Method for fermenting apple vinegar by compounding phellinus igniarius metaplasts with medicinal and edible raw materials

Abstract

The invention discloses a method for fermenting apple vinegar by compounding phellinus igniarius postbiotics with medicinal and edible raw materials, and relates to an apple vinegar fermentation production process by using acetobacter pasteurianus CL247, CGMCC No.33924, phellinus igniarius postbiotics and medicinal and edible raw materials (astragalus membranaceus, dogwood and cinnamon) which have definite hereditary characteristics and proprietary intellectual property rights as fermentation medium components. The oriented regulation and control of flavor substances and functional components of the apple vinegar are realized.

Description

Technical Field

[0001] This invention relates to a method for fermenting apple cider vinegar using a compound medicinal and edible raw material, Phellinus linteus. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] As research into fruit vinegar deepens, more and more researchers are focusing on the various functional properties of this new type of vinegar product, including health benefits, therapeutic effects, and nutritional value. Research into its nutritional components and its development are showing a year-on-year upward trend. Fruit vinegar, as a sour condiment made primarily from fruit, is rich in nutrients and offers certain health benefits.

[0004] In recent years, with the increasing health awareness of the public, the demand for various healthy and green beverages, including fruit vinegar products, has been continuously increasing. The development trend of fruit vinegar shows changes in several directions. First, research is being conducted on its nutritional value. Fruit vinegar contains various amino acids and vitamins needed by the human body, providing rich nutrition. Therefore, some manufacturers have launched fruit vinegar products containing probiotics, hoping to help improve the balance of intestinal flora, aid digestion, or assist in the treatment of common intestinal problems such as indigestion. Second, there is innovation in flavor. To meet consumers' taste preferences, some producers have launched fruit vinegars with different flavors, such as lemon, grape, and apple. In addition, some brands are mixing fruit vinegar with other beverages to launch unique drinks. With consumers pursuing a healthy lifestyle, more and more people are choosing to buy sugar-free and low-calorie products. Therefore, some manufacturers have launched sugar-free and low-calorie fruit vinegar products. In general, the development trend of fruit vinegar products is towards rich nutrition and innovative flavors, with consumers' pursuit of a healthy lifestyle further driving the development of fruit vinegar products.

[0005] In August 2024, the Shanxi Provincial Society of Food Nutrition and Health drafted a group standard for "Herbal Vinegar," defining it as: a brewed vinegar made primarily from starchy and sugary grains or fruits and vegetables, and supplemented with one or more plants and edible fungi suitable for blending with vinegar according to national regulations on food and medicine homology or new food ingredients, or as saccharification and fermentation agents, through solid-state or liquid fermentation. This group standard provides a standard basis for the definition and research of food and medicine homology fruit vinegar.

[0006] Phellinus linteus is a medicinal and edible fungus widely distributed in Asian countries such as China, South Korea, and Japan. It contains various bioactive components, such as polysaccharides, terpenes, and flavonoids, which have been proven to possess strong biological activity. Phellinus linteus has multiple functions and clinical therapeutic effects, including anti-cancer, anti-inflammatory, antioxidant, anti-diabetic, immunomodulatory, blood-activating and hemostatic, and phlegm-resolving and diarrhea-relieving properties. It can prevent and treat various diseases such as influenza, cancer, and diabetes.

[0007] With the growing popularity of healthy eating concepts, products derived from both medicinal and edible sources are gaining market favor. Fruit vinegar, as a healthy beverage, combines the sweetness of fruit with the tangy flavor of vinegar, making it a popular choice among consumers. This invention aims to develop a new fruit vinegar product with clearly defined flavor and metabolic components by incorporating the traditional Chinese medicinal herb Sanghuang (Phellinus linteus) into the fermentation process of fruit vinegar.

[0008] Analysis of the existing implementation schemes most similar to the present invention: (1) According to the literature review, the article "Gao Xingen, Shi Jinglue. Development and research of Sanghuang health-care fruit vinegar [J]. China Condiments, 2008, 33(12):5961+83" reported the process of fermenting Sanghuang polysaccharide and apple. It is to use cellulase to treat Sanghuang dried mycelium to obtain Sanghuang extract, and then use commercial yeast and acetic acid bacteria for fermentation.

[0009] (2) Existing solutions mostly use single plant extracts (such as astragalus or cinnamon) and lack optimization of multi-component compound ratios, resulting in low DPPH scavenging rate. For example, the patent "A Kind of Sanghuang Lingzhi Vinegar and Its Processing Technology" (application number 202210573417.1) uses the fruiting bodies of Sanghuang, Lingzhi and straw mushroom, which are ground and mixed with pure water to form a slurry. Saccharifying enzymes and dry yeast are added for alcoholic fermentation. The brewing raw material is millet, and solid-state acetic acid fermentation is used.

[0010] (3) The invention patent "A preparation process of Sanghuang vinegar beverage" (application number 201810080031.0) uses Sanghuang fungus sticks as raw materials, which are crushed to obtain Sanghuang fungus powder, and then edible alcohol is added for acetic acid fermentation. High-temperature amylase with an activity of 40 million U / g and saccharifying enzyme with an activity of 100,000 U / g are added to obtain Sanghuang saccharified extