CN117322629B - Preparation method of horseshoe lipid-lowering composition based on fermentation enzymolysis technology - Google Patents

Preparation method of horseshoe lipid-lowering composition based on fermentation enzymolysis technology Download PDF

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CN117322629B
CN117322629B CN202311567588.4A CN202311567588A CN117322629B CN 117322629 B CN117322629 B CN 117322629B CN 202311567588 A CN202311567588 A CN 202311567588A CN 117322629 B CN117322629 B CN 117322629B
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horseshoe
lipid
traditional chinese
chinese medicine
polysaccharide
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CN117322629A (en
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敬思群
谢镇蔚
刘立鑫
吴泽桐
何锦业
吴世荣
方铭深
卢祺俊
郭伯旭
丘乐萍
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Shaoguan University
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Abstract

The invention discloses a preparation method of a horseshoe lipid-lowering composition based on a fermentation enzymolysis process, wherein horseshoe skin is subjected to wall breaking enzymolysis to prepare horseshoe skin polysaccharide, hawthorn, lotus seeds, coix seeds and radix puerariae powder are subjected to decoction and fermentation to extract rich substances, the rich substances are mixed with tremella liquid functional colloid and freeze-dried to prepare traditional Chinese medicine functional substances, cereal raw materials are subjected to fermentation treatment of rich polypeptides and mixing with horseshoe dices and preservatives to prepare the horseshoe lipid-lowering composition, other additives are not required to be added, the horseshoe lipid-lowering composition has light sweetness, waste utilization of the horseshoe skin is realized, the compound effect of active ingredients of bean cereal polypeptides, horseshoe skin polysaccharide and medicinal and edible food is realized, the prepared horseshoe lipid-lowering composition is a functional food raw material, accords with the concept and environmental protection concept of lipid-lowering functional food, can be directly eaten, can also be added into foods such as porridge and the like to increase lipid-lowering effect, and has light sweetness.

Description

Preparation method of horseshoe lipid-lowering composition based on fermentation enzymolysis technology
Technical Field
The invention relates to the technical field of enzymolysis fermentation products, in particular to a preparation method of a horseshoe lipid-lowering composition based on a fermentation enzymolysis process.
Background
Along with the improvement of the living standard of people in China, people pay more and more attention to the quality of life and healthy life, and have higher requirements on food, which is a greater challenge for the processing and technological innovation of food in China. Under the background, people have requirements on the mouthfeel and basic quality of foods, and are favored for functional foods with the effects of reducing blood sugar, reducing blood fat and the like, and research on the direction of functional foods beneficial to the health and longevity of people from the viewpoints of natural science, human science and the like is carried out on the functional foods, so that the pursuit of the functional foods by people is gradually trend.
The water chestnut is used as a food with homology of medicine and food, has the effects of reducing blood fat, resisting cancer, resisting oxidation and the like, and is also a good nutritional food with blood fat reduction, wherein the water chestnut porridge is prepared by mixing raw materials with other beans and grains and food materials with homology of medicine and food; the functional food contains various active substances, but the general functional food in the market has a very strong effect, because the functional food contains macromolecular substances such as polysaccharide, protein and the like which are difficult to dissolve and digest, and some active substances which are easy to inactivate are easy to lose the effect in the preparation process due to the preparation process such as heating and boiling, so that the effect of the functional food is very limited; in addition, in order to adapt to the taste of consumers, additives such as xylitol and the like are generally required to be additionally added to achieve the sweet effect, and as functional food, excessive xylitol is eaten to raise triglyceride in blood to cause coronary atherosclerosis, and some toxic substances are easily accumulated in the water chestnut skin, and as waste generated in the production process, both raw food and cooked food are generally discarded and used as fertilizers, and more starch is contained, but researches show that the water chestnut skin polysaccharide contained in the water chestnut skin polysaccharide has the health-care performances of lipid reduction, oxidation resistance and the like, the water chestnut skin polysaccharide is complex and has strong heat stability, and the traditional ultrasonic heating assisted enzymolysis and chemical reagent extraction precipitation process has influence on the polysaccharide structure easily to damage the polysaccharide activity and also easily inactivate enzymes, so that the water chestnut skin polysaccharide extracted by a more mild, effective and high-purity extraction method has a certain research significance on the utilization of the water chestnut skin polysaccharide.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a preparation method of the horseshoe lipid-lowering composition based on a fermentation enzymolysis process.
