Sodium hyaluronate drink, preparation method and application thereof

Abstract

The present application relates to the technical field of food processing, and particularly relates to a sodium hyaluronate drink and a preparation method thereof. The drink comprises a plant extract and sodium hyaluronate; the mass ratio of the plant extract to the sodium hyaluronate is (3.2-3.8):3; the plant extract and the sodium hyaluronate are dissolved in water to prepare a 0.08-0.12 mg / mL drink, namely the sodium hyaluronate drink. The two natural sugar substitutes in the technical scheme of the present application do not participate in the carbon metabolic process of the human body, do not produce glycosylation with hemoglobin, and the pancreas secretes a large amount of insulin under the stimulation of pheromones, which helps to regulate the internal balance of the human body. Under the joint regulation of the duodenum and the gallbladder, the probiotics become active, the pathogenic bacteria such as proteus and pseudomonas are inhibited by butyrate produced in the metabolic process of probiotics, and the number is relatively reduced, the inflammation in the intestinal tract is reduced, the flora is more stable, and the immunity can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food processing, and in particular to a sodium hyaluronate drink and a preparation method thereof. BACKGROUND

[0002] Sugar has a high demand in the food industry. With the progress of industrial technology and the improvement of living standards, the demand for sugar has also risen. However, we cannot ignore the negative effects of sugar, such as diabetes, obesity, and hyperglycemia. The intestinal flora communicates with the enteric nervous system and the central nervous system in a bidirectional manner. Due to fluctuations in blood sugar balance, the hypothalamic-pituitary-adrenal axis is overactive, and cortisol secretion is increased. The intestinal microecological balance is broken, the intestinal microbial flora is imbalanced, some beneficial bacteria disappear, the abundance of butyrate-producing bacteria decreases, and the abundance of various pathogenic bacteria increases.

[0003] Sugar substitutes belong to chemical sweeteners, such as sucralose, aspartame, and acesulfame potassium. The existing sugar substitutes as flavorings do not contain almost no calories and do not cause negative effects such as diabetes, obesity, and hyperglycemia. However, these sugar substitutes have impure taste and often have bitter and metallic tastes, especially when used alone. The bitterness is very heavy when a certain amount is reached, and the taste is very poor, which limits their application. Moreover, these sugar substitutes do not contain any nutritional ingredients and do not have health benefits.

[0004] Monk fruit glycosides V (MV) and steviosides are currently two popular natural sugar substitutes. They not only have high sweetness, but also do not contain calories and do not directly participate in metabolism in the body. They also have certain pharmacological effects. The main components and sweeteners in steviosides are rebaudioside A (RA) and stevioside (STV), which account for 50-60% and 20-30% of steviosides, respectively. Since STV has a special bitter aftertaste, removing a small amount of STV to obtain high-purity RA can help improve the quality of steviosides. However, the molecular structures of STV and RA are very similar, making separation difficult. In addition, after entering the mouth, the residence time in the mouth is long, which can easily stimulate the oral mucosa. SUMMARY

[0005] The technical solution of the present application aims to solve the problems of difficulty in extracting effective components and strong stimulation to cells during the consumption of natural sugar substitutes. The present application provides a sodium hyaluronate drink and a preparation method thereof.

[0006] The main purpose of the present application is to:

[0007] I. Efficiently purifying sugar substitutes by biological extraction;

[0008] II. Reducing the stimulation of sugar substitutes by microbial metabolism;

[0009] III. Using the optimized sugar substitute to regulate the microecological balance in the flora.

[0010] To achieve the above object, the application adopts the following technical scheme.

[0011] A sodium hyaluronate drink,

[0012] The drink comprises a plant extract and sodium hyaluronate;

[0013] The mass ratio of the plant extract and sodium hyaluronate is (3.2-3.8):3;

[0014] The plant extract and sodium hyaluronate are dissolved in water to prepare a drink with a concentration of 0.08-0.12 mg / mL, i.e. a sodium hyaluronate drink.

[0015] Preferably,

[0016] The plant extract is a stevia extract and a monk fruit extract;

[0017] The mass ratio of the stevia extract and the monk fruit extract is (1.4-1.6):(1.8-2.2).

[0018] A preparation method of a sodium hyaluronate drink,

[0019] The stevia extract, the monk fruit extract and sodium hyaluronate are mixed in a certain proportion, and distilled water is used for dilution to prepare a sodium hyaluronate drink.

