Post-generation fermented beverage using hericium erinaceus by-products and preparation method of post-generation fermented beverage

Abstract

The invention relates to the technical field of food processing and waste recycling, and particularly discloses a metabiotics fermented beverage using hericium erinaceus byproducts and a preparation method thereof. According to the method, waste mushroom residues generated by hericium erinaceus cultivation and leftovers generated by sporocarp processing are used as raw materials, and the functional beverage rich in metabiotics is obtained through a differentiated biological enzymolysis technology and a targeted gradient fermentation technology. According to the invention, high-valued and full utilization of by-products in the hericium erinaceus industry is realized, and the obtained beverage is rich in nutrition and unique in flavor, has the health benefits of postbiotics, and provides an innovative approach for recycling related wastes.

Description

Technical Field

[0001] This invention belongs to the fields of food engineering technology, microbial fermentation and comprehensive utilization of agricultural product processing by-products. Specifically, it relates to a method for preparing a post-biotic beverage by using waste mycelium from Hericium erinaceus cultivation and fruiting body processing by-products as raw materials, through enzymatic hydrolysis and lactic acid bacteria fermentation. Background Technology

[0002] Hericium erinaceus (monkey head mushroom) is a valuable edible and medicinal fungus, rich in bioactive components such as polysaccharides, polypeptides, and terpenes, and has attracted much attention in the food, health, and pharmaceutical fields. With the rapid development of its large-scale cultivation and intensive processing, the industry inevitably generates a large amount of by-products. These mainly include two categories: firstly, the cultivation substrate after fruiting body harvesting (i.e., spent mushroom substrate), which accounts for more than 60% of the total substrate; secondly, processing waste such as substandard mushrooms, stems, and debris generated during the processing of fruiting bodies into dried products, canned goods, slices, etc., which accounts for about 15%-30% of the raw materials.

[0003] Although these byproducts are often considered waste or treated only for low-value purposes (such as discarding or composting), they actually contain considerable nutritional and functional value. Besides incompletely utilized lignocellulose, waste mushroom substrate is rich in mycelial proteins, mycelial polysaccharides, and various metabolic intermediates derived from the degradation and transformation of Hericium erinaceus mycelium. While processing waste may not meet commercial requirements in terms of appearance, its chemical composition is highly similar to that of genuine Hericium erinaceus, also rich in characteristic proteins, polysaccharides (such as β-glucan), various amino acids, and minerals. Therefore, the systematic and high-value comprehensive utilization of these two types of byproducts can not only alleviate the environmental pressure caused by industrial solid waste treatment but also turn waste into treasure, fully tapping their potential economic value. This is a key link in promoting the green, circular, and sustainable development of the Hericium erinaceus industry.

[0004] Chinese invention patent CN108432996A discloses a Hericium erinaceus lactic acid bacteria fermented beverage and its preparation method. The Hericium erinaceus lactic acid bacteria fermented beverage prepared by this invention is orange-yellow in color, uniform in color, cloudy, without sedimentation or layering, has a sweet and sour taste, rich flavor, and has the unique aroma of Hericium erinaceus and lactic acid bacteria fermentation, without any unpleasant odors. This method mainly uses Hericium erinaceus fruiting bodies (genuine mushrooms) as raw materials.

[0005] Currently, research on the utilization of Hericium erinaceus by-products mainly focuses on the extraction of single components (such as extracting polysaccharides from waste materials) or simple composting, resulting in low added value and insufficient utilization. There are no reports of synergistically utilizing these two by-products from different sources with complementary components to develop a directly edible functional beverage.

[0006] Meanwhile, the functional food market is developing rapidly. Epigenetics, as preparations of non-living microorganisms and their components (including inactivated bacterial cells, cell fragments, and metabolites) that are beneficial to host health, have become a current research hotspot due to their advantages such as high stability, good safety, well-defined targets, and no need for cold chain preservation. Fermenting nutrient-rich plant-based raw materials with lactic acid bacteria, followed by inactivation treatment, is one of the effective methods for preparing functional beverages rich in epigenetics.

[0007] Therefore, developing a method to efficiently utilize waste mycelium residue and processing by-products from Hericium erinaceus and transform them into functional beverages rich in post-biotics is of great significance for increasing the added value of the Hericium erinaceus industry, realizing the full utilization of waste, and meeting the market demand for new healthy beverages. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for high-value utilization of Hericium erinaceus by-products. Specifically, it involves the preparation of post-biotic fermented beverages using Hericium erinaceus by-products. This method aims to realize the resource utilization and productization of waste, and to prepare a new type of beverage that is rich in nutrients, has a mellow flavor, and has health benefits.

