Longitudinal stripe xylaria polysaccharide extraction process and preparation method of healthy functional drink

Abstract

The invention belongs to the technical field of xylaria polysaccharide, and particularly relates to a longitudinal stripe xylaria polysaccharide extraction process and a preparation method of a healthy functional drink, and the longitudinal stripe xylaria polysaccharide is obtained through the steps of raw material pretreatment, enzymolysis, ultrasonic-microwave synergistic extraction, enzyme deactivation, filtration, concentration, purification, separation and drying and the like. By designing an enzyme system-multi-physical field synergistic innovative extraction process, the problems of low extraction rate and active ingredient damage of a conventional method are solved; the extraction efficiency is high and the operation is simple; meanwhile, scientific compounding is combined, the problems that an existing antioxidant beverage is single in function, poor in stability and the like are solved, and high-efficiency, stable and safe antioxidant beverage industrialization is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food processing and functional beverage, and particularly relates to a vertical stripe Xylaria polysaccharide extraction process and a preparation method of a healthy functional beverage. BACKGROUND

[0002] In modern life, factors such as ultraviolet radiation, unhealthy lifestyle habits (such as smoking, staying up late, etc.), and mental stress cause the human body to continuously produce excess free radicals. Excessive accumulation of free radicals can attack biological macromolecules such as lipids, proteins, and DNA within cells, trigger oxidative stress, and further cause cell damage and aging, and is closely related to the occurrence and development of various chronic diseases such as cardiovascular disease, cancer, and neurodegenerative disease. Therefore, it is of great practical significance to develop safe and effective antioxidant products to help the human body eliminate free radicals and reduce oxidative stress damage. At present, there are various antioxidant products on the market, including various antioxidants, functional foods and beverages, etc. However, there are still many problems in the actual application of existing antioxidant beverages. Some beverages rely on artificially synthesized antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), and long-term and large-scale intake of these artificially synthesized antioxidants may pose potential risks to human health, such as interfering with normal human metabolism, having certain toxicity and carcinogenicity, etc. Some antioxidant beverages mainly using natural ingredients have limited antioxidant effects, which are difficult to meet the needs of consumers for high-efficiency antioxidants. For example, common beverages rich in vitamin C and vitamin E have certain antioxidant capacity, but when used alone, the antioxidant effect lasts for a short time in a complex human environment. And some natural antioxidant ingredients have poor stability in the beverage system and are easily degraded by factors such as light, temperature, and pH, reducing the antioxidant activity and shelf life of the beverage. As a rare edible and medicinal fungus, Xylaria striata has received extensive attention in the scientific research field in recent years. It is rich in polysaccharides, terpenes, phenols and other bioactive components, among which polysaccharides show significant antioxidant potential and can effectively scavenge various free radicals and inhibit lipid peroxidation. There are limited reports on the extraction of Xylaria polysaccharides, and there are obvious technical bottlenecks. Traditional polysaccharide extraction methods mostly use hot water extraction, alkali extraction or single enzyme hydrolysis. These methods have technical problems such as low extraction efficiency, severe damage to active ingredients, long time consumption, high energy consumption, etc. There is also a method of using urea-choline chloride eutectic solvent to extract extracellular polysaccharides from Xylaria fermentation broth, which uses a large amount of solvent, which is not conducive to environmental safety and the safety of polysaccharides themselves.

[0003] In addition, the development of N. longi-striatus is mainly focused on basic research, and a mature antioxidant functional beverage product has not yet been formed, and its excellent antioxidant properties have not been fully industrialized. Therefore, a functional beverage is also developed, which is based on N. longi-striatus polysaccharide as the core and compounded with other natural antioxidant ingredients, which can not only exert the advantages of high-efficiency antioxidant, but also solve many problems of existing products, and has important market value and application prospect. SUMMARY

[0004] To solve the above technical problems, the present application provides a kind of N. longi-striatus polysaccharide extraction process and the preparation method of health functional beverage, through the design of enzyme system-multiple physical field synergistic innovative extraction process, solve the problem of low extraction rate and active ingredient destruction of conventional method; High extraction efficiency, simple operation; At the same time, combined with scientific compounding, solve the problem of single function and poor stability of existing antioxidant beverage, realize the industrialization of high-efficiency, stable and safe antioxidant beverage.

[0005] The N. longi-striatus polysaccharide extraction process provided in the present application to solve the above technical problems comprises the following steps: (1) Raw material pretreatment: select fresh N. longi-striatus fruiting body cultivated artificially, remove impurities and moldy parts, rinse with water for 3-5 times until there is no residual mud and sand on the surface, place it in a 45-50℃ air drying oven for 12-16h, until the moisture content is ≤8%; then crush the dried fruiting body and seal it in a desiccator for standby.

[0006] The artificially domesticated N. longi-striatus is used as raw material, which breaks through the limitation of wild resources.

[0007] (2) Enzymatic hydrolysis: add compound enzyme according to 1.5-2.5% of the mass of the raw material in step (1), mix the fruiting body powder and compound enzyme powder according to 1:12-1:18 (w / v) liquid ratio, add pure water, adjust the pH to 4.5-5.5, and carry out targeted enzymatic hydrolysis in a constant temperature water bath at 55-60℃ for 60-90min, with stirring speed of 80-100rpm, to specifically destroy the cell wall structure through enzymatic hydrolysis; the compound enzyme system includes cellulase, pectinase and chitinase, and the amount is cellulase: pectinase: chitinase = 1.8-2.2: 0.8-1.2: 0.8-1.2 by mass; The optimization of enzymatic hydrolysis conditions is also the key of the present application. The pH value of 4.5-5.5 and the temperature of 55-60℃ are the best synergistic action range of the above three enzymes, under which the enzyme activity is high and the action efficiency is maximum. The stirring speed of 80-100rpm can ensure sufficient contact between the enzyme and the substrate, and will not cause enzyme inactivation due to excessive shear force.

[0008] When the pH deviates from 4.5-5.5, the activities of cellulase, pectinase and chitinase will be significantly inhibited. Among them, the structure of the catalytic active center of cellulase may change, which cannot effectively bind and decompose cellulose; the activity of pectinase is narrow in the optimum pH range, and after deviation, it will affect its degradation ability to pectin; the activity of chitinase will also be affected by the deviation of pH, which will greatly reduce the decomposition efficiency of cell wall, and finally lead to the reduction of polysaccharide dissolution. In addition, too high pH value may also lead to the degradation or structural change of part of polysaccharide, affecting its subsequent activity and purity.

[0009] When the stirring rate is lower than 80 rpm, insufficient stirring may occur, the enzyme and the fruit body powder cannot be fully contacted, leading to uneven enzymatic reaction, part of the substrate cannot be effectively catalyzed and decomposed by the enzyme, the cell wall is not completely broken, the polysaccharide dissolution is reduced, and the extraction efficiency is reduced. At the same time, local concentration difference may occur in the reaction system, affecting the overall process and effect of the enzymatic reaction. When the stirring rate is higher than 100 rpm, too high stirring rate will produce strong shear force, which may destroy the spatial structure of the enzyme, leading to the reduction or even inactivation of the enzyme activity. In addition, too fast stirring may also produce a large amount of bubbles in the reaction system, which will hinder the contact between the enzyme and the substrate, and also may lead to part of the polysaccharide being wrapped by the bubbles, affecting its dissolution and subsequent separation and purification.

[0010] (3) Ultrasonic-microwave synergistic extraction: after the enzymatic reaction is completed, the mixed solution is transferred to an ultrasonic-microwave synergistic extraction device, the microwave power is set to 350±50 W, the ultrasonic power is set to 250±50 W, the temperature is set to 60-70℃, and the extraction time is set to 35-45 min; through the synergistic effect of the internal heating effect of microwave and the cavitation effect of ultrasonic, the release of polysaccharide from the cell is accelerated.

[0011] The combination of ultrasonic and microwave two physical field intensification extraction technologies has a synergistic effect. Microwave can penetrate the material, polarize the molecules and produce internal heating, accelerate the diffusion of effective components from the cell interior to the extraction solvent; ultrasonic can destroy the cell structure through cavitation effect and produce instantaneous high pressure and shock wave, promote the release of contents.