[0020] Preferably,

[0021] The stevia extract is prepared by microbial extraction;

[0022] The monk fruit extract is monk fruit glycoside V;

[0023] The sodium hyaluronate is a glucosaminoglucuronic acid.

[0024] Preferably,

[0025] The stevia extract is prepared by the following method:

[0026] A microbial environment is cultivated, stevia leaves are used as a nutrient source and are put in for fermentation treatment, the fermentation liquid is filtered and sterilized to prepare a stevia extract.

[0027] Preferably,

[0028] The microbial environment is a microbial environment composed of Pichia pastoris and Bacillus, the temperature is controlled at 16-25 ℃, the proportion of Pichia pastoris is 20-30 %, and the concentration of the microbial agent is 1×10 8 -2×108 CFU / g;

[0029] The stevia leaves are new leaves and / or old leaves and / or fallen leaves, with no damage, mold spots and rot on the surface, and broken to 40-60 mesh.

[0030] As a preferred option

[0031] The fermentation process was carried out at 30–35 °C with a pH value controlled at 5–8, and fermented in the dark for 24–48 h.

[0032] As a preferred option

[0033] The stevia extract, monk fruit extract, and sodium hyaluronate were mixed evenly at a mass ratio of (1.4-1.6):(1.8-2.2):3.

[0034] An application of sodium hyaluronate in beverages.

[0035] The sodium hyaluronate beverage is used as a sugar substitute, or as an adjunct treatment for diabetes and / or dry skin and / or constipation and / or tooth decay and / or depression, or to improve human metabolism.

[0036] The core of this invention lies in utilizing microbial extraction and purification of sugar substitutes, and regulating their unique structure through microbial metabolism to maintain metabolic balance within a specific bacterial community. In the human body with high blood sugar levels, the gut microbiota, acting as the body's second brain, experiences a disruption in the balance of pheromones secreted by various glands due to elevated blood sugar. This leads to intestinal microecological imbalance, gut microbiota dysbiosis, the disappearance of some beneficial bacteria, a decrease in butyrate-producing bacteria, and an increase in the abundance of various pathogenic bacteria. Simultaneously, restoring internal balance requires controlling dietary sugar intake; however, the lack of sweet stimulation can lead to an addictive dependence on dopamine, causing negative emotions such as anxiety, unease, and irritability due to the secretion of free electrons by some glands, thus hindering the restoration of internal balance. Therefore, this invention provides a sodium hyaluronate beverage with a certain degree of sweetness that can assist in maintaining the balance of the human internal environment.

[0037] Steviol glycosides, derived from stevia, are natural, high-sweetness, low-calorie sweeteners widely used in the food additive field due to their unique flavor, and also possess certain pharmacological effects. The main components and sweeteners of steviol glycosides are rebaudioside A and steviol glycosides. However, steviol glycosides have a distinctive bitter aftertaste; therefore, during the production process, removing a small amount of steviol glycosides to obtain high-purity rebaudioside A helps improve the quality and pharmacological effects of steviol glycosides. This invention utilizes a microbial system composed of Pichia pastoris and Bacillus to oxidize and cleave glycosidic bonds of some steviosides through microbial growth and metabolism, thereby reducing the stevioside content in the extract. Simultaneously, it deactivates the active sites on steviosides, reducing their stimulation of cell synapses and constructing a β-sheet structure with glycosides at its core, further reducing the stimulation potential. After biomodification by the microbial system, only soluble sugars and their derivatives, along with α-pheromones, remain in the solution. The remaining fibers and other polysaccharides become solid residues, carbon dioxide, and water. Only filtration and sterilization of the filtrate are needed to obtain a stevia extract with high pharmacological efficacy.

[0038] The restoration of the body's internal circulation requires the interaction between food and drugs. In the technical solution of this invention, the effect of beverages on oral absorption is due to changes in gastrointestinal physiological variables. Furthermore, beverages may have a direct impact on absorption, including the combination of drugs and beverage components. The interaction between beverages and drugs further depends on the type of food ingested and the nature of the administered formulation.

[0039] Changes in gastrointestinal pH are a factor affecting bioavailability, potentially influencing the dissolution, solubility, and stability of active ingredients in beverages. Furthermore, individual differences exist in gastric pH levels, significantly influenced by dietary calorie content. However, daily diet has little impact on intestinal and colonic pH. In this invention, stevia glycosides undergo microbial metabolism to enhance their bioavailability. This increased bioavailability delays gastric emptying, promoting satiety, and stimulates insulin production in the pancreas, stimulating the central nervous system to reduce glucagon secretion. Simultaneously, microbial pheromones enter systemic circulation through the gastric mucosa, increasing visceral blood flow after consuming the beverage. This enhances the absorption and bioavailability of hypoglycemic drugs, as a larger portion of the drug bypasses the liver and evades metabolism along with the microbial pheromones, maximizing lymphatic drug transport and reducing the time it takes for the drug to reach its target.