The technical content of the invention is as follows:
The invention provides a preparation method of a horseshoe lipid-lowering composition based on a fermentation enzymolysis process, which comprises the following steps of: crushing the bean and cereal mixed raw materials, regulating the pH to 3-4, heating by microwaves, regulating the pH to be neutral, fermenting, sterilizing, adding water chestnut skin polysaccharide, traditional Chinese medicine functional substances and epsilon-polylysine, uniformly stirring, adding cooked water chestnut pieces, and performing ultrahigh-pressure sterilization to obtain a finished product;
The fermentation is that glutamic acid accounting for 3 to 5 percent of the mass of the mixture and Neurospora crassa accounting for 0.8 to 1.2 percent of the inoculum size are added for 2 to 4 hours at normal temperature;
the soybean cereal contains abundant polypeptides and is starch food, the intermolecular acting force of the protein is destroyed by microwave heating under an acidic condition, so that the structure is loose, the fermentation effect is facilitated, the glutamic acid is a nutritional ingredient, the activity of Neurospora crassa can be improved, the abundant polypeptides are obtained by the Neurospora crassa fermentation, the blood fat reducing effect can be improved, the starch can be decomposed by amylase carried by the soybean cereal, the resistant starch content is improved, the taste is improved, and the effect is improved.
In the preparation method, the dosage of each component is as follows: 0.5-1.5 parts of water chestnut skin polysaccharide, 12-14 parts of traditional Chinese medicine functional substances, 20-26 parts of bean and grain mixed raw materials, 5-7 parts of water chestnut butyl and 30-50 parts of water and 0.18 g/L of epsilon-polylysine;
The bean and cereal mixed raw materials are in mass ratio of 3-5:3:2-5:20-30 parts of red beans, peanuts, chickpeas and silk seedling rice. The preparation method of the traditional Chinese medicine functional substance comprises the following steps:
Step 1: preparing a traditional Chinese medicine active mixed solution;
Step 2: cutting tremella, adding 2-3 times of enzyme reaction liquid for soaking enzymolysis, boiling, mixing with the active mixed liquid of traditional Chinese medicines, and freeze-drying to obtain the traditional Chinese medicine functional substance;
the enzyme reaction solution is prepared by mixing 0.05-0.1mol/L citric anhydride, 0.06-0.8mol/L papain and citric acid-sodium citrate buffer solution.
The colloid in the tremella is released in the boiling process, is rich in tremella polysaccharide, contains acidic heteropolysaccharide, neutral heteropolysaccharide, mural polysaccharide and acidic oligosaccharide, and is added with enzyme reaction solution for reaction, the enzyme activity under the acidic system is high, a small amount of enzyme can enable the polysaccharide to be more completely separated out, the chemical structure of the polysaccharide contains uronic acid, esterification reaction is carried out with citric anhydride under the action of the enzyme reaction solution, the polysaccharide is subjected to charge modification, the digestion resistance of the polysaccharide is improved, tremella liquid functional colloid is obtained, the tremella liquid functional colloid is mixed with traditional Chinese medicine active ingredients under the precursor with the efficacy of reducing blood fat, the effect of the traditional Chinese medicine functional substance obtained through freeze drying can be improved, and the effect of effectively reducing blood fat is achieved.
The preparation method of the traditional Chinese medicine active mixed solution comprises the steps of adding water with the volume multiple of 1-3 into a traditional Chinese medicine mixture, stirring the mixture into a mixture, boiling, sterilizing, cooling and carrying out compound fermentation to obtain the traditional Chinese medicine active mixed solution;
the composite fermentation is carried out by adding the mass ratio of 0.5-1 g/L of inoculation amount to 3-5:1-3:1, the Neurospora crassa, the saccharomycetes and the streptococcus thermophilus are fermented for 2 to 4 hours at normal temperature.
The traditional Chinese medicine mixture comprises the following components in parts by weight: 2-4:4-7:8-12 of hawthorn, lotus seeds, coix seeds and kudzuvine root powder;
the whole powder of the hawthorn, lotus seed and coix seed and the kudzuvine root powder contains rich functional components, such as flavonoid substances, polysaccharide, fatty acid, amino acid and the like, and has the effects of reducing blood sugar, blood fat and the like;
The fermentation is carried out by compounding three probiotics, the probiotics improves the digestion and absorption capacity of intestinal tracts, and the fermentation obtains functional traditional Chinese medicine active mixed liquor by saccharification, amino acid release, small molecule polypeptide conversion and other actions on the traditional Chinese medicine mixture.
