JPH034768A - Lactic acid bacterial preparation - Google Patents

Abstract

PURPOSE:To obtain a lactic acid bacterial preparation, composed of digested microbial cells of Streptococcus lactis Lister and effective in preventing cancer and other various diseases by use thereof as part of daily foods and beverages. CONSTITUTION:The objective preparation which is preferably a dialytic part of digested microbial cells of Streptococcus lactis Lister and an acidic fraction extractable with ethyl acetate.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to the use of lactic acid bacteria (Streptococcus lactis Lister), which has useful physiological activity and is used for food.

(Current status and problems to be solved by the invention) Cancer, which is the biggest cause of death in our country, is mostly caused by the stimulation of chemical substances in food, that is, carcinogens induce canceration, which is then promoted. It is said that cancer is established after passing through the go stage of being exposed to cancer and developing symptoms.

Based on this idea, the ingestion of carcinogenic substances contained in food is unavoidable, but the formation of cancer can be prevented by substances that suppress this in the second promotion process, that is, anti-promotional active substances. If we can treat cancer, it will be possible to prevent cancer.

That is, there are carcinogenic substances in foods such as pentsupyrene in smoked foods, and there are substances such as nitrous acid that combine with amines contained in other foods to form carcinogenic substances called nitrosamines. Cancer develops when some kind of promoter acts on tissue cells that have been induced to become cancerous by carcinogens, so by suppressing this promotion process, , cancer prevention becomes possible. In particular, in Japan, stomach cancer accounts for 35% of cases (in 1976), and this is said to be largely influenced by food.

According to epidemiological studies, the tea-producing areas of Shizuoka Prefecture have some of the lowest incidences of stomach cancer, and a causal relationship with green tea drinking has been proven. That is, it is known that green tea also has anti-promotional activity.

In the same spirit as No. 1111, we conducted a diligent search for a more powerful and economical anti-promotional active agent in foods, and as a result, we completed the present invention.

In recent years, in the medical field, the delicate balance between oxidation and antioxidant reactions that govern many life phenomena has been disrupted, and the phenomenon in which living organisms tend to oxidize has come to be called ``oxidative stress.'' It has been shown that the reaction can also promote oxidative processes that are dangerous for the organism at the same time.

This is the basis of many biological and pathophysiological phenomena, and is deeply related to various medical problems such as inflammation, aging, canceration, and drug effects. It has been revealed that this "oxidative stress" can be practically monitored as biochemical indicators such as 8-hydroxydeoxyguanosine, thymine glycol, and thymidine glycol in 24-hour urine. In addition, it has been found that this index changes with age, and it has been speculated that oxidative stress may be exacerbated by lifestyle and nutrition, leading to various diseases. Until now, the link between oxidative stress and disease and effective treatment methods based on this link have not been investigated. The present inventor searched for an effective antioxidant substance while controlling oxidative stress, and found that the present lactic acid bacteria digest product has physiologically high antioxidant properties. In other words, completely different from the conventional method of using lactic acid bacteria, they completed an epoch-making invention of using lactic acid bacteria that have been digested and killed by enzyme treatment (lysozyme, chitalase, etc.).

At present, the existence of diabetes in adults, estimated to be between 2 and 3 million people, is a matter of great concern.This disease cannot be treated with symptomatic treatment, but with scientific research based on the true cause. As a result of many years of research, we have come to the conclusion that the state of oxidative stress may be related to the onset of the disease, and we believe that it is possible to find a solution to the disease. As a result of our search for antioxidants, we hope to contribute to this therapeutic field by providing a substance that enhances the antioxidant activity of Streptococcus lactis cells.

In addition, diseases caused by oxidative stress include diabetes (II), autoimmune diseases, inflammation, rheumatism, edema, cataracts, ischemia, stroke, cancer, myocardial infarction, hypertension, ulcers, and Alzheimer's disease. , Parkinson's disease, senile muscular atrophy, and osteoarthritis.

