TWI740073B - Lactic acid bacteria crystal composition capable of promoting intestinal stem cell proliferation, antiviral, anti-inflammatory and anti-allergic effects and preparation method thereof. - Google Patents

Abstract

The present invention provides a lactic acid bacteria crystal composition coated with algae fermentation broth, which has ability of acid resistance and choline resistance. The composition has efficacies of promoting intestinal stem cell proliferation, antiviral, anti-inflammatory and anti-allergy, and a preparation method thereof.

Description

Milk with the effects of promoting the proliferation of intestinal stem cells, antiviral, anti-inflammatory and anti-allergic effects Acid bacteria crystal composition and preparation method thereof

The present invention provides a lactic acid bacteria crystal composition coated with an algae fermented liquid, which is produced in a purely natural way, using the algae fermented liquid produced by fermented fermentation as a colloid source for protecting bacteria, so that the composition has the ability to resist gastric acid and bile And this composition takes a variety of lactic acid bacteria as the main body, which can provide the benefits of multiple beneficial bacteria at one time. At the same time, the algae fermented liquid colloid can cooperate with the lactic acid bacteria to promote the proliferation of intestinal stem cells, anti-virus, anti-inflammatory and anti-allergic lactic acid bacteria crystal combination物 and its preparation method.

Lactic acid bacteria have a positive effect on the human body. From 1961 to 1998, a total of 143 human clinical trials of lactic acid bacteria have been completed and published in well-known academic journals. The number of people tested reached 7,526, and there were no negative results reported. The main positive effects that have been reported include: (1) improve the nutritional quality of food, (2) promote vitamin synthesis and enzyme production, (3) stabilize the intestinal flora and remove intestinal bacteria, (4) produce antibacterial substances , Enhance host immunity, (5) reduce cholesterol content, (6) reduce the risk of colorectal cancer, (7) inhibit tumors, etc.

The most commonly used lactic acid bacteria to be tested by scientists is Lactobacillus acidophilus (commonly known as bacteria A), followed by Lactobacillus casei (commonly known as bacteria C), and other bacteria that are more commonly tested are still There are Bifidobacterium (Bifidobacterium sp., commonly known as B bacteria, also known as Bifidobacterium or Bifidobacterium), Lactobacillus bulgaricus, Streptococcus thermophilus, and the latter two are Two strains of lactic acid bacteria must be added in the process of making yogurt from fresh milk.

However, lactic acid bacteria products are prone to lose their activity after digestion in the gastrointestinal tract. Some bacteria can reach the intestines but do not have multiple functions. It is currently known that the modified bacteria can have acid and alkali resistance and also have physiological activities. However, some scientists still There are doubts about strain improvement. Therefore, lactic acid bacteria generally have the shortcomings of instability, inability to heat and acid, will change with environmental changes and poor storage conditions, resulting in reduced efficacy. They must be refrigerated at low temperatures. How to increase the stability and acid and alkali resistance of lactic acid bacteria products to achieve their efficacy is an urgent need to solve problem.

According to the literature The Anti-Inflammatory Effect of Algae-Derived Lipid Extracts on Lipopolysaccharide(LPS)-Stimulated Human THP-1 Macrophages.Marine drugs.Vol 13,P5402-5424, the antibacterial, acid-fast and antibacterial properties of Lactobacillus isolated from Koozeh cheese In the study of bile tolerance, 28 Lactobacillus strains were isolated from three groups of Lactobacillus (such as Lactobacillus plantarum, Lactobacillus casei and Lactobacillus delbrueckii) for bile tolerance, acid tolerance and antibacterial Performance, it turns out that only one isolate, Lactobacillus casei, can tolerate acid and bile salts and has anti-monocytic proliferation antibacterial activity. It can be considered as a natural probiotic product, but additional tests are still needed to prove it. Lactic acid bacteria do not have the ability to tolerate choline and acid, and it takes a lot of time to screen and test, which severely limits the application of lactic acid bacteria.

