood pressure and diastole after 4 hours from the administration. Any decrease in blood pressure was observed. On the other hand, in the control group in which only water for injection was administered, neither change in systolic blood pressure nor diastolic blood pressure was observed after administration.

  Dairy lactic acid bacteria beverage (product of the present invention) was produced by mixing and homogenizing 200 ml of syrup prepared by adding 1 g of stabilizer to 46 g of liquid sugar to 100 ml of lactic acid bacteria culture solution obtained by the above operation. .

  By this invention, the new dairy product lactic acid bacteria drink which has functionality can be provided, and this invention may be utilized greatly in the field of food.

Claims (2)

In a method for producing a dairy lactic acid bacteria beverage in which the number of living lactic acid bacteria is 10 million / ml or more,
After inoculating Lactobacillus casei as a dairy raw material, cultivating Lactobacillus casei for 20 to 30 hours after the inoculation, inoculate Lactobacillus helveticus into the culture, and further mixing for 1 to 2 days A method for producing a dairy lactic acid bacteria beverage characterized by culturing. The method for producing a dairy lactic acid bacteria beverage according to claim 1, wherein Lactobacillus helveticus is inoculated 20 to 24 hours after inoculating Lactobacillus casei in the dairy raw material.

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