TW201540307A - Anti-glycosylation agent, life-extending agent and anti-aging agent - Google Patents

Abstract

The subject of the instant disclosure is to provide an anti-glycosylation agent, a life-extending agent and an anti-aging agent comprising fermented metabolites of a given lactobacillus. The anti-glycosylation agent, the life-extending agent and the anti-aging agent according to an embodiment of the present invention are characterized in containing fermented metabolites of a lactobacillus, Lactococcus lactis subsp. cremoris (H-61) as an active ingredient.

Description

Anti-glycation agent, life extension agent and aging inhibitor

The present invention relates to an anti-glycation agent, a life extension agent and an aging inhibitor containing a fermentation metabolite of a specific lactic acid bacterium.

In the context of welcoming an aging society and promoting health, attention has been focused on the suppression of aging and the extension of life. For example, Patent Document 1 discloses a life extension agent and an antioxidant function improving agent containing astaxanthin as an active ingredient.

In addition, probiotics are widely known as functional foods that promote health. Probiotics are generally defined as "active microbial supplements which have a positive effect on host animals by improving the balance of the host intestinal flora" (see Non-Patent Document 1). According to this definition, the live bacteria of the probiotic lactic acid bacteria reach the intestinal tract alive, and stimulate the movement of the intestinal flora, thereby improving the intestinal immunity and exerting a beneficial effect. For example, Patent Document 2 discloses a lactic acid bacterium which inhibits aging by suppressing the decrease in bone density or the occurrence of skin ulcer.

Alternatively, even a dead cell exhibits a beneficial effect, and there is also an opinion that the definition of probiotics is expanded (see Non-Patent Document 2). For example, Patent Document 2 discloses that the dead cells have the same effect as the living cells.

Therefore, in recent years, in addition to probiotics, a biological database has been proposed (see Non-Patent Document 3). The biological database is defined as direct, or through the intestinal flora, immune stimulation, lowering cholesterol, lowering blood pressure, regulating intestinal function, anti-tumor effect, anti-thrombosis, hematopoietic effects, biological regulation, biological defense, disease Prevention, recovery, control of active food ingredients such as aging. That is, the biological database, unlike probiotics, not only acts on the intestinal flora, but also directly acts on intestinal immunity, and can be expected to promote health. In view of the above definition, dead cells can also achieve the above beneficial effects as a biological database.

In addition, as an example of the biological database, in addition to the bacterial component of the lactic acid bacteria containing dead bacteria or the like, a lactic acid bacteria fermentation metabolite, a mixture of these, and the like can be cited.

[Previous Technical Literature] [Patent Document]

Patent Document 1 Patent No. 4604207

Patent Document 2, JP-A-2012-72132

[Non-patent literature]

Non-Patent Document 1 Fuller, (1989) J. Appl. Bacteriol., 66, 365-378

Non-Patent Document 2 Salminen et al., (1999) Trends Food Sci. Technol., 10, 107-110

Non-Patent Document 3 Guanggang Contents Probiotic. Prebiotic. Biogenic consortium Japan Bifidobacterium Center June 2006

Here, the lactic acid bacteria fermentation metabolite has less knowledge about a specific action because it has a component different from the cell body.

In view of the above, it is an object of the present invention to provide an anti-glycation agent, a life extension agent and an aging inhibitor containing a fermentation metabolite of a specific lactic acid bacterium.

In order to achieve the above object, the anti-glycation agent according to one embodiment of the present invention has a feature of containing a fermentation metabolite of the Lactococcus lactis subsp. cremoris H-61 strain as an active ingredient.

The anti-glycation agent of the present invention has a very high activity for inhibiting the production of advanced glycation end-products (AGEs) due to the fermentation metabolite containing the above-mentioned lactic acid bacteria as an active ingredient. AGEs are substances derived from reducing sugars such as Glucose (glucose) and which are non-enzymatically reacted with the amino group of the protein (Millard reaction), and are called causes of myocardial infarction, cerebral infarction, osteoporosis, cataract, and complications of diabetes. substance. This hair The anti-glycation agent of Ming can prevent the occurrence of these diseases by inhibiting the formation of AGEs.