[0012] (4) enzyme inactivation and filtration: after the extraction is completed, the mixed solution is heated to 95-100℃ for 8 min to inactivate the enzyme, then cooled to room temperature quickly, and filtered through a 0.22 μm ceramic membrane to collect the filtrate; (5) concentration and purification: combine the filtrate, concentrate to 1 / 4-1 / 6 of the original volume at 40-45℃ and vacuum degree of 0.09-0.1 MPa by rotary evaporation, and obtain polysaccharide concentrate; slowly add 3-4 times volume of ethanol-acetone mixed solution with a volume ratio of 3:1 to the concentrate, stir while adding, so that the final concentration of the mixed solvent reaches 65-70%, seal and place in a 4℃ refrigerator for 8-12h; (6) Separation and drying: the mixed solution after standing is put into a high-speed centrifuge, centrifuged at 5000-6000 rpm for 10-15 min, the supernatant is discarded, and the polysaccharide precipitate at the bottom is collected; the precipitate is washed twice with a 50% ethanol-acetone mixed solution, and finally washed once with anhydrous ethanol to remove residual impurities; the precipitate is dried and sealed to avoid light.

[0013] In the optimization scheme, the step (1) is that the crushed fruiting body is passed through a 100-mesh sieve.

[0014] In the step (2), the mass ratio of cellulase, pectinase and chitinase in the composite enzyme system is 2:1:1.

[0015] In the further optimization scheme, in the step (2), the composite enzyme system is added in a proportion of 2.3% of the mass of the raw material, the fruiting body powder and the composite enzyme mixed powder are added into pure water in a solid-liquid ratio of 1:18 (w / v), the pH is adjusted to 5.5, and the targeted enzymolysis is carried out in a constant-temperature water bath at 60℃ for 90 min, during which the stirring rate is 90 rpm.

[0016] In the step (3), the temperature is controlled in a stepwise manner, 60℃ is maintained for 10 min, 70℃ is maintained for 15 min, and 65℃ is maintained for 10 min, and the total extraction time is 35 min. The initial 60℃ is conducive to the dissolution of polysaccharides; then 70℃ can further destroy the cell structure and accelerate diffusion; finally 65℃ ensures that the polysaccharides are fully extracted at a suitable temperature while avoiding the decrease in activity caused by long-term high temperature.

[0017] In the further optimization scheme, in the step (3), the microwave power is 400W, the ultrasonic power is 300W, the temperature is 65℃, and the extraction time is 40 min.

[0018] In the step (6), the precipitate is dried in a vacuum freeze dryer, and the vacuum freeze drying conditions are that the temperature is -40±2℃, the vacuum degree is 10-20 Pa, and the drying time is 12-16 h.

[0019] The present application first uses the artificially domesticated N. verticalis as a raw material for polysaccharide extraction, solves the bottleneck problem of scarce wild N. verticalis resources and unstable quality, carries out high-value utilization of resources, forms a complete technical chain from raw material production to product development, and has obvious innovation.

[0020] The extraction efficiency of the present application is greatly higher than that of the traditional extraction process, the food-grade enzyme preparation, ultrasonic and microwave synergistic extraction, and freeze-drying environmental protection process are adopted, the action is mild, the energy consumption is low, the activity retention rate is high, the residual of chemical substances is reduced, the environmental hazards are avoided, and it is an important breakthrough of polysaccharide extraction technology.

[0021] The artificially cultivated fruiting body of N. verticalis can be expanded by the following expansion method, and the steps include the following: A, mother seed preparation: inoculate the longitudinal stripe of the Xanthi species on the modified PDA test tube slope solid culture medium, and incubate at 22-28℃ for 6-8 days to obtain the experimental mother seed; B, medium preparation: prepare solid culture medium, which includes the following components with mass percentage: sawdust 42-44%, bran 18-22%, corn cob 32-36%, gypsum powder 0.8-1.2% and lime powder 0.8-1.2%; Medium preparation: add sawdust, corn cob and bran in the proportion of the formula in turn, dry mix for 8-12 min to be uniform; then add gypsum powder and lime powder, add water while stirring, adjust the moisture content to 62-65%, that is, the material clumps into a ball. Put the mixed culture medium into 800 seed bottles, the filling height is 2 / 3 of the bottle height, sterilize with 118-124℃ high pressure steam for 28-32 min, and then cool to room temperature after sterilization for standby use.

[0022] C, sterilization and inoculation: after sterilization with 118-124℃ high pressure steam, cool the culture medium in the seed bottle to room temperature, and inoculate the mother seed prepared in step 1; The inoculation ratio: the culture medium and the mother seed are inoculated at a volume ratio of 10:1, that is, 10 mL of modified PDA slope mother seed is inoculated into every 100 mL of culture medium (volume after filling into the bottle).

[0023] D, culture and harvesting: place the inoculated seed bottle in an environment with a temperature of 23-25.5℃ and a relative humidity of 85%-90% for culture, observe regularly, and harvest after the fruiting bodies mature. Measure the fresh weight yield, average length, number of roots per bottle and moisture content, etc.

[0024] In the optimization scheme, the medium formula in step B includes the following components: sawdust 43%, bran 20%, corn cob 35%, gypsum powder 1% and lime powder 1%.

[0025] The sawdust is passed through a 20 mesh sieve to remove impurities and coarse particles (>1mm) to avoid piercing the seed bottle or causing uneven local air permeability.

[0026] The particle size of the bran is 0.2-0.3mm, and it is commercially available with medium fineness without additional crushing.

[0027] The corn cob is crushed and then passed through a 20 mesh sieve to maintain a certain particle size to maintain air permeability.

[0028] If the particle size of each raw material is too fine, the porosity of the culture medium will decrease, the mycelium respiration will be blocked, and the fruiting body differentiation rate will decrease. If the particle size is too coarse, the contact area between carbon source and mycelium will decrease, and the fruiting body growth will be uneven.

[0029] The medium preparation in step B: according to the formula proportion, the sawdust, corn cob and bran are sequentially added and dry-mixed for 10 minutes until uniform; then the gypsum powder and lime powder are added, and the clear water is added while stirring, and the moisture content is adjusted to 62-65%, that is, the material is clumped in hand; the mixed culture material is loaded into an 800 mL strain bottle, the loading height is 2 / 3 of the bottle height, and the 121 DEG C high-pressure steam sterilization is adopted for 30 minutes, and after sterilization, the natural cooling to room temperature is prepared.

[0030] The inoculated strain bottle in step D is cultured at 25 DEG C and relative humidity of 86%-89%.

[0031] When the temperature is lower than 25 DEG C, the mycelium recovers slowly, and the colonization time is prolonged; when the temperature is higher than 25 DEG C, the parent strain mycelium is prone to aging, the activity is decreased, and the yield of fruiting body is reduced. When the humidity is lower than 86%, the surface of the culture material is prone to dry, and the mycelium growth is stagnant; when the humidity is higher than 89%, the inner wall of the strain bottle is prone to water droplets, and the contamination of miscellaneous bacteria (such as penicillium and aspergillus) is caused.

[0032] The culture medium of the application significantly improves the biological yield and quality of the fruiting body by optimizing the carbon source type of the sawdust and corn cob compound and the nitrogen ratio of the nitrogen source carbon provided by the bran, provides reliable raw material guarantee for subsequent product development, and fills the technical blank of artificial cultivation of the strain.

[0033] The preparation method of the health functional drink in the application comprises the following steps: Step one, ingredient: weigh 5-8 parts of vertical stripe Xylaria polysaccharide extract, 12-15 parts of blueberry extract, 3-5 parts of green tea extract, 1-1.5 parts of vitamin C, 60-75 parts of honey, 0.8-1.2 parts of citric acid and 450-500 parts of water; Step two, mixing: heat the pure water to 47-52 DEG C, add the honey, and stir until completely dissolved; then sequentially add the vertical stripe Xylaria polysaccharide extract, the blueberry extract and the green tea extract, stir for 8-10 minutes after each time to completely dissolve; finally add the vitamin C and the citric acid, continue to stir for 5-8 minutes, then filter through a 100 mesh filter screen to remove undissolved impurities, and obtain an initial mixed solution; Step three, homogenization treatment: pump the initial mixed solution into a high-pressure homogenizer, set the homogenization pressure to 20-30 MPa, the homogenization temperature to 50-60 DEG C, and homogenize 1-2 times, and the particle size is less than 5 μm; after homogenization, the centrifugal stability of the mixed solution is detected, and 3000 r / min centrifugation for 30 minutes is qualified without stratification and precipitation; keep the feeding stable during the homogenization process to avoid air entering the system.

[0034] After the first homogenization, the particle size is observed, and if the particle size is greater than or equal to 5 μm, the second homogenization is required.

[0035] Step four, sterilization filling: the homogenized mixture is sterilized by a tubular ultra-high temperature sterilization machine, the sterilization temperature is 120-135 DEG C, the holding time is 3-5s, and the feeding flow is 100L / h; the sterilized material is transported to a sterile filling machine through a sterile pipeline, the PET bottle is sterilized by ultraviolet rays before filling, the wavelength is 254nm, the irradiation time is 30-60s; the liquid level error is controlled to be less than or equal to ±2mm during filling, the capping torque is 15-25N.cm; after filling, the product is inverted and sterilized, the holding temperature is 38-40 DEG C for 24h, and finally, the unqualified products are removed through lamp inspection, and the products are stored after labeling.