[0009] The technical solution of the present invention is as follows: One objective of this invention is to provide a method for preparing a post-biotic fermented beverage using Hericium erinaceus byproducts, comprising the following steps: S1. Dry and pulverize the waste mushroom substrate from Hericium erinaceus cultivation to obtain waste mushroom substrate powder; process the by-products from Hericium erinaceus fruiting body processing into pulp according to their morphology; S2. Mix waste bacterial bran powder, microalgae powder and water, add compound plant hydrolytic enzyme for the first enzymatic hydrolysis, inactivate the enzyme and then add protease for the second enzymatic hydrolysis, filter after the enzymatic hydrolysis is completed to obtain waste bacterial bran compound enzymatic hydrolysate containing microalgae. S3. After sterilizing the waste bacterial bran compound enzymatic hydrolysate containing *Microcystis aeruginosa*, inoculate with *Lactobacillus plantarum* at an inoculation rate of 1×10⁻⁶. 6 ~1×10 7 CFU / mL; the initial fermentation temperature was 35℃, and the fermentation time was 24 h. The fermentation was carried out according to the following temperature gradient: 0-6 h 35℃, 6-9 h 37℃, 9-12 h 39℃, 12-15 h 41℃, 15-18 h 43℃, 18-24 h 45℃; the dissolved oxygen content was controlled at 0-12 h 30-35%, 12-18 h 25-30%, and 18-24 h 20-25%; nutrient solution A was obtained after fermentation. S4. Add protease to the slurry obtained from the processing waste of Hericium erinaceus fruiting bodies for the third enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated and filtered to obtain the enzymatic hydrolysate of the fruiting body waste. S5. Add 4~6 g / L of algal oil derived from Schizochytrium or Cryptodinium kowti to the enzymatic hydrolysate of fruiting body by-products. The algal oil contains ≥38% docosahexaenoic acid. Emulsify and mix to obtain an emulsified mixture. S6. Inoculate the obtained emulsified mixture with *Lactobacillus paracasei* at a concentration of 1 × 10⁻⁶. 6 ~1×10 7 CFU / mL; the initial fermentation temperature was 35℃, and the fermentation time was 24 h. The fermentation was carried out according to the following temperature gradient: 0-6 h 37℃, 6-12 h 39℃, 12-15 h 41℃, 15-18 h 43℃, 18-24 h 45℃; the dissolved oxygen content was controlled at 0-12 h 30-35%, 12-18 h 25-30%, and 18-24 h 20-25%; nutrient solution B was obtained after fermentation. S7. Mix nutrient solution A with nutrient solution B, add inorganic salts, and inoculate with Streptococcus thermophilus for fermentation. The inoculum size is 1×10⁻⁶. 6 ~1×10 7 CFU / mL, anaerobic fermentation at 42-45℃ for 12-24 h; S8. After fermentation, the fermentation liquid is sterilized, homogenized, and bottled to obtain the post-fermented beverage.

[0010] Furthermore, in step S1, the particle size of the waste mushroom bran powder is ≤150 μm, and the moisture content of the waste mushroom bran powder is ≤10%.

[0011] Furthermore, the compound plant hydrolytic enzyme added in step S2 includes cellulase (enzyme activity ≥5000 U / g) and hemicellulase (enzyme activity ≥5000 U / g), with a weight ratio of cellulase to hemicellulase of 2:1. The amount of compound plant hydrolytic enzyme added is 0.5%-2.0% of the dry weight of the waste bacterial bran powder. The pH of the first enzymatic hydrolysis reaction is 4.5-5.5, the temperature is 45-55℃, and the enzymatic hydrolysis time is 2-4 hours.

[0012] Furthermore, the protease added in step S2 is a neutral protease (enzyme activity ≥10000 U / g) or a flavor protease (enzyme activity ≥10000 U / g), and the amount of protease added is 1.0%-3.0% of the dry weight of the waste bacterial bran powder. The pH of the second enzymatic hydrolysis reaction is 6.5-7.5, the temperature is 50-55℃, and the enzymatic hydrolysis is carried out for 1-3 hours.

[0013] Furthermore, in step S2, the dry matter content of the waste bacterial bran powder is 120-135 g / L, and the dry matter content of the microalgae powder is 12-14 g / L.

