Snail enzymolysis liver-protecting oral liquid based on intestinal bionic fermentation and preparation method of snail enzymolysis liver-protecting oral liquid

Abstract

The invention belongs to the field of health care products, and particularly discloses a snail enzymolysis liver protection oral liquid based on intestinal bionic fermentation and a preparation method thereof, the snail enzymolysis liver protection oral liquid comprises the following components by weight: 3-5% of a snail digestive gland extract, 30-40% of a probiotic fermented Chinese herbal medicine liquid, 5-8% of a liposome embedding protective agent, 10-15% of a flavoring agent, and the balance of purified water; the probiotic fermented Chinese herbal medicine liquid is prepared from 8-12% of radix puerariae, 5-8% of hovenia dulcis thunb, 3-5% of chicory and 2-3% of radix salviae miltiorrhizae through multi-strain symbiotic fermentation; an intestinal microenvironment is simulated through a three-stage gradient bionic fermentation technology, active ingredients of Chinese herbal medicines are fully released by utilizing symbiotic fermentation of multiple strains and are converted into easy-to-absorb substances such as small molecular peptides and short-chain fatty acids, and meanwhile, the release rate of mycoprotein and coenzyme is increased through low-temperature and ultrahigh-pressure wall breaking; by combining a liposome embedding technology, active ingredients such as glycerol kinase in the digestive gland of the snail are protected, gastric acid damage is avoided, and liver targeted delivery is realized.

Description

Technical Field

[0001] The invention belongs to the field of health care products, and particularly relates to a snail enzymatic hydrolysis liver-protecting oral liquid based on intestinal biomimetic fermentation and a preparation method thereof. Background Art

[0002] With the continuous development of modern medicine and bioengineering technology and the continuous improvement of people's living standards, people are increasingly concerned about their health. As a result, various biological health products have flooded into the market, especially various nutritional health oral liquids, which have a very large market share. However, looking at the various oral liquids currently popular on the market, the vast majority are simply nutritional supplements with a single function, making it difficult to meet consumer demand for such products and their functions.

[0003] As an important metabolic organ in the human body, the liver is easily damaged by factors such as alcohol, drugs and environmental toxins, leading to diseases such as fatty liver and liver damage.

[0004] However, the macromolecules of herbal medicines in traditional liver-protecting oral liquids are not easily absorbed by the human body and have low bioavailability. In addition, the alcohol-detoxifying ingredients need to be transformed in the intestines to be effective, resulting in low enrichment efficiency of the active ingredients in the liver and slow onset of effect. At the same time, they cannot provide exogenous glycerol kinase, and their health care performance for diseases such as fatty liver and liver damage is not ideal. Summary of the Invention

[0005] The purpose of the present invention is to provide a snail enzymatic hydrolysis liver-protecting oral liquid based on intestinal biomimetic fermentation and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation comprises the following components in weight percentage: 3-5% snail digestive gland extract, 30-40% probiotic fermented Chinese herbal medicine liquid, 5-8% liposome embedding protective agent, 10-15% flavoring agent, and the balance being purified water;

[0008] The probiotic fermented Chinese herbal medicine liquid is prepared by symbiotic fermentation of multiple strains with 8-12% of kudzu root, 5-8% of hovenia dulcis fruit, 3-5% of chicory and 2-3% of salvia miltiorrhiza.

[0009] Preferably, the flavoring agent includes at least one of erythritol and oligofructose.

[0010] The preparation method of snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation comprises:

[0011] S1. Put the mixed extract of Pueraria root, Hovenia dulcis fruit, Chicory and Salvia miltiorrhiza into a primary fermentation tank, inoculate Lactobacillus plantarum, and ferment at 37°C and pH 2.0-3.0 for 24 hours;

[0012] S2. Transfer the fermented product obtained in step S1 to a secondary fermentation tank, inoculate Lactobacillus acidophilus and Bacillus subtilis, and ferment at pH 6.8-7.4 for 24 hours;

[0013] S3, transferring the fermented product obtained in step S2 to a tertiary fermentation tank, inoculating bifidobacteria and yeast, and fermenting at a pH of 5.5-6.5 for 24 hours to obtain a fermentation broth;

[0014] S4, subjecting the fermentation liquid to low-temperature ultrahigh-pressure homogenization to obtain a cell-broken fermentation liquid;

[0015] S5, enzymatically hydrolyzing and ultrafiltering the snail digestive gland, and then embedding the snail digestive gland with a liposome-embedding protective agent to obtain a snail digestive gland extract;

[0016] S6. Mix the broken fermentation liquid with the snail digestive gland extract at 4° C. under nitrogen protection, add flavoring agents, and aseptically fill to obtain an oral solution.

[0017] Preferably, the inoculation amount of the Lactobacillus plantarum is 5% of the mixed extract.

[0018] Preferably, the mass ratio of Lactobacillus acidophilus to Bacillus subtilis is 3:2.

[0019] Preferably, the mass ratio of the bifidobacteria to the yeast is 2:1.

[0020] Preferably, the low-temperature ultrahigh-pressure homogenization treatment pressure is 1500 bar, the number of treatments is 3 times, and the temperature is 0-20°C.

[0021] Preferably, the particle size of the liposome-embedded protective agent is ≤200 nm.

[0022] Preferably, the bacterial protein yield of the cell wall-broken fermentation broth is ≥18%, the proportion of small molecule peptides with a molecular weight of <1000Da is >75%, and the glycerol kinase activity in the snail digestive gland extract is ≥450U / g.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention simulates the acidic-neutral-weakly acidic microenvironment of the intestine through three-stage gradient bionic fermentation technology, utilizes multi-strain symbiotic fermentation to fully release the active ingredients of Chinese herbal medicines and convert them into easily absorbable substances such as small molecule peptides and short-chain fatty acids, while improving the release rate of bacterial proteins and coenzymes through low-temperature ultra-high pressure wall breaking; combined with liposome encapsulation technology, it protects active ingredients such as glycerol kinase in the snail digestive gland, avoids gastric acid damage and realizes liver-targeted delivery; the oral liquid finally prepared can synergistically promote alcohol metabolism, repair the intestinal mucosal barrier to reduce alcohol absorption, block fat accumulation in the liver and enhance the antioxidant capacity of liver cells, and has the advantages of high ingredient activity, strong stability, high bioavailability and significant liver protection effect. DETAILED DESCRIPTION

[0025] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the specification.

[0026] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0027] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0028] Example 1: This example provides a snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation, comprising the following components in percentage by weight: 4% snail digestive gland extract, 35% probiotic fermented Chinese herbal medicine liquid, 6% liposome encapsulation protective agent, 12% flavoring agent, and the balance being purified water;

[0029] The probiotic fermented Chinese herbal medicine liquid is prepared by symbiotic fermentation of multiple strains with 10% of kudzu root, 7% of hovenia dulcis fruit, 4% of chicory and 3% of salvia miltiorrhiza.

[0030] Specifically, the flavoring agent includes erythritol and oligofructose in a mass ratio of 1:1.

[0031] The preparation method of snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation comprises:

[0032] S1. Place the mixed extract of Pueraria root, Hovenia dulcis fruit, Chicory, and Salvia miltiorrhiza in a primary fermentation tank, inoculate with 5% Lactobacillus plantarum → MRS medium, and ferment at 37°C and pH 2.5 for 24 hours;

[0033] The mixed extract comprises: decocting Pueraria root, Hovenia dulcis fruit, Chicory and Salvia miltiorrhiza in water for 3 times (material-liquid ratio 1:10), combining the filtrates and concentrating the filtrates to a specific gravity of 1.20 (measured at 60°C).

[0034] S2. The fermented product obtained in step S1 was transferred to a secondary fermentation tank, inoculated with Lactobacillus acidophilus and Bacillus subtilis (total inoculum amount 5%) → MRS+LB mixed medium, and fermented at 37°C, pH 7, and aeration rate of 0.2 vvm for 24 hours;

[0035] The mass ratio of Lactobacillus acidophilus to Bacillus subtilis is 3:2.

[0036] S3, the fermentation product obtained in step S2 was transferred to a tertiary fermentation tank, inoculated with bifidobacteria and yeast (total inoculation amount 6%) → modified MRS medium, and fermented at 37°C, pH 6 for 24 hours to obtain a fermentation broth in an anaerobic environment;

[0037] The mass ratio of bifidobacteria to yeast was 2:1.

[0038] S4, subjecting the fermentation liquid to low-temperature ultrahigh-pressure homogenization to obtain a cell-broken fermentation liquid;

[0039] The low-temperature ultra-high-pressure homogenization treatment pressure is 1500 bar, the number of treatments is 3 times, and the temperature is 4°C.