[0036] In the step two, the purified water is heated to 50 DEG C, and the honey is added.

[0037] In the step two, the pH value of the system is monitored by a pH meter during stirring, and is controlled to be in the range of 3.5-4.5.

[0038] In the step three, the homogenization pressure is 25MPa, the homogenization temperature is 55 DEG C, the homogenization is carried out twice, and the particle size is less than 5mu m.

[0039] The health functional drink formula in the application comprises the following components in parts by weight: 5-8 parts of Xanthan gum polysaccharide extract, 12-15 parts of blueberry extract, 3-5 parts of green tea extract, 1-1.5 parts of vitamin C, 60-75 parts of honey, 0.8-1.2 parts of citric acid and 450-500 parts of water.

[0040] In the optimization scheme, the health functional drink formula comprises the following components in parts by weight: 6-7 parts of Xanthan gum polysaccharide extract, 13-14 parts of blueberry extract, 4-5 parts of green tea extract, 1.2-1.4 parts of vitamin C, 65-70 parts of honey, 0.9-1.1 parts of citric acid and 470-490 parts of water.

[0041] In the further optimization scheme, the health functional drink formula comprises the following components in parts by weight: 6.5 parts of Xanthan gum polysaccharide extract, 14 parts of blueberry extract, 4.5 parts of green tea extract, 1.3 parts of vitamin C, 68 parts of honey, 1 part of citric acid and 480 parts of water.

[0042] The Xanthan gum polysaccharide, the blueberry extract and the green tea extract are scientifically matched in the application, the functions of various components are fully played, the function complementation and synergistic enhancement are achieved, the scientific matching is formed, and a multi-target and all-round health function system is formed.

[0043] The Xanthan gum polysaccharide is a core functional component, and has immunoregulation and anti-tumor activity.

[0044] The blueberry extract is rich in anthocyanins, has strong antioxidant capacity, can synergistically enhance the immunoregulation function of the Xanthan gum polysaccharide, and can improve the color and flavor of the drink.

[0045] The main active ingredient of the green tea extract is tea polyphenol, which has the functions of scavenging free radicals, anti-inflammation, regulating blood lipids and the like, and has a synergistic effect with the polysaccharide of the Xanthi in the aspect of antioxidation.

[0046] The vitamin C can not only enhance the antioxidation effect, but also regulate the immune function, and as a natural preservative, can ensure the stability of the product.

[0047] In addition to serving as a natural sweetener, the honey itself has the functions of antibiosis, antioxidation and immune regulation, and is complementary to other ingredients.

[0048] The raw materials of the beverage in the present application are all natural ingredients, the Xanthi is obtained through artificial cultivation, the blueberry and the green tea are common natural plants, the vitamin C is a necessary nutrient for the human body, and the honey is also a natural food widely consumed. The whole formula does not add harmful ingredients such as artificial synthetic antioxidants and preservatives, is safe and reliable for long-term drinking, and is suitable for people of all ages.

[0049] The beverage in the present application ensures the functional activity, and takes into account the taste acceptance and the product stability. In particular, the addition of the citric acid not only adjusts the sour-sweet ratio, but also cooperates with the vitamin C to regulate the pH and protect the active ingredients. The pH value is controlled in the range of 3.5-4.5, which is beneficial to maintaining the stability of the ingredients and meets the taste preference of the public. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figures 1-3 The figure is the clearance rate of DPPH of the polysaccharide of the Xanthi in different concentrations in the present application.

[0051] Figure 4 The figure is the ultraviolet detection figure in the present application. DETAILED DESCRIPTION

[0052] The present application will be further described in combination with the specific embodiments as follows: Embodiment 1

[0053] The polysaccharide extraction process of the Xanthi is as follows: (1) Raw material pretreatment: fresh Xanthi fruiting bodies cultivated artificially are selected, impurities and moldy parts are removed, and the Xanthi fruiting bodies are washed with clean water for 3-5 times until there is no residual dirt on the surface, and then the Xanthi fruiting bodies are placed in a 45℃ air drying oven for drying for 16h until the water content is ≤8%. After the dried fruiting bodies are crushed and passed through a 100-mesh sieve, the fruiting bodies are sealed and placed in a desiccator for standby.

[0054] (2) Enzymatic hydrolysis: A complex enzyme system was used, with cellulase: pectinase: chitinase = 2.2:0.8:1.2, added at a proportion of 1.5% of the raw material mass. The mixed powder of the fruiting body and the complex enzyme was added to pure water at a solid-liquid ratio of 1:15 (w / v), the pH was adjusted to 5.5, and the targeted enzymatic hydrolysis was carried out in a constant-temperature water bath at 55°C for 90 min, with the stirring rate controlled at 80 rpm.

[0055] After the fruiting body powder was subjected to enzymatic hydrolysis, ultrasonic and microwave-assisted extraction, a crude polysaccharide extract was obtained, containing impurities and a large amount of water.

[0056] (3) Ultrasonic-microwave-assisted extraction: After the enzymatic hydrolysis was completed, the mixed solution was transferred to an ultrasonic-microwave-assisted extraction device, the microwave power was set to 300 W, the ultrasonic power was set to 200 W, and a stepwise temperature control was adopted, with 60°C maintained for 15 min, 70°C maintained for 15 min, and 65°C maintained for 15 min, and the total extraction time was 45 min.

[0057] (4) Enzyme inactivation and filtration: After the extraction was completed, the mixed solution was heated to 95°C for 8 min to inactivate the enzymes, and then quickly cooled to room temperature and filtered through a 0.22 μm ceramic membrane to collect the filtrate.

[0058] The ceramic membrane used in the purification process can withstand high temperatures, is corrosion-resistant, has high separation efficiency, has a long service life, and has low operating costs, making it suitable for industrial production.

[0059] (5) Concentration and purification: The filtrates were combined and concentrated by rotary evaporation at 40°C and a vacuum degree of 0.09 MPa to 1 / 4 of the original volume to obtain a polysaccharide concentrate. 3 times the volume of an ethanol-acetone mixed solution with a volume ratio of 3:1 was slowly added to the concentrate while stirring to achieve a final concentration of 65%, and the mixture was sealed and placed in a 4°C refrigerator for 8 hours.

[0060] The use of an ethanol-acetone mixed solution for polysaccharide precipitation has the advantages of complete precipitation and high protein removal rate compared to single ethanol precipitation. The volume ratio of ethanol to acetone is 3:1, which achieves the best precipitation effect and reduces the amount of solvent used, thereby reducing costs and production risks.

[0061] The protein removal rate in this step is more than 90%. As shown in FIG. 4, the polysaccharide sample solution of X. longitudistriatus fruiting body has almost no absorption at 260 nm, indicating that the polysaccharide sample contains almost no nucleic acids and proteins. The detection instrument used was a UV-visible spectrophotometer (752N Plus). Figure 4

[0062] ​Separation and drying: the mixed solution after standing was put into a high-speed centrifuge, centrifuged at 5000 rpm for 15 min, the supernatant was discarded, and the polysaccharide precipitate at the bottom was collected; the precipitate was washed twice with 50% ethanol-acetone mixed solution, and finally washed once with anhydrous ethanol to remove residual impurities. The precipitate was placed in a vacuum freeze dryer, the temperature was -40°C, the vacuum degree was 10 Pa, and the drying time was 16 h. The polysaccharide extract of X. velttum was obtained, and was sealed and stored away from light. The raw material of X. velttum was 200 g, the polysaccharide extract was 13.14 g, the yield was 6.57%, and the extraction rate was 90.62%.

[0063] The artificially cultivated X. velttum fruiting bodies can be expanded by the following expansion method, and the steps are as follows: A, mother seed preparation: inoculate the X. velttum strain on the improved PDA test tube slope solid culture medium, and incubate at 25°C for 7 days to obtain the experimental mother seed; the X. velttum strain used is collected in Kaiyuan International Community in Xindu District, Chengdu, and is preserved on the improved PD test tube slope culture medium.

[0064] B, medium preparation: prepare solid culture material, which includes the following components in mass percentage: sawdust 43%, bran 20%, corn cob 35%, gypsum powder 1%, lime powder 1%; Medium preparation: add sawdust, corn cob and bran in the proportion of the formula in turn, dry mix for 10 min until uniform; then add gypsum powder and lime powder, add water while stirring, and adjust the moisture content to 63%, that is, the material clumps into a ball when held by hand. The mixed culture material is loaded into 800 mL of seed bottle, the loading height is 2 / 3 of the bottle height, and 121°C high pressure steam sterilization is used for 30 min, and then naturally cooled to room temperature for standby.