[0014] Furthermore, the protease added in step S4 is a neutral protease or a complex protease, and the amount of protease added is 1.0%-3.0% of the dry weight of the waste material. The pH of the third enzymatic hydrolysis reaction is 6.5-7.5, the temperature is 50-55℃, and the enzymatic hydrolysis time is 2-4 hours.

[0015] Furthermore, in step S7, the volume ratio of nutrient solution A to nutrient solution B is 1:(0.5-2).

[0016] Furthermore, the inorganic salt added in step S7 is one or a mixture of potassium dihydrogen phosphate, magnesium sulfate, and ammonium citrate, and the amount of inorganic salt added is 0.05%-0.2% of the mass of the mixture.

[0017] Furthermore, the metabiotic components include one or more of the following: inactivated bacterial cells, bacterial cell fragments, and metabolites.

[0018] Compared with the prior art, the present invention has the following advantages: 1. This invention is the first to use two main types of solid waste in the Hericium erinaceus (monkey head mushroom) industry—cultivation waste substrate and processing by-products—as core raw materials. Addressing the fundamental differences in composition and structure between the waste substrate, rich in cellulose, hemicellulose, and mycelial protein, and the by-products, rich in fruiting body protein and polysaccharides, a differentiated stepwise enzymatic hydrolysis process is employed. For the waste substrate, a two-step method of "first cellulase / hemicellulase cell disruption, then protease hydrolysis" is used to efficiently release soluble fiber, oligosaccharides, and amino acids. For the by-products, targeted protease hydrolysis is employed. This stepwise enzymatic hydrolysis method is highly targeted, enabling more complete and efficient release of active ingredients and nutrients from different raw materials, providing an optimized and nutritionally balanced substrate for subsequent fermentation.

[0019] 2. This invention utilizes *Lactobacillus plantarum* and *Lactobacillus paracasei* for gradient-temperature fermentation of enzymatically hydrolyzed bacterial culture waste and processing by-products, respectively. After mixing, *Streptococcus thermophilus* is inoculated for a secondary high-temperature fermentation to increase the active ingredients. The hydrolysis of *Microcystis aeruginosa* powder releases algal oil rich in EPA and DHA algal oil derived from *Schizochytrium* or *Cryptodinium coelestii*, which protect the cell structure and antioxidant components of *Lactobacillus plantarum* and *Lactobacillus paracasei* during the gradient-temperature process, and also increases the content of polyunsaturated fatty acids in the product. The product is rich in various postbiotic components, including inactivated bacterial cells, bacterial fragments, and metabolites. Compared with existing live bacteria beverages, this postbiotic makes the product more stable, and the different enzymatic hydrolysis methods construct high-quality, fully nutritious substrates, resulting in higher raw material utilization. Its health benefits are clear, directly targeting the currently popular functional demands in the food industry such as intestinal health and immune regulation, giving it strong market recognition and competitiveness. Detailed Implementation

