TWI785854B - A lactobacillus strain for producing hyaluronic acid, and an application and a composition thereof - Google Patents

Abstract

The present invention provides novel strains of lactic acid bacteria that can effectively produce hyaluronic acid. The selected lactic acid bacteria is Lactobacillus paracasei CMU566, the deposit number is BCRC911070. The present invention also provides human tests that can support the practicability and effectiveness of the present invention, and provides the composition and use of the lactic acid strain.

Description

A kind of lactic acid bacteria strain producing hyaluronic acid, its use and composition

The present invention relates to a probiotic strain and its use, in particular to a novel compound lactic acid bacteria strain and its use in the production of hyaluronic acid.

Hyaluronic acid (Hyaluronan, Hyaluronic acid), also known as hyaluronic acid, glass uronic acid, glass sugar carbonic acid, is a sugar composed of disaccharides (D-glucuronic acid and N-acetyl glucosamine) basic structure Aminoglycans. Because hyaluronic acid has very good water absorption and moisturizing properties, and has viscoelasticity, it also has good biocompatibility to the human body, and does not cause allergic reactions to antigens. Therefore, hyaluronic acid can be widely used in food, dairy products, beverages, pharmaceuticals, cosmetics, etc. cosmetics and medical supplies.

In the human body, hyaluronic acid widely exists in connective tissue, epithelial tissue and nerve tissue, and in the form of hyaluronic acid in the skin, umbilical cord and vitreous humor. A normal human body with an average weight of 75 kg contains about 16 grams of hyaluronic acid, of which about one-third It is decomposed and re-synthesized every day, but hyaluronic acid will gradually decrease with age. Therefore, the younger the skin or joints contain more hyaluronic acid, so the skin is softer, more elastic and the joints are lubricated. When the hyaluronic acid decreases, the skin will gradually lose its water retention capacity. In addition to the need to replenish due to aging, if the work pressure Factors such as strong strength, staying up late, unbalanced eating habits, ultraviolet rays, or working in an air-conditioned room for a long time can also easily cause the loss of hyaluronic acid and skin moisture.

Hyaluronic acid has been widely used in medicine. For example, injection of hyaluronic acid into the knee joint can improve degenerative osteoarthritis, and moisturizing products containing sodium hyaluronate active ingredients can be used to relieve dry skin (such as atopic dampness). xeroderma due to rashes). For some cancers, the concentration of hyaluronic acid in the patient's body can reflect the prognosis. Therefore, hyaluronic acid is also used as a marker of prostate cancer and breast cancer. Hyaluronic acid can also be used to monitor the course of cancer. Meanwhile, hyaluronic acid has been used to synthesize bioscaffolds with healing function. The hyaluronic acid in this scaffold and the fibronectin associated with it can promote the migration of cells to the wound. This effect is especially important for diabetics whose wounds are not easy to heal. In cataract surgery and other surgeries, hyaluronic acid is also used to promote postoperative tissue repair, and it can also help the fluid in the eye tissue to help protect the eyes.

Currently commercially available edible hyaluronic acid mainly comes from the fermentation of cockscombs and Streptococcus epidemics. Among them, the source of cockscomb extract is widely used in joint and skin health food appeals. However, hyaluronic acid extracted from cockscomb still has doubts about zoonotic virus infection in terms of food safety, and the price is quite expensive, so it cannot be widely used in health food supply. The general public eats it. In addition, Streptococcus epidemics is an epidemic pathogenic bacterium that can cause a variety of animal infections. The main clinical symptoms are sepsis, meningitis, and arthritis. Therefore, hyaluronic acid from this source has risks in consumption.

In order to solve the above problems, the present invention provides a novel lactic acid bacterial strain, which is selected from healthy newborns and is generally recognized as safe strain (GRAS), and the lactic acid bacterial strain can effectively produce hyaluronic acid. The lactic acid bacteria strain is Lactobacillus paracasei ( Lactobacillus paracasei ) CMU566 (registration number is BCRC911070).

The present invention also provides a lactic acid bacterium strain for the production of hyaluronic acid composition, wherein the lactic acid bacterium strain is Lactobacillus paracasei ( Lactobacillus paracasei ) CMU566 (registration number is BCRC911070), and the lactic acid bacterium strain is active or inactive strains.

The present invention further provides a composition for producing hyaluronic acid, comprising: Lactobacillus paracasei (Lactobacillus paracasei) CMU566 (registration number: BCRC911070) lactic acid bacteria strain producing hyaluronic acid, wherein these strains can produce hyaluronic acid in the intestinal tract, and can produce hyaluronic acid in the intestinal tract. The hyaluronic acid produced by the intestinal tract can be absorbed through the intestinal tract to increase the content of hyaluronic acid in the blood; and the strain can survive and multiply in the intestinal tract to increase the total number of the lactic acid bacteria strain, which can be an active or inactive strain.

In one embodiment of the present invention, the composition can be used as food, beverage, dietary supplement, dairy product, medicine, beauty care product and medical product, etc.

The present invention also provides a use of a lactic acid bacterial strain, which is to apply the lactic acid bacterial strain of the present invention or its metabolites to the preparation of foods, beverages, dietary supplements, dairy products, medicines, beauty care products and medical products that increase hyaluronic acid. Supplies etc.

The isolated and purified lactic acid bacteria strains of the present invention are screened from healthy newborns, and this The strain has good resistance to bile salts and gastric acid, so it can pass through the human digestive tract and enter the intestinal tract to grow smoothly. More particularly, the lactic acid bacteria strain of the present invention can produce hyaluronic acid in the intestinal tract, so that the content of hyaluronic acid in the human body can be increased after taking it. Therefore, the lactic acid bacteria strain of the present invention or its metabolites can be applied to food, beverages, dairy products, medicines, beauty care products and medical supplies, etc.

Figure 1 is a schematic diagram showing the comparison of various bacterial strains with hyaluronic acid production potential.

Figure 2 shows the results of the acid resistance test of various lactic acid bacteria strains in artificial simulated gastric juice.

Fig. 3 is a schematic diagram showing the results of the bile salt tolerance test of various strains of lactic acid bacteria in artificial simulated intestinal fluid.

Fig. 4 is a schematic diagram showing the results of various lactic acid bacteria strains adsorbing to intestinal cells.

Fig. 5 is a schematic diagram showing the test results of the bactericidal activity of various lactic acid bacteria strains on Escherichia coli.

Fig. 6 is a schematic diagram showing the hyaluronic acid production concentration curve detected by the lactic acid bacteria strain of the present invention in simulated artificial intestinal juice at different fermentation times.

Fig. 7 shows the bar diagram of the average hyaluronic acid concentration in the blood of the CMU566 lactic acid bacteria of the present invention in the human experiment.

In order to enable those who are familiar with the general skills in the technical field of the present invention to further understand the present invention, several preferred embodiments of the present invention are enumerated below, and in conjunction with the attached drawings, the constitutional content and desired achievement of the present invention are described in detail. The effect.

The lactic acid bacteria strain CMU566 isolated and purified in the present invention is screened from healthy newborns. After purification and culture and strain identification, the lactic acid bacteria strain CMU566 is Lactobacillus paracasei , which is deposited in the Hsinchu Food Industry Development Research Institute. The serial number is BCRC911070.

The lactic acid bacteria strain CMU566 of the present invention is derived from a large number of lactic acid bacteria collected from healthy newborns and breast milk. Then, the hyaluronic acid analysis method is used to test and screen the strains that can ferment and produce hyaluronic acid in large quantities, and the lactic acid bacteria strain CMU566 has the highest hyaluronic acid production. Then, the simulated human digestive tract experiment proved that the lactic acid bacteria strain CMU566 has good resistance to gastric acid and intestinal bile salts, so it can pass through the human digestive tract smoothly. Finally, the intestinal adsorption capacity of the lactic acid bacteria strain CMU566 was analyzed, and it was confirmed that it can enter the intestinal tract. reproductive growth. In addition, it has been proved that the lactic acid bacteria strain CMU566 of the present invention has the potential to improve intestinal flora, maintain joint lubrication, and increase skin elasticity. Therefore, it can be made into food, beverage, dietary supplement, milk Products, pharmaceuticals, beauty care products and medical supplies, etc.

(1) Lactic acid bacteria collection

The sources of the various lactic acid bacteria tested in this experiment include: purchased from the Biological Resource Conservation and Research Center (BCRC, Taiwan, Hsinchu), self-screened from a number of hospitals and confinement centers in the central region, etc. Feces of newborn babies, Adult feces, fruits, traditional kimchi food and animal sources, etc. After these lactic acid bacteria strains were isolated, they were preliminarily confirmed as lactic acid bacteria by Catalase test, Gram stain, microscopic examination, and experimental analysis of motility. These lactic acid bacteria strains were cultured with Lactobacilli MRS broth (containing 0.02% L-cysteine) at 37°C for 24 hours, and then stored in a -70°C refrigerator with 15% glycerol MRS broth.