In another study of algae, the polysaccharides and polyphenols in algae have anti-oxidation, anti-cancer, anti-coagulation, anti-inflammatory, anti-viral, and anti-hyperlipidemia effects. In order to obtain these active substances, the industry usually uses hot water, Solvent, acid hydrolysis, enzyme hydrolysis and other extraction methods, but these extraction methods are usually accompanied by some limiting factors, including: high cost, complicated process and residual toxicity of solvents. Another study found that the algae fermentation process can release more algae active ingredients, such as: the unique seaweed peptides, amino acids, and polyphenol compounds that are not easily obtained by general extraction methods can be fully released through the fermentation process, so that The nutrients in the product are richer, and after a long time of fermentation, the nutrients in the seaweed can be decomposed into small molecules, which is more conducive to biological absorption and utilization, but usually algae polysaccharides are used as the main raw material, and there are no probiotics in terms of efficacy. Improve intestines, repair mucosal tissues and other functions.

Therefore, the development of a lactic acid bacteria crystal composition that is suitable for the general public and has acid and choline resistance, and maintains the effects of anti-inflammatory, anti-virus and mucosal repair is the most important problem that needs to be solved at this stage.

In order to solve the above-mentioned problems, the present invention adopts a purely natural process, and uses the algae fermented liquid produced by fermenting as a source of colloid to protect the bacteria, so that the lactic acid bacteria have the ability to resist acid and choline and there is no doubt about the safety of use and food, and The present invention takes multiple lactic acid bacteria as the main body and can provide the benefits of multiple beneficial bacteria at one time. At the same time, the algae fermented liquid colloid can synergize the action of lactic acid bacteria, so that the effect can simultaneously promote intestinal stem cell proliferation, anti-virus, anti-inflammatory and anti-allergic.

In an embodiment of the present invention, a lactic acid bacteria crystal composition with acid and choline resistance, wherein the composition comprises: lactic acid bacteria and algae fermented liquid, the algae fermented liquid is a colloidal structure, which is coated outside the lactic acid bacteria, The lactic acid bacteria crystal shell is formed.

In an embodiment of the present invention, the lactic acid bacteria crystal composition, wherein the colloidal structure contains sugar molecules, the sugar molecules are small molecule sugars produced after degradation of α-glucan or β-glucan, and the sugar molecules are coated The lactic acid bacteria form the lactic acid bacteria crystal shell.

In an embodiment of the present invention, the lactic acid bacteria crystal composition, wherein the lactic acid bacteria comprise Lactobacillus rhamnosus , Lactobacillus plantarum , Lactobacillus acidophilus or Lactobacillus sporogenes strains.

In an embodiment of the present invention, in the lactic acid bacteria crystal composition, the mixing ratio of the lactic acid bacteria is that the content of Lactobacillus rhamnosus is 3×10 ⁸ CFU/g~5×10 ⁸ CFU/g, and the content of Lactobacillus plantarum is 3×10 ⁸ CFU/g~5×10 ⁸ CFU/g, Lactobacillus acidophilus content is 3×10 ⁸ CFU/g~5×10 ⁸ CFU/g, and Lactobacillus sporogenes content is 3×10 ⁸ CFU/g~5×10 ⁸ CFU /g.

In an embodiment of the present invention, the lactic acid bacteria crystal composition, wherein the algae fermented liquid contains sea fungus, agaric, ulva, chlorophyllum, sea grape or pueraria lobata fermented fermented liquid, a preferred In the embodiment, the algae ferment system is sea fungus fermented liquid.

In an embodiment of the present invention, in the lactic acid bacteria crystal composition, the mixing volume ratio of the algae fermented liquid and the lactic acid bacteria is 50:1 to 70:1.

In an embodiment of the present invention, in the lactic acid bacteria crystal composition, the mixing volume ratio of the algae fermented liquid and the lactic acid bacteria is preferably 60:1.

According to an embodiment of the present invention, a lactic acid bacteria crystal composition with acid and choline resistance is used for the efficacy application, wherein the efficacy includes the effects of promoting intestinal stem cell proliferation, antiviral, anti-inflammatory and anti-allergic effects.

In one embodiment of the present invention, a method for preparing the algae fermented liquid of the acid-resistant and choline-resistant lactic acid bacteria crystal composition is: (1) the initial extraction ferment step: after the algae is squeezed, A is added (2) Compound fermentation step: add B yeast and C lactic acid bacteria for 390 days compound fermentation; (3) Storage fermentation step: add D acetic acid bacteria for 30 days fermentation And (4) Chelated fermentation step: add magnesium and calcium compounds, carry out 30-day fermentation, and use bacteria to convert calcium and magnesium into organic state; during the fermentation process, the bacteria are added to 10 ⁷ ~ 10 ⁸ CFU /ml, the inoculum volume is 1.5% by volume; the fermentation temperature is controlled at 26-32℃.

