JPH0799986A - Cell containing lactobacillic acid in high concentration and its production - Google Patents

Abstract

PURPOSE:To obtain a cell of a bacterium belonging to the genus Lactococcus containing lactobacillic acid in high concentration, where the lactobacillic acid can suppress absorption of fats and so can prepare a fat absorption inhibitor and a food or a drink therefrom, and to provide a production method therefor. CONSTITUTION:A cell of a bacterium belonging to the genus Lactococcus, capable of producing lactobacillic acid, is cultured in a medium whose pH is controlled at fixed pH near neutrality by using a neutralizing agent in the culture period to produce a cell containing a large amount of lactobacillic acid therein. A fat absorption inhibitor, or a fat absorption inhibiting food or drink containing the cell or containing fats and oils produced from the cell is provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a bacterium containing a high-concentration lactobacillic acid, which is a C 19 fatty acid containing a cyclopropane ring, a method for producing the bacterium, and a method for using the bacterium. The present invention relates to absorption-suppressing foods and fat absorption-suppressing agents.

[0002]

2. Description of the Related Art Lactobacillic acid (2-hexylcyclopropane)
decanoic acid) is a cell wall / cell membrane-constituting lipid found by Hofmann et al. in 1954 from Lactobacillus cells. This fatty acid has an odd number of carbon atoms and has a cyclopropane ring and thus has a very low melting point of 33.6 to 35 ° C. [Dictionary of Organ]
ic Compounds, 5th. edit. vol.3, 2946 (1982)]. Its structural formula is shown in Chemical formula 1.

[0003]

[Chemical 1]

It has been reported so far that many bacteria, including Lactobacillus cells, contain lactobacillic acid. It has been reported that the content of lactobacillic acid contained in Lactococcus is about 25% of the total fatty acid composition. See also Schmitt et al. [Milchwissenschaft 4
4, 556 (1989)], it has been reported that the leuconostoc cells contain lactobacillic acid in 8 to 50% of the total fatty acid composition. Under normal culture conditions, the content of lactobacillic acid contained in Lactococcus is about 25% of the total fatty acid composition, and at most 44%.
[Shchleifer, KH et al. Syste
Appl. Microbiol. 6, 183-195 (1985)]. A lipid having a cyclopropane ring such as lactobacillic acid is known as a lipid that is difficult to be absorbed, and it is industrially desired to produce a large amount of lactobacillic acid, a lipid containing the same, a bacterial cell or the like.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a microbial cell or fat containing a large amount of lactobacillic acid having a specific physiological activity as a fatty acid as described above. Another object of the present invention is to provide a method for efficiently producing cells or fats and oils containing a large amount of lactobacillic acid. A further object of the present invention is to provide a fat absorption-suppressing food or drink or a drug containing a large amount of such bacterial cells or lactobacillic acid.

[0006]

Means for Solving the Problems The present inventor was examining the constituent fatty acids in the cells of lactic acid bacteria of the genus Lactococcus, and found that the cells of the genus Lactococcus were cultured in a medium in which the pH was controlled to be constant. Then, the fatty acid content in the bacterial cells was increased, and the yield of the bacterial cells themselves was also increased, and it was found that the yield of lactobacillic acid was dramatically increased, and the above problems were successfully solved.

The present invention relates to the genus Lactococcu
By culturing cells belonging to ( s) under pH control,
The present invention relates to a method for producing bacterial cells, which comprises containing lactobacillic acid in a high concentration. The present invention also relates to a method for producing a lactobacillic acid-containing oil or fat, which comprises collecting an oil or fat containing lactobacillic acid at a high concentration from such a bacterium. Furthermore, the present invention relates to foods and drinks and pharmaceuticals containing microbial cells containing lactobacillic acid in such a high concentration and oils and fats thereof.

The lactic acid bacteria belonging to the genus Lactococcus used in the present invention belong to the genus Lactococcus [Schleife
r, KH et al. System. Appl. Microbiol. 6 183 (198
5)] Any of the bacterial species capable of producing lactobacillic acid can be used. Examples of such bacterial species include Lactococcus lactis subspecies lactis and Lactococcus lactis subspecies cremoris. But especially Lactococcus lactis subspecies cremoris SBT 049
It is preferable to use 5 strains (Microtechnology Research Institute No. 10053) because lactobacillic acid can be contained in the cells at a high concentration.