The water chestnut skin polysaccharide is prepared by cleaning water chestnut skin, sterilizing, crushing, performing ultrasonic wall breaking treatment for 30-40min by using an enzyme buffer solution with the volume multiple of 2-4, standing for reaction for 1-3h, centrifuging, removing supernatant, concentrating, performing secondary enzymolysis on a concentrated mixture obtained by concentrating by using a compound enzyme with the mass fraction of 6-8%, filtering, and drying to obtain water chestnut skin polysaccharide;
the enzyme buffer solution is prepared by mixing 0.05-0.1mol/L cellulase and 0.06-0.8mol/L papain with PBS buffer solution;
the mass ratio of the complex enzyme is 1-3:1 and laccase;
in the preparation process of the water chestnut skin polysaccharide, enzyme buffer solution keeps the activity to continuously act through substances such as wall breaking enzyme, buffer solution and the like, the ultrasonic wave and enzymolysis are combined, so that the polysaccharide such as starch and the like are separated out and decomposed, the polymeric linkage bond of the polysaccharide is decomposed into micromolecular polysaccharide, toxic substances are removed by centrifugation, and the polysaccharide is further decomposed through compound enzymolysis, so that the insoluble polysaccharide is fully decomposed, the content of water-soluble sulfated polysaccharide in the water chestnut skin is improved, the water solubility and the biological activity are further improved, and the prepared water chestnut skin polysaccharide has the lipid-lowering effect and is safe and nontoxic.
Advantageous effects
According to the preparation method of the horseshoe lipid-lowering composition based on the fermentation enzymolysis process, other processes are matched on the basis of fermentation enzymolysis, lipid-lowering activity enrichment of bean cereal raw materials, traditional Chinese medicine raw materials and horseshoe skin raw materials is realized, the two fermentation processes respectively decompose components such as bean cereal proteins, fatty acids and polypeptides in homologous food materials treated by the processes, and insoluble substances in foods are digested, more nutrients are reacted on the basis of keeping original nutrients, and the content of short-chain fatty acids and amino acid components are increased; the enzyme buffer solution can enable enzymolysis reaction to be carried out stably under physical energy, and the complex enzyme is combined to separate out and decompose macromolecular polysaccharide to generate more water-soluble polysaccharide, so that toxic substances are removed in the process, meanwhile, no chemical reagent pollution and no denaturation effect are generated on the molecular structure of the polysaccharide, and the obtained water chestnut skin polysaccharide is safe and harmless, is rich in water chestnut fragrance, can provide light sweet taste, has the effect of reducing blood fat, and replaces xylitol, white granulated sugar and other additives in a general water chestnut lipid-lowering composition; the enzyme reaction solution utilizes the characteristic of polysaccharide to separate out and react and modify tremella polysaccharide, so that the tremella polysaccharide forms a functional substance with load protection and lipid-lowering activity; the preparation process is based on biological reaction, accords with the green concept, and effectively enhances the activity of lipid-lowering components of various raw materials.
The horseshoe lipid-lowering composition obtained by the preparation method is a functional food raw material, breaks through the defects of easy loss of active ingredients of basic raw materials and insufficient efficacy, utilizes the horseshoe skin which is originally kitchen waste or production waste, realizes the compound effect of active ingredients of bean cereal polypeptide, horseshoe skin polysaccharide and medicinal and edible food, and accords with the concept and environmental protection concept of lipid-lowering functional food, and the prepared horseshoe lipid-lowering composition can be directly eaten or added into food such as porridge to increase lipid-lowering efficacy.
Drawings
FIG. 1 is a statistical plot of the effects of the test group on protein and triglyceride levels in Drosophila bodies;
FIG. 2 is a statistical chart of the active ingredients of example 1 and comparative example 3;
FIG. 3 is a graph showing the detection of water-soluble polysaccharide content of water chestnut skin polysaccharide and water chestnut skin crude polysaccharide.
Detailed Description
The application is described in further detail below with reference to specific embodiments and the accompanying drawings, it being understood that these embodiments are only for the purpose of illustrating the application and not for the purpose of limiting the same, and that various modifications of the application, which are equivalent to those skilled in the art, will fall within the scope of the appended claims after reading the present application.