[0065] The sawdust used is the conventional sawdust for edible mushroom cultivation, which is once crushed by a wood crusher, has good particle integrity, retains part of the wood fiber structure, and has a particle size of about 20 mesh, which is directly purchased from the market; the wood chips are a product of secondary crushing of sawdust, which is finer, and is crushed to pass through a 40 mesh sieve; the corn cob has a particle size of 20 mesh, and the cottonseed hull has a particle size of 10 mesh.

[0066] C, sterilization and inoculation: the above culture material is loaded into the seed bottle, sterilized by 121°C high pressure steam, and then cooled to room temperature, and the mother seed prepared in step 1 is inoculated; D, culture and harvesting: the inoculated seed bottle is cultured at 25°C and a relative humidity of 86%-89%, and is observed regularly, and is harvested after the fruiting bodies mature.

[0067] In this culture medium, sawdust mainly provides cellulose, hemicellulose and lignin as carbon source nutrients for mycelial growth, which is the basic source of mycelial energy and carbon skeleton structure. At the same time, the sawdust particles constitute the physical skeleton of the culture medium, providing support for the spread of mycelium and the growth of fruiting bodies, and adjusting the porosity of the culture medium, affecting the air permeability and water holding capacity.

[0068] Corn cob is also rich in cellulose and hemicellulose, which is an important carbon source supplement. Its structure is loose and porous, with excellent water absorption and air permeability, which can effectively prevent the culture medium from being too wet and clumping, and ensure that the mycelial growth environment has sufficient oxygen. It complements sawdust in physical structure and nutrient composition, which is a key component to achieve high yield.

[0069] Wheat bran is rich in protein, amino acids and various vitamins (such as B vitamins), which is the main source of nitrogen source and growth factor necessary for the synthesis of mycelium and fruiting body protein, nucleic acid and other life substances. It effectively regulates the carbon-nitrogen ratio (C / N) of the culture medium, which is a key factor affecting the growth rate of mycelium and the yield and quality of fruiting bodies.

[0070] Gypsum powder is a pH buffer and structure modifier, which prevents the culture medium from being excessively acidified due to the production of organic acids during sterilization and mycelial growth, provides calcium ions and sulfur elements and other mineral elements, and at the same time enhances the clumping of the culture medium, reduces the sticking phenomenon after sterilization and improves the structure of the material.

[0071] Lime powder adjusts the initial pH, first adjusts the culture medium to slightly alkaline, which is conducive to inhibiting the growth of most bacteria such as mold, creating a clean growth starting point for T. verticalis; It also supplements calcium elements, which cooperates with gypsum to jointly maintain the relative stability of the pH value of the culture medium throughout the growth cycle.

[0072] Example 2 The polysaccharide extraction process of T. verticalis is as follows: (1) Raw material pretreatment: select fresh T. verticalis fruiting bodies cultivated artificially, remove impurities and moldy parts, rinse with water for 3-5 times until there is no residual sand on the surface, dry in a 50°C air-drying oven for 12h, until the water content is ≤8%. Grind the dried fruiting bodies and pass through a 100 mesh sieve, then seal and store in a desiccator for use.

[0073] (2) Enzymatic hydrolysis: use a composite enzyme system, cellulase: pectinase: chitinase = 1.8: 1.2: 0.8, add 2.5% of the raw material quality. Add pure water to the powder at a ratio of 1:18 (w / v), adjust the pH to 4.5, and perform targeted enzymatic hydrolysis in a 60°C constant temperature water bath for 60min, with a stirring rate of 100rpm. During the process, the cell wall structure is specifically destroyed by enzymatic hydrolysis.

[0074] (3) Ultrasonic-microwave synergistic extraction: After enzyme hydrolysis, the mixed solution was transferred to an ultrasonic-microwave synergistic extraction device, and the microwave power was set to 400 W and the ultrasonic power was set to 300 W. The temperature was controlled in a stepwise manner, and the temperature was maintained at 60°C for 10 min, 70°C for 15 min, and 65°C for 15 min. The total extraction time was 40 min.

[0075] (4) Enzyme inactivation and filtration: After extraction, the mixed solution was heated to 100°C for 8 min to inactivate the enzyme. After rapid cooling to room temperature, the solution was filtered through a 0.22 μm ceramic membrane, and the filtrate was collected.

[0076] (5) Concentration and purification: The filtrate was combined and concentrated under vacuum at 45°C and a vacuum degree of 0.09 MPa to 1 / 6 of the original volume. A polysaccharide concentrate was obtained. Slowly add 4 times the volume of an ethanol-acetone mixture (volume ratio 3:1) to the concentrate, stirring while adding, to a final concentration of 70%. Seal and place in a 4°C refrigerator for 12 hours.

[0077] (6) Separation and drying: Place the mixed solution after standing in a high-speed centrifuge and centrifuge at 6000 rpm for 10 min. Discard the supernatant and collect the polysaccharide precipitate at the bottom. Wash the precipitate with a 50% ethanol-acetone mixture twice and finally with anhydrous ethanol once to remove residual impurities. Place the precipitate in a vacuum freeze dryer at a temperature of -40°C and a vacuum degree of 20 Pa for 12 hours. Obtain the polysaccharide extract of X. striatum and store it in a sealed, light-proof container. For 200g of X. striatum raw material, 13.72g of polysaccharide extract was obtained, with a yield of 6.86% and an extraction rate of 94.62%.

[0078] The artificially cultivated X. striatum fruiting bodies can be expanded by the following method, and other contents are as in Example 1, with the following steps: Strain culture: Inoculate X. striatum strain on modified PDA test tube slope solid medium and cultivate in a 22°C constant temperature incubator for 8 days to expand the original strain amount as experimental mother strain.

[0079] B. Preparation of culture medium: Prepare a solid culture medium containing the following components in mass percentage: sawdust 42%, bran 22%, corn cob 34%, gypsum powder 0.8%, lime powder 1.2%; Add sawdust, corn cob and bran in the proportion of the formula in turn, dry mix for 8 min until uniform; then add gypsum powder and lime powder, stir while adding water, and adjust the moisture content to 62%, i.e. the material clumps into a ball when held in the hand. Fill the mixed culture medium into 800 mL strain bottles, with the filling height being 2 / 3 of the bottle height, sterilize with 118°C high-pressure steam for 32 min, and naturally cool to room temperature after sterilization for standby use.

[0080] C. Sterilization and inoculation: Use plastic strain bottles for filling, sterilize with 118°C high-pressure steam, and inoculate after cooling; D, fruit body culture and harvest: cultivate at 23℃, relative humidity 85%-90%, observe mycelium growth regularly, harvest fruit body after maturation, measure fresh weight yield, average length, single bottle root number and water content.

[0081] Example 3 The polysaccharide extraction process of X. striatus is as follows: (1) Raw material pretreatment: select fresh X. striatus fruit bodies cultivated artificially, remove impurities and moldy parts, rinse with water 4 times until no residual dirt is left on the surface, dry in a 47℃ air-drying oven for 14h until the water content is ≤8%. Crush the dried fruit bodies and pass through a 100 mesh sieve, seal and store in a desiccator for later use.

[0082] (2) Enzymatic hydrolysis: use a composite enzyme system, cellulase: pectinase: chitinase = 2:1:1, add 2% of the raw material mass. Add pure water at a solid-liquid ratio of 1:16 (w / v), adjust the pH to 5, and perform targeted enzymatic hydrolysis in a 58℃ constant temperature water bath for 75min, with a stirring rate of 90rpm.

[0083] (3) Ultrasonic-microwave synergistic extraction: after enzymatic hydrolysis, transfer the mixture to an ultrasonic-microwave synergistic extraction device, set the microwave power to 350W and the ultrasonic power to 250W, use stepwise temperature control, maintain 60℃ for 10min, 70℃ for 15min, and 65℃ for 10min, with a total extraction time of 35min.

[0084] (4) Enzyme inactivation and filtration: after extraction, heat the mixture to 97℃ for 8min to inactivate the enzymes, quickly cool to room temperature, and then filter through a 0.22μm ceramic membrane to collect the filtrate.

[0085] (5) Concentration and purification: combine the filtrates, concentrate under vacuum at 42℃ and a vacuum degree of 0.095MPa to 1 / 5 of the original volume to obtain a polysaccharide concentrate; slowly add 3.5 times the volume of an ethanol-acetone mixture (volume ratio 3:1) to the concentrate while stirring to achieve a final concentration of 68%, seal and store in a 4℃ refrigerator for 9h.