[0040] The beneficial effects of this invention are:

[0041] The two natural sugar substitutes in this invention do not participate in the human body's sugar metabolism process and do not undergo glycosylation with hemoglobin. The pancreas, stimulated by pheromones, secretes a large amount of insulin, which helps the body to regulate its internal balance. Under the joint regulation of the duodenum and gallbladder, probiotics become active, and pathogenic bacteria such as Proteus and Pseudomonas are inhibited by butyrate produced during the metabolism of probiotics, resulting in a relative reduction in their numbers. Inflammation in the intestines is reduced, the flora becomes more stable, and the body's immune system is enhanced. Detailed Implementation

[0042] The present invention will be further described clearly and in detail below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0043] Unless otherwise specified, all raw materials used in the embodiments of the present invention are commercially available or obtainable by those skilled in the art; unless otherwise specified, all methods used in the embodiments of the present invention are methods mastered by those skilled in the art.

[0044] Example 1: A method for preparing a sodium hyaluronate beverage.

[0045] The method includes:

[0046] (1) A microbial environment composed of Pichia pastoris and Bacillus was cultured at a temperature of 16 ℃. After culture, the proportion of Pichia pastoris community was 20% and the concentration of inoculum was 1×10. 8 CFU / g, Stevia leaves without surface damage, mold spots and rot are crushed to 40 mesh as new leaves and / or old leaves and / or fallen leaves as a nutrient source and introduced into the microbial environment. Fermentation is carried out at 30 ℃ with pH controlled at 5 and in the dark for 48 h. After filtration, the fermentation liquid is sterilized to prepare Stevia extract.

[0047] (2) Stevia extract, mogroside V with a purity of ≥95% and sodium hyaluronate are mixed evenly in a mass ratio of 1.4:1.8:3 and diluted with distilled water to a concentration of 0.08 mg / mL to obtain sodium hyaluronate beverage.

[0048] The sodium hyaluronate beverage prepared in the examples was tested, and the specific characterization results are as follows:

[0049] Cell compatibility: 3000 oral epithelial cells were collected as standard samples, added to 96-well plates for adhesion, and incubated with the culture medium of sodium hyaluronate beverage prepared in the example for 24 h. Then, 10 μL of CCK-8 was added and incubated for 1 h. This group was recorded as the detection group. A blank control group was also set up. The absorbance was measured at 450 nm using a SPARK microplate reader.

[0050] High-glucose blood compatibility: The sodium hyaluronate beverage prepared in the example was placed in a 1 mL centrifuge tube, 20 μL of high-glucose red blood cells were added, and the mixture was co-cultured at 37°C for 4 h. After centrifugation at 3000 r for 10 min, the absorbance was measured at 542 nm using an ELISA reader, and the hemolysis rate was calculated.

[0051] Antibacterial activity: Streptococcus mutans and Staphylococcus aureus were cultured in vitro in a laboratory environment. 10 mL of bacterial culture was transferred to a 50 mL centrifuge tube and centrifuged at 12,000 rpm for 3 min. The culture was then washed twice with PBS buffer and diluted to 10. 5 Then, 200 μL of the sodium hyaluronate beverage prepared in the example was added to a 24-well plate and incubated in a 37 ℃ incubator for 36 h. After that, it was diluted with PBS buffer and spread on plates, cultured separately for 24 h, and the number and density of colonies were recorded to calculate the antibacterial effect.

[0052] In summary, the above tests showed that: (1) In the cell compatibility test, compared with the blank control group, the sodium hyaluronate beverage prepared in the example did not produce toxic effects on oral epithelial cells, and the cells proliferated under the promotion of residual microbial pheromones. The beverage prepared in the example also has the potential to promote the repair of oral ulcer mucosa. (2) After centrifugation and precipitation of red blood cells, the absorbance of the sodium hyaluronate beverage was measured by an enzyme-linked immunosorbent assay (ELISA) reader. The hemolysis rate was 0.73%, which is less than 5%, indicating that no hemolytic reaction occurred. (3) According to the technical statistical analysis, the sodium hyaluronate beverage had an antibacterial rate of 99.16% against Staphylococcus aureus and 73.43% against Streptococcus mutans. This indicates that the sodium hyaluronate beverage has a strong antibacterial effect against both Staphylococcus aureus and Streptococcus mutans.