[0020] The present invention will be further described below with reference to preferred embodiments. The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values. These ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed herein.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0022] Example 1 This embodiment provides a post-biotic fermented beverage utilizing Hericium erinaceus byproducts, and the specific steps for its preparation are as follows: S1. The waste mushroom substrate after the cultivation of Hericium erinaceus is dried in hot air at 60℃ until the moisture content is ≤8%, and then crushed through a 150-mesh sieve (particle size ≤100 μm) to obtain waste mushroom substrate powder; the fresh by-products (mushroom stems, broken mushrooms) generated after slicing Hericium erinaceus fruiting bodies are directly added to deionized water for pulping, and the dry matter content of the pulp is adjusted to 100 g / L and the pH of the pulp is 7.0. S2. Take waste mushroom bran powder, microalgae powder and 7.5 L of deionized water and mix them. The dry matter content of the waste mushroom bran powder is 133 g / L and the dry matter content of the microalgae powder is 12 g / L. Adjust the pH to 5.0 and add a compound plant hydrolytic enzyme consisting of 2 g cellulase (6000 U / g) and 1 g hemicellulase (5500 U / g) (total addition is 1.5% of the dry weight of waste mushroom bran powder). Stir at 50℃ and 200 rpm for 3 h for enzymatic hydrolysis. After inactivating the enzyme at 95℃ for 10 min, cool to 52℃, adjust the pH to 7.0, add 2.5 g flavor protease (12000 U / g, addition is 2.5% of the dry weight of waste mushroom bran powder), and enzymatic hydrolyze for 2 h. After the enzymatic hydrolysis is completed, centrifuge at 4000 rpm for 15 minutes and take the supernatant to obtain the compound enzymatic hydrolysate. S3. Sterilize the waste bacterial bran compound enzymatic hydrolysate containing *Microcystis aeruginosa* at 121℃ for 15 minutes, cool to 35℃, and inoculate with *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum GDMCC 1.140) was used to set the initial viable count to 5 × 10⁻⁶. 6CFU / mL; the initial fermentation temperature was 35℃, and the fermentation was carried out for 24 h according to the following temperature gradient: 0-6h 35℃, 6-9h 37℃, 9-12h 41℃, 12-15h 43℃, 15-18h 43℃, 18-24h 45℃; dissolved oxygen (DO) was controlled by stirring speed: 0-12h DO 32%, 12-18h DO 28%, 18-24h DO 22%. After 24 hours of fermentation, nutrient solution A was obtained. S4. Neutral protease (11000 U / g) was added to the slurry prepared from the by-products of Hericium erinaceus fruiting body processing. The neutral protease was 2.0% of the weight of the by-products in the slurry. The slurry was enzymatically hydrolyzed at 53°C for 3 hours, inactivated at 95°C for 10 minutes, and filtered to obtain the enzymatic hydrolysate of the fruiting body by-products. S5. Add algal oil from Schizochytrium to the enzymatic hydrolysate of fruiting body by-products at a rate of 5 g / L. The algal oil contains ≥38% docosahexaenoic acid. Use a high-speed shear emulsifier (10000 rpm, 5 min) to fully emulsify it and obtain an emulsified mixture. S6. Sterilize the emulsified mixture at 121°C for 15 minutes, cool to 37°C, and inoculate with *Lactobacillus paracasei* (…). Lacticaseibacillus paracasei (GDMCC 1.159), to achieve an initial viable count of 5 × 10⁻⁶. 6 The fermentation concentration was CFU / mL, with an initial fermentation temperature of 35℃ and a fermentation time of 24 h. The fermentation was carried out according to the following temperature gradient: 0-6h 37℃, 6-12h 39℃, 12-15h 43℃, 15-18h 43℃, and 18-24h 45℃. The dissolved oxygen (DO) content was controlled as follows: 0-12h DO 33%, 12-18h DO 28%, and 18-24h DO 23%. After 24 hours of fermentation, nutrient solution B was obtained. S7. Mix nutrient solution A and nutrient solution B at a volume ratio of 1:1 without sterilization; add potassium dihydrogen phosphate and ammonium citrate to the mixture, with a total addition of 0.2% of the mixture's mass; inoculate with Streptococcus thermophilus (… Streptococcus thermophilu s GDMCC 1.2800), to achieve an initial viable count of 5 × 10⁻⁶. 6 CFU / mL, and static (anaerobic) fermentation was carried out at 43℃ for 18 hours; S8. After fermentation, the fermentation broth is pasteurized at 85℃ for 30 min. Then, it is homogenized twice under 20 MPa pressure and aseptically filled to obtain the post-fermented beverage product.

