JPS62258323A - Agent for suppressing increase of serum cholesterol level - Google Patents

Abstract

PURPOSE:To obtain the titled agent effective in suppressing the increase of serum cholesterol level caused by intake of food, by culturing lactic bacteria belonging to Lactobacillus genus or Bifidobacterium genus and using the obtained cultured product or microbial cell as an active component. CONSTITUTION:A lactic bacterial strain belonging to Lactobacillus genus [e.g. Lactobacillus acidophilus SBT-2056 (FERM P-8744)] or Bifidobacterium genus [Bifidobacterium longum SBT-2933R (FERM P-8743)] is cultured and the obtained cultured product or microbial cell is used as an active component of the objective agent. The cultured product or bacterial cell suppresses the alimentary cholesterol increase caused by the intake of cholesterol-rich food (e.g. yolk, butter, etc.). The suppressing agent may be administered by adding about 2-5wt% freeze-dried cell, etc., to a cholesterol-rich food and taking the agent simultaneous with the food or before or after meals.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an agent for suppressing the increase in serum cholesterol level that is used to suppress the increase in serum cholesterol level associated with the intake of foods containing high cholesterol.

In recent years, with the spread of high-fat diets, the incidence of adult diseases such as hyperlipidemia and arteriosclerosis has increased, and this phenomenon is particularly prevalent among middle-aged and elderly people. It extends not only to Yoshitoshi but also to Yoshitoshi. As a result, there is even a tendency to avoid eating fat, especially animal fat.

Originally, most of the cholesterol in a healthy person's body was thought to be derived from biosynthesis, but as mentioned above, today's intake of animal fat has increased, and dietary cholesterol has become a major source of cholesterol in the body, that is, serum cholesterol. It is becoming increasingly involved in changes in cholesterol levels.

On the other hand, cholesterol plays an important role in the synthesis of bile acids and hormones, and as a component of cell membranes.

Based on the above, a diet that properly controls dietary cholesterol is emphasized, and several diets have been proposed for this purpose, but no effective means of reducing in vivo cholesterol levels using dietary cholesterol has yet been reported. do not have.

The present invention was made in view of the situation on the ridge, and when ingesting high cholesterol-containing foods such as egg yolks and butter, it is possible to simultaneously ingest these foods orally. An object of the present invention is to provide a serum cholesterol increase inhibitor for effectively suppressing the upper limit of dietary cholesterol based on foods.

The present inventors have found that a culture or bacterial cells obtained by culturing lactic acid bacteria belonging to the genus Lactobacillus and Bifidobacterium have the above-mentioned effect of suppressing the rise in serum cholesterol, and have succeeded in solving the above-mentioned problem.

The present invention will be explained in detail below.

The present invention is also characterized by an agent for suppressing the increase in serum cholesterol, which uses as an active ingredient a culture or bacterial cells obtained by culturing lactic acid bacteria belonging to the genus Lactobacillus or Bifidobacterium.

An example of the lactic acid bacteria belonging to the genus Lactobacillus used in the present invention to solve the problem is Lactobacillus acidophilus, and an example of the lactic acid bacteria belonging to the genus Bifidobacterium is Bifidobacterium longum. The strains of these microorganisms have been deposited with the Microbial Research Institute under the following deposit numbers.

Lactobacillus acidophilus SB? -2056 stocks-
・-・Microtechnology Research Institute No. 8744 Bifidobacterium longum SB? -2933R strain--Feikoken Bacterium No. 8743 These microorganisms are known as useful bacteria in the intestinal tract and are extremely safe for the human body.

The properties of the above-mentioned bacterial strain are shown in Table 1.
Determinative Bacteriology
The properties of the above microorganisms are similar to those described in "The World of Intestinal Bacteria" by Mitsuoka and "The World of Intestinal Bacteria" by Mitsuoka.

The inhibitory effect on serum cholesterol elevation of each microorganism of Tamade used in the present invention can be confirmed by the following screening method.

Screening method: (a) Preparation of test feed Add 0.5% yeast extract to 10% Hemoto skim milk medium,
After sterilization at 121°C for 10 minutes, the medium was inoculated with 2% of various lactic acid bacteria, cultured at each optimum temperature for 16 hours, and then the culture was freeze-dried.

The dried product of each culture thus obtained was added to 20% of the feed of the following formulation (Table 2) that causes hyperlipidemia,
Subjected to animal experiments using rats.

As a control, feed to which the same amount of skim milk powder was added instead of the above culture was used.