All materials and reagents of the invention are materials and reagents of the conventional market unless specified otherwise.
Example 1 preparation method of Water chestnut lipid-lowering composition based on fermentation enzymolysis technology
Firstly, selecting raw materials and cleaning the raw materials: selecting high-quality raw materials with complete particles, no mildew or rot, no decay or deterioration, natural color, no impurity and no strange odor, washing, removing impurities, and draining water for later use;
20 parts by weight of the composition are as follows: 3:2: crushing 20 red beans, peanuts, chickpeas and corn mixed raw materials of silk seedling rice, regulating the pH to 3, carrying out microwave heating, wherein the microwave heating temperature is 100 ℃, the microwave heating time is 1h, the power is 550W, regulating the pH to be neutral, fermenting, adding glutamic acid accounting for 4% of the mass of the mixture and Neurospora crassa with the inoculation amount of 1% to ferment for 2h at 30 ℃, sterilizing, adding boiled 30 parts of water and 5 parts of water chestnut, cooling, mixing 0.5 part of water chestnut skin polysaccharide, 12 parts of traditional Chinese medicine functional substances and 0.18 g/L of epsilon-polylysine uniformly, and carrying out ultrahigh-pressure sterilization: the pressure is 420MPa, the dwell time is 12min, the temperature is 60 ℃, and the finished product is obtained.
The preparation method of the traditional Chinese medicine functional substance comprises the following steps:
Step 1: preparing a traditional Chinese medicine active mixed solution: the weight ratio of the components is 3:2:4:8, adding 1 volume multiple of water into the whole powder of the hawthorn, lotus seeds and coix seeds and the root of kudzu vine powder, stirring into a mixture, boiling for 1h, sterilizing, cooling, and adding the inoculation amount of 0.5g/L into the mixture, wherein the mass ratio is 3:1:1, the Neurospora crassa, the saccharomycetes and the streptococcus thermophilus are fermented for 2 hours at the temperature of 26 ℃ to obtain the traditional Chinese medicine active mixed solution.
Step 2: cutting tremella, adding 2 times of enzyme reaction liquid for soaking enzymolysis, boiling, mixing with the active mixed liquid of traditional Chinese medicines, and freeze-drying to obtain the traditional Chinese medicine functional substance; the enzyme reaction solution is prepared by mixing 0.05mol/L citric anhydride, 0.06mol/L papain and 10mmol/L citric acid-sodium citrate buffer solution.
The water chestnut skin polysaccharide is prepared by cleaning water chestnut skin, sterilizing, crushing, performing ultrasonic wall breaking treatment for 30min by using an enzyme buffer solution with the volume multiple of 2 times of the water chestnut skin, standing for reaction for 1h, centrifuging, removing supernatant, and concentrating to obtain a concentrated mixture with the mass fraction of 6% which is 1:1, carrying out secondary enzymolysis on glucosidase and laccase, filtering and drying to obtain horseshoe polysaccharide; the enzyme buffer was a mixture of 0.05mol/L cellulase and 0.06mol/L papain with 1 XPBS buffer.
Example 2 preparation method of Water chestnut lipid-lowering composition based on fermentation enzymolysis technology
Firstly, selecting raw materials and cleaning the raw materials: selecting high-quality raw materials with complete particles, no mildew or rot, no decay or deterioration, natural color, no impurity and no strange odor, washing, removing impurities, and draining water for later use;
23 parts by weight of the composition are as follows: 3:3: crushing 25 red beans, peanuts, chickpeas and corn mixed raw materials of silk seedling rice, regulating the pH to 4, carrying out microwave heating, wherein the microwave heating temperature is 100 ℃, the microwave heating time is 1h, the power is 550W, regulating the pH to be neutral, fermenting, adding glutamic acid accounting for 4% of the mass of the mixture and Neurospora crassa with the inoculation amount of 1% to ferment for 2h at 30 ℃, sterilizing, adding 40 parts of boiled water and 6 parts of water chestnut, cooling, mixing 1 part of water chestnut skin polysaccharide, 13 parts of traditional Chinese medicine functional substances and 0.18 g/L epsilon-polylysine uniformly, and carrying out ultrahigh-pressure sterilization: the pressure is 420MPa, the dwell time is 12min, the temperature is 60 ℃, and the finished product is obtained.