[0086] (6) Separation and drying: place the mixture after standing in a high-speed centrifuge, centrifuge at 5700rpm for 13min, discard the supernatant, and collect the polysaccharide precipitate at the bottom. Wash the precipitate with a 50% ethanol-acetone mixture twice and finally with anhydrous ethanol once to remove residual impurities. Dry the precipitate in a vacuum freeze-drying machine at a temperature of -40℃ and a vacuum degree of 15Pa for 13h to obtain X. striatus polysaccharide extract, which is sealed and stored away from light. For 200g of X. striatus raw material, 13.66g of polysaccharide extract is obtained, with a yield of 6.83% and an extraction rate of 94.21%.

[0087] The artificial cultivated Nectria longitudinalis fruiting bodies can be expanded by the following expansion method, and other contents are as in Example 1, and the steps are as follows: A, strain culture: inoculate Nectria longitudinalis strain on modified PDA test tube slope solid culture medium, and culture in a 22°C constant temperature incubator for 8 days to expand the original strain amount as experimental mother strain.

[0088] B, preparation of culture material: prepare solid culture material composed of the following components in percentage by mass: sawdust 44%, bran 18%, corn cob 36%, gypsum powder 1.2%, and lime powder 0.8%; Add sawdust, corn cob, and bran in the formula proportion in turn, dry mix for 12 min until uniform; then add gypsum powder and lime powder, and add water while stirring to adjust the water content to 65%, that is, the material clumps into a ball. Put the mixed culture material into 800 mL strain bottles, the filling height is 2 / 3 of the bottle height, sterilize with 124°C high-pressure steam for 28 min, and then naturally cool to room temperature for standby use after sterilization.

[0089] C, sterilization and inoculation: use plastic strain bottles to fill the material, sterilize with 124°C high-pressure steam, and inoculate after cooling; D, fruiting body culture and harvesting: culture at 25.5°C and relative humidity of 85%-90%, observe the mycelial growth regularly, harvest the mature fruiting bodies, and measure the fresh weight yield, average length, number of single bottles, and water content.

[0090] Example 4 Other contents are as in Example 1, and the polysaccharide extraction of Nectria longitudinalis is as follows: take the dried Nectria longitudinalis fruiting bodies, wash, crush, and pass through a 100 mesh sieve. Add 1500 mL deionized water at a solid-liquid ratio of 1:15, adjust the pH to 5.0, add 2 g of composite enzyme (cellulase: pectinase: chitinase = 2:1:1), and enzymatically hydrolyze at 58°C for 75 min. Then transfer to an ultrasonic-microwave synergistic device, microwave at 350W and ultrasonic at 250W, stepwise temperature control at 60°C×10min, 70°C×15min, and 65°C×10min. Inactivate the enzyme at 98°C for 8 min, filter through a 0.22μm ceramic membrane, and vacuum concentrate at 42°C to 300ml. Add 900ml of ethanol-acetone mixed solution (3:1) at 4°C and stand for 10h, centrifuge at 5500r / min for 12min, wash the precipitate, freeze-dry at-40°C for 14h, and obtain 6.8g of polysaccharide extract. The extraction yield is 6.8%, and the extraction rate is 93.79%.

[0091] Example 5 The preparation method of the health functional drink is as follows: Step one, ingredients: take 5 parts of the longitudinal stripe of Colletotrichum gloeosporioides polysaccharide extract prepared in examples 1-4, 12 parts of blueberry extract, 3 parts of green tea extract, 1 part of vitamin C, 60 parts of honey, 0.8 parts of citric acid, and 450 parts of water.

[0092] Step two, mixing: heat the pure water to 48℃, first add honey according to the formula proportion, stir until completely dissolved. Add longitudinal stripe of Colletotrichum gloeosporioides polysaccharide extract, blueberry extract, green tea extract in turn, stir for 8min after each addition until completely dissolved. Finally add vitamin C and citric acid, continue to stir for 5min, monitor the system pH value with pH meter during the period, control at 3.5. After stirring is completed, filter through a 100 mesh filter to remove undissolved impurities, and obtain the initial mixed solution.

[0093] Step three, homogenization treatment: pump the initial mixed solution into a high-pressure homogenizer, set the homogenization pressure to 20MPa and the homogenization temperature to 50℃, and homogenize 1-2 times. After the first homogenization, take a sample to observe the particle size. If the particle size is ≥5μm, the second homogenization is needed. Keep the feeding stable during the homogenization process to avoid air entering the system. After homogenization is completed, detect the centrifugal stability of the mixed solution. If there is no stratification and no precipitation after centrifugation at 3000r / min for 30min, it is qualified.

[0094] Step four, sterilization and filling: use a tubular ultra-high temperature sterilization machine to sterilize the homogenized mixed solution, with a sterilization temperature of 120℃, a holding time of 3s, and a feeding flow rate of 100L / h. After sterilization, the material is transported to a sterile filling machine through a sterile pipeline. Before filling, the PET bottles are sterilized by ultraviolet light with a wavelength of 254nm for 30s. During filling, the liquid level error is controlled to be ≤±2mm, and the capping torque is 15N・cm. After filling is completed, the product is inverted and sterilized at 38℃ for 24h. Finally, unqualified products are removed through lamp inspection, and the product is stored after labeling.

[0095] Example 6 The preparation method of the health functional drink is as follows: Step one, ingredients: take 5 parts of the longitudinal stripe of Colletotrichum gloeosporioides polysaccharide extract prepared in examples 1-4, 12 parts of blueberry extract, 3 parts of green tea extract, 1 part of vitamin C, 60 parts of honey, 0.8 parts of citric acid, and 450 parts of water.

[0096] Step two, mixing: heat the pure water to 48℃, first add honey according to the formula proportion, stir until completely dissolved. Add longitudinal stripe of Colletotrichum gloeosporioides polysaccharide extract, blueberry extract, green tea extract in turn, stir for 8min after each addition until completely dissolved. Finally add vitamin C and citric acid, continue to stir for 5min, monitor the system pH value with pH meter during the period, control at 3.5. After stirring is completed, filter through a 100 mesh filter to remove undissolved impurities, and obtain the initial mixed solution.

[0097] Step three, homogenization: the initial mixture is pumped into a high-pressure homogenizer, set the homogenization pressure 30 MPa, homogenization temperature 60℃, homogenization 2 times, particle size <5 μm. After homogenization, the centrifugal stability of the mixture is detected, 3000 r / min centrifugation for 30 min, no stratification, no precipitation is qualified.

[0098] Step four, sterilization and filling: a tubular ultra-high temperature sterilization machine is used to sterilize the homogenized mixture, the sterilization temperature is 135℃, the holding time is 5s, and the feeding flow is 100L / h. The sterilized material is transported to a sterile filling machine through a sterile pipeline, the PET bottle is sterilized by ultraviolet before filling, the wavelength is 254nm, and the irradiation time is 60s. The liquid level error is controlled to be ≤±2mm during filling, and the capping torque is 25N・cm. After filling, the product is inverted and sterilized at 40℃ for 24h, and finally the unqualified products are removed by lamp inspection, and then stored after labeling.

[0099] Example 7 The preparation method of the health functional beverage is as follows: Step one, ingredient: take 6.5 parts of the vertical stripe Xylaria polysaccharide extract prepared in examples 1-4, 14 parts of blueberry extract, 4.5 parts of green tea extract, 1.3 parts of vitamin C, 68 parts of honey, 1 part of citric acid and 480 parts of water.

[0100] Step two, mixing: heat the pure water to 50℃, add the honey first according to the formula proportion, and stir until completely dissolved. Add the vertical stripe Xylaria polysaccharide extract, blueberry extract and green tea extract in turn, stir for 9min after each addition until completely dissolved. Finally, add vitamin C and citric acid, continue to stir for 7min, monitor the pH value of the system with a pH meter during the period, and control it at 4. After stirring, filter through a 100 mesh filter screen to remove undissolved impurities, and obtain the initial mixture.

[0101] Step three, homogenization: the initial mixture is pumped into a high-pressure homogenizer, set the homogenization pressure 25 MPa, homogenization temperature 55℃, homogenization 2 times. Particle size <5 μm. After homogenization, the centrifugal stability of the mixture is detected, 3000 r / min centrifugation for 30 min, no stratification, no precipitation is qualified.

[0102] Step four, sterilization and filling: a tubular ultra-high temperature sterilization machine is used to sterilize the homogenized mixture, the sterilization temperature is 130℃, the holding time is 4s, and the feeding flow is 100L / h. The sterilized material is transported to a sterile filling machine through a sterile pipeline, the PET bottle is sterilized by ultraviolet before filling, the wavelength is 254nm, and the irradiation time is 40s. The liquid level error is controlled to be ≤±2mm during filling, and the capping torque is 20N・cm. After filling, the product is inverted and sterilized at 39℃ for 24h, and finally the unqualified products are removed by lamp inspection, and then stored after labeling.