This strain was isolated by the present inventors from the Nordic viscous fermented milk villi, and its isolation method and mycological properties are described in JP-A-3-229702. This strain was initially deposited as Streptococcus cremoris SBT 0495 (Deposit No. Lactococcus lactis subsp.
cremoris) SBT 0495 (Accession No. Microtech Lab.
Has been deposited with the Institute of Microbial Technology, Institute of Industrial Technology (currently Institute of Biotechnology). The main mycological properties are shown below for reference.

[0010]

Next, a method for producing lactobacillic acid-rich cells using these lactic acid bacteria will be described. As the culture medium for lactic acid bacteria of the genus Lactococcus, a milk culture medium or a culture medium containing milk components, or a semi-synthetic or synthetic culture medium not containing these components can be used. As such a culture medium, the following medium of Nakajima et al. [J. Dairy Sci. 73 1473 (1990)] can be exemplified.

Whey agar medium [glucose 10 g, galactose 10 g, cheese whey powder (manufactured by Snow Brand Milk Products) 80 g,
A medium prepared by mixing 15 g of agar and 1 liter of distilled water to adjust the pH to 6.8]. Whey permeate medium [10% reduced skim milk at 115 ° C
After heat sterilizing for 15 minutes at 50 ° C, add 50 mg of filter-sterilized actinase E (trade name) per 1 liter of skim milk, 37
The enzyme was inactivated by heating at 0 ° C for 16 hours and heating at 100 ° C for 5 minutes. This supernatant is processed by ultrafiltration (UF) (model SE
P-1013, manufactured by Asahi Kasei) and the permeate obtained is 115 ° C.
Heat sterilize for 15 minutes to prepare the medium].

In the culture, the lactic acid produced by the lactic acid bacterium is neutralized, and the pH of the culture is adjusted to near neutral (pH 5 to 8, preferably 5.5 to 6.5), which is good for the growth of the lactic acid bacterium.
It is carried out by a constant controlled neutralization culture. As the neutralizing agent used at this time, any neutralizing agent can be used as long as it does not affect the culture of lactic acid bacteria. Ammonia is preferable as such a neutralizing agent, but sodium hydroxide, potassium hydroxide and the like can also be exemplified.

Lactobacillus high concentration lactococcus cells can be used as they are or in a dried state. The method of collecting oils and fats from the lactobacillic acid high-concentration bacterial cells is generally to dry and pulverize or crush the lactobacillic acid high-concentration bacterial cells. This is extracted with a fat solvent. The solvent to be used is usually safe even when used as food or medicine, and examples thereof include n-hexane and 2-propanol. Extraction is performed by shaking with such a solvent at a temperature not too high such as room temperature. Next, the extracted solvent layer is collected and the solvent is removed by distillation under reduced pressure or the like to obtain fats and oils.

By adding the oil or fat or lactobacillic acid thus obtained to various foods and drinks, absorption of fat can be suppressed. Also, lactobacillic acid can be extracted from these cells by an appropriate method and used. The lactobacillic acid is determined by adding hydrochloric acid / methanol to the lyophilized product of the cells and hydrolyzing at 100 ° C. for 3 hours, and then measuring by gas chromatography. Confirmation of lactobacillic acid can be performed by analysis of a mass spectrometer connected to gas chromatography.

These lactic acid bacterium containing a high concentration of lactobacillic acid may be added as a raw material for food and drink. Any food or drink may be used. Examples thereof include fermented milk, dairy products such as cheese and butter, drinks such as drink yogurt and lactic acid bacteria beverages, confectionery such as butter cakes and breads. Addition to fermented milk or a lactic acid bacterium drink can also be carried out by adding recovered cells to a fermented product. Further, an extract obtained by extracting lactobacillic acid from cells or lactobacillic acid itself can be added. Furthermore, the cells themselves, the extract or lactobacillic acid can be used as a medicine. As the medicine, a dried product of bacterial cells can be made into a powder form or an extract containing lactobacillic acid can be encapsulated. These are orally administered and used as fat absorption inhibitors. Dosage is 10 per adult per day
Administer 0 mg to 5 g in several divided doses.

The contents of the invention will be specifically described below, but the present invention is not limited to this description.