The preparation method of the traditional Chinese medicine functional substance comprises the following steps:
Step 1: preparing a traditional Chinese medicine active mixed solution: the weight ratio of the components is 4:3:6:10, adding water with the volume multiple of 2 into the whole powder of the hawthorn, lotus seeds and coix seeds and the root of kudzu vine powder, stirring to form a mixture, boiling for 1h, sterilizing, cooling, and adding the inoculation amount of 0.8g/L according to the mass ratio of 4:2:1, the Neurospora crassa, the saccharomycetes and the streptococcus thermophilus are fermented for 3 hours at the temperature of 26 ℃ to obtain the traditional Chinese medicine active mixed solution.
Step 2: cutting tremella, adding 2 times of enzyme reaction liquid for soaking enzymolysis, boiling, mixing with the active mixed liquid of traditional Chinese medicines, and freeze-drying to obtain the traditional Chinese medicine functional substance; the enzyme reaction solution is prepared by mixing 0.081mol/L of citric anhydride, 0.07mol/L of papain and 10mmol/L of citric acid-sodium citrate buffer solution.
The water chestnut skin polysaccharide is prepared by cleaning water chestnut skin, sterilizing, crushing, performing ultrasonic wall breaking treatment for 35min by using an enzyme buffer solution with the volume multiple of 3 times of the water chestnut skin, standing for 2h, centrifuging, removing supernatant, and concentrating to obtain a concentrated mixture with the mass fraction of 7% which is 2:1, carrying out secondary enzymolysis on glucosidase and laccase, filtering and drying to obtain horseshoe polysaccharide; the enzyme buffer was a mixture of 0.08mol/L cellulase and 0.07mol/L papain with 1 XPBS buffer.
Example 3 preparation method of Water chestnut lipid-lowering composition based on fermentation enzymolysis technology
Firstly, selecting raw materials and cleaning the raw materials: selecting high-quality raw materials with complete particles, no mildew or rot, no decay or deterioration, natural color, no impurity and no strange odor, washing, removing impurities, and draining water for later use;
26 parts by weight of the composition are 5:3:5: crushing 30 red beans, peanuts, chickpeas and corn mixed raw materials of silk seedling rice, regulating the pH to 4, carrying out microwave heating, wherein the microwave heating temperature is 100 ℃, the microwave heating time is 1h, the power is 550W, regulating the pH to be neutral, fermenting, adding glutamic acid accounting for 4% of the mass of the mixture and Neurospora crassa with the inoculation amount of 1% to ferment for 2h at 30 ℃, sterilizing, adding 50 parts of boiled water and 7 parts of water chestnut, cooling, mixing 1.5 parts of water chestnut skin polysaccharide, 14 parts of traditional Chinese medicine functional substances and 0.18 g/L of epsilon-polylysine, uniformly stirring, and carrying out ultrahigh-pressure sterilization: the pressure is 420MPa, the dwell time is 12min, the temperature is 60 ℃, and the finished product is obtained.
The preparation method of the traditional Chinese medicine functional substance comprises the following steps:
Step 1: preparing a traditional Chinese medicine active mixed solution: the weight ratio of the components is 5:4:7:12, adding 3 volume times of water into the whole powder of the hawthorn, lotus seeds and coix seeds and the root of kudzu vine powder, stirring to form a mixture, boiling for 1h, sterilizing, cooling, and adding the inoculation amount of 1g/L with the mass ratio of 5:3:1, the Neurospora crassa, the saccharomycetes and the streptococcus thermophilus are fermented for 4 hours at the temperature of 26 ℃ to obtain the traditional Chinese medicine active mixed solution.
Step 2: cutting tremella, adding 3 times of enzyme reaction liquid for soaking enzymolysis, boiling, mixing with the active mixed liquid of traditional Chinese medicines, and freeze-drying to obtain the traditional Chinese medicine functional substance; the enzyme reaction solution is prepared by mixing 0.1mol/L citric anhydride, 0.8mol/L papain and 10mmol/L citric acid-sodium citrate buffer solution.
The water chestnut skin polysaccharide is prepared by cleaning water chestnut skin, sterilizing, crushing, performing ultrasonic wall breaking treatment for 40min by using an enzyme buffer solution with the volume multiple of 4 times of the water chestnut skin, standing for 3h, centrifuging, removing supernatant, and concentrating to obtain a concentrated mixture with the mass fraction of 8% which is 3:1, carrying out secondary enzymolysis on glucosidase and laccase, filtering and drying to obtain horseshoe polysaccharide; the enzyme buffer was a mixture of 0.1mol/L cellulase and 0.8mol/L papain with 1 XPBS buffer.