The above-mentioned fermentation metabolite may also contain no bacterial cells.

The fermentation metabolite of the present invention has a very high anti-glycation effect even if it does not contain a bacterial cell.

The fermentation metabolite may be produced by fermenting the lactic acid bacteria in a medium containing at least one of soybean milk, milk, goat milk, and sheep milk.

The life extension agent according to one embodiment of the present invention is characterized in that a fermentation metabolite of the Lactococcus lactis subsp. cremoris H-61 strain is used as an active ingredient.

The life extension agent of the present invention can prolong the life of the living body and can increase the period from birth to death.

In addition, the extension of life can also be due to anti-glycation.

As described above, the above-mentioned fermentation metabolite has an anti-glycation action, and can prevent the occurrence of a disease caused by heart disease, brain disease, and the like, and prolongs life due to anti-glycation.

Further, the above-mentioned fermentation metabolite may not contain a bacterial cell.

The fermentation metabolite may be produced by fermenting the lactic acid bacteria in a medium containing at least one of soybean milk, milk, goat milk, and sheep milk.

An aging inhibitor according to an embodiment of the present invention has a lactic acid bacteria Lactococcus lactis subsp. cremoris H-61 The fermentation metabolite of the strain is characterized as an active ingredient.

Further, the above-mentioned fermentation metabolite has an anti-glycation action, and can prevent the occurrence of a disease caused by heart disease, brain disease, or the like, suppressing aging, and may also be resistant to saccharification.

Fig. 1 is a graph showing the life curve of nematodes cultured in a culture solution containing the fermentation metabolite of the present invention.

Hereinafter, embodiments of the present invention will be described.

According to the present invention, the inventors have extensively studied the fermentation metabolites of the lactic acid bacteria Lactococcus lactis subsp. cremoris H-61 strain (hereinafter referred to as H-61 strain), which have anti-glycation action and longevity. The prolonged action and the aging-inhibiting effect caused by these are completed. In the previous study, the lactic acid bacteria's autologous inhibition of aging and health promotion was obtained (see Patent Document 2, etc.). However, the bacterial cells are extremely rare, or there is almost no knowledge about the fermentation metabolites of the lactic acid bacteria which do not contain the cells. According to the present invention, it has been found that the fermentation metabolite of the lactic acid bacterium is self-contained, and the remarkable anti-glycation action, the prolonged life effect, and the aging-suppressing action accompanying the unpredictable action of the bacterium are completed.

The H-61 strain of the present invention is attached to the patent microbiology center of the independent administrative company product evaluation technology base mechanism, and the trust number is NITE P-92 (refer to Patent Document 2). In addition, the H-61 strain mentioned above is also attached to the Agricultural Bioresource Gene Bank of the Institute of Agricultural Bioresources, an independent administrative agency, number MAFF. No.400007.

The fermentation metabolite of the present invention is a substance which removes the cells after the H-61 strain is fermented under any conditions, and the cells are very little or do not contain the cells. That is, the fermentation metabolite of the present invention mainly contains lactic acid, various amino acids, and the like. The "bacterial-free" fermentation metabolite refers to a filtrate which is substantially free of cells, for example, after fermentation, by filtering a culture solution with a filter having a diameter of 0.1 to 0.5 μm or less, and a treated product of the filtrate. Also included is a substance that homogenizes the culture solution in a conventional manner prior to filtration.

The fermentation medium used for the production of the fermentation metabolite of the present invention may be, for example, milk, soy milk, goat milk, sheep milk or a mixture of these. Thus, the fermentation efficiency of the H-61 strain can be improved.

The fermentation metabolite of the present invention can also be produced, for example, by culturing the above strain according to a usual method, fermenting it, sterilizing, and filtering the fermentation broth. The fermentation conditions are not particularly limited. For example, the above-mentioned fermentation metabolite may be a material obtained by fermentation at a fermentation temperature of from 20 ° C to 40 ° C. Further, the above-mentioned fermentation metabolite may be a substance obtained by fermentation for 70 hours or more, preferably 70 hours to 700 hours.

The fermentation broth is heat-sterilized, suspended, and filtered according to a usual method. Filtration, as described above, uses a filter having a diameter of 0.1 to 0.5 um or less. Moreover, it can be filtered by a filter having a larger diameter and then filtered by the above filter.