(2) Screening and analysis of strains capable of producing hyaluronic acid and their tolerance test

The present invention measures the content of hyaluronic acid according to the carbazole method (Bitter and Muir, 1962, Anal. Biochem. 4, 330-334). The lactic acid bacteria strains collected above were fermented with the MRS culture medium for 48 hours, and the culture medium was filtered with a sterile filter membrane, and the filtrate was precipitated with 4 times the volume of 95% alcohol for 3 times to remove non-sugar components, and then the precipitate Dissolve in an equal volume of buffer solution. Take 1 milliliter (mL) of the sample and standard solution into each test tube and place it on ice, add 3 ml of reagent A (dissolve 0.381 g of sodium tetraborate in 400 ml of sulfuric acid, keep away from light and refrigerate), and move the test tube to a boiling water bath After 10 minutes, place on ice to cool to room temperature, then add 0.1 ml of reagent B (reagent B: dissolve 0.15 g of carbazole in 100 ml of ethanol, keep in dark and refrigerated), move the test tube to a boiling water bath for 15 Minutes, then placed on ice to cool to room temperature, and finally measured the absorbance with a spectrophotometer at a wavelength of 525 nanometers (nm).

The results of the experiment found that the hyaluronic acid production of the lactic acid strain CMU566 screened from healthy newborns was the highest. To further analyze whether the CMU566 lactic acid strain can safely reach the intestinal tract and exert the function of hyaluronic acid secretion, a simulation test of the human digestive tract was carried out, including the gastric acid resistance of the strain Sex test and bile salt tolerance test, as well as intestinal absorption capacity. The experimental steps are as follows:

(1) gastric acid resistance test

The analysis method was modified with reference to the research report of Zavaglia et al. (1998). Take 1 ml of the lactic acid bacteria liquid activated by overnight culture, wash once with pH 7.2 phosphate buffer solution, and then relyse the bacteria with deionized water, take 100 microliters (μl) of the above bacterial liquid and add them to different pH value (pH2.0, 2.5 and 7.2) in 9.9 ml sterile phosphate buffer solution. After the bacteria solution and the buffer solution are fully mixed, place in an incubator at 37°C with a rotation speed of 80rpm. After 3 hours, take out 1 ml of phosphate buffer solution with pH 7.2 for serial dilution, and use MRS agar medium (MRS agar) at 37°C. Cultivate for 48 hours, and calculate the number of surviving lactic acid bacteria.

(2) Intestinal bile salt resistance test

The effect of bile salts on the growth of lactic acid bacteria was modified according to the methods described by Lin et al. (2006) and Gilliland and Walker (1990). 100 microliters of the activated lactic acid bacteria cultured overnight were taken and added to 10 milliliters of MRS broth or MRS broth (MRS broth) with 0.3% (w/v) ox gall (ox gall, Sigma) added. After incubating at 37°C for 8 hours, take 1 ml of bacterial liquid, add 9 ml of pH 7.2 phosphate buffer solution (PBS) for serial dilution, and use the pour plate count method (pour plate count method) to mix culture with MRS agar medium to analyze the number of bacteria , to evaluate the number growth of tested lactic acid bacteria in the presence of bile salts. In addition, take out 1 ml of bacterial solution and centrifuge at 0, 2 and 4 hours, redissolve and suspend in phosphate buffer solution, then use a spectrophotometer to analyze its OD 600 nm absorbance value, and calculate the bile salt tolerance (%), the formula is as follows Shown: Bile tolerance(%)=(Increment of OD 600 nm in MRS broth with bile salt/increment of OD 600 nm in MRS broth without bile salt)×100.