In one embodiment of the present invention, the method for preparing the algae lactic acid bacterium having resistance to acid and choline ability of the crystalline composition of the fermentation broth Po, wherein the concentration of lactic acid 10 ⁸ CFU / ml, was inoculated in an amount of 1.5% by volume, a concentration of ¹⁰⁷ yeast CFU/ml, the inoculum volume is 1.5% by volume, and the number of bacteria must be sufficient to become the dominant bacteria species.

In an embodiment of the present invention, the method for preparing the algae fermented liquid of the acid-resistant and choline-resistant lactic acid bacteria crystal composition, wherein the bacteria A added in the initial extraction ferment step is the yeast Saccharomyces cerevisiae .

In one embodiment of the present invention, the method for preparing the algae fermented liquid of the lactic acid bacteria crystal composition with acid and choline resistance ability, wherein the B yeast added in the compound fermenting step is Saccharomycopsisfibufigera , Pichia membramefaciens , Schizosaccharomyespombe yeast.

In an embodiment of the present invention, the method for preparing the algae fermentation liquid of the lactic acid bacteria crystal composition with acid and choline resistance ability, wherein the C lactic acid bacteria added in the compound fermentation step are Lactobacillus plantarum , Lactobacillus delbrueckii , Lactococcuslactis , Lactococcus acidophillus, Bifidobacterium bifidum lactic acid bacteria.

In an embodiment of the present invention, the method for preparing an algae fermentation liquid of a lactic acid bacteria crystal composition with acid and choline resistance, wherein the D acetic acid bacteria added in the storage fermentation step are Acetobacterhansenii , Acetobacterxylinum , Acetobactor suboxydans acetic acid bacteria.

In an embodiment of the present invention, a method for preparing a lactic acid bacteria crystal composition with acid and choline resistance includes: (1) providing an algae fermented liquid, and the production method of the algae fermented liquid is as described in the previous process; 2) Provide a lactic acid bacteria, the lactic acid bacteria is selected from the group consisting of Lactobacillus rhamnosus , Lactobacillus plantarum , Lactobacillus acidophilus and Lactobacillus sporogenes ; (3) mixing step, mixing the algae fermented liquid with the lactic acid bacteria in a volume ratio of 60:1 And (4) a high-speed emulsification step. After the mixed solution completed in step (3) is emulsified at a speed of 1000~3000rpm through a high-speed emulsifying head, the algae fermented liquid colloidal structure that has completed the fermentation process is coated on the outer layer of the lactic acid bacteria , The lactic acid bacteria crystal shell is formed, and the lactic acid bacteria crystal composition is completed.

The structure of algae colloid is dominated by sugar molecules. The source of sugar molecules is the small molecule sugars produced by the degradation of glucan. During the emulsification stage, a shell is formed around the lactic acid bacteria by coating or bonding to produce lactic acid bacteria crystals; the lactic acid bacteria crystal combination It has acid and choline resistance, promotes the proliferation of intestinal stem cells, antiviral, anti-inflammatory and anti-allergic effects.

Figure 1 shows the crystal shell composition of lactic acid bacteria coated with algae fermented liquid.

Figure 2 shows a photograph of the lactic acid bacteria crystal composition imaged by a compound microscope with a magnification of 400 times.

Figure 3 shows the acid tolerance test of lactic acid bacteria. The experiment is divided into two groups: (1) a mixture of four lactic acid bacteria and (2) a crystal composition of four lactic acid bacteria + algae fermentation solution. The values are expressed as mean ± standard deviation, ^{* *} P<0.01 means that (2) the crystal composition of the four lactic acid bacteria + algae fermented liquid has a significant difference compared with (1) the value of the four lactic acid bacteria strains in the acid tolerance test. It is confirmed that the survival rate of lactic acid bacteria is greatly reduced after being destroyed by simulating gastric acid through hydrochloric acid without treatment. However, the lactic acid bacteria crystal composition coated with algae fermented liquid still retains a good survival rate even if it is destroyed by gastric acid, that is, through the algae fermented liquid package. Cover structure, can provide lactic acid bacteria resistance to acid corrosion.