[0023] Example 2 This embodiment provides a post-biotic fermented beverage utilizing Hericium erinaceus byproducts, and the specific steps for its preparation are as follows: S1. The waste mushroom substrate after the cultivation of Hericium erinaceus is dried in hot air at 60℃ until the moisture content is ≤8%, and then crushed through a 150-mesh sieve (particle size ≤100 μm) to obtain waste mushroom substrate powder; the fresh by-products (mushroom stems, broken mushrooms) generated after slicing Hericium erinaceus fruiting bodies are directly added to deionized water for pulping, and the dry matter content of the pulp is adjusted to 100 g / L and the pH of the pulp is 7.0. S2. Take waste mushroom bran powder, microalgae powder and deionized water, the dry matter content of waste mushroom bran powder is 125 g / L and the dry matter content of microalgae powder is 13 g / L. Adjust the pH to 4.5, add a compound plant hydrolytic enzyme composed of cellulase (6000 U / g) and hemicellulase (5500 U / g), the total amount added is 0.5% of the dry weight of waste mushroom bran powder, stir at 50℃ and 200 rpm for 2 h for enzymatic hydrolysis; after inactivating the enzyme at 95℃ for 10 min, cool to 55℃, adjust the pH to 7.5, add flavor protease (12000 U / g, the amount added is 1.0% of the dry weight of waste mushroom bran powder), and enzymatic hydrolyze for 3 h; after the enzymatic hydrolysis is completed, centrifuge at 4000 rpm for 15 minutes, take the supernatant to obtain the compound enzymatic hydrolysate; S3. Sterilize the waste bacterial bran compound enzymatic hydrolysate containing *Microcystis aeruginosa* at 121℃ for 15 minutes, cool to 35℃, and inoculate with *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum GDMCC 1.140) was used to set the initial viable count to 5 × 10⁻⁶. 6 CFU / mL; the initial fermentation temperature was 35℃, and the fermentation was carried out for 24 h according to the following temperature gradient: 0-6h 35℃, 6-9h 37℃, 9-12h 41℃, 12-15h 43℃, 15-18h 43℃, 18-24h 45℃; dissolved oxygen (DO) was controlled by stirring speed: 0-12h DO 32%, 12-18h DO 28%, 18-24h DO 22%. After 24 hours of fermentation, nutrient solution A was obtained. S4. Add a complex protease (11000 U / g) to the slurry obtained from the processing by-products of Hericium erinaceus fruiting bodies. The complex protease is 2.0% of the dry weight of the by-products in the slurry. Enzymatic hydrolysis is carried out at 50℃ for 4 hours, enzyme inactivation is carried out at 95℃ for 10 minutes, and the mixture is filtered to obtain the enzymatic hydrolysate of the fruiting body by-products. S5. Add algal oil derived from *Cryptodinium coccineum* to the enzymatic hydrolysate of fruiting body by-products at a rate of 4.5 g / L. The algal oil contains ≥38% docosahexaenoic acid. Use a high-speed shear emulsifier (10000 rpm, 5 min) to fully emulsify it and obtain an emulsified mixture. S6. Sterilize the emulsified mixture at 121°C for 15 minutes, cool to 37°C, and inoculate with *Lactobacillus paracasei* (…). Lacticaseibacillus paracasei (GDMCC 1.159), to achieve an initial viable count of 5 × 10⁻⁶. 6The fermentation concentration was CFU / mL, with an initial fermentation temperature of 35℃ and a fermentation time of 24 h. The fermentation was carried out according to the following temperature gradient: 0-6h 37℃, 6-12h 39℃, 12-15h 43℃, 15-18h 43℃, and 18-24h 45℃. The dissolved oxygen (DO) content was controlled as follows: 0-12h DO 33%, 12-18h DO 28%, and 18-24h DO 23%. After 24 hours of fermentation, nutrient solution B was obtained. S7. Mix nutrient solution A and nutrient solution B at a volume ratio of 1:0.5 without sterilization; add potassium dihydrogen phosphate to the mixture at a concentration of 0.05% of the mixture's mass; inoculate with Streptococcus thermophilus (… Streptococcus thermophilu sGDMCC 1.2800) was used to set the initial viable count to 5 × 10⁻⁶. 6 CFU / mL, and static (anaerobic) fermentation was carried out at 42℃ for 24 hours; S8. After fermentation, the fermentation broth is pasteurized at 85℃ for 30 min. Then, it is homogenized twice under 20 MPa pressure and aseptically filled to obtain the post-fermented beverage product.