[0053] In addition, the microecological balance of the sodium hyaluronate beverage prepared in the examples was tested: the microbiota was cultivated in an environment with a temperature of 37 ℃ and a pH of 7.2, and divided into two groups with a bacterial concentration greater than 3 × 10⁻⁶ in each group. 8 CFU / g, Group A received 20 mL of 16.5 mmol / L glucose solution per hour, and Group B received 20 mL of the sodium hyaluronate beverage prepared in the example per hour. After 48 h, the types and quantities of some microorganisms in the bacterial community were measured, and the characterization results are as follows:

[0054]

[0055] Analysis of the above characterization results shows that the sodium hyaluronate beverage prepared in the embodiments has an increased content of beneficial intestinal bacteria such as Bacteroides, Eubrobacterium, and Bifidobacterium, and a decreased abundance of pathogenic bacteria such as Proteus and Pseudomonas. This demonstrates that the sodium hyaluronate beverage prepared in the embodiments of the present invention has the function of positively regulating the intestinal microbiota.

[0056] Example 2: A method for preparing a sodium hyaluronate beverage.

[0057] The method includes:

[0058] (1) A microbial environment composed of Pichia pastoris and Bacillus was cultured at a temperature of 21 ℃. After culture, the proportion of Pichia pastoris community was 25% and the concentration of inoculum was 1.5×10. 8 CFU / g, Stevia leaves without surface damage, mold spots and rot are crushed to 50 mesh as new leaves and / or old leaves and / or fallen leaves as a nutrient source and introduced into the microbial environment. Fermentation is carried out at 33 ℃ with pH controlled at 7 and in the dark for 36 h. After filtration, the fermentation liquid is sterilized to prepare Stevia extract.

[0059] (2) Stevia extract, mogroside V with a purity of ≥95% and sodium hyaluronate are mixed evenly in a mass ratio of 1.5:2:3 and diluted with distilled water to a concentration of 0.1 mg / mL to obtain sodium hyaluronate beverage.

[0060] The sodium hyaluronate beverage prepared in the examples was tested, and the specific characterization results are as follows:

[0061] Cell compatibility: 3000 oral epithelial cells were collected as standard samples, added to 96-well plates for adhesion, and incubated with the culture medium of sodium hyaluronate beverage prepared in the example for 24 h. Then, 10 μL of CCK-8 was added and incubated for 1 h. This group was recorded as the detection group. A blank control group was also set up. The absorbance was measured at 450 nm using a SPARK microplate reader.

[0062] High-glucose blood compatibility: The sodium hyaluronate beverage prepared in the example was placed in a 1 mL centrifuge tube, 20 μL of high-glucose red blood cells were added, and the mixture was co-cultured at 37°C for 4 h. After centrifugation at 3000 r for 10 min, the absorbance was measured at 542 nm using an ELISA reader, and the hemolysis rate was calculated.

[0063] Antibacterial activity: Streptococcus mutans and Staphylococcus aureus were cultured in vitro in a laboratory environment. 10 mL of bacterial culture was transferred to a 50 mL centrifuge tube and centrifuged at 12,000 rpm for 3 min. The culture was then washed twice with PBS buffer and diluted to 10. 5Then, 200 μL of the sodium hyaluronate beverage prepared in the example was added to a 24-well plate and incubated in a 37 ℃ incubator for 36 h. After that, it was diluted with PBS buffer and spread on plates, cultured separately for 24 h, and the number and density of colonies were recorded to calculate the antibacterial effect.

[0064] In summary, the above tests showed that: (1) In the cell compatibility test, compared with the blank control group, the sodium hyaluronate beverage prepared in the example did not produce toxic effects on oral epithelial cells, and the cells proliferated under the promotion of residual microbial pheromones. The beverage prepared in the example also has the potential to promote the repair of oral ulcer mucosa. (2) After centrifugation of red blood cells, the absorbance of the sodium hyaluronate beverage was measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the hemolysis rate was 0.71%, which is less than 5%, indicating that no hemolytic reaction occurred. (3) According to the technical statistical analysis, the sodium hyaluronate beverage had an antibacterial rate of 99.22% against Staphylococcus aureus and 73.50% against Streptococcus mutans. This indicates that the sodium hyaluronate beverage has a strong antibacterial effect against both Staphylococcus aureus and Streptococcus mutans.