Figure 4 shows the choline tolerance test of lactic acid bacteria. The experiment is divided into two groups: (1) four strains of lactic acid bacteria mixture group and (2) four strains of lactic acid bacteria + algae fermentation liquid crystal composition. The values are expressed as mean ± standard deviation. ^{** *} P<0.001 indicates that (2) the four lactic acid bacteria + algae fermentation liquid crystal composition in the choline tolerance test is significantly different from (1) the values of the four lactic acid bacteria strains.

Figure 5 shows the intestinal stem cell proliferation test of lactic acid bacteria. The experiment is divided into five groups of samples: (1) Control-control group, (2) a mixture of four lactic acid bacteria, (3) algae extract, (4) algae fermented liquid And (5) Four strains of lactic acid bacteria + algae fermented liquid crystal composition, the value is expressed as the mean ± standard deviation, ^* P<0.05, ^** P<0.01, ^*** P<0.001 indicates that the intestinal stem cell survival rate test neutral Compared with the control group, there are significant differences. The results confirmed that the survival rate of lactic acid bacteria without treatment was greatly reduced after being destroyed by Oxoid to simulate choline, but the lactic acid bacteria crystal composition coated with algae fermented liquid, even if destroyed by choline, still maintained a good survival rate.

Figure 6 shows the antiviral test of lactic acid bacteria. The experiment is divided into six groups of samples: (1) Control-control group, (2) Virus-challenge group, (3) four lactic acid bacteria mixture-challenge group, (4) ) Algae extract-challenge group, (5) algae fermentation liquid-challenge group and (6) four lactic acid bacteria + algae fermentation liquid crystal-challenge group. Values are expressed as mean ± standard deviation, ^* P<0.05, ^** P<0.01, ^*** P<0.001 indicates that there is a significant difference in the antiviral efficacy test compared with (2) Virus-challenge group value; the results show After mixing, the general lactic acid bacteria have a significant protective effect on the vero renal epithelial cells, which can protect the vero renal epithelial cells from virus infection; the algae extract can enhance the protection of the cells after being fermented; the lactic acid bacteria are fermented with algae The crystal composition coated with liquid can provide the highest protection and reach the highest significance; it also has an effect on virus inhibition. Also, (6) four strains of lactic acid bacteria + algae fermentation liquid crystals-challenge group lactic acid bacteria through algae fermentation It has the highest inhibitory effect after being coated with ferment liquid.

Figure 7 shows an anti-inflammatory human trial. 12 subjects were randomly taken according to the recommended method every day for 5 weeks. The feedback questionnaire was filled out every week and the score was calculated. The higher the score, the better the improvement. (N=12)

Example 1: Fermentation process of algae fermented liquid

The algae fermented liquid is fermented with magnesium and calcium in the fermenting process. After 540 days, 12 strains of 4 yeasts, 5 lactic acid bacteria, and 3 acetic bacteria are fermented to convert the magnesium and calcium into an organic state. Lactic acid bacteria are added to make the algae fermented liquid coat the outer layer of lactic acid bacteria to provide biological utilization.

The production process of the algae fermented liquid of the present invention is divided into three stages of implantation of strains, a total of four fermenting stages, stage 1: initial extraction ferment, after the algae is squeezed, 1 strain of yeast Saccharomyces cerevisiae is added for 90 days of initial extraction yeast; stage 2: composite Po fermentation stage, yeast such as add 3: Saccharomycopsisfibufigera, Pichia membramefaciens, Schizosaccharomyespombe, such as lactic acid and 5: Lactobacillus plantarum, Lactobacillus delbrueckii, Lactococcuslactis , Lactococcusacidophillus, Bifidobacterium bifidum, composite Po fermentation for 390 days Phase 3: Storage fermentation stage, add 3 strains of acetic acid bacteria such as: Acetobacterhansenii , Acetobacterxylinum , Acetobactorsuboxydans , for 30 days fermentation; Phase 4: Chelate fermentation stage, add magnesium and calcium compounds, for 30 days fermentation, use The bacteria convert the calcium and magnesium elements into organic state. During the fermentation process, the bacteria are added at 10 ⁷ ~ 10 ⁸ CFU/ml, the inoculation amount is 1.5% by volume, and the fermentation temperature is controlled at 26-32°C to produce algae broth. Fermented liquid.