(3) Intestinal adhesion test

This analysis method refers to the research steps of scholars such as Lin et al. (2006) and Sarem et al. (1996).

a. Human intestinal cell line Caco-2 was treated with 1 ml of 0.05% trypsin (Trypsin) for 5 minutes, and the cells were gently tapped on the culture flask by hand, and distributed in 96-well culture plates, each hole Add 1 x 10 ⁴ cells to medium, and change 200 μl of fresh DMEM medium every day.

b. Then add 20 microliters of lactic acid bacteria solution to the cells, and then culture for 1 hour to allow the lactic acid bacteria to attach.

c. After the lactic acid bacteria attach for 1 hour, remove the medium, wash 5 times with phosphate buffer solution to remove unattached lactic acid bacteria, add 100 microliters, 10% formaldehyde solution (formalin), 30 minutes, fix the cells and bacteria, and then Wash 3 times with phosphate buffer solution, and finally stain with 100 microliters of crystal violet (Crystal violet), and rinse quickly with a small amount of 75% alcohol after five minutes to remove the staining agent on the cells.

d. Use a phase-contrast microscope to observe the adsorption of lactic acid bacteria to intestinal epithelial cells, observe and calculate the adsorption of lactic acid bacteria in 50 cells randomly under different microscope fields (randomized microscopic areas), and finally calculate the average adsorption of lactic acid bacteria per cell quantity.

(3) Bactericidal test against Escherichia coli

The method used was modified from the procedure of Koga et al. (2002) and Lorca et al. (2001). Inoculate the screened lactic acid bacteria into 10 ml of MRS culture medium, after 48 hours of fermentation, add 100 microliters of Escherichia coli bacteria solution and mix evenly, take 100 microliters of co-culture bacteria at 0, 2, and 4 hours respectively The solution was serially diluted 10 times in sterile phosphate buffer solution, smeared with Coliform medium, cultured at 37°C, and counted the number of surviving E. coli bacteria after 2 days.

(4) Strain identification

Microbes of different genera must have their unique nucleic acid sequences. In the case of lactic acid bacteria, The identification of bacterial species is usually carried out by 16S rDNA gene sequence. Therefore, the lactic acid bacteria strain CMU566 of the present invention was commissioned by the Food Industry Development Research Institute of the Foundation to analyze the 16S rDNA sequence (as shown in the sequence identification code: 1), and it was identified as Lactobacillus paracasei (Lactobacillus paracasei), which is now deposited in Hsinchu Food Industry Development Institute Biological Resources Conservation and Research Center, registration number is BCRC911070. The analysis results are shown in the strain identification report attached together with the present invention.

(5) Analysis of hyaluronic acid production concentration of lactic acid bacteria strain CMU566 at different fermentation times

In order to observe the hyaluronic acid production and secretion curve of the lactic acid bacteria strain CMU566 in the intestinal juice, the lactic acid bacteria strain CMU566 was cultured in the simulated artificial intestinal juice prepared by MRS medium. At the time point of 72 hours, the fermentation broth was collected for hyaluronic acid concentration analysis.

(6) Effectiveness test of lactic acid bacteria strain CMU566 in human body In order to confirm the effectiveness of the lactic acid bacteria strain CMU566 on human body, a human clinical trial was conducted on 12 people, aged between 30 and 50 years old, and conducted by self-comparison , the subjects took the embedded protected CMU566 lactic acid bacteria powder containing more than 1*10 ¹⁰ CFU dose once a day. On the 0th, 1st, 3rd, 5th and 7th day of taking, the serum samples of the subjects were collected for analysis with the human hyaluronic acid immunoassay kit. The experimental procedure is to add 100 microliters of hyaluronic acid standard solutions of different concentrations and the serum samples of the subjects into each hole of the coating plate coated with hyaluronic acid antibody, and incubate at 37°C for 2 hours. Then remove the liquid in the hole, then add 100 microliters of biotin-antibody (Biotin-antibody), and then incubate at 37°C for 1 hour to react, then add HRP (horseradish peroxidase)-avidin, and incubate at 37°C for 1 hour After washing 5 times to remove unbound reagents, add 100 microliters of TMB substrate to react for 30 minutes, add 50 microliters to stop the color reaction, and measure the absorbance at 450nm wavelength with an immunoenzyme analyzer within 5 minutes .

Experimental results

Please refer to Figure 1, which shows the results of comparing strains with hyaluronic acid production potential. The data shows that the lactic acid strain CMU566 has the highest hyaluronic acid production.

Please refer to Figure 2, which shows the acid resistance test of various strains of lactic acid bacteria with higher production of hyaluronic acid cultured in artificial simulated gastric juice. The data show that the lactic acid bacteria strain CMU566 has very good gastric acid tolerance.