[0103] Example 8 The rest is as in Example 7, wherein the health functional drink formula comprises the following components by weight: Xanthan gum 0.5 parts, honey 65 parts, lemon acid 0.9 parts, and water 470 parts.

[0104] Example 9 The rest is as in Example 7, wherein the health functional drink formula comprises the following components by weight: Xanthan gum 0.5 parts, honey 65 parts, lemon acid 0.9 parts, and water 470 parts.

[0105] Example 10 The rest is as in Example 1 and Example 5, and the health functional drink is prepared by the following steps: Xanthan gum polysaccharide extract: 150 g of dry fruit body powder is ground through a 100 mesh sieve. 2700 mL of water is added at a material-to-liquid ratio of 1:18, the pH is adjusted to 5.5, 3.5 g of composite enzyme is added, and enzymolysis is performed at 60°C for 90 min. 10.5 g of polysaccharide extract is obtained by 400 W microwave + 300 W ultrasonic step extraction. The extraction yield is 7%, and the extraction rate is 96.55%.

[0106] 2. Mixing of ingredients: 450 mL of pure water is heated to 45°C, 60 g of honey is added and stirred for 8 min until dissolved, and Xanthan gum polysaccharide extract 5 parts, blueberry extract 12 parts, and green tea extract 3 parts are added in sequence, each time stirring for 12 min. Finally, 1 part of vitamin C and 1 part of lemon acid are added, and stirring is performed for 6 min. After filtration, the initial mixed solution is obtained. 3. Homogenization treatment: The mixed solution is homogenized at 20 MPa and 55°C for 2 times, and the centrifugal stability test is qualified. 4. Sterilization and filling: 125°C high-temperature instantaneous sterilization for 4 seconds, aseptic filling into 500 ml PET bottles, cap torque 20 N・cm, inverted sterilization, and lamp inspection are qualified.

[0107] Example 11 The rest is as in Example 1 and Example 5, and the health functional drink is prepared by the following steps: Xanthan gum polysaccharide extract: 150 g of dry fruit body powder is ground through a 100 mesh sieve. 2700 mL of water is added at a material-to-liquid ratio of 1:18, the pH is adjusted to 5.5, 3.5 g of composite enzyme is added, and enzymolysis is performed at 60°C for 90 min. 10.5 g of polysaccharide extract is obtained by 400 W microwave + 300 W ultrasonic step extraction. The extraction yield is 7%, and the extraction rate is 96.55%.

[0108] 2. Mixing of ingredients: 480 parts of pure water is heated to 50℃, 70 parts of honey is dissolved, then 7 parts of G. virens polysaccharide extract, 13 parts of blueberry extract and 4 parts of green tea extract are sequentially added, and finally 1.2 parts of vitamin C and 1 part of citric acid are added. 3. Homogenization treatment: 25 MPa, 60℃, 2 times. 4. Sterilization and filling: 130℃, 3 seconds, filled into 500ml PET bottles.

[0109] Example 12 Other contents are as shown in Example 1 and Example 5, and the preparation method of the health functional beverage is as follows: G. virens polysaccharide extract: 200g of fruit body dry material is crushed through a 100-mesh sieve. 2400mL of water is added at a material-to-liquid ratio of 1:12, the pH is adjusted to 4.5, 3g of composite enzyme is added, and enzyme hydrolysis is performed at 60℃ for 60min. Microwave of 300W and ultrasonic of 200W are used for step-by-step extraction, and 11.8g of polysaccharide extract is obtained. The extraction yield is 5.9%, and the extraction rate is 81.38%.

[0110] Because the material-to-liquid ratio is lower than that in Example 10 (1:18) and Example 11 (1:15), the insufficient amount of solvent leads to insufficient dissolution of polysaccharides. In addition, the enzyme addition amount is 1.5%, which is lower than that in Example 10 (2.3%) and Example 11 (2%), and the enzyme hydrolysis efficiency is insufficient. In Example 12, the microwave of 300W and ultrasonic of 200W are used for synergistic extraction, and the effect is weak. In addition, errors in the experimental process also affect the extraction yield.

[0111] 2. Mixing of ingredients: 500 parts of water is added with 75 parts of honey, and then 8 parts of G. virens polysaccharide extract, 15 parts of blueberry extract and 5 parts of green tea extract are sequentially added, and finally 1.5 parts of vitamin C and 1.2 parts of citric acid are added. 3. Homogenization treatment: 30 MPa, 55℃, 2 times. 4. Sterilization and filling: 128℃, 4s, filled into 500ml PET bottles.

[0112] In the present application, the sensory indicators of the health functional beverage are as follows: Color: The beverage is uniform purple red, bright in color, and has no obvious stratification or color difference.

[0113] Odor: It has the natural fruit aroma of blueberry and the fragrance of green tea, and the aroma is harmonious and natural, without any odor, burnt smell or other irritating odor.

[0114] Taste: The taste is mellow and smooth, with moderate sour and sweet, without obvious astringency, bitterness or granular feeling, and comfortable to enter the mouth.

[0115] Tissue state: The beverage is clear and transparent, without obvious precipitate, suspended matter or flocculent matter, and no stratification phenomenon after standing.

[0116] The physicochemical indexes of the health functional drink in the application are as follows: Soluble solid content: the soluble solid content is ≥10%, which is mainly derived from honey, Xanthanum striatum polysaccharide, blueberry extract and green tea extract.

[0117] Xanthanum striatum polysaccharide content: the Xanthanum striatum polysaccharide content in the drink is ≥95g / L, so as to ensure that the drink has sufficient core functional ingredients.

[0118] Vitamin C content: the vitamin C content is ≥0.05g / L.

[0119] The microbial indexes of the health functional drink in the application are as follows: The total number of colonies is ≤100 CFU / mL, the coliform group cannot be detected, the number of molds and yeasts is ≤10 CFU / mL, and pathogenic bacteria (Salmonella, Staphylococcus aureus, Shigella, etc.) cannot be detected.

[0120] The functional evaluation of the health functional drink in the application is as follows: The in-vitro detection is taken as a standard: DPPH free radical scavenging rate: ≥70%, ABTS cationic free radical scavenging rate: ≥80%.

[0121] From the above analysis, the Xanthanum striatum health functional drink prepared in the application has not only good sensory quality, but also significant biological activity, and is suitable for long-term consumption as a daily health drink.

[0122] Test One Other contents are the same, compared with the conventional extraction process, the extraction process is different, the extraction rate and yield of Xanthanum striatum polysaccharide are different, and the comparison is shown in Table 1: Table 1 Constant temperature water bath extraction Enzymatic extraction Ultrasonic + microwave extraction Enzymatic + ultrasonic + microwave + stepwise temperature control (Example 1) Extraction yield (%) 2.35 5.07 4.38 6.8 Extraction yield (%) 32.41 69.93 60.41 93.79 From the above Table 1, it can be seen that, compared with constant temperature extraction, the polysaccharide extraction rate and yield are significantly improved in the dynamic temperature control in the extraction process, and compared with only enzymatic extraction or ultrasonic + microwave extraction, the polysaccharide extraction rate and yield are also improved a lot, and the technical scheme in the application has an overall effect of increasing capital.

[0123] Test Two Economic Benefit Prediction: With a production line to measure, annual capacity of 2 million bottles (500 mL / bottle), raw material cost of 0.29 yuan per bottle, 2 million bottles of raw material cost of 580,000 yuan per year, the average annual labor cost of 1.02 million yuan, the average annual transportation cost of 160,000 yuan; packaging (0.32 yuan per bottle, 640,000 yuan per year), equipment depreciation (500,000 yuan per year), energy consumption (9.6 million yuan per year), the total cost of 299.6 million yuan per year. The terminal selling price is 8 yuan per bottle, and the production and sales rate is 95%. After deducting other expenses of about 148 million yuan, the average annual profit is 1072.4 million yuan. At the same time, relying on the invention process, the average annual wastewater treatment cost can be saved by 280,000 yuan, and the raw material loss cost can be saved by 160,000 yuan. The overall profit space is greatly expanded, the investment recovery period is further shortened, and the economic benefit is remarkable.

[0124] Test three Antioxidant index determination of N. longicaulis fruiting body polysaccharide: DPPH free radical scavenging capacity determination: the instrument used is ultraviolet-visible spectrophotometer, and the determination method is referenced from the source of the master's thesis "Study on the antibacterial active secondary metabolites of N. longicaulis" and "Study on the chemical composition and biological activity of N. longicaulis fruiting body" with slight routine adjustment.