Example 1 A lactic acid bacterium of the genus Lactococcus containing a high concentration of lactobacillic acid was prepared by the following method.
10% reduced skim milk / pronase decomposition medium was prepared by Nakajima et al.
A growth medium (the above whey permeate medium) was prepared according to the method of [J. Dairy Sci. 73 1473 (1990)]. The prepared main culture medium was sterilized in a jar fermenter and pre-cultured in the same culture medium, Lactococcus lactis subsp. Cremoris SBT 0495 strain (Microtechnology Research Institute
No.) was inoculated at 1%. Using ammonia as a neutralizing agent,
The culture was incubated at 30 ° C. for 24 hours while keeping the pH of the medium at 5.5. After culturing, the bacterial cells are collected, and after freeze-drying, the bacterial cell yield,
The lactobacillic acid content in the cells was measured. The results are shown in Table 1. As a control, the same Lactococcus
Using Lactis subsp. Seacres cremoris SBT 0495 strain, static culture was performed at 30 ° C. in the same medium as above without adjusting the pH during the culture period, and the obtained bacterial cells were used for the culture period during the culture period. The pH changed from 6.8 to 4.5.

The analysis results of the lipid extracted from the Lactococcus cells prepared by this method by gas chromatography / mass spectrometry are shown in FIG. As a result of analysis with a mass spectrometer, it was confirmed that the fatty acid of the present lipid was lactobacillic acid having a cyclopropane ring and 19 carbon atoms. Table 1
As shown in, the lactobacillic acid content per total fatty acid extracted from the dried bacterial cells was 25.8% in the case of the bacterial cells prepared by static culture (control), while the cultivated by controlling the pH. However, it rose to 63.9%. Since the yield of dried cells increased 10 times, the yield of lactobacillic acid per medium increased 32 times.

[0019]

[Table 1] ───────────────────────────────── pH control culture Static culture (Example 1) (control) ) ───────────────────────────────── Dry cell yield 2.2g 0.2g (per liter of medium) Lactobacillic acid 63.9% 25.8% (per total fatty acid) Lipid content 12% 10% Yield of lactobacillic acid (per liter of medium) 168.7mg 5.2mg ───────────── ────────────────────

[0020]

Example 2 The microbial cells containing lactobacillic acid at a high concentration obtained by the method of Example 1 were lyophilized to obtain dried microbial cells. The cells were kneaded into butter at a ratio of 10% to prepare a fat absorption-suppressing fermented butter. That is, 10 kg of cream (40% fat rate) obtained from milk by a known method was sterilized by a conventional method, and 700 g of a skim milk culture of Lactococcus lactis subspecies lactis was added thereto,
It was cultured and fermented at 10 ° C for 16 hours. This fermented cream was cherned to obtain 4.5 kg of fermented butter grains. To the butter granules, 450 g of freeze-dried cells containing the above-mentioned lactobacillic acid at a high concentration was added and worked to obtain a fermented butter for suppressing fat absorption. The fermented butter obtained had a diacetyl content of 1.25 ppm, an acetaldehyde content of 1.3 ppm, and a whey pH.
Was 5.5, and it was a fermented butter containing lactobacillic acid, which was of good quality and had a rich fermented flavor and suppressed fat absorption.

[0021]

Example 3 Lactococcus cells having a high content of lactobacillic acid obtained in Example 1 were fed to rats to measure the triglyceride content in serum. The feed given to the rats has the following composition.

[0022]

[Table 2] ───────────────────────────────── Feed A Feed B ────────── ───────────────────────── Casein 20 20 Lard 10 10 Corn oil 10 10 Vitamin mix 0.85 0.85 AIN76 (trade name) Salt mix 4 4 AIN76 (trade name) Cellulose 3 3 Choline chloride 0.2 0.2 Sodium cholate 0.125 0.125 Sucrose 51.825 41.825 Bacteria 0 10 ──────────── ──────────────────────

The above-mentioned feed was fed to 6 F-244 rats (12 weeks old, male) per group for 1 week. After fasting for 6 hours, the amount of triglyceride in serum was measured with a triglyceride measurement kit (manufactured by Kyowa Medix).

No difference was observed in the feed intake between the two groups during the above-mentioned feed administration. Table 3 shows the amount of triglyceride in serum. The values in the table show the average value ± standard deviation.