[0065] In addition, the microecological balance of the sodium hyaluronate beverage prepared in the examples was tested: the microbiota was cultivated in an environment with a temperature of 37 ℃ and a pH of 7.2, and divided into two groups with a bacterial concentration greater than 3 × 10⁻⁶ in each group. 8 CFU / g, Group A received 20 mL of 16.5 mmol / L glucose solution per hour, and Group B received 20 mL of the sodium hyaluronate beverage prepared in the example per hour. After 48 h, the types and quantities of some microorganisms in the bacterial community were measured, and the characterization results are as follows:

[0066]

[0067] Analysis of the above characterization results shows that the sodium hyaluronate beverage prepared in the embodiments has an increased content of beneficial intestinal bacteria such as Bacteroides, Eubrobacterium, and Bifidobacterium, and a decreased abundance of pathogenic bacteria such as Proteus and Pseudomonas. This demonstrates that the sodium hyaluronate beverage prepared in the embodiments of the present invention has the function of positively regulating the intestinal microbiota.

[0068] Example 3: A method for preparing a sodium hyaluronate beverage.

[0069] The method includes:

[0070] (1) A microbial environment composed of Pichia pastoris and Bacillus was cultured at a temperature of 25 ℃. After culture, the proportion of Pichia pastoris community was 30% and the concentration of inoculum was 2×10. 8CFU / g, Stevia leaves without surface damage, mold spots and rot are crushed to 60 mesh as new leaves and / or old leaves and / or fallen leaves as a nutrient source and introduced into the microbial environment. Fermentation is carried out at 35 ℃ with pH controlled at 8 and in the dark for 24 h. After filtration, the fermentation liquid is sterilized to prepare Stevia extract.

[0071] (2) Stevia extract, mogroside V with a purity ≥95% and sodium hyaluronate are mixed evenly in a mass ratio of 1.6:2.2:3 and diluted with distilled water to a concentration of 0.12 mg / mL to obtain sodium hyaluronate beverage.

[0072] The sodium hyaluronate beverage prepared in the examples was tested, and the specific characterization results are as follows:

[0073] Cell compatibility: 3000 oral epithelial cells were collected as standard samples, added to 96-well plates for adhesion, and incubated with the culture medium of sodium hyaluronate beverage prepared in the example for 24 h. Then, 10 μL of CCK-8 was added and incubated for 1 h. This group was recorded as the detection group. A blank control group was also set up. The absorbance was measured at 450 nm using a SPARK microplate reader.

[0074] High-glucose blood compatibility: The sodium hyaluronate beverage prepared in the example was placed in a 1 mL centrifuge tube, 20 μL of high-glucose red blood cells were added, and the mixture was co-cultured at 37°C for 4 h. After centrifugation at 3000 r for 10 min, the absorbance was measured at 542 nm using an ELISA reader, and the hemolysis rate was calculated.

[0075] Antibacterial activity: Streptococcus mutans and Staphylococcus aureus were cultured in vitro in a laboratory environment. 10 mL of bacterial culture was transferred to a 50 mL centrifuge tube and centrifuged at 12,000 rpm for 3 min. The culture was then washed twice with PBS buffer and diluted to 10. 5 Then, 200 μL of the sodium hyaluronate beverage prepared in the example was added to a 24-well plate and incubated in a 37 ℃ incubator for 36 h. After that, it was diluted with PBS buffer and spread on plates, cultured separately for 24 h, and the number and density of colonies were recorded to calculate the antibacterial effect.

[0076] In summary, the above tests showed that: (1) In the cell compatibility test, compared with the blank control group, the sodium hyaluronate beverage prepared in the example did not produce toxic effects on oral epithelial cells, and the cells proliferated under the promotion of residual microbial pheromones. The beverage prepared in the example also has the potential to promote the repair of oral ulcer mucosa. (2) After centrifugation and precipitation of red blood cells, the absorbance of the sodium hyaluronate beverage was measured by an enzyme-linked immunosorbent assay (ELISA) reader. The hemolysis rate was 0.73%, which is less than 5%, indicating that no hemolytic reaction occurred. (3) According to the technical statistical analysis, the sodium hyaluronate beverage had an antibacterial rate of 99.16% against Staphylococcus aureus and 73.43% against Streptococcus mutans. This indicates that the sodium hyaluronate beverage has a strong antibacterial effect against both Staphylococcus aureus and Streptococcus mutans.