Table 2 (匈t%) (b) Animal experiment SD male and female rats (4 weeks old) were pre-fed for 7 days on commercial feed (CE-2: Nippon Flare ■), and then divided into groups so that their body weights were approximately averaged. The mice were divided into groups of 8, and each group was given free access to each of the above feeds.

Thereafter, on the 7th day when the serum cholesterol level of the rats reached almost the maximum level, blood was collected from the tail vein of each rat and the serum cholesterol level was measured. The measured values were subjected to statistical processing using the multiple comparison method of Dunnett et al.

As a result of the above experiment, a significant effect (P<0.05) was observed on the Lactobacillus bulgaricus seedlings, Bifidobacterium longum strains, and Lactobacillus acidophilus strains among the lactic acid seedlings used. Ta.

In addition, although there was no significant difference, strains of Lactobacillus casei also tended to suppress the increase in serum cholesterol.

However, among these strains, Lactobacillus acidophilus SB? -2056 strain had the strongest suppressive effect, followed by Bifidobacterium longum SBT~
It was observed that the same effect of the 2933R strain was strong.

Next, a method for preparing the serum cholesterol increase inhibitor according to the present invention will be explained.

The microorganisms that have the effect of suppressing the increase in serum cholesterol obtained by the above screening method are added to a medium containing whole milk, skim milk, buttermilk, whey, or powders thereof at a concentration of 0.1 to 0.5%. The cells are then sterilized at 121°C for 10 minutes, then inoculated and cultured at a temperature of 35-42°C, and the resulting culture is washed as is or after centrifugation, washed, and lyophilized.

The concentration of the medium used for the above culture should be selected depending on the medium components used. For example, when using skim milk, the concentration of the medium should be 5 to 12% in terms of solids. The mixture is neutralized with 1 to 2N ammonia water so that the pH during cultivation is 6.5 to 6.8, and then used for cultivation.

The present invention contains as an active ingredient the culture or freeze-dried Kokutai collected by centrifugation as described above. It may be added and ingested at the same time, or it may be ingested before or after ingesting the above food.

As mentioned above, the present invention exhibits the effect of suppressing the increase in serum cholesterol associated with the intake of high-cholesterol-containing foods, but also has the advantage of lowering the arteriosclerotic index and suppressing obesity.

EXAMPLES The present invention and its effects will be specifically explained below with reference to Examples.

Example 1 Preparation of serum cholesterol upper limit inhibitor: pH of 8% concentration reduced cheese whey was adjusted to 6.8-7 with IN-ammonia water.
．． After adjusting to 0 and removing undissolved matter by centrifugation,
The mixture was heated for 15 minutes using a Kotsuho sterilizer, and the precipitate was removed by centrifugation again. Sodium ascorbate O, OS% and yeast extract 0.2% are added to the whey thus obtained.
The added material was dispensed into a culture vessel, sterilized in an autoclave at 121°C for 15 minutes, and used as a culture medium.

Next, the above medium was inoculated with 2% Lactobacillus acidophilus strain 5BT-2056 (Kaiko Kenkanyori No. 8744), which had been precultured in the same medium, and cultured at 37°C for 18 hours. During culture, the medium was neutralized with IN-ammonia water using a pH stat so that the pH of the medium was 6.5 to 6.8. After culturing, the bacterial cells were collected by centrifugation, washed twice with physiological saline, dispersed in 10% reduced skim milk containing 1% monosodium glutamate, and freeze-dried. The obtained freeze-dried Kokutai was used as a sample of a serum cholesterol increase inhibitor. The number of bacteria in the above bacteria is 1
01°/g.

Serum cholesterol rise suppression and obesity suppression test using the above increase inhibitor sample: The above sample was added to feed loaded with cholesterol in the formulation shown in Table 3 and fed to experimental animals by the following method to determine serum cholesterol levels. It was measured. In addition, as a control, the same amount of skim milk powder was added in place of the above sample and the same measurement was conducted.

Table 3 (wt%) (Note) Reverberon combination.

Test method SD male and female rats (withdrawal) were preliminarily fed for 7 days with commercially available feed (GE-2, manufactured by Nippon Talea Co., Ltd.), and then divided into groups of 8 rats so that their body weights were approximately averaged. Each of the above feeds was allowed to be consumed ad libitum. Then on the 5th day, 8th day and 122
On the 122nd day, blood was collected from the tail vein and the serum cholesterol level was measured. On the 122nd day, the animals were fasted for 24 hours. On the 133rd day, the blood was collected at the beginning of the episode and the serum cholesterol level was measured. The average daily feed intake of rats was 17.2±0 in the control group.
．． 4g, and the test group was 17.1±0.6g,
Cholesterol loading was 172 ± 4 mg and 171, respectively.
It was ±61wg.