Example 2: Method for preparing a composition in which algae fermented liquid is coated on a lactic acid bacteria crystal shell

The method of the present invention for preparing a composition in which an algae fermented liquid is coated on a lactic acid bacteria crystal shell, step 1: a mixing step, the algae fermented liquid that has completed the fermenting process and 1.5% of four lactic acid bacteria solutions (such as: Lactobacillus rhamnosus , Lactobacillus plantarum , Lactobacillus acidophilus , Lactobacillus sporogenes, etc.) mixing and step 2: a high-speed emulsification step, the mixed solution is emulsified by a high-speed emulsifying head, so that the algae fermented liquid that has completed the fermentation process is coated with the colloidal structure On the outer layer of the lactic acid bacteria, a lactic acid bacteria crystal shell is formed to complete the lactic acid bacteria crystal composition. The lactic acid bacteria crystal composition has the ability of acid resistance and choline resistance, promotes the proliferation of intestinal stem cells, has antiviral, anti-inflammatory and anti-allergic effects.

Example 3: Acid resistance and choline resistance test of lactic acid bacteria crystal composition

The acid resistance and choline resistance experiments are divided into two groups: (1) a mixture of four lactic acid bacteria, (2) Four strains of lactic acid bacteria + algae fermented liquid crystal composition

1. Acid resistance test

Four strains of lactic acid bacteria were cultured in MRS medium. After culturing at 37°C for 48 hours, each 0.1ml of the bacterial solution was mixed, and 5N HCl was added to adjust the pH to 2. After culturing at 37°C for four hours, the sequence was diluted and coated, and cultivated for 24 hours. Count the colonies after hours; in addition, four strains of lactic acid bacteria + algae fermentation broth crystallization composition were cultured in MRS medium, cultured at 37°C for 48 hours, then 0.4ml was taken, and 5N HCl was added to adjust the pH to 2, and cultured at 37°C for four hours The post-sequence was diluted and plated, and the colonies were counted after 24 hours of cultivation. The surviving lactic acid bacteria can form colonies normally, so as to evaluate the number of viable bacteria.

The results of the acid tolerance test are shown in Figure 3, showing that (2) the number of viable acid-resistant bacteria of the four lactic acid bacteria + algae fermented liquid crystal composition is significantly higher than (1) the four lactic acid bacteria mixture ( ^** P<0.01), It is confirmed that the survival rate of lactic acid bacteria is greatly reduced after being destroyed by HCl to simulate the destruction of gastric acid without treatment. However, the lactic acid bacteria crystal composition coated with algae fermented liquid still maintains a good survival rate even if it is destroyed by gastric acid, that is, through the algae fermented liquid package Cover structure, can provide lactic acid bacteria resistance to acid corrosion.

2. Choline resistance test:

Four strains of lactic acid bacteria were cultured in MRS medium. After culturing at 37℃ for 48 hours, each 0.1ml of the bacterial solution was mixed, and Oxoid saturated solution (Oxoid is bile extract) was added at a concentration of 0.3%, and cultured at 37℃ for four hours The post-sequence was diluted and plated, and the colonies were counted after 24 hours of culture; 4 strains of lactic acid bacteria + algae fermented liquid crystals were cultured in MRS medium, incubated at 37°C for 48 hours, 0.4ml was taken, and Oxoid saturated solution (Oxoid is bile) Extract), added at a concentration of 0.3%, cultured at 37°C for four hours and then serially diluted and plated. After 24 hours of culture, the colonies were counted. The surviving lactic acid bacteria can form colonies normally, so as to evaluate the number of viable bacteria.

The results of the choline tolerance test are shown in Figure 4, showing that (2) the number of viable choline-resistant bacteria of the four lactic acid bacteria + algae fermented liquid crystal composition is significantly higher than (1) the four lactic acid bacteria mixture ( ^*** P<0.001), it was confirmed that the survival rate of lactic acid bacteria without treatment was greatly reduced after simulating choline destruction by Oxoid, but the lactic acid bacteria crystal composition coated with algae ferment solution, even if destroyed by choline, still maintained a good survival rate. That is, through the algae fermented liquid coating structure, it can provide the anticholinergic ability of lactic acid bacteria.