[0077] In addition, the microecological balance of the sodium hyaluronate beverage prepared in the examples was tested: the microbiota was cultivated in an environment with a temperature of 37 ℃ and a pH of 7.2, and divided into two groups with a bacterial concentration greater than 3 × 10⁻⁶ in each group. 8 CFU / g, Group A received 20 mL of 16.5 mmol / L glucose solution per hour, and Group B received 20 mL of the sodium hyaluronate beverage prepared in the example per hour. After 48 h, the types and quantities of some microorganisms in the bacterial community were measured, and the characterization results are as follows:

[0078]

[0079] Analysis of the above characterization results shows that the sodium hyaluronate beverage prepared in the embodiments has an increased content of beneficial intestinal bacteria such as Bacteroides, Eubrobacterium, and Bifidobacterium, and a decreased abundance of pathogenic bacteria such as Proteus and Pseudomonas. This demonstrates that the sodium hyaluronate beverage prepared in the embodiments of the present invention has the function of positively regulating the intestinal microbiota.

[0080] Application Example 1: Zebrafish depression was established by inducing a group of zebrafish to undergo treatment with 40 μg / mL reserpine for 20 min daily for 14 consecutive days. Four test groups were established: a blank group, a beverage group, an induction group, and a combined group. The blank group consisted of 10 healthy zebrafish in clean water without any treatment. The beverage group consisted of 10 zebrafish with depression treated with the beverage prepared in Example 2. The induction group consisted of 10 zebrafish with depression induced by reserpine in clean water. The combined group consisted of 10 zebrafish with depression treated with reserpine in the beverage prepared in Example 2. Changes in the depressive behavior of the zebrafish were observed after 14 days. The results are as follows:

[0081]

[0082] Zebrafish exhibiting depressive-like behavior primarily stayed in the lower half of the aquarium, while normal zebrafish explored the top of the tank. The above tests recorded the number of times zebrafish explored the top, the time spent exploring the top, the delay in reaching the top, and the total distance traveled during top exploration to assess their depressive-like behavior. The tests were repeated three times over five minutes. Analysis of the NTT behavioral observation data showed that the induced group zebrafish were almost entirely active at the bottom of the tank, while the zebrafish in the remaining three groups swam back and forth between the top and bottom and were more active. Furthermore, the induced group zebrafish showed significant differences in top exploration time, top exploration distance, number of top explorations, and delay in reaching the top compared to the drink group, the control group, and the combined group, while there was no significant difference between the drink group and the control group.

[0083] In addition, shallow water experiments were conducted simultaneously on four groups. Water was added to four 10×20×20 cm square pools, with the waterline 2 cm from the bottom. Each group was placed in the pool, and the fish in each group were photographed for 5 minutes, with three repetitions. The swimming speed, minimum distance between groups, and average distance between groups of zebrafish were recorded. The results are as follows:

[0084]

[0085] In shallow water experiments, zebrafish with depression generally show reluctance to socialize and exhibit significant anxiety-like behavior in the submerged aquarium. Analysis of the behavioral observation data from the shallow water experiments revealed significant differences in the minimum distance, average speed, and average distance between the induced group zebrafish and the drinking group, control group, and composite group. However, there was no significant difference between the drinking group and the control group. This indicates that the zebrafish in the induced group were unwilling to socialize and swam faster in shallow water, exhibiting anxiety-like behavior.

[0086] Based on the combined results of the two tests, the data analysis concludes that long-term immersion of zebrafish in the aqueous solution of the beverage prepared by the technical solution of this invention will not cause mental illness in zebrafish, and the beverage prepared by the technical solution of this invention can also effectively prevent depression.

[0087] Comparative Example 1: A commercially available sodium hyaluronate beverage contains 20% sodium hyaluronate and the remainder is distilled water. It was subjected to the same testing and characterization as the example, and the characterization results are as follows.

[0088] Summary of test results: (1) In the cell compatibility test, the commercially available sodium hyaluronate beverage did not produce a toxic effect on oral epithelial cells and increased the water content of oral epithelial cells, demonstrating good water retention properties. (2) After centrifugation and precipitation of red blood cells, the absorbance of the sodium hyaluronate beverage was measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the hemolysis rate was found to be 22.4%, which is greater than 5%, indicating a hemolytic reaction. (3) According to the technical statistical analysis, the sodium hyaluronate beverage had an antibacterial rate of 49.19% against Staphylococcus aureus and 33.33% against Streptococcus mutans. This indicates that the sodium hyaluronate beverage has an antibacterial effect against both Staphylococcus aureus and Streptococcus mutans.