[0024] Example 3 The difference between this embodiment and Embodiment 1 is that: This embodiment provides a post-biotic fermented beverage utilizing Hericium erinaceus byproducts, and the specific steps for its preparation are as follows: S1. The waste mushroom substrate after the cultivation of Hericium erinaceus is dried in hot air at 60℃ until the moisture content is ≤8%, and then crushed through a 150-mesh sieve (particle size ≤100 μm) to obtain waste mushroom substrate powder; the fresh by-products (mushroom stems, broken mushrooms) generated after slicing Hericium erinaceus fruiting bodies are directly added to deionized water for pulping, and the dry matter content of the pulp is adjusted to 100 g / L and the pH of the pulp is 7.0. S2. Take waste mushroom bran powder, microalgae powder and deionized water, the dry matter content of waste mushroom bran powder is 120 g / L and the dry matter content of microalgae powder is 14 g / L. Adjust the pH to 5.5, add a compound plant hydrolytic enzyme composed of cellulase (6000 U / g) and hemicellulase (5500 U / g), the total amount added is 2.0% of the dry weight of waste mushroom bran powder, stir at 50℃ and 200 rpm for 4 h for enzymatic hydrolysis; after inactivating the enzyme at 95℃ for 10 min, cool to 50℃, adjust the pH to 6.5, add flavor protease (12000 U / g, the amount added is 3.0% of the dry weight of waste mushroom bran powder), and enzymatic hydrolyze for 3 h; after the enzymatic hydrolysis is completed, centrifuge at 4000 rpm for 15 minutes, take the supernatant to obtain the compound enzymatic hydrolysate; S3. Sterilize the waste bacterial bran compound enzymatic hydrolysate containing *Microcystis aeruginosa* at 121℃ for 15 minutes, cool to 35℃, and inoculate with *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum GDMCC 1.140) was used to set the initial viable count to 5 × 10⁻⁶.6 CFU / mL; the initial fermentation temperature was 35℃, and the fermentation was carried out for 24 h according to the following temperature gradient: 0-6h 35℃, 6-9h 37℃, 9-12h 41℃, 12-15h 43℃, 15-18h 43℃, 18-24h 45℃; dissolved oxygen (DO) was controlled by stirring speed: 0-12h DO 32%, 12-18h DO 28%, 18-24h DO 22%. After 24 hours of fermentation, nutrient solution A was obtained. S4. Neutral protease (11000 U / g) was added to the slurry obtained from the processing by-products of Hericium erinaceus fruiting bodies. The neutral protease was 3.0% of the dry weight of the by-products in the slurry. The mixture was enzymatically hydrolyzed at 55°C for 2 hours, then inactivated at 95°C for 10 minutes. The mixture was filtered to obtain the enzymatic hydrolysate of the fruiting body by-products. S5. Add algal oil derived from *Cryptodinium coccineum* to the enzymatic hydrolysate of fruiting body by-products at a rate of 4.5 g / L. The algal oil contains ≥38% docosahexaenoic acid. Use a high-speed shear emulsifier (10000 rpm, 5 min) to fully emulsify it and obtain an emulsified mixture. S6. Sterilize the emulsified mixture at 121°C for 15 minutes, cool to 37°C, and inoculate with *Lactobacillus paracasei* (…). Lacticaseibacillus paracasei (GDMCC 1.159), to achieve an initial viable count of 5 × 10⁻⁶. 6 The fermentation concentration was CFU / mL, with an initial fermentation temperature of 35℃ and a fermentation time of 24 h. The fermentation was carried out according to the following temperature gradient: 0-6h 37℃, 6-12h 39℃, 12-15h 43℃, 15-18h 43℃, and 18-24h 45℃. The dissolved oxygen (DO) content was controlled as follows: 0-12h DO 33%, 12-18h DO 28%, and 18-24h DO 23%. After 24 hours of fermentation, nutrient solution B was obtained. S7. Mix nutrient solution A and nutrient solution B at a volume ratio of 1:2 without sterilization; add magnesium sulfate to the mixture at a rate of 0.1% of the mixture's mass; inoculate with Streptococcus thermophilus (…). Streptococcus thermophilu (s GDMCC1.2800), to achieve an initial viable count of 5 × 10⁻⁶. 6 CFU / mL, and static (anaerobic) fermentation was carried out at 45℃ for 12 hours; S8. After fermentation, the fermentation broth is pasteurized at 85℃ for 30 min. Then, it is homogenized twice under 20 MPa pressure and aseptically filled to obtain the post-fermented beverage product.

[0025] Comparative Example 1 The difference between this embodiment and Embodiment 1 is that only waste substrate is used, and a single fermentation method is employed. The specific steps are as follows; S1. Same as in Example 1.

[0026] S2. Take 100g of waste bacterial bran powder and mix it with 8L of water (dry matter content 125 g / L), without adding microalgae powder; after adjusting the pH, perform the same compound enzymatic hydrolysis as in Example 1; filter to obtain the enzymatic hydrolysate.

[0027] S3. After sterilization of the enzymatic hydrolysate, only Lactobacillus plantarum (same strain as in Example 1) is inoculated, and fermentation is carried out for 24 hours at a constant temperature of 37°C and without precise control of dissolved oxygen (only natural dissolved oxygen is provided by constant stirring at 150 rpm) to obtain the fermentation broth.

[0028] S4. The fermentation broth obtained in S3 is directly pasteurized.

[0029] S5. After sterilization, homogenize and fill to obtain control beverage 1.

[0030] Comparative Example 2 The difference between this embodiment and Embodiment 1 is that only fruiting body waste is used, and ordinary fermentation is employed. The specific steps are as follows: S1. Prepare the enzymatic hydrolysate of fruiting body by-products in the same manner as in Example 1.

[0031] S2. After sterilizing the enzymatic hydrolysate of the fruiting body by-products, inoculate it with a common commercial yogurt starter (Lactobacillus bulgaricus: Streptococcus thermophilus = 1:1) and ferment at a constant temperature of 42℃ for 6 hours to obtain the fermentation broth.