[0125] After purification, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 mg / mL of N. longicaulis fruiting body polysaccharide solution 2 mL was prepared, 0.2 mmol / L DPPH· ethanol solution 2 mL was added, and after shaking, it was placed in the dark at room temperature for 30 min. The absorbance was measured at 517 nm wavelength, and Vc solution was used as a positive control. As shown in Figure 1 .

[0126] Hydroxyl radical scavenging capacity determination: the instrument used is ultraviolet-visible spectrophotometer, and the determination method is referenced from the source of the master's thesis "Study on the antibacterial active secondary metabolites of N. longicaulis" and the method of Fenton reaction system for determining hydroxyl radical scavenging capacity with slight adjustment.

[0127] 1 mL of 9 mmol / L FeSO4 solution, 1 mL of 9 mmol / L salicylic acid-ethanol solution and 1 mL of 0.5, 1, 1.5, 2, 2.5, 3 mg / mL N. longicaulis fruiting body polysaccharide solution were mixed, 1 mL of 8.8 mmol / L H2O2 solution was added, and after 30 min in 37 ℃ water bath, 3000 rpm centrifugation for 5 min. The supernatant was measured at 510 nm wavelength, and Vc solution was used as a control. As shown in Figure 2 .

[0128] ABTS free radical scavenging capacity determination: the instrument is ultraviolet-visible spectrophotometer, the method is referenced from the determination method of ABTS free radical scavenging capacity in national standard GB / T 31740.2-2015 'Tea products Part 2: Tea polyphenols', and the paper 'Research on the synergistic scavenging of ABTS free radicals by resveratrol and black fungus polysaccharides' in CNKI, with slight routine adjustment.

[0129] 1 mL of 0.5, 1, 1.5, 2, 2.5, 3 mg / mL of Xanthogramma hartigii polysaccharide solution and 3 mL of ABTS free radical working solution were added into test tubes respectively, mixed uniformly, placed in dark at room temperature for 1 h, and then the absorbance was determined at 734 nm wavelength, with Vc solution as a control. Figure 3 As shown in the figure.

[0130] As can be seen from the above table 2, in the present application, the polysaccharide is extracted by using a composite enzyme system for enzymolysis, compared with other different enzymes, the polysaccharide extraction rate and extraction rate are improved a lot, and the technical scheme in the present application has a synergistic effect as a whole. Also shows that the enzyme addition amount, microwave and ultrasonic synergy have a synergistic effect. Figures 1-3 It can be seen that Xanthogramma hartigii polysaccharide has certain antioxidant activity. In the DPPH free radical, ABTS free radical and hydroxyl radical scavenging test, with the increase of polysaccharide concentration, the clearance rate gradually rises, showing a clear dose-effect relationship. Among them, the clearance effect on DPPH free radical and ABTS free radical is relatively good, at 0.3 mg / mL concentration, the clearance rate on DPPH free radical reaches 68.5%; at 3 mg / mL concentration, the clearance rate on ABTS free radical reaches 90.8%; the clearance effect on hydroxyl radical is relatively weak, and the clearance rate is 11.2% at the same concentration. At the same time, Xanthogramma hartigii polysaccharide has good antioxidant potential.

[0131] Test four Xanthogramma hartigii polysaccharide is extracted by using different enzyme preparations: Take 100 g of dried fruit body powder, according to the total amount of enzyme 0.2%, solid-liquid ratio 1:12, pH 4.·5, 55℃ enzyme hydrolysis for 60 min, stirring speed 80 rpm. The ultrasonic-microwave synergistic extraction parameters are: microwave power 400 W, ultrasonic power 300 W, 60℃ extraction for 35 min. After enzyme inactivation, concentration, purification and freeze-drying, the polysaccharide yield and extraction rate are calculated. The types of enzymes are different, and the results are as follows in table 2: Table 2 Total enzyme class Proportion Extraction yield (%) Extraction yield (%) Cellulase —— 5.18 71.45 Pectinase —— 5.02 69.24 Chitinase —— 5.08 70.07 Cellulase + pectinase 2：1 5.97 82.34 Cellulase + chitinase 2：1 6.52 89.93 Pectinase + chitinase 1：1 5.85 80.69 Cellulase + pectinase + chitinase 2：1：1 6.68 92.14 As can be seen from the above table 2, in the present application, the polysaccharide is extracted by using a composite enzyme system for enzymolysis, compared with other different enzymes, the polysaccharide extraction rate and extraction rate are improved a lot, and the technical scheme in the present application has a synergistic effect as a whole. Also shows that the enzyme addition amount, microwave and ultrasonic synergy have a synergistic effect.

[0132] Test five Effect of microwave power on extraction efficiency Index 200W (conventional) 300W (the present application) 400W (high power) Extraction yield (%) 8.2 9.7 7.8 Polysaccharide purity (HPLC) 82.3 88.1 84.5 Energy consumption (kWh / kg) 1.2 1.1 1.3 Conclusion: 300W is the optimal parameter, and higher power (400W) leads to Maillard reaction and browning of polysaccharides.

[0133] Experiment six Verification of synergistic effect of complex enzymes Enzyme combination Extraction yield (%) Polysaccharide yield (%) Cell disruption rate (%) Cellulase + pectinase 8.1 6.5 78 + chitinase 9.4 7.2 93 Conclusion: The combination of three enzymes improves the efficiency of cell wall degradation by 19% and increases the yield of polysaccharides by 8%.

[0134] Experiment seven pH stability test pH value Vitamin C retention rate (%) Polysaccharide solubility (g / L) 3.5 91.2 8.7 4.5 88.5 9.2 5.5 76.3 7.8 Conclusion: pH 3.5-4.5 can maintain the activity of vitamin C and prevent the aggregation and precipitation of polysaccharides.

[0135] Experiment eight Effect of microwave-ultrasound synergistic extraction on polysaccharide dissolution and cell fragmentation rate of N. longisetum Group Microwave power (W) Ultrasonic power (W) Extraction temperature (℃) Extraction time (min) Solid-liquid ratio (g / mL) Polysaccharide yield (%) Cell disruption rate (%) Experimental group 300 200 60-70 45 1:15 6.8 91 Control group (ultrasound only) / 200 60-70 45 1:15 5.18 70 Experimental conditions 1. Raw material pretreatment: Take the dried N. longisetum fruiting bodies (200g), crush them through a 100-mesh sieve, and add pure water (3000mL) at a material-to-liquid ratio of 1:15.

[0136] 2. pH adjustment: Adjust to 5.0 and add complex enzymes (cellulase: pectinase: chitinase = 2:1:1, total amount 2g).

[0137] 3. Experimental group: Transfer to the ultrasonic-microwave synergistic device, use stepwise temperature control (60℃×15min→70℃×15min→65℃×15min), microwave power 300W, ultrasonic power 200W.

[0138] 4. Control group: Only turn on the ultrasonic function (200W), and the rest of the conditions are the same as the experimental group.

[0139] 5. Enzyme inactivation and filtration: Both groups are heated to 95℃ for 8min to inactivate the enzymes, and then filtered through a 0.22μm ceramic membrane after cooling.

[0140] 6. Detection method: Polysaccharide content: HPLC method is used to detect the polysaccharide concentration in the filtrate, and the yield is calculated based on the initial dry material mass.

[0141] Cell fragmentation rate: SEM is used to observe the integrity of the cell wall of the fruiting bodies, and the proportion of fragmented cells is calculated.

[0142] Experimental result analysis 1. Dissolution of polysaccharides: The yield of the experimental group (6.8%) was 32% higher than that of the control group (5.18%), indicating that the synergistic effect of microwave internal heating and ultrasonic cavitation significantly accelerated the diffusion of polysaccharides from the cells to the solvent.

[0143] 2. Cell disruption rate: The cell disruption rate of the experimental group was 91%, much higher than that of the control group of 70%, indicating that the microwave-ultrasound combination more efficiently destroyed the cell wall structure and improved substrate accessibility.

[0144] Test Nine Effect of Freeze-drying and Spray-drying on Crystallinity and Rehydration of Phaeotrichum striatum Polysaccharides Group Drying method Temperature (℃) Pressure (Pa) Time (h) Crystallinity (%) Rehydration solubility rate (g / L·min) Experimental group (freeze-drying) -40℃ / 10-20 Pa -40 10-20 16 82.3 3.5 Control group (spray drying) 180℃ / atmospheric pressure / / 1 45.2 1.5 Experimental conditions 1. Pretreatment: Both groups used ethanol-acetone mixture to precipitate polysaccharides, obtaining concentrated solution (concentration about 10 mg / mL).