Example 4: Intestinal stem cell proliferation test of lactic acid bacteria crystal composition

The intestinal stem cell proliferation experiment is divided into five groups of samples, namely:

(1) Control-control group

(2) Mixture of four lactic acid bacteria

(3) Algae extract

(4) Algae fermented liquid

(5) Four strains of lactic acid bacteria + algae fermented liquid crystal composition

ISCs are intestinal stem cells. The samples are (1) Control-control group, (2) a mixture of four lactic acid bacteria, (3) algae extract (without algae fermentation), (4) algae fermentation solution and (5) Four strains of lactic acid bacteria + algae fermented liquid crystal composition were co-cultured with SCIs intestinal stem cells to analyze the ability of each sample to proliferate intestinal stem cells. ISCs intestinal stem cells can differentiate into mucosal cells and epidermal cells to achieve wound repair and repair mucosal tissue And other capabilities. In the experiment, ISCs intestinal stem cells were planted in 96-well plates, 50,000cell/well, cultured for 24 hours and then replaced with medium (with 200μg/ml samples and no samples respectively), cultured for 24 and 48 hours, and survived by MTT assay Rate test.

The results of the intestinal stem cell proliferation test of the lactic acid bacteria crystal composition are shown in Figure 5, which shows It is shown that at 24 hours and 48 hours of co-cultivation, the uncoated lactic acid bacteria proliferation rate is up to about 15%. The proliferation rate of (3) the algae extract is similar to (2) the lactic acid bacteria mixture; but the algae is fermented (4) ) The algae fermented liquid can increase the micro-proliferation ability by about 50%; and (5) After the lactic acid bacteria and the algae fermented liquid are combined to form a crystal composition, the proliferation effect reaches about 75%, which is a significant difference, which means that the lactic acid bacteria and the algae fermented liquid There is an additive effect.

Example 5: Antiviral test of lactic acid bacteria crystal composition

The antiviral experiment of the lactic acid bacteria crystal composition is divided into six groups of samples, namely:

(1) Control-control group

(2) Virus-challenge group

(3) Mixture of four lactic acid bacteria-challenge group

(4) Algae extract-challenge group

(5) Algae fermented liquid-challenge group

(6) Four strains of lactic acid bacteria + algae fermented liquid crystals-challenge group

Using vero renal epithelial cells as a model, challenge with Enterovirus 71. The experiment was carried out in two ways:

1. Protective effect on vero renal epithelial cells:

The vero renal epithelial cells were seeded in 96-well plates, 50,000cell/well, cultured for 24 hours and then replaced the medium (with 200μg/ml samples and no samples respectively), after 4 hours of culture, enterovirus 71 challenge was added, 24 One hour later, the viability of vero renal epithelial cells was determined by MTT assay. That is, (1) Control-control group, (2) Virus-challenge group, (3) four strains of lactic acid bacteria mixture-challenge group, (4) algae extract-challenge group, (5) algae fermentation Liquid-challenge group, (6) four strains of lactic acid bacteria +Algae fermentation liquid crystals-the challenge group samples are co-cultured with vero renal epithelial cells to test whether each sample has a protective effect on vero renal epithelial cells and can resist virus infection.

2. Resistance to viruses

Plant vero renal epithelial cells in a 96-well plate, 50,000cell/well, culture for 24 hours, change the medium (without samples), and then add enterovirus 71 (mix or not mix with 200μg/ml each sample respectively to make the virus Co-cultivation with each sample for 4 hours) challenge, 24 hours later, the cell viability was determined by MTT assay. Co-culture the virus and cells, test samples (1) Control-control group, (2) Virus-challenge group, (3) four strains of lactic acid bacteria mixture-challenge group, (4) algae extract-challenge group, ( 5) Whether the algae fermented liquid-challenge group, (6) whether the four lactic acid bacteria + algae fermented liquid crystals-the challenge group has the effect of resisting the virus and inhibiting the infection of the virus.