[0089]

[0090] Analysis of the above characterization results shows that commercially available sodium hyaluronate beverages can promote the growth of microorganisms, but cannot positively guide or regulate the microbial community.

[0091] Comparative Example 2: A method for preparing a sodium hyaluronate beverage, the specific preparation method is the same as in Example 2, except that the preparation process of stevia extract, which is unique to this invention, is changed, and an equal amount of brewer's yeast is used instead of Pichia pastoris for beverage preparation. The specific operation is as follows:

[0092] A method for preparing a sodium hyaluronate beverage.

[0093] The method includes:

[0094] (1) A microbial environment composed of Saccharomyces cerevisiae and Bacillus was cultured at a temperature of 21 ℃. After culture, the proportion of Saccharomyces cerevisiae was 25% and the concentration of inoculum was 1.5×10⁻⁶. 8 CFU / g, Stevia leaves without surface damage, mold spots and rot are crushed to 50 mesh as new leaves and / or old leaves and / or fallen leaves as a nutrient source and introduced into the microbial environment. Fermentation is carried out at 33 ℃ with pH controlled at 7 and in the dark for 36 h. After filtration, the fermentation liquid is sterilized to prepare Stevia extract.

[0095] (2) Stevia extract, mogroside V with a purity of ≥95% and sodium hyaluronate are mixed evenly in a mass ratio of 1.5:2:3 and diluted with distilled water to a concentration of 0.1 mg / mL to obtain sodium hyaluronate beverage.

[0096] The beverage obtained was characterized and tested in the same parts as in Example 2, and the results are as follows.

[0097] Summary of tests: (1) In the cell compatibility test, oral epithelial cells caused rejection of the sodium hyaluronate beverage prepared in the comparative ratio, which poses a significant biosafety risk. (2) After centrifugation of red blood cells, the absorbance of the sodium hyaluronate beverage prepared in the comparative ratio was measured by an enzyme-linked immunosorbent assay (ELISA) reader. The hemolysis rate was 53.4%, which is greater than 5%, indicating a hemolytic reaction. This is because the core oligosaccharide of the disaccharide and polysaccharide chain of the yeast contains α-1,3 glycosidic bonds, which are easily involved in the glycosylation of proteins, leading to changes in cell membrane permeability. (3) According to the technical statistical analysis, the sodium hyaluronate beverage had an inhibition rate of 91.07% against Staphylococcus aureus and 64.25% against Streptococcus mutans. This indicates that the sodium hyaluronate beverage has a strong antibacterial effect against both Staphylococcus aureus and Streptococcus mutans.

[0098] Comparative Example 3: A method for preparing a sodium hyaluronate beverage, the specific preparation method is the same as in Example 2, except that the stevia extract unique to this invention is not prepared, and an equal amount of steviol glycosides are used instead of stevia extract to prepare the beverage. The specific operation is as follows:

[0099] A method for preparing a sodium hyaluronate beverage.

[0100] The method includes:

[0101] Stevioside, mogroside V with a purity ≥95% and sodium hyaluronate were mixed evenly in a mass ratio of 1.5:2:3 and diluted with distilled water to a concentration of 0.1 mg / mL to obtain a sodium hyaluronate beverage.

[0102] The beverage obtained was characterized and tested in the same parts as in Example 2, and the results are as follows.

[0103] Summary of tests: (1) In the cell compatibility test, oral epithelial cells showed an adverse reaction to the sodium hyaluronate beverage prepared in the comparative ratio, posing a significant biosafety risk. (2) After centrifugation and precipitation of red blood cells, the absorbance of the sodium hyaluronate beverage prepared in the comparative ratio was measured by an enzyme-linked immunosorbent assay (ELISA) reader. The hemolysis rate was 43.5%, which is greater than 5%, indicating a hemolytic reaction. This reaction is likely to participate in protein glycosylation, leading to changes in cell membrane permeability. (3) According to the technical statistical analysis, the sodium hyaluronate beverage showed an inhibition rate of 84.07% against Staphylococcus aureus and 63.67% against Streptococcus mutans. This indicates that the sodium hyaluronate beverage has a strong antibacterial effect against both Staphylococcus aureus and Streptococcus mutans.