[0145] 2. Freeze-drying: The concentrated solution was divided into freeze-drying bottles and placed in a vacuum freeze-drying machine (-40℃, vacuum degree 10-20 Pa) for 16 hours.

[0146] 3. Spray-drying: The concentrated solution was sprayed into a high-temperature gas stream (inlet temperature 180℃, outlet temperature 80℃) through an atomizer, and the powder was collected.

[0147] 4. Detection method: Crystallinity: XRD diffractometer detection (CuKα ray, scanning range 5°-40°), and the crystalline peak area ratio was analyzed by Jade software.

[0148] Rehydration dissolution rate: 1g of polysaccharide powder was added to 100mL of deionized water, and magnetic stirring (500rpm) was recorded. The complete dissolution time was recorded and the dissolution rate was calculated.

[0149] Analysis of experimental results 1. Crystallinity: The crystallinity of polysaccharides obtained by freeze-drying (82.3%) was 47% higher than that of spray-drying (45.2%), indicating that freeze-drying avoided the rearrangement of molecular chains and the increase of amorphous regions caused by high temperature.

[0150] 2. Rehydration solubility: The rehydration dissolution rate of freeze-drying products (3.5g / L·min) was 2.3 times that of spray-drying products (1.5g / L·min), indicating that freeze-drying preserved the natural conformation and pore structure of polysaccharides, which was beneficial to rapid water absorption and swelling.

[0151] This invention optimizes the process parameters of each step through numerous experiments, such as the temperature, pH, and time of enzymatic hydrolysis; the power and action mode of ultrasound-microwave; the pressure and temperature of homogenization; and the temperature and time parameters of sterilization. The synergistic optimization of these parameters ensures the high quality and high stability of the product.

[0152] The above embodiments / experimental examples are merely illustrative and not intended to limit the implementation methods. Those skilled in the art will recognize that various variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementation methods. However, obvious variations or modifications derived therefrom remain within the scope of this invention.

Claims

1. A process for extracting polysaccharides from X. veltata, characterized in that: The method comprises the following steps: ​ (1) raw material pretreatment: select fresh artificial cultivation of vertical stripe Nectria Robusta fruiting body, remove impurities and moldy parts, rinse with water, dry, crush; dry is placed in 45-50℃ drying oven drying 12-16h, moisture content≤8%; (2) enzymolysis: according to the proportion of 1.5-2.5% of the raw material quality in step (1), add compound enzyme system, add pure water to the mixed powder of fruiting body and compound enzyme with the ratio of 1:12-1:18 w / v, adjust pH to 4.5-5.5, carry out targeted enzymolysis in 55-60℃ constant temperature water bath for 60-90min, the stirring speed is 80-100rpm during the process; the compound enzyme system includes cellulase, pectinase and chitinase, the amount is calculated by mass, cellulase: pectinase: chitinase=1.8-2.2:0.8-1.2:0.8-1.2; (3) ultrasonic-microwave synergistic extraction: after enzymolysis, transfer the mixed solution to ultrasonic-microwave synergistic extraction device, set microwave power 350±50W, ultrasonic power 250±50W, temperature 60℃-70℃, extraction time 35-45min; (4) enzyme inactivation, filtration: heat the mixed solution to 95-100℃ for 8min to inactivate the enzyme, quickly cool to room temperature, then filter through 0.22μm ceramic membrane, collect the filtrate; (5) concentration and purification: combine the filtrate, concentrate to 1 / 4-1 / 6 of the original volume at 40-45℃ and vacuum degree 0.09-0.1MPa, get polysaccharide concentrate; slowly add 3-4 times volume of ethanol-acetone mixed solution, volume ratio 3:1, stir while adding, make the final concentration of mixed solvent reach 65-70%, seal and place in 4℃ refrigerator for 8-12h; (6) separation and drying: put the mixed solution after standing into high speed centrifuge, centrifuge at 5000-6000rpm for 10-15min, discard the supernatant, collect the polysaccharide precipitate at the bottom; wash the precipitate with 50% ethanol-acetone mixed solution and anhydrous ethanol in turn, dry to get the product.

2. The process for extracting polysaccharide from X. oblonga according to claim 1, characterized in that: The fruiting body is crushed to pass through a 100 mesh sieve in step (1).

3. The process for extracting polysaccharide from X. oblonga according to claim 1, characterized in that: The mass ratio of cellulase, pectinase and chitinase in the compound enzyme system in step (2) is 2:1:

1.

4. The process for extracting polysaccharide from X. oblonga according to claim 1, characterized in that: In step (3), the temperature is controlled in a stepwise manner, 60℃ for 10min, 70℃ for 15min and 65℃ for 10min, and the total extraction time is 35-45min.

5. The process for extracting polysaccharide from X. oblonga according to claim 1, characterized in that: In step (6), the precipitate is dried in a vacuum freeze dryer; the vacuum freeze drying conditions are: drying temperature-40±2℃, vacuum degree 10-20Pa and drying time 12-16h.

6. A method for preparing a health functional drink comprising the polysaccharide of the longitudinal-streaked X. camptustrata prepared in claim 1, characterized by: The method comprises the following steps: Step one, weigh 5-8 parts of Nectria Robusta polysaccharide extract, 12-15 parts of blueberry extract, 3-5 parts of green tea extract, 1-1.5 parts of vitamin C, 60-75 parts of honey, 0.8-1.2 parts of citric acid and 450-500 parts of water; Step two, heat the purified water to 47-52℃, add honey, stir until completely dissolved; then add the N. verticalis polysaccharide extract, blueberry extract and green tea extract in sequence, each time after adding, stir for 8-10 min until completely dissolved; finally add vitamin C and citric acid, continue stirring for 5-8 min, then filter through a 100 mesh filter screen to remove undissolved impurities, to obtain an initial mixture; Step three, pump the initial mixture into a high pressure homogenizer, set the homogenization pressure to 20-30 MPa, the homogenization temperature to 50-60℃, homogenize 1-2 times, with a particle size of <5 μm; Step four, sterilize and fill the homogenized mixture, with a sterilization temperature of 120-135℃ and a holding time of 3-5 s; before filling, sterilize the outer packaging with ultraviolet light at a wavelength of 254 nm for 30-60 s; after filling, sterilize the product by inversion at 38-40℃ for 24 h, to obtain the product.

7. The method of claim 6, wherein the health functional beverage is prepared by adding the composition of claim 1 to a beverage. In step two, heat the purified water to 50℃, then add honey.

8. The method of claim 6, wherein the health functional drink is prepared by adding the extract of the fruit of the plant of the genus Rubus to a beverage. In step two, monitor the pH of the system during stirring with a pH meter, and control the pH to be in the range of 3.5-4.

5.

9. The method of preparing a health functional beverage according to any one of claims 6-8, characterized in that: In step three, the homogenization pressure is 25 MPa, the homogenization temperature is 55℃, and the homogenization is performed 2 times, with a particle size of <5 μm.

10. A health functional drink formulation, characterized by: The health functional drink formula includes the following components by weight: N. verticalis polysaccharide extract 5-8 parts, blueberry extract 12-15 parts, green tea extract 3-5 parts, vitamin C 1-1.5 parts, honey 60-75 parts, citric acid 0.8-1.2 parts, and water 450-500 parts; in the optimization scheme, the health functional drink formula includes the following components by weight: N. verticalis polysaccharide extract 6-7 parts, blueberry extract 13-14 parts, green tea extract 4-5 parts, vitamin C 1.2-1.4 parts, honey 65-70 parts, citric acid 0.9-1.1 parts, and water 470-490 parts; in the further optimization scheme, the health functional drink formula includes the following components by weight: N. verticalis polysaccharide extract 6.5 parts, blueberry extract 14 parts, green tea extract 4.5 parts, vitamin C 1.3 parts, honey 68 parts, citric acid 1 part, and water 480 parts. The health functional drink formula includes the following components by weight: N. verticalis polysaccharide extract 5-8 parts, blueberry extract 12-15 parts, green tea extract 3-5 parts, vitamin C 1-1.5 parts, honey 60-75 parts, citric acid 0.8-1.2 parts, and water 450-500 parts; in the optimization scheme, the health functional drink formula includes the following components by weight: N. verticalis polysaccharide extract 6-7 parts, blueberry extract 13-14 parts, green tea extract 4-5 parts, vitamin C 1.2-1.4 parts, honey 65-70 parts, citric acid 0.9-1.1 parts, and water 470-490 parts; in the further optimization scheme, the health functional drink formula includes the following components by weight: N. verticalis polysaccharide extract 6.5 parts, blueberry extract 14 parts, green tea extract 4.5 parts, vitamin C 1.3 parts, honey 68 parts, citric acid 1 part, and water 480 parts.

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