The fermentation metabolite of the present invention is contained in the above filtrate. Further, the above filtrate may be suitably subjected to deethanolization treatment by an evaporator or depending on the form of ingestion. Heat sterilization treatment, etc. The above filtrate is hereinafter referred to as a fermentation metabolite-containing liquid.

The anti-glycation agent of the present invention has a feature of containing the above-mentioned fermentation metabolite as an active ingredient. The above anti-glycation agents have very high inhibitory activity against Advanced Glycation End-products (AGEs) production. AGEs are substances which are produced by a non-enzymatic reaction (Millard reaction) with a reducing sugar such as Glucose (glucose). The accumulation of AGEs in blood vessels becomes a cause of myocardial infarction and cerebral infarction, and accumulation of bone in the bone becomes a cause of osteoporosis, and accumulation in the eye becomes a cause of cataract. In addition, AGEs are also known as the cause of diabetic complications such as diabetic retinopathy and diabetic nephropathy. In other words, inhibition of the generation of AGEs can effectively suppress the occurrence of heart disease, brain diseases, diabetic complications, and the like, and is associated with suppression of aging and prolonged life.

The life extension agent of the present invention has a feature of containing the above-mentioned fermentation metabolite as an active ingredient. The life extension agent of the present invention can prolong the life of the living body and can increase the period from birth to death. The life extension agent of the present invention can increase the average life of a living organism by, for example, 1.1 to 1.5 times. One of the reasons for the extended life of the life extension agent of the present invention is anti-glycation, and other factors can be considered for various effects.

The aging inhibitor of the present invention has a feature of containing the above-mentioned fermentation metabolite as an active ingredient. As described above, it is possible to suppress the occurrence of vascular diseases, brain diseases, and the like associated with aging due to accumulation of AGEs. Therefore, one of the causes of the aging inhibition of the aging inhibitor of the present invention is anti-glycation, and others can be considered to be produced by various effects.

The anti-glycation agent, the life-extending agent, and the aging inhibitor of the present invention can be formulated by various methods depending on the purpose of use, and for example, a method of freeze-dried powder, spray-dried powder, or suspension in a liquid can be applied. Further, a suitable additive may be added along with the formulation.

The anti-glycation agent, the life-extending agent, and the aging inhibitor of the present invention may be combined so that the fermentation metabolite-containing solution ingests 0.001 to 2 ml per 1 kg of body weight per day, preferably 0.01 ml to 0.2 ml. The dose can be adjusted depending on the age of the person to be administered (animal), the degree of the symptoms, and the form of administration. Further, the anti-glycation agent, the life-extending agent, and the aging inhibitor of the present invention can be used as fermented milk by using the above-mentioned fermentation metabolite-containing liquid cultured in milk or soybean milk.

[Examples]

(Example 1)

The soybean milk is poured into a 500 ml container and heat-sterilized by a usual method, and cooled to be used as a soy milk medium. The H-61 strain was cultured in advance for 7 days in accordance with a usual method. Then, the cultured H-61 strain was inoculated into a soybean milk medium, cultured at about 35 ° C for a day and night, and the cells were propagated to 1 × 10 ⁹ CFU/ml. Then, the mixture was heat-sterilized according to a usual method, and ethanol homogenization treatment was added thereto, and filtration was performed once. This primary filtration solution was filtered twice with a 0.2 μm diameter hollow fiber membrane. The secondary filtrate was subjected to deethanolization treatment by an evaporator, 90% of lactic acid was added, and heat sterilization was carried out by a usual method. Thus, the fermentation metabolite-containing liquid of Example 1 was completed.

(Example 2)

Example 2. A fermentation metabolite-containing liquid was produced in the same manner as in Example 1 except that the milk medium was replaced with milk and milk (skimmed milk powder).

(Test Example 1 AGEs production inhibitory activity measurement)

The measurement of the inhibitory activity of AGEs production was carried out by using Example 1 and Example 2.