The results of the antiviral test of the lactic acid bacteria crystal composition are shown in Figure 6, showing (3) a mixture of four lactic acid bacteria-challenge group ( ^** P<0.01), (4) algae extract-challenge group ( ^* P< 0.05), (5) Algae fermentation liquid-challenge group ( ^** P<0.01), (6) Four strains of lactic acid bacteria + algae fermentation liquid crystals-challenge group ( ^*** P<0.001) and (2) Virus-challenge group has a significant protective effect on vero renal epithelial cells; the results show that the general lactic acid bacteria have a significant protective effect on vero renal epithelial cells after mixing, and can protect vero renal epithelial cells from virus infection; After the algae extract is fermented, it can enhance the protective power of the cells; the crystalline composition of the lactic acid bacteria coated with the algae fermented liquid can provide the highest protective power and reach the highest significance; it also has an effect on the virus inhibition, and is also effective Lactic acid bacteria have the highest inhibitory effect after being coated with algae fermented liquid.

Example 5: Anti-inflammatory test of lactic acid bacteria crystal composition

12 subjects used the lactic acid bacteria crystal combination according to the recommended dosage and recommended method It lasts for 5 weeks. Questionnaire feedback will be conducted every Friday, and the improvement effect will be counted by scoring method.

The anti-inflammation test of the lactic acid bacteria crystal composition was conducted through a questionnaire survey, and the results are shown in Figure 7. It is shown that the use of lactic acid bacteria crystals for 5 weeks can improve the symptoms of seborrheic dermatitis and allergic rhinitis.

The content of the questionnaire is as follows: Name: Product used:, Days of use this week: Days Description: For the physical condition after using the product this week, please tick the most appropriate □ based on the actual situation and feelings.

1. General physiological conditions:

Height: Weight:

2. Skin condition:

3. Resistance status:

4. Body circulation status:

5. Gastrointestinal function:

Average number of bowel movements per day:

6. Liver function status:

7. If you have other physical conditions or want to specify, please discuss:

Claims (21)