[0025]

[Table 3] (Number of samples = 6 each) ──────────────────────── Feed A Feed B ──────────── ───────────── 102 ± 19mg / dl 82 ± 11mg / dl ────────────────────────

When the triglyceride content in the serum in the above table was statistically tested, it was found that the risk rate was 5% and there was a significant difference between the feed group B. As a result, the effect of administration of Lactobacillus-rich Lactococcus cells was confirmed.
As shown in the results, the triglyceride content in the serum of rats fed with the cells containing lactobacillic acid at a high concentration was low. From these results, it was revealed that Lactococcus cells containing lactobacillic acid at a high concentration suppress the lipid uptake into blood.

[0027]

Example 4 1 g of lactobacillic acid-rich cells obtained by the method of Example 1 was mixed with 5 g of lactose and molded into granules to prepare a fat absorption inhibitor containing lactobacillic acid as an active ingredient. .

[0028]

[Example 5] The dried product of the cells containing a high concentration of lactobacillic acid of Example 1 obtained by culturing while controlling the pH of the culture medium to near neutral was sonicated. 1 liter of n-hexane was added to 100 g of the obtained disrupted cells, and the mixture was sufficiently extracted at room temperature with shaking. Then, the extracted solvent layer was distilled under reduced pressure to remove the solvent. 11.9 g of oil and fat was obtained. When the fatty acid of this oil was quantified, it was found that it contained 7.56 g of lactobacillic acid, and it was confirmed that this was a high concentration.

[0029]

EFFECT OF THE INVENTION Wood & Reiser [J. Am, Oil Chem, Soci.
42 315 (1965)], when a lipid having a cyclopropane ring was administered to rats, a fatty acid having a cyclopropane ring was detected in adipose tissue, and the β-oxidation enzyme, which is a fatty acid absorption system, was detected by cyclopropane. It was revealed that it was blocked at the ring position. This suggests that lacitobacillic acid also readily inhibits β-oxidation enzymes. That is, lactobacillic acid is expected to be a raw material for fat absorption-suppressing food and drink, as a fatty acid that is difficult to be absorbed. In addition, a Lactococcus bacterium containing a high content of lactobacillic acid can also be used as a fat absorption-suppressing food or drink because fat absorption is suppressed.

[Brief description of drawings]

FIG. 1 shows the results of analysis by a gas chromatography / mass spectrometer of lipids extracted from Lactococcus cells prepared by the method of the present invention.

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[Procedure amendment]

[Submission date] November 11, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0029

[Correction method] Change

[Correction content]

[0029]

EFFECT OF THE INVENTION Wood & Reiser [J. Am, O
il Chem, Soci. 42 315 (196
5)] was administered to rats, a fatty acid having a cyclopropane ring was detected in the adipose tissue, and the β-oxidation enzyme, which is a fatty acid absorption system, was located at the position of the cyclopropane ring. It became clear that it was blocked by. Therefore, it is inferred that the La transfected Bachirin acid inhibits easily β oxidative enzyme.
That is, lactobacillic acid is expected to be a raw material for fat absorption-suppressing food and drink, as a fatty acid that is difficult to be absorbed. In addition, a Lactococcus bacterium containing a high content of lactobacillic acid can also be used as a fat absorption-suppressing food or drink because fat absorption is suppressed.

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Claims (6)

[Claims] 1. A bacterium belonging to the genus Lactococcus (Lactococcus sp.), Which is cultivated in a medium controlled to have a constant pH around neutral during the culturing period and contains lactobacillic acid at a high concentration. 2. A bacterium belonging to the genus Lactococcus sp. And having the ability to produce lactobacillic acid is cultivated in a medium in which the pH is controlled to be around neutral and the lactobacillic acid is contained in the bacterium. A method for producing a bacterium containing a high concentration of lactobacillic acid, which comprises collecting the bacterium in a high concentration. 3. The method according to claim 2, wherein the bacterial cell is Lactococcus lactis subspecies cremoris SBT 0495 strain (Ministry of Industrial Science and Technology No. 10053). 4. A method for producing a lactobacillic acid-containing oil or fat, which comprises collecting the lactobacillic acid-containing oil or fat from the bacterium obtained according to claim 2. 5. A fat absorption-suppressing food or drink containing the microbial cell obtained according to claim 1 or a lactobacillic acid-containing oil or fat obtained therefrom. 6. A fat absorption inhibitor containing the bacterium obtained according to claim 1 or a lactobacillic acid-containing oil or fat obtained therefrom as an active ingredient.