The above test results are as shown in the attached Figure 1,
The cholesterol levels of the test group given the inhibitor sample of the present invention were significantly lower on the 8th and 122nd days compared to the control group.
The effect of suppressing the increase was observed (based on Dunnett's multiple comparison).

Furthermore, as shown in Figure 2, the test group showed a tendency to have lower cholesterol levels at the time of autopsy after fasting.

Example 2 The inhibitor was prepared in the same manner as described in Example 1, except that the bacterial cells obtained by culturing and harvesting were sterilized with a Kotsuho sterilizer and then freeze-dried. prepare the sample,
In addition, a suppression test was conducted.

The results are shown in the attached Figure 3, and even when sterilized bacterial cells were used, the effect of suppressing the increase in serum cholesterol was observed.

Example 3 Lactobacillus acidophilus 5BT-2056 strain (
Microtechnical Research Institute No. 8744) and Bifidobacterium longum 5OT-2933R strain (Microtechnology Research Institute No. 874)
No. 3) was inoculated at 2% each into a medium with the following composition.
Culture was carried out for a period of time, and the resulting culture was directly lyophilized and used as an inhibitor sample.

Medium composition: Skim milk powder 10 t% Yeast extract 0.5 wt% Water 89.5 wt% 10 at 121℃
It was sterilized for minutes before use.

The sample prepared as described above was added to the cholesterol-loaded feed having the formulation shown in Table 4, and a serum cholesterol increase suppression test was conducted in the same manner as described in Example 1. The average daily feed intake of rats was 17.1±0.0 for the test group cultured with the 5BT-2056 strain.
3g, 17.6± for 5OT-2933R strain culture
0.4 g, and the cholesterol loads were 171 and 176±4 mg, respectively. On the other hand, in the control group, the average intake was 17.6±0.4g, and the cholesterol load was 176±4B.

The test results are shown in Figure 4, and the serum cholesterol levels of the test group were as follows on the 5th day of the 5BT-2056 strain culture.
In the 5LIT-2933R strain culture, the levels were significantly lower than those in the control group on day 133 (according to Dunnett's multiple comparison), clearly demonstrating an effect of suppressing the increase in serum cholesterol.

In addition, the cholesterol level at autopsy after fasting was also 5th.
As seen in the figure, the test group showed a tendency to be lower.

Example 4 In Example 3, 5BT-2056 strain and 5BT-
The test was conducted in the same manner as described in Example 3, except that each culture obtained by culturing the 2933R strain was sterilized in a Kotsuho sterilizer for 15 minutes and then lyophilized and used as the inhibitor sample. . The average daily feed intake of rats was 17.5% in the test group using the 5BT-2056 culture.
4±0.4g, the test group using 5BT-2933R culture weighed 17.6±1.8g, and the control group weighed 17.7g.
It was ±0.4g. In addition, the loaded cholesterol amount is
In case of 5OT-2056, 179±4a+g, 5B
In the case of T-2933R, it was 176±8 g, and in the control group it was 177±4 Illg.

The results of the test are as shown in Figure 6, and the serum cholesterol levels of the test group were significantly lower than the control group on the 5th day in all cases (according to Dunnett's multiple comparison), indicating that the serum cholesterol level did not increase. A suppressive effect was observed.

In addition, the cholesterol level at the time of autopsy after fasting was also 7th.
As seen in the figure, the test group showed a tendency to be lower.

In other words, it can be seen that there is no effect on the effectiveness even if a sterilized culture is used.

Example 5 This example shows the results of testing the effect of the inhibitor of the present invention on lowering the arteriosclerotic index.

Using the same procedure as described in Example 3, Bifidobacterium longum 5OT-2933R strain
The culture obtained by culturing No. 43) was divided into two parts, one of which was used as a sample, and the other was sterilized for 20 minutes in a Kotsuho sterilizer and then freeze-dried and used as a sample.

Test method: SD rats (4 weeks old) were fed commercially available feed (manufactured by Nippon Talea Co., Ltd.,
After 5 days of pre-breeding with CE-2), the animals were divided into groups of 8 so that their body weights were approximately averaged, and each group was fed cholesterol-loaded feed with the composition shown in Table 5, with 20% of the sample prepared above. The rats were raised for 12 days with the supplements given to them ad libitum. Next, they were fasted for 24 hours, and blood was collected at the beginning of the 133rd day, and total cholesterol and HDL in the serum were measured.
(Ilight Density Lipoprotein
) The amount of cholesterol was measured. As a control, the same amount of skim milk powder was added instead of the above sample and the same measurement was conducted.