(Means for determining the issue as N) Regarding the evaluation of oxidative stress, the applicant of the present invention has established a method for measuring oxidative stress in the "method for measuring the degree of aging" that was previously proposed. I) by method
The correlation between the oxidative damage of NA and the central office was clarified, leading to the completion of the present invention.

Lactic acid bacteria Streptococcus lactis in the present invention
Lister includes ATCCPA'r$ strain 19435 and its related strains.

For example, Streptococcus lactis can be cultured using 1% glucose, 0.87% peptone, 0.4% yeast extract, 0.3% sodium citrate,
Static culture was performed at 37° C. for 24 hours in an aqueous medium containing 0.25% sodium bicarbonate, the cells were collected by centrifugation, washed three times with physiological saline, and freeze-dried.

For enzymatic treatment of the bacterial cells, the bacterial cells were dispersed in water to a concentration of 10%, and treated with lysozyme at 30 μg/m°C for 3 hours at 37°C with gentle stirring.

A digestibility of more than 80% was obtained as measured by turbidity at 660 nm.

This product was freeze-dried to obtain a lactic acid bacteria digest (1).

Furthermore, add 1 g of this digested material to 10 nl of phosphate buffer (
I OmM, pt17. O) dispersed in “Chitarase” (enzyme agent obtained from Rhizoctonia solani culture,
K.I. Kasei Co., Ltd., Fukuda-cho, Iwata-gun, Shizuoka Prefecture) 5m
g, and the reaction was carried out at 37°C for 12 hours. A freeze-dried product of this digest (digest I+) was used for the test.

(Experimental Example 1) Anti-promotional test This digested product (1) was extracted with ethanol, and its anti-promotional activity was examined using a detradecanoyl phorbol acetate (TPA) carcinogenic promotion system.

That is, anti-promotional activity was detected by using J B 6 cultured cells derived from bulb C mouse epidermis and observing the inhibitory effect on "T' PΔ-induced soft agar colony formation."

Fractionation of active ingredients in the digested product of Streptococcus lactis: The alcohol extracted fraction of the digested product (T) was dialyzed and divided into the dialyzed fraction and the non-dialyzed fraction, and the activity was found.
For Komi I, the acidic fraction was obtained by liquid-liquid partitioning with ethyl acetate. That is, the ethyl acetate if soluble acidic fraction of the dialysable fraction of the digested Streptococcus lactis was packed into a silica gel column, and both fractions eluted with ethyl acetate were examined for activity.

First, the digested product was 1 μg/mI for clone 22 for J 13 E5 cells! , growth at higher concentration t<1+
harm was seen. Furthermore, in the JB6 cell in vitro carcinogenic promotion detection system, '[)Δ-induced photocarcinogenic promotion was reduced by about 50% (Figure 1).

Moreover, since this is a tumor-forming JB6 cell and does not suppress its gene expression, it specifically suppressed the process of carcinogenesis promotion.

Furthermore, when an ethyl acetate extract of green tea was used as a control, the activity was 40% or more.

From the above, it can be said to be a powerful anti-promotional active agent, and since it cultivates lactic acid bacteria and utilizes the bacterial cells, it is extremely economical.

(Experiment Example 2) Antioxidant test A preparation containing lactic acid bacteria digested product (2.8 g of sugars, 16 g of lactic acid bacteria digested product, 1 package of 4 g in total, 0.04 g of citric acid) was administered three times a day for one week. 24 days before and after administration
8-hydroxydeoxyguanosine in the urine was measured and expressed as a molar ratio to creatinine.

As a result of conducting tests on 5 healthy people, the average Yli value was 0°074, 8 hydroxyguanosine 1d before using lactic acid bacteria.
All of the high values showed a decreasing trend (Figure 2).

(Experimental Example 3) Life extension effect test As test animals, two groups of 5-week-old stroke-prone spontaneously high surface pressure rats (SHR3P, male of a strain with a slow increase in blood pressure) were used. Powdered feed ("Funabashi SP" manufactured by Funabashi Farm) was administered, and the □ experimental group was given 15% equivalent of digestive lactic acid bacteria.