A lactic acid bacteria crystal composition, wherein the composition comprises: a lactic acid bacteria and an algae fermented liquid, the algae fermented liquid is a colloidal structure, the colloidal structure coats the lactic acid bacteria to form a lactic acid bacteria crystal shell, the lactic acid bacteria The crystal shell has the characteristics of being acid-resistant or choline-resistant; wherein the preparation method of the algae fermented liquid is as follows: (1) the initial extraction and fermentation step: After the algae is squeezed, the yeast Saccharomyces cerevisiae is added for 90 days of the initial extraction and fermentation; (2) Compound fermentation step: adding yeast and lactic acid bacteria for 390 days of compound fermentation; (3) storage fermentation step: adding acetic acid bacteria for 30 days of fermentation; and (4) chelating fermentation step: adding Magnesium and calcium compounds are fermented for 30 days, and the organic acids produced by the bacteria are used to convert the calcium and magnesium elements into an organic state; where steps (1) to (3) add strains of 107~108CFU/ml, and the inoculum is 1.5% by volume; the temperature of step (1)~(4) is controlled at 26-32℃; the yeast strain added in the compound fermentation step is selected from the group consisting of Saccharomycopsisfibufigera, Pichia membramefaciens and Schizosaccharomyespombe yeast; The lactic acid bacteria added in the compound fermentation step are selected from the group consisting of Lactobacillus plantarum, Lactobacillus delbrueckii, Lactococcuslactis, Lactococcus acidophillus and Bifidobacterium bifidum; the acetic acid bacteria added in the storage fermentation step are selected from the group consisting of Acetobacterethansenii, Acetobacterethansenii, Acetobacterethansenii The group; wherein the colloidal structure contains a sugar molecule, the sugar molecule is a small molecule polysaccharide of less than 10,000 daltons. According to the lactic acid bacteria crystal composition described in item 1 of the scope of patent application, the polysaccharide is α-glucan or β-glucan. The lactic acid bacteria crystal composition described in item 1 of the scope of patent application, wherein the lactic acid bacteria strain is Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus or Lactobacillus sporogenes strain. The lactic acid bacteria crystal composition described in item 3 of the scope of patent application, wherein the mixing ratio of the lactic acid bacteria is that the content of Lactobacillus rhamnosus is 3×10 ⁸ CFU/g~5×10 ⁸ CFU/g, and the content of Lactobacillus plantarum is 3×10 ⁸ CFU/g~5×10 ⁸ CFU/g, Lactobacillus acidophilus content is 3×10 ⁸ CFU/g~5×10 ⁸ CFU/g, Lactobacillus sporogenes content is 3×10 ⁸ CFU/g~5×10 ⁸ CFU /g. The lactic acid bacteria crystal composition as described in item 1 of the scope of patent application, wherein the algae fermented liquid is the fermented liquid of sea fungus, rock cauliflower, ulva, chlorophyll, sea grape, or kudzu. According to the lactic acid bacteria crystal composition described in item 1 of the scope of patent application, the mixing ratio of the algae fermented liquid and the lactic acid bacteria is 50:1 to 70:1. A method for preparing the algae fermented liquid of the lactic acid bacteria crystal composition as in Patent Scope 1, including: (1) the initial extraction and fermentation step: After the algae is squeezed, the yeast Saccharomyces cerevisiae is added for 90-day early extraction and fermentation; (2) ) Compound fermentation step: adding yeast and lactic acid bacteria for 390 days of compound fermentation; (3) storage fermentation step: adding acetic acid bacteria for 30 days of fermentation; and (4) chelating fermentation step: adding magnesium and Calcium compounds are fermented for 30 days, and the organic acids produced by the bacteria are used to convert calcium and magnesium elements into an organic state. Among them, steps (1)~(3) add strains of 10 ⁷ ~ 10 ⁸ CFU/ml, and the amount of inoculation It is 1.5% by volume; the temperature of fermentation in steps (1)~(4) is controlled at 26-32℃. The method for preparing algae fermented liquid as described in item 7 of the scope of patent application, wherein the yeast strain added in the compound fermenting step is selected from the group consisting of Saccharomycopsisfibufigera, Pichia membramefaciens and Schizosaccharomyespombe yeast. The method for preparing algae fermented liquid as described in item 7 of the scope of patent application, wherein the lactic acid bacteria added in the compound fermenting step are selected from the group consisting of Lactobacillus plantarum, Lactobacillus delbrueckii, Lactococcuslactis, Lactococcus acidophillus and Bifidobacterium bifidum. The method for preparing algae fermented liquid as described in item 7 of the scope of patent application, wherein the acetic acid bacteria added in the storage fermenting step are selected from the group consisting of Acetobacterhansenii, Acetobacterxylinum and Acetobactorsuboxydans acetic acid bacteria. A method for preparing the lactic acid bacteria crystal composition as described in item 1 of the scope of patent application, comprising: (1) providing an algae fermented liquid, and the method for manufacturing the algae fermented liquid is as described in the patent scope.

7 manufacturing processes; (2) provide a lactic acid bacteria selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus sporogenes; (3) mixing step, the algae fermented liquid and the lactic acid bacteria Mix at a volume ratio of 50:1 to 70:1; and (4) high-speed emulsification step, emulsify the mixed solution completed in step (3) through a high-speed emulsifying head at a speed of 1000-3000 rpm to make the algae that has completed the fermentation process The fermented fermentation liquid is coated on the outer layer of the lactic acid bacteria to form a lactic acid bacteria crystal shell to complete the lactic acid bacteria crystal composition; wherein, the coating rate of the lactic acid bacteria crystal composition is judged by using a common probiotic bacteria coating rate detection method. A use of a lactic acid bacteria crystal composition as in Patent Scope 1 for preparing a medicine for promoting the proliferation of intestinal stem cells. The use described in Patent Scope 12, wherein the use includes increasing the cell survival rate of intestinal stem cells by 50%-85%. A use of the lactic acid bacteria crystal composition as in Patent Scope 1 for the preparation of antiviral drugs. The use as described in patent scope 14, wherein the use is to protect cells against virus infection. The use as described in Patent Scope 14, wherein the use is the effect of inhibiting the ability of the virus to infect the virus. The use as described in patent scope 14, wherein the virus is Enterovirus 71. A use of the lactic acid bacteria crystal composition as in Patent Scope 1 for the preparation of anti-inflammatory drugs. The use described in patent scope 18, wherein the inflammation is seborrheic dermatitis. A use of the lactic acid bacteria crystal composition as in Patent Scope 1 for the preparation of anti-allergic drugs. The use described in patent scope 20, wherein the allergy is allergic rhinitis.

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