[0032] S3. The fermentation broth was pasteurized, homogenized, and bottled to obtain control beverage 2.

[0033] Activity test To verify the significant advantages of the technical solution of this invention in enhancing the bioactivity of products, the in vitro bioactivity of beverage samples prepared in the above five embodiments was determined, and the results are compared in the table below: Table 1: Comparison of in vitro bioactivity of beverages from various examples

[0034] Note: IC 50 The value represents the sample concentration required to achieve a 50% inhibition effect; the smaller the value, the stronger the activity.

[0035] Comparative analysis: Regarding antioxidant activity: the DPPH free radical scavenging ability (IC50) of the three embodiments of the present invention 50 The 2.1-2.4 mg / mL concentration was significantly superior to the two comparative examples (IC50). 50(4.5-5.8 mg / mL). This is attributed to: ① the combined enzymatic hydrolysis of waste bacterial bran and *Microcystis aeruginosa*, releasing richer phenols, flavonoids, and algal antioxidants; ② the stepped temperature fermentation process (especially the later high-temperature stage) promoted the production of more antioxidant metabolites (such as short-chain fatty acids, bacteriocins, and reducing substances) by *Lactobacillus plantarum* and *Lactobacillus paracasei*; ③ the synergistic effect of the final mixed fermentation further enhanced the overall antioxidant capacity of the system. Comparative Example 1 lacked *Microcystis aeruginosa* and synergistic fermentation, resulting in a single type of antioxidant; Comparative Example 2 lacked a targeted probiotic fermentation process, resulting in limited improvement in antioxidant activity.

[0036] Regarding the potential for lowering blood sugar: the inhibitory capacity of the present invention on α-glucosidase (IC50) 50 (3.8-4.2 mg / mL) was significantly stronger than comparative examples 1 and 2 (IC50). 50 (7.2-8.5 mg / mL). Its advantages mainly stem from: ① Complex enzymatic hydrolysis breaks down dietary fiber and other polysaccharides in waste into oligosaccharides and oligopeptides with potential inhibitory activity; ② Specific probiotic fermentation (especially the fermentation of fruiting body by-products by *Lactobacillus paracasei*) can produce specific peptides and organic acids, and these postbiotic components have been proven to have good α-glucosidase inhibitory effects. In contrast, single fermentation or ordinary fermentation used in comparison cannot produce the same types and quantities of active inhibitors.

[0037] Regarding its potential to lower blood lipids: This invention exhibits a significant advantage in inhibiting pancreatic lipase activity (IC50). 50 4.5-4.9 mg / mL), far exceeding the comparative ratio (IC50). 50 (9.1-11.3 mg / mL). Its core technology lies in: ① the addition of DHA-rich algal oil: DHA itself has the function of regulating lipid metabolism; ② the fermentation of the emulsified algal oil system by *Lactobacillus paracasei* under a specific temperature gradient may produce metabolites that can bind to or alter the conformation of lipases, thereby strongly inhibiting their hydrolysis of fats. Comparative Example 1 contains no lipids or related fermentation products; Comparative Example 2 did not add algal oil and used common bacterial strains for fermentation, therefore it has the lowest potential for lowering blood lipids.

[0038] Conclusion: A comparison of the five embodiments clearly demonstrates that the "Preparation Method of an Epigenetic Fermented Beverage Utilizing Hericium erinaceus By-products" provided by this invention, through a series of synergistic processes including full utilization of waste components (mushroom bran + *Microcystis aeruginosa*, fruiting body waste + algal oil), multi-stage targeted enzymatic hydrolysis, and multi-strain stepwise temperature- and oxygen-controlled fermentation, can maximize the release and transformation of active precursor substances in the raw materials and enrich a large number of highly active epigenetic components. The final product exhibits significant and comprehensive advantages in the three core bioactivity indicators of antioxidation, blood sugar reduction, and blood lipid reduction, far exceeding the control product prepared using conventional single raw materials or ordinary fermentation processes. This fully demonstrates the outstanding innovation and application value of this invention in the field of functional food development and high-value utilization of edible fungi waste.