Groups of 6 to 7 animals were kept in an animal breeding room controlled at 22±1°C. The animals were allowed free access to feed and were kept until death. After 10 weeks of age, blood was collected from the tail vein at regular intervals to obtain serum and red blood cell fractions.
1 was expressed as the amount of 13A reactant by the Yagi fluorescence method.The red blood cell fraction was lysed to obtain a crude enzyme solution, and the glutathione peroxidase activity was measured by the method of P. D. Wagner et al. It is shown as the activity value in 1 mI2 of blood.

(Example 1) (1) Creation of Streptococcus lactis cells 1.5
% skim milk powder, 0.8% peptone, 0.4% yeast extract, and 0.3% sodium citrate for 24 hours at 37°C with gentle stirring.
Next, centrifugation was performed at 10,000 rpm for 10 minutes, and the mixture was washed three times with physiological saline and freeze-dried.

(2) Preparation of Digested Bacterial Cells M/] Bacterial cells were suspended in 0 phosphate buffer to a solid content of 10%, and digestion was performed at 25 μg/m of lysozyme at 37° C. with stirring for 3 hours. . 78% Digestion rate 6 (3) (TPA-induced carcinogenic promotion inhibition test using al-digested bacterial cells Soft agar colony number (%) was 45, so the inhibition rate was 55%)
%.

(3) (b) Effects on urinary 8-hydroxydeoxyguanosine IJ (tJA urinary disease NIDI) 2 cases of M patients After taking 2 g of digested bacterial cells per day suspended in milk for 1 week, the blood sugar level and blood sugar levels increased for 24 hours. Measurements were performed using urine.

(Example 2) 1 kg of digested lactic acid bacteria (digested material ■) digested with lysozyme,
Dissolved in 10mM phosphate buffer (pH 7, 0) IO+, added chitalase IOg, and reacted at 37°C for 8 hours. After completion, freeze-dried product (digest II) was prepared using a commercially available powdered feed (manufactured by Funabashi Farm, Funabashi Farm). 5t)) was added in an amount equivalent to 15% and mixed well to prepare experimental feed.
In I), 6 animals were in the control group and 7 animals were in the experimental group, and a survival effect test and measurements of serum lipid peroxide and red blood cell giltathione peroxidase activity were conducted.

Number of raw boxes A significant difference was observed between the control group and the 5% risk level.

In other words, lifespan was extended.

Next, lipid peroxide in serum and glutathione peroxidase in red blood cells were measured separately in 5 to 7 animals per group.

When used as part of a diet, it has an excellent effect on preventing cancer and other diseases associated with aging.

[Brief explanation of the drawing]

Figure 1 is a graph of the influence of digestive bacteria on i''PA-induced photocancer promotion, and Figure 2 is a graph of the effect of digestive bacteria on oxidative damage. Regarding serum lipid peroxides, However, the experimental group showed a significantly higher activity in glutathione peroxidase activity in the red blood cell membrane.This result suggests that the survival effect observed in the experimental group was due to the increase in lipid peroxidase activity. It is thought that this is related to the low and high activity of glutathione peroxidase, and that this is based on the anti-promotional activity and antioxidant properties of the lactic acid bacteria digest. (Effect of the invention)

Claims (1)

[Scope of Claims] 1. A lactic acid bacteria preparation consisting of a bacterial digest of Streptococcus lactis Lister. 2. A dialyzable portion of a bacterial digest of Streptococcus lactis Lister, which is acidic and extractable with ethyl acetate. The lactic acid bacteria preparation 3 according to claim 1, wherein lysozyme (EC3,
21, 17) and/or "chitalase" 4. The lactic acid bacterium preparation according to claim 1, wherein the objective physiological activity is anti-promotional activity. Product 5: The lactic acid bacterium preparation according to claim 1, wherein the desired physiological activity is antioxidant and alleviates related diseases. 6: The desired physiological activity is a life-prolonging effect. Lactic acid bacteria preparation according to claim 1