[0104]

[0105] Analysis of the above characterization results shows that although the beverage prepared in the comparative proportion can promote the growth of beneficial bacteria, its inhibitory effect on pathogenic bacteria is not as strong as that of the beverage prepared in the example. In addition, because the beverage prepared in the comparative proportion is hemolytic, it is prone to biological hazards during use.

[0106] Comparative Example 4: A method for preparing a sodium hyaluronate beverage, the specific preparation method is the same as in Example 2, except that the preparation process of stevia extract, which is unique to this invention, is changed, and the fermentation time of the stevia extract is changed to prepare the beverage. The specific operation is as follows:

[0107] A method for preparing a sodium hyaluronate beverage.

[0108] The method includes:

[0109] (1) A microbial environment composed of Pichia pastoris and Bacillus was cultured at a temperature of 21 ℃. After culture, the proportion of Pichia pastoris community was 25% and the concentration of inoculum was 1.5×10. 8 CFU / g, Stevia leaves without surface damage, mold spots and rot are crushed to 50 mesh as new leaves and / or old leaves and / or fallen leaves as a nutrient source and introduced into the microbial environment. Fermentation is carried out at 33℃ with pH controlled at 7 and in the dark for 72 h. After filtration, the fermentation liquid is sterilized to prepare Stevia extract.

[0110] (2) Stevia extract, mogroside V with a purity of ≥95% and sodium hyaluronate are mixed evenly in a mass ratio of 1.5:2:3 and diluted with distilled water to a concentration of 0.1 mg / mL to obtain sodium hyaluronate beverage.

[0111] The beverage obtained was characterized and tested in the same parts as in Example 2, and the results are as follows.

[0112] In summary, the above tests showed that: (1) In the cell compatibility test, compared with the blank control group, the sodium hyaluronate beverage prepared in the example did not produce a toxic effect on oral epithelial cells. (2) After centrifugation of red blood cells, the absorbance of the sodium hyaluronate beverage was measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the hemolysis rate was 1.45%, which is less than 5%, indicating that no hemolytic reaction occurred. (3) According to the technical statistical analysis, the sodium hyaluronate beverage had an inhibition rate of 99.57% against Staphylococcus aureus and 75.33% against Streptococcus mutans. This indicates that the sodium hyaluronate beverage has a strong antibacterial effect against both Staphylococcus aureus and Streptococcus mutans.

[0113]

[0114] Analysis of the above characterization results shows that due to the excessive fermentation time of stevia extract, Bacillus subtilis grew excessively, causing some of the sugar substitutes and their derivatives to undergo fibrotic changes. This prevented the microbial community from absorbing and converting the fibrotic sugar substitutes, ultimately failing to achieve the positive guidance of microorganisms and the inhibitory effect on pathogenic bacteria.

Claims

1. A sodium hyaluronate beverage, characterized in that, The beverage contains plant extracts and sodium hyaluronate; The mass ratio of the plant extract to sodium hyaluronate is (3.2-3.8):3; The plant extract and sodium hyaluronate are dissolved in water to prepare a beverage with a concentration of 0.08–0.12 mg / mL, which is called sodium hyaluronate beverage. The plant extracts are stevia extract and monk fruit extract; The mass ratio of stevia extract to monk fruit extract is (1.4-1.6):(1.8-2.2). The preparation method of the beverage is as follows: Stevia extract, monk fruit extract and sodium hyaluronate were mixed in a certain proportion and diluted with distilled water to prepare a sodium hyaluronate beverage. The stevia extract was prepared by microbial extraction; The monk fruit extract is monk fruit glycoside V; The stevia extract was prepared by the following method: A microbial environment was cultivated, stevia leaves were used as a nutrient source for fermentation, filtered, and the fermentation liquid was sterilized to prepare stevia extract. The microbial environment is composed of Pichia pastoris and Bacillus, with the temperature controlled at 16–25°C. The Pichia pastoris community accounts for 20–30%, and the inoculum concentration during cultivation is 1×10⁻⁶. 8 ~2×10 8 CFU / g; The stevia leaves are new leaves and / or old leaves and / or fallen leaves, with no damage, mold spots and rot on the surface, and are broken to 40-60 mesh; The fermentation process was carried out at 30–35 °C with a pH value controlled at 5–8, and fermented in the dark for 24–48 h.

2. An application of the sodium hyaluronate beverage as described in claim 1, characterized in that, The sodium hyaluronate beverage is used as a sugar substitute.

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