[0039] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing a post-biotic fermented beverage using Hericium erinaceus byproducts, characterized in that, Includes the following steps: S1. Dry and crush the waste mushroom substrate from Hericium erinaceus cultivation to obtain waste mushroom substrate powder. Separate the by-products from Hericium erinaceus fruiting body processing into pulp according to their morphology. S2. Mix waste bacterial bran powder, microalgae powder and water, add compound plant hydrolytic enzyme for the first enzymatic hydrolysis, inactivate the enzyme and then add protease for the second enzymatic hydrolysis, filter after the enzymatic hydrolysis is completed to obtain waste bacterial bran compound enzymatic hydrolysate containing microalgae. S3. After sterilizing the waste bacterial bran compound enzymatic hydrolysate containing *Microcystis aeruginosa*, inoculate it with *Lactobacillus plantarum* and ferment it according to the following temperature gradient: 0-6 h 35℃, 6-9 h 37℃, 9-12 h 39℃, 12-15 h 41℃, 15-18 h 43℃, 18-24 h 45℃; the dissolved oxygen content is controlled at 0-12 h 30-35%, 12-18 h 25-30%, and 18-24 h 20-25%; nutrient solution A is obtained after fermentation. S4. Add protease to the slurry obtained from the processing waste of Hericium erinaceus fruiting bodies for the third enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated and filtered to obtain the enzymatic hydrolysate of the fruiting body waste. S5. Add algal oil derived from Schizochytrium or Cryptodinium kowti to the enzymatic hydrolysate of fruiting body by-products, emulsify and mix to obtain an emulsified mixture. S6. After inoculating the obtained emulsified mixture with *Lactobacillus paracasei*, fermentation was carried out according to the following temperature gradient: 0-6 h 37℃, 6-12 h 39℃, 12-15 h 41℃, 15-18 h 43℃, 18-24 h 45℃; dissolved oxygen was controlled at 0-12 h 30-35%, 12-18 h 25-30%, and 18-24 h 20-25%; nutrient solution B was obtained after fermentation. S7. Mix nutrient solution A and nutrient solution B, add inorganic salts, and inoculate with Streptococcus thermophilus for fermentation; S8. After fermentation, the fermentation liquid is sterilized, homogenized, and bottled to obtain the post-fermented beverage.

2. The method for preparing a post-biotic fermented beverage using Hericium erinaceus by-products according to claim 1, characterized in that, In step S1, the particle size of the waste mushroom bran powder is ≤150 μm and the moisture content of the waste mushroom bran powder is ≤10%.

3. The method for preparing a post-biotic fermented beverage using Hericium erinaceus by-products according to claim 1, characterized in that, The compound plant hydrolytic enzyme added in step S2 includes cellulase and hemicellulase, wherein the ratio of cellulase to hemicellulase is 2:1 by weight, and the amount of compound plant hydrolytic enzyme added is 0.5%-2.0% of the dry weight of waste bacterial bran powder; the pH of the first enzymatic hydrolysis reaction is 4.5-5.5, the temperature is 45-55℃, and the enzymatic hydrolysis time is 2-4 hours.

4. The method for preparing a post-biotic fermented beverage using Hericium erinaceus by-products according to claim 1, characterized in that, The protease added in step S2 is a neutral protease or a flavor protease. The amount of protease added is 1.0%-3.0% of the dry weight of the waste bacterial bran powder. The pH of the second enzymatic hydrolysis reaction is 6.5-7.5, the temperature is 50-55℃, and the enzymatic hydrolysis time is 1-3 hours.

5. The method for preparing a post-biotic fermented beverage using Hericium erinaceus by-products according to claim 1, characterized in that, In step S2, the dry matter content of the waste bacterial bran powder is 120-135 g / L, and the dry matter content of the microalgae powder is 12-14 g / L.

6. The method for preparing a post-biotic fermented beverage using Hericium erinaceus by-products according to claim 1, characterized in that, The protease added in step S4 is a neutral protease or a complex protease. The amount of protease added is 1.0%-3.0% of the dry weight of the waste material. The pH of the third enzymatic hydrolysis reaction is 6.5-7.5, the temperature is 50-55℃, and the enzymatic hydrolysis time is 2-4 hours.

7. The method for preparing a post-biotic fermented beverage using Hericium erinaceus by-products according to claim 1, characterized in that, In step S7, the volume ratio of nutrient solution A to nutrient solution B is 1:(0.5-2).

8. The method for preparing a post-biotic fermented beverage using Hericium erinaceus by-products according to claim 1, characterized in that, In step S7, the inorganic salt is one or more of potassium dihydrogen phosphate, magnesium sulfate, and ammonium citrate, and the total amount added is 0.05%-0.2% of the mass of the mixture.

9. A postbiotic fermented beverage made from Hericium erinaceus by-products prepared by any one of claims 1 to 8, characterized in that, The metabiotic components include one or more of the following: inactivated bacterial cells, bacterial cell fragments, and metabolites.

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