First, prepare the sample. A fermentation metabolite-containing solution (hereinafter referred to as a stock solution) related to each of Example 1 and Example 2 as a sample of the test example, and a liquid in which the respective stock solutions were diluted 10-fold with a buffer solution and a 100-fold diluted liquid ( Refer to Table 1) below. The buffer was phosphate buffer (pH 7.4).

For each sample, the AGEs production inhibitory activity was measured by a fluorescence assay. In other words, 0.5 mL of an aqueous solution of 360 mg/mL of glucose, 0.5 mL of a 40 mg/mL BSA (Bovine Serum Albumin) aqueous solution, 2.5 mL of a phosphate buffer (pH 7.4), and 0.5 mL of pure water were added to generate 0.5 mL of each sample. Measuring solution. A control solution containing 0.5 mL of pure water instead of the sample was prepared. Next, each of the measurement liquid and the control liquid was cultured at 60 ° C for 40 hours. Thereafter, the measurement liquid and the control liquid after the incubation were measured for fluorescence intensity at a excitation wavelength of 270 nm and a fluorescence wavelength of 440 nm in a fluorescence spectrophotometer. The decrease rate of the fluorescence intensity at the time of adding each sample was calculated as the AGEs generation inhibition rate with respect to the fluorescence intensity of the control liquid.

Table 1 is a graph showing the results of the test examples. As shown in the table, in both of Example 1 and Example 2, AGEs production inhibitory activity was observed. The lower the dilution rate, the higher the inhibition rate of AGEs production, and the same results were obtained in Example 1 using soybean milk fermentation and Example 2 using milk fermentation. Therefore, this experimental example can be confirmed. The anti-glycation activity is produced by the fermentation metabolite of H-61 of the present invention.

In the stock solutions of Example 1 and Example 2, the AGEs production inhibition rate was almost 100%. Therefore, it was confirmed that the fermentation metabolite of H-61 of the present invention has a very high AGEs production inhibitory activity.

(Test Example 2 Determination of nematode life)

The lifespan of the nematodes in Examples 1 and 2 was determined.

The nematode (Caenorhabditis elegans) is used as a biological model with a shorter life span of 3-4 weeks (culture temperature 20 °C), and the whole genome has been deconstructed. Much research has been done on the relationship between aging and oxidative stress, as well as to explore substances that have an impact on life. In this experimental example, a fer-15 mutant strain was used. The fer-15 mutant has the characteristic that it cannot produce the next generation at 25 ° C or higher, and has the benefit of not being mixed with the next generation of nematodes at the measurement life.

First, prepare the S-medium of the composition of Table 2. The composition of the Trace metals solution in S-medium is shown in Table 3. Escherichia coli OP-50 strain as a bait was suspended in S-medium as a culture solution of nematodes. The nematode was cultured to adult (20 ° C, 200 rpm) with the culture solution. After, as shown As shown in Fig. 4, the nematode was recovered in the S-buffer, and after washing, the body of the nematode was dissolved with NaOH solution and liquid chlorine bleach (trade name Haiter Kao Co., Ltd.), and the eggs were recovered from the body. The recovered eggs were incubated at 20 ° C for one night to incubate. The hatched L1 larvae were cultured in a culture flask at 26 ° C and 100 rpm with E. coli OP-50 strain as a bait. At this time, each of the culture flasks was adjusted to 2000. Ampicillin and each sample were added to the culture flask on the 5th day after egg recovery. For each sample, the fermentation metabolite-containing liquid (stock solution) of Example 1 and Example 2 was added, and the final concentration in the culture solution was diluted 11-fold and 101-fold diluted. Add pure water to the control solution. Thereafter, a certain amount of the culture solution was taken every several days, and the survival number of the nematode was investigated. The survival number on the fifth day was taken as 100%, and the survival curve was drawn, and the average life and the longest life were calculated.

1 is a graph showing a life curve of a nematode, and Table 5 is a graph showing an average life, a longest life, and a life extension rate of a nematode. The life extension rate, as shown by these graphs showing the average life of each sample when the average life of the control liquid is 100%, is intentionally extended for controlling the lifespan of the liquid nematode, and the examples of the present invention confirmed the life extension effect. In particular, with reference to the life extension rate, the material of Example 1 after 11-fold dilution was extended by 30% or more, the substance of Example 1 after 101-fold dilution, and the substance of Example 2 after 11-fold dilution, 20 More than % extended the average life. Further, 10% or more of the substance of Example 2 after the 101-fold dilution extended the average life.