Comparative example 1
Comparative example 1 differs from example 1 in that the comparative example 1 uses a preparation method of water chestnut skin polysaccharide, which comprises the steps of cleaning water chestnut skin, sterilizing, crushing, adding 8% of cellulase and pectase with a mass ratio of 1:1 and 4 volume times of FBS buffer solution, carrying out ultrasonic heating for 40min, heating at 60 ℃, then carrying out reflux extraction for 2h at 40 ℃, inactivating enzyme, centrifuging, taking supernatant, adding absolute ethyl alcohol, freezing, crystallizing, carrying out suction filtration, and then drying to obtain water chestnut skin polysaccharide; the others are unchanged.
Comparative example 2
Comparative example 2 is different from example 1 in that comparative example 2 does not perform fermentation treatment on the medium active mixed liquid, and the others are unchanged.
Comparative example 3
Comparative example 3 is different from example 1 in that comparative example 3 does not prepare a Chinese medicinal functional substance, and the raw materials are uniformly mixed with water according to the same weight ratio of the examples and then decocted to obtain the Chinese medicinal nutrient, and the other materials are unchanged.
Comparative example 4
Comparative example 4 is different from example 1 in that comparative example 4 does not process and ferment the cereal raw material of beans, directly adds water with a mass multiple of 3 times to cook for 1 hour, and the other is unchanged.
Comparative example 5
Comparative example 5 differs from example 1 in that comparative example 5 replaces horseshoe polysaccharide with an equivalent amount of xylitol.
1. Drosophila lipid-lowering experiment
Preparation of Drosophila culture medium
(1) And (3) solution A: taking 20g of sucrose and 3g of agar in a beaker, adding 300mL of distilled water into the beaker, fully stirring and uniformly mixing the materials, and heating and boiling the materials until the sucrose and the agar are completely melted;
(2) And (2) liquid B: taking 33g of corn flour, 21.24g of maltose and 4.5g of soybean powder in a beaker, adding 200mL of distilled water into the beaker, and fully stirring and uniformly mixing the materials;
(3) Slowly adding the solution B into the solution A, continuously stirring, boiling to paste, stopping heating, dissolving 0.125g of methyl parahydroxybenzoate into 1.25mL of absolute ethyl alcohol, adding the AB mixed solution, and cooling.
(4) And (3) after the temperature of the AB pasty mixed solution is reduced to 70 ℃, adding 12.5g of yeast powder, uniformly stirring, adding 3.5mL of propionic acid, fully stirring, and immediately packaging in a clean culture tube, wherein the thickness is 2cm. After complete solidification, the culture tube is inverted into an incubator and can be used after 24 hours.
Drosophila anesthesia mode
(1) Taking out the Drosophila culture tube, and tapping the culture tube to collect the Drosophila at the bottom of the culture tube.
(2) And (5) removing the bottle stopper of the culture tube, and transferring the drosophila in the culture tube into a clean culture tube.
(3) And (3) dripping a small amount of diethyl ether to the prepared cotton plug, rapidly sealing the pipe orifice by using the plug, waiting for 1-2 minutes, and ending anesthesia when all the drosophila are no longer moving.
(4) And pouring the drosophila on a clean test bed, and carrying out male-female identification.
Feeding and collecting drosophila
And (3) transferring male drosophila melanogaster to a fresh common drosophila melanogaster culture medium, culturing for three days, selecting 30 drosophila melanogaster with good activity and state from each tube, and freezing and killing the rest drosophila melanogaster at-20 ℃ in a refrigerator.
Establishment of high-fat induced drosophila model
The Drosophila high fat model is provided with a normal control group, a positive control group, a model group and an administration group. Three day old male Drosophila were randomly collected into 11 groups of 3 tubes each, 30 per tube. The normal control group is fed with normal culture medium, the other groups are fed with high fat culture medium for 3 days, and then the normal control group is transferred to the corresponding formula culture medium for 4 days, and the experimental groups and the culture medium formula are shown in table 1. After the end of feeding, drosophila was sacrificed in a-20deg.C refrigerator and the index was determined using a kit.
Table 1 drosophila experimental grouping table
Index monitoring and method
According to the method for providing the kit, the protein and triglyceride contents in drosophila melanogaster are respectively measured, and the results are shown in table 2 and fig. 1, wherein A is a normal control group, B is a model control group, C is a positive control group, D is a low dose group of the lipid-lowering composition of water chestnut, E is a dose group in the lipid-lowering composition of water chestnut, F is a high dose group of the lipid-lowering composition of water chestnut, G is a control group 1, H is a control group 2, I is a control group 3, J is a control group 4, and K is a control group 5.
TABLE 2 Drosophila total protein and triglyceride content in high fat model group）
Note that: compared with the normal control group, the model control group has the "+" of P less than 0.05, namely the difference is obvious; p < 0.01, i.e. the difference is very significant; comparing the administration group with the model control group, wherein '#' is P < 0.05, and the difference is obvious; "##" is P < 0.01, the difference is very remarkable
From the experimental results in table 2 and fig. 1, it can be seen that the lipid-lowering composition of water chestnut prepared by the invention has remarkable lipid-lowering effect by the influence of different dosages of the lipid-lowering composition of water chestnut on the total protein content and the triglyceride content of drosophila melanogaster, and the effect is enhanced along with the increase of the addition amount of the lipid-lowering composition of water chestnut; the prepared water chestnut skin polysaccharide is used for replacing xylitol, compared with other methods for preparing water chestnut polysaccharide, the lipid-lowering effect is obviously improved, the bean cereal is treated and fermented, the traditional Chinese medicine functional components are extracted, fermented and mixed with tremella glue, and compared with the traditional decoction, the prepared water chestnut lipid-lowering composition has a lipid-lowering effect.
2. Detection of the active ingredients of the Chinese medicinal effects and Chinese medicinal nutrients of the water chestnut lipid-lowering composition
The content of unsaturated fatty acid, amino acid, flavonoid substances and polysaccharide in the traditional Chinese medicine functional substances and the traditional Chinese medicine nutrient substances prepared in the example 1 and the comparative example 3 are further verified, so that the lipid-lowering efficacy of the traditional Chinese medicine functional substances and the traditional Chinese medicine nutrient substances is further verified; wherein, the total polysaccharide is determined by phenol-sulfuric acid colorimetric method, the total flavone is determined by liquid chromatography, and the amino acid is determined by external standard method of amino acid automatic analyzer. The absence of fatty acids as determined by gas chromatography; the above measurement results are summarized in FIG. 2;
As can be seen from fig. 2, the content of the functional components of the traditional Chinese medicine is improved compared with that of the traditional Chinese medicine nutrient substances, which shows that the invention fully utilizes and enhances the effective components compared with the common traditional Chinese medicine decoction method by fermenting the whole powder of hawthorn, lotus seeds and coix seeds and the kudzu vine root powder, preparing tremella glue by enzymolysis, mixing the tremella glue, and maintaining the active substances on the basis of reacting more active substances.
3. Detection of water chestnut skin polysaccharide
(1) Water-soluble polysaccharide content detection
The water-soluble polysaccharide content contained in the water chestnut skin polysaccharide prepared in the example 1 is detected by a weight method: filtering and drying the concentrated mixture before secondary enzymolysis to obtain crude polysaccharide of the water chestnut skin, respectively weighing 1g of water chestnut skin polysaccharide and crude polysaccharide sample of the water chestnut skin, placing the water chestnut skin polysaccharide and crude polysaccharide sample of the water chestnut skin into a flask, heating in a water bath, taking out, cooling, diluting, filtering, adding absolute ethyl alcohol for ultrasonic action to obtain precipitate, washing the precipitate, drying, weighing to obtain the polysaccharide content in the sample, wherein the polysaccharide content is the ratio of water-soluble polysaccharide to the total polysaccharide sample, and the detection result is shown in figure 3;
According to the preparation method of the water chestnut skin polysaccharide, disclosed by the application, the water chestnut skin is further decomposed on the basis of wall breaking enzymolysis, and meanwhile, the high-molecular sugar is degraded, so that the water-soluble polysaccharide content is high, the water-soluble polysaccharide is soluble in water, the sweet taste is provided, and the utilization rate of the water chestnut skin is improved.