Please refer to Figure 3, which shows that lactic acid bacteria with higher production of hyaluronic acid were cultured in artificial simulated intestinal fluid to test the bile salt tolerance test of each strain. The data show that the lactic acid bacteria strain CMU566 has very good bile salt tolerance, so it can grow stably in intestinal fluid.

Please refer to Figure 4, which shows the adsorption test of lactic acid bacteria with higher hyaluronic acid production to intestinal cells. Experiments have confirmed that the lactic acid bacteria strain CMU566 has the ability to adsorb intestinal cells.

Please refer to Figure 5, which shows the bactericidal ability test of lactic acid bacteria with higher production of hyaluronic acid on Escherichia coli. Experiments have confirmed that the lactic acid bacteria strain CMU566 has excellent bactericidal ability against pathogenic bacteria.

Please refer to Figure 6, which shows that the lactic acid bacteria strain CMU566 is simulated artificially In the intestinal juice, the production concentration curve of hyaluronic acid was detected at different fermentation times. Experiments show that the lactic acid bacteria strain CMU566 can reach the highest hyaluronic acid production concentration of 1460 micrograms per milliliter (μg/mL) within 24 hours of fermentation.

Please refer to Figure 7, which shows the average hyaluronic acid concentration in the blood of 12 subjects after taking the lactic acid bacteria strain CMU566 of the present invention on the 0th, 1st, 3rd and 5th day. Experiments have confirmed that the average concentration of hyaluronic acid in the blood of the subjects gradually increases with time, showing that the lactic acid bacteria strain CMU566 can indeed continuously produce hyaluronic acid in the human intestine, and can be absorbed by the intestine at the same time, increasing the concentration of hyaluronic acid in the blood.

In summary, the lactic acid bacteria strain CMU566 of the present invention can produce high-concentration hyaluronic acid, and the strain has gastric acid resistance, intestinal bile salt resistance and intestinal cell adsorption ability, so it can smoothly reach the intestinal tract. Therefore, it can be made into foods, beverages, dietary supplements, dairy products, medicines, beauty care products and medical supplies that can effectively increase hyaluronic acid in the body.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

【Biological Material Storage】

Lactobacillus paracasei ( Lactobacillus paracasei ) CMU566 strain was deposited in the Food Industry Development Institute of the Foundation on August 12, 2021, and the deposit number is BCRC911070.

Claims (9)

A lactic acid bacteria strain producing hyaluronic acid, wherein the lactic acid bacteria strain is Lactobacillus paracasei ( Lactobacillus paracasei ) CMU566, deposited in Taiwan Hsinchu Food Industry Development Institute Biological Resource Conservation and Research Center, registration number is BCRC911070. A lactic acid bacterium strain is used in the preparation of a composition for the production of hyaluronic acid, wherein the lactic acid bacterium strain is Lactobacillus paracasei (Lactobacillus paracasei), deposited in Taiwan Hsinchu Food Industry Development Institute Biological Resource Preservation and Research Center, CMU566 deposit number is BCRC911070. The use as described in item 2 of the scope of the patent application, wherein the lactic acid bacteria strain is an active or inactivated strain. The use as described in item 2 of the scope of the patent application, wherein the lactic acid bacteria strain produces metabolites after fermentation and culture, and the metabolites are used to produce hyaluronic acid compositions. A composition for producing hyaluronic acid, comprising: hyaluronic acid-producing lactic acid bacteria strain or its metabolites, the lactic acid bacteria strain is Lactobacillus paracasei ( Lactobacillus paracasei ), deposited in Taiwan Hsinchu Food Industry Development Institute Biological Resource Preservation and Research Center , CMU566 registration number is BCRC911070. The composition described in item 5 of the patent application, wherein the lactic acid bacteria strain can produce hyaluronic acid in the intestinal tract, and the hyaluronic acid produced in the intestinal tract can be absorbed through the intestinal tract to increase the content of hyaluronic acid in the blood. The composition as described in item 5 of the patent application, wherein the lactic acid bacteria strain can effectively multiply in the intestinal tract to increase the number of the lactic acid bacteria strain. The composition as described in item 5 of the patent application, wherein the lactic acid bacteria strain is an active or inactivated strain. The composition as described in item 5 of the scope of the patent application, wherein the composition can be food, beverage, dietary supplement, dairy product, medicine, beauty care product and medical product, etc.

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