【table 5】

Further, it can be seen that the higher the concentration of the fermentation metabolite-containing liquid in the culture solution, the longer the life is prolonged. Thus, it was confirmed that the life extension in the present experimental example was due to the fermentation metabolite of the present invention.

In addition, it was confirmed that Example 1 which was fermented in a medium containing soybean milk had a slightly higher life extension effect than Example 2 which was fermented in a medium containing milk.

From the above, it was confirmed that the fermentation metabolite of the present invention has a very high anti-glycation action and life extension effect. The prolongation of life is thought to be achieved by various mechanisms of action, and as a mechanism of action, it is thought to be related to anti-glycation. In addition, it is also considered to be active against other antioxidants and tyrosinase Properties, collagenase inhibitory activity, lipase inhibitory activity, angiotensin-converting enzyme inhibitory activity, and the like.

In Example 1 and Example 2, respectively, a medium containing soybean milk and milk was used, but even a medium containing goat milk or sheep milk having the same constitution as milk, or a fermentation metabolite of fermented H-61 strain in other medium was used. The same results as in Example 1 and Example 2 were obtained.

Since the fermentation metabolite of the present invention has a life-prolonging action and an anti-glycation action, it can be said to have an aging-suppressing action associated with these.

(examine)

With reference to Patent Document 1, it is reported that the living cells and the dead cells of the above H-61 strain have an effect of suppressing a decrease in bone density, an effect of suppressing the occurrence of skin ulcers, and an effect of suppressing aging caused by these. In this case, the components contained in the living cells and the dead cells, that is, the proteins, nucleic acids, and other minor components mainly constituting the cells act on the intestinal immunity, and the above effects are found. However, the bone density reduction inhibitor, the skin ulcer inhibitor, and the aging inhibitor according to Patent Document 1 collect and culture the above-mentioned H-61 strain and use the washed cells (see [0015] [0018] of Patent Document 1 [ Since 0020] paragraph), the fermentation metabolite produced by the above H-61 strain is not substantially contained.

According to the present invention, it has been confirmed that the fermentation metabolite from which the cells are removed has a very high anti-glycation action and a prolonged life effect. Of course, the components contained in the cells and the fermentation metabolites are very different, and the cells contain proteins and lipids which mainly constitute the cells, whereas the fermentation metabolites mainly contain lactic acid and various amino acids. Therefore, the fermented metabolite of the present invention is produced at least by a mechanism different from that of the bacterial cell. It has a significant anti-glycation effect and prolongs life. Thus, the fermentation metabolite of lactic acid bacteria has an effect of inhibiting aging, and the like, and the present invention exhibits this new knowledge.

Claims (9)

An anti-glycation agent characterized by comprising a fermentation metabolite of a lactic acid bacteria Lactococcus lactis subsp. cremoris H-61 strain as an active ingredient. The anti-glycation agent according to claim 1, wherein the fermentation metabolite does not contain a bacterial cell. The anti-glycation agent according to claim 1 or 2, wherein the fermentation metabolite is produced by fermenting the lactic acid bacteria in a medium containing at least one of soybean milk, milk, goat milk, and sheep milk. A life extender characterized by comprising a fermentation metabolite of a lactic acid bacteria Lactococcus lactis subsp. cremoris H-61 strain as an active ingredient. The life extension agent as recited in claim 4, wherein the life extension is caused by anti-glycation. The life extension agent according to claim 4 or 5, wherein the fermentation metabolite does not contain a bacterial cell. The life extension agent according to any one of claims 4 to 6, wherein the fermentation metabolite is produced by fermenting the lactic acid bacteria in a medium containing at least one of soybean milk, milk, goat milk, and sheep milk. An aging inhibitor characterized by comprising a fermentation metabolite of a lactic acid bacteria Lactococcus lactis subsp. cremoris H-61 strain as an active ingredient. The aging inhibitor according to claim 8, wherein the inhibition of aging is caused by Anti-glycation.