(2) Toxicity detection
The experimental method comprises the following steps: selecting healthy transgenic zebra fish embryos with the same cell cycle, placing the embryos into a culture solution in a 24-hole culture plate, dividing the culture solution into three groups, wherein each group is provided with 10 parallel holes, each group contains 200 mug/mL of the water chestnut polysaccharide prepared in the embodiment 1 and the comparative example 1, the other group is provided with 4 parallel holes, a raw material reagent is not added additionally to prepare a blank test, each hole contains 1 embryo, culturing the embryos in an incubator at 26 ℃ for 95 hours, and observing the survival condition of the embryos;
The results show that the zebra fish embryos in the holes of the example 1 are good in state, have no lesions and death, are not different from the blank control group, and the zebra fish embryos of the comparative example 1 have 70% of dead without heartbeat; the water chestnut skin polysaccharide prepared by the method has higher purity and no toxic substance residue.

Claims (4)

1. The preparation method of the horseshoe lipid-lowering composition based on the fermentation enzymolysis process is characterized by comprising the following steps of: crushing the bean and cereal mixed raw materials, regulating the pH to 3-4, carrying out microwave heating, regulating the pH to be neutral, fermenting, sterilizing, adding boiled water and diced water, cooling, mixing the water chestnut skin polysaccharide, the traditional Chinese medicine functional substances and epsilon-polylysine, uniformly stirring, and carrying out ultrahigh-pressure sterilization to obtain a finished product;
the fermentation is that glutamic acid accounting for 3 to 5 percent of the mass of the mixture and Neurospora crassa with the inoculation amount of 0.8 to 1.2g/L are added for 2 to 4 hours at normal temperature;
The water chestnut skin polysaccharide is prepared by cleaning water chestnut skin, sterilizing, crushing, performing ultrasonic wall breaking treatment for 30-40min by using an enzyme buffer solution with the volume multiple of 2-4, standing for reaction for 1-3h, centrifuging, removing supernatant, concentrating, performing secondary enzymolysis on a concentrated mixture obtained by concentrating by using a compound enzyme with the mass fraction of 6-8%, filtering, and drying to obtain water chestnut skin polysaccharide;
The horseshoe lipid-lowering composition is prepared from the following components in parts by weight: 0.5-1.5 parts of water chestnut skin polysaccharide, 12-14 parts of traditional Chinese medicine functional substances, 20-26 parts of bean and grain mixed raw materials, 5-7 parts of water chestnut butyl and 30-50 parts of water and 0.18 g/L of epsilon-polylysine;
the preparation method of the traditional Chinese medicine functional substance comprises the following steps:
Step 1: preparing a traditional Chinese medicine active mixed solution;
Step 2: cutting tremella, adding 2-3 times of enzyme reaction liquid for soaking enzymolysis, boiling, mixing with the active mixed liquid of traditional Chinese medicines, and freeze-drying to obtain the traditional Chinese medicine functional substance;
the preparation method of the traditional Chinese medicine active mixed solution comprises the steps of adding water with the volume multiple of 1-3 into a traditional Chinese medicine mixture, stirring the mixture into a mixture, boiling, sterilizing, cooling and carrying out compound fermentation to obtain the traditional Chinese medicine active mixed solution;
The traditional Chinese medicine mixture comprises the following components in parts by weight: 2-4:4-7:8-12 of hawthorn, lotus seeds, coix seeds and kudzuvine root powder;
the enzyme buffer solution is prepared by mixing 0.05-0.1mol/L cellulase and 0.06-0.8mol/L papain with PBS buffer solution;
The bean and cereal mixed raw materials are in mass ratio of 3-5:3:2-5:20-30 parts of red beans, peanuts, chickpeas and silk seedling rice.
2. The method for preparing the horseshoe lipid-lowering composition based on the fermentation and enzymolysis process according to claim 1, wherein the enzyme reaction solution is prepared by mixing 0.05-0.1mol/L of citric anhydride, 0.06-0.8mol/L of papain and citric acid-sodium citrate buffer solution.
3. The preparation method of the horseshoe lipid-lowering composition based on the fermentation enzymolysis process as claimed in claim 1, wherein the composite fermentation is carried out with the mass ratio of 0.5-1g/L of added inoculation amount of 3-5:1-3:1, the Neurospora crassa, the saccharomycetes and the streptococcus thermophilus are fermented for 2 to 4 hours at normal temperature.
4. The preparation method of the horseshoe lipid-lowering composition based on the fermentation and enzymolysis process as claimed in claim 1, wherein the mass ratio of the complex enzyme is 1-3:1 and laccase.
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