Table 5 (Currency t%) The average daily feed intake of rats was 19.0 ± 0.4 g for food that was dried without sterilization, and 19.0 ± 0.9 g for food that was dried after sterilization. , 19.1± in control
It was 0.5g.

In addition, the cholesterol loading amount is 190 for unkilled seedlings.
±4) 11g, the sterilized one was 190±9111g, and the control one was 191±5mg. The result is the 8th
As shown in the figure, HD of the test group given the inhibitor of the present invention.
The L-cholesterol levels in all cases tended to be the same or higher than those in the control group, and therefore, as seen in Figure 9, the arteriosclerosis index also decreased significantly (P<0.05).

HDL Cholesterol Amount Example 6 Lactobacillus acidophilus strain 5BT-2056 (Feikoken Bibori No. 8744) was sterilized at 121° C. for 15 minutes and added to the following Br1gg's Liver Broth.
2% of the culture solution pre-cultured in the same medium was inoculated and incubated at 37°C under anaerobic conditions with carbon dioxide aeration, while maintaining the pt+ of the medium at 6.5 to 6.8 (neutralized with IN aqueous ammonia). de1
Culture was performed for 6 hours.

After completing the composition culture of Br1gg's Liver Broth, the culture was centrifuged at 10,000 g for 15 minutes to collect the bacteria. It was dispersed in 10% reduced skim milk containing 1% sodium glutamate (wet weight) and freeze-dried. Using the obtained freeze-dried bacterial cells as an inhibitor sample, animal experiments were conducted in the same manner as described in Example 5, and the amount of HDL cholesterol and arteriosclerosis index were measured. In addition, cholesterol-loaded feed with the formulation shown in Table 6 was used as the feed.

Table 6 (%) The average daily feed intake of rats was 17.1 ± 0 in the test group.
．． 6g and 17.1±0.4g in the control group, and the cholesterol loading was 171±6mg and 171g, respectively.
It was ±4 mg.

As shown in Fig. 1θ, the above test results show that the HD of the test group
The amount of L cholesterol tends to be the same or higher than that of the control group, and therefore, as seen in Figure 11,
The arteriosclerosis index also decreased significantly (P<0.05) in the test group.

Example 7 This example shows the results of testing the obesity suppressing effect of the inhibitor of the present invention.

When adipose tissue accumulation was measured in an animal experiment using the 5BT-2933R strain (Feikoken Bibori No. 8743) described in Example 5, the amount of accumulation was significantly suppressed as shown in Figure 12. .

Example 8 This example shows an example of incorporating the inhibitor according to the present invention into a high cholesterol-containing food.

■ Fermented butter milk fat 78.8 (wt%) Salt 1.2 2 Freeze-dried bacterial cell water obtained in Example 1 18 ■ Butter cake butter 24 (wt%)
Light flour 24 Sand tri 24 Whole egg
24 Obtained in Example 3 4 Freeze-dried culture flavoring A little Mayonnaise Salad oil 65.0 (wt%) Egg yolk 17.0 Vinegar
10.0 Obtained in Example 4 3 Freeze-dried spice 4.38 Monosodium glutamate 0.6 Complex chemical seasoning (WP) 0.02

[Brief explanation of drawings]

The attached figures illustrate the test results of the serum cholesterol increase inhibitor according to the present invention, and FIGS. 1 and 2 show Example 1, FIG. 3 shows Example 2, and FIGS. Example 3
, FIGS. 6 and 7 show the test results of Example 4, FIGS. 8 and 9 show the test results of Example 5, and FIGS. 10 and 11 show the test results of Example 6. Moreover, FIG. 12 shows the test results in Example 7.

Claims (4)

[Claims] (1) A serum cholesterol increase inhibitor containing a culture or bacterial cells obtained by culturing lactic acid bacteria belonging to the genus Lactobacillus or Bifidobacterium as an active ingredient. (2) The serum cholesterol increase inhibitor according to claim (1), wherein the lactic acid bacteria belonging to the genus Lactobacillus is Lactobacillus acidophilus. (3) Claim No. 1 in which the lactic acid bacterium belonging to the genus Bifidobacterium is Bifidobacterium longum.
) The serum cholesterol increase inhibitor described in item ). (4) The serum cholesterol increase inhibitor according to claim (1), which is obtained by freeze-drying the culture or bacterial cells to form a powder.