[0040] S5, hydrolyzing the snail digestive gland with cellulase (50°C, pH 5.0, 2h), ultrafiltration with a 10kDa membrane, and embedding with a liposome-embedded protective agent to obtain the snail digestive gland extract;

[0041] The particle size of the liposome-embedded protective agent was 185±15 nm (Malvern particle size analyzer), and the mass ratio of phospholipid to cholesterol was 7:3.

[0042] S6. Mix the broken fermentation liquid with the snail digestive gland extract at 4°C under nitrogen protection, add flavoring agents and aseptically fill to obtain the oral liquid. Use a 0.22 μm filter membrane for sterilization during filling.

[0043] The bacterial protein yield of the broken cell fermentation broth is 21.3% (Kjeldahl method), the small molecular peptides with a molecular weight of <1000Da account for 82.5% (HPLC-MS detection), and the glycerol kinase activity in the snail digestive gland extract is 503U / g (colorimetric detection).

[0044] The oral liquid product obtained above was tested, and the test data are as follows:

[0045]

[0046]

[0047] Conclusion: The proportion of small molecule peptides meets the requirements, the liposome particle size is ≤200nm, and the bacterial protein yield is >18%, which meets the needs.

[0048] The results of the comparison of the components of the fermentation broth before and after cell wall breaking are as follows:

[0049] Element Content before cell wall breaking Content after cell wall breaking Release rate bacterial proteins 8.7％ 21.3％ ↑145％ Short-chain fatty acids (acetic acid) 0.5mg / g 2.1mg / g ↑320％ <![CDATA[Glycerol kinase coenzyme (NAD + )]]> Not detected 15.3U / g 100％

[0050] Verification of liposome encapsulation protection effect:

[0051] The embedded group and the non-embedded group were treated in a simulated gastric fluid environment (pH 2.0, 2h). The residual activity rate of glycerol kinase in the non-embedded group was 12%, while that in the embedded group was 91%.

[0052] Example 2: This example provides a snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation, comprising the following components in percentage by weight: 5% snail digestive gland extract, 40% probiotic fermented Chinese herbal medicine liquid, 8% liposome encapsulation protective agent, 10% flavoring agent, and the balance being purified water;

[0053] The probiotic fermented Chinese herbal medicine liquid is prepared by symbiotic fermentation of multiple strains with 8% of kudzu root, 5% of hovenia dulcis fruit, 3% of chicory and 2% of salvia miltiorrhiza.

[0054] Specifically, the flavoring agent includes erythritol.

[0055] The preparation method of snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation comprises:

[0056] S1: Root of Pueraria, Hovenia dulcis, Cichorium chinense, and Salvia miltiorrhiza were decocted twice with water at a material-liquid ratio of 1:12. The filtrates were combined and concentrated to a specific gravity of 1.25 (measured at 60°C) to obtain a mixed extract.

[0057] The mixed extract is placed in a primary fermentation tank, inoculated with 5% (based on the weight of the mixed extract) of Lactobacillus plantarum, and anaerobically fermented for 24 hours at 37° C. and pH 2.0 using MRS medium as a substrate to simulate the gastric environment. Lactobacillus plantarum secretes cellulase to hydrolyze the cell walls of Pueraria lobata / chicory, releasing the embedded flavonoid aglycones.

[0058] Effect: Exposure rate of herbal active ingredients increased by 80%, and bitter substances were partially degraded.

[0059] S2: The fermentation product obtained in step S1 was transferred to a secondary fermentation tank, inoculated with Lactobacillus acidophilus and Bacillus subtilis (mass ratio 3:2, total inoculum size 5%), and fermented in MRS+LB mixed medium at 37°C, pH 6.8 for 24 hours with an aeration rate of 0.15 vvm to simulate the environment of the small intestine. In this neutral environment, Bacillus subtilis secretes protease / lipase, hydrolyzes macromolecular proteins into small peptides (e.g., pueraria flavonoids → genistein), and emulsifies the lipids into droplets;

[0060] Effect: Generates small peptides with a molecular weight of <1000Da (accounting for >75%), which can be directly absorbed into the blood through the sublingual mucosa.

[0061] S3: The fermentation product obtained in step S2 is transferred to a tertiary fermentation tank, inoculated with bifidobacteria and yeast (mass ratio 2:1, total inoculum amount 6%), and anaerobically fermented at 37°C and pH 5.5 for 24 hours using modified MRS medium as the substrate to obtain a fermentation broth. The weakly acidic anaerobic conditions simulate the colon environment and drive bidirectional metabolism: bifidobacteria convert genistein into equol (alcohol detoxification activity ↑10 times); and yeast synthesizes glutathione precursors to enhance the liver's antioxidant capacity;

[0062] Effect: The concentration of the key active substance, equol, reaches 0.8mg / mL.

[0063] S4: The fermentation broth was subjected to ultrahigh pressure homogenization treatment three times at a pressure of 1500 bar and 20°C to obtain a broken cell fermentation broth. The ultrahigh pressure shear force caused the bacterial cell wall to disintegrate, releasing the intracellular glycerol kinase coenzyme (NAD + ) and bacterial protein peptides;

[0064] Effect: Improve glycerol kinase activity and bacterial protein yield.

[0065] S5: The snail digestive gland was enzymatically hydrolyzed with cellulase (55°C, pH 5.5, enzymatic hydrolysis for 1.5 hours), ultrafiltered through a 10kDa membrane, and encapsulated with a liposome-encapsulated protective agent with a particle size of ≤200nm (phospholipid to cholesterol mass ratio of 7:3, particle size 190±10nm) to obtain the snail digestive gland extract. The phospholipid bilayer (particle size ≤200nm) encapsulated the snail glycerol kinase to avoid damage by gastric acid and was targeted for release through the liver sinusoidal endothelial gap.

[0066] S6: The broken fermentation broth was mixed with the snail digestive gland extract at 4°C under nitrogen protection, erythritol was added, and the mixture was sterilized through a 0.22 μm filter membrane and aseptically filled to obtain an oral solution.

[0067] The oral liquid product obtained above was tested, and the test data are as follows:

[0068] Test items result Detection method Proportion of small molecule peptides (<1000Da) 78.3％ HPLC-MS (size exclusion chromatography) Liposome particle size 190±10nm Dynamic Light Scattering Bacterial protein yield 19.5％ Kjeldahl method Glycerol kinase activity 462U / g Colorimetry

[0069] Conclusion: The proportion of small molecule peptides meets the requirements, the liposome particle size is ≤200nm, and the bacterial protein yield is >18%, which meets the needs.

[0070] The results of the comparison of the components of the fermentation broth before and after cell wall breaking are as follows:

[0071] Element Content before cell wall breaking Content after cell wall breaking Release rate bacterial proteins 9.2％ 19.5％ ↑112％ Short-chain fatty acids (acetic acid) 0.3mg / g 1.8mg / g ↑500％ <![CDATA[Glycerol kinase coenzyme (NAD + )]]> Not detected 12.7 U / g 100％

[0072] Verification of liposome encapsulation protection effect:

[0073] The embedded group and the non-embedded group were treated with simulated intestinal fluid environment (pH 7.5, 37°C, 2h). Among them, the residual activity rate of glycerol kinase in the non-embedded group was 18%, and the residual activity rate in the embedded group was 89%.

[0074] Through three-stage gradient fermentation (acidic → neutral → weakly acidic) to simulate the intestinal microenvironment, the synergistic effect between the effective ingredients of Chinese herbal medicines and bacterial metabolites is enhanced, and the release rate of small molecule peptides after cell wall breaking is significantly improved;

[0075] Liposome encapsulation technology ensures the stability of active ingredients such as glycerol kinase in the snail digestive gland in the gastrointestinal tract.

[0076] Example 3: The oral solution obtained in Example 1 was tested for efficacy using a SD rat model (n=10). The test data are shown in the following table:

[0077] Group Peak blood alcohol level (mg / dL) Time to peak (min) Clearance to baseline time Blank control group 185±21 30 >240min The present invention group 121±15* 45 90 minutes*

[0078] Results: Fifteen minutes after sublingual administration, the blood ethanol concentration decreased by 38%.

[0079] The oral solution obtained in Example 1 was tested for efficacy using an alcoholic fatty liver model (4 weeks). The test data are shown in the following table:

[0080] index Model control group The present invention group Improvement rate serum triglycerides 4.54±0.3mmol / L 1.62±0.2mmol / L* ↓64.3％ Liver fatty degeneration area 56.4±5.1％ 14.2±3.3％* ↓74.8％ Liver tissue glycerol kinase activity 18.7±2.1U / g 48.9±4.5U / g* ↑161％

[0081] Results: Pathological sections showed a significant decrease in hepatocyte lipid droplets (hematoxylin-eosin staining).

[0082] The present invention simulates the intestinal microenvironment through three-stage gradient fermentation (acidic → neutral → weakly acidic), completing efficient conversions that the human body is unable to complete (for example, Hovenia dulcis and Pueraria root are fermented into equol (a small molecule phenol) by Bifidobacteria in the colon, directly activating liver alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), increasing the harmless metabolic rate of ethanol → acetaldehyde → acetic acid by 2.1 times, and reducing blood alcohol concentration within 15 minutes). It bypasses the patient's damaged digestion / absorption system, directly reaches the target organs, and accelerates the process of alcohol detoxification.

[0083] The short-chain fatty acids produced by fermentation repair the intestinal mucosal barrier, reduce alcohol absorption, and reduce the burden on the liver from the source;

[0084] Glycerol kinase in snail digestive gland extract (encapsulated in liposomes to avoid gastric acid) is delivered to the liver, phosphorylating glycerol to 3-phosphoglycerol, which combines with fatty acid to form triacylglycerol for energy, thus blocking the pathological chain of "fatty acid accumulation in the liver → fatty liver" described in PPT.

[0085] The bacterial protein peptides (<380Da) released by high-pressure cell wall breaking upregulate the activity of superoxide dismutase (SOD) in hepatocytes and neutralize alcohol free radicals.

[0086] It will be appreciated that in the development of any actual embodiment, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will, for those of ordinary skill having the benefit of this disclosure, be a routine undertaking of design, fabrication, and production without undue experimentation.

[0087] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Snail enzymatic liver protection oral liquid based on intestinal biomimetic fermentation, characterized by: The invention comprises the following components in weight percentage: 3-5% of snail digestive gland extract, 30-40% of probiotic fermented Chinese herbal medicine liquid, 5-8% of liposome embedding protective agent, 10-15% of flavoring agent, and the balance is purified water; The probiotic fermented Chinese herbal medicine liquid is prepared by symbiotic fermentation of multiple strains with 8-12% of kudzu root, 5-8% of hovenia dulcis fruit, 3-5% of chicory and 2-3% of salvia miltiorrhiza.

2. The snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation according to claim 1, characterized in that: The flavoring agent includes at least one of erythritol and oligofructose.

3. The method for preparing the snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation according to any one of claims 1 to 2, characterized in that: include: S1. Put the mixed extract of Pueraria root, Hovenia dulcis fruit, Chicory and Salvia miltiorrhiza into a primary fermentation tank, inoculate Lactobacillus plantarum, and ferment at 37°C and pH 2.0-3.0 for 24 hours; S2. Transfer the fermented product obtained in step S1 to a secondary fermentation tank, inoculate Lactobacillus acidophilus and Bacillus subtilis, and ferment at pH 6.8-7.4 for 24 hours; S3, transferring the fermented product obtained in step S2 to a tertiary fermentation tank, inoculating bifidobacteria and yeast, and fermenting at a pH of 5.5-6.5 for 24 hours to obtain a fermentation broth; S4, subjecting the fermentation liquid to low-temperature ultrahigh-pressure homogenization to obtain a cell-broken fermentation liquid; S5, enzymatically hydrolyzing and ultrafiltering the snail digestive gland, and then embedding the snail digestive gland with a liposome-embedding protective agent to obtain a snail digestive gland extract; S6. Mix the broken fermentation liquid with the snail digestive gland extract at 4° C. under nitrogen protection, add flavoring agents, and aseptically fill to obtain an oral solution.

4. The method for preparing the snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation according to claim 3, characterized in that: The inoculation amount of the plant lactobacillus is 5% of the mixed extract.

5. The method for preparing the snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation according to claim 3, characterized in that: The mass ratio of the Lactobacillus acidophilus to the Bacillus subtilis is 3:

2.

6. The method for preparing the snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation according to claim 3, characterized in that: The mass ratio of the bifidobacteria to the yeast is 2:

1.

7. The method for preparing the snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation according to claim 3, characterized in that: The low-temperature ultra-high-pressure homogenization treatment pressure is 1500 bar, the number of treatments is 3 times, and the temperature is 0-20°C.

8. The method for preparing the snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation according to claim 3, characterized in that: The particle size of the liposome-embedded protective agent is ≤200 nm.

9. The method for preparing the snail enzymatic liver-protecting oral liquid based on intestinal biomimetic fermentation according to claim 3, characterized in that: The bacterial protein yield of the cell wall-broken fermentation broth is ≥18%, the proportion of small molecule peptides with a molecular weight of <1000Da is >75%, and the glycerol kinase activity in the snail digestive gland extract is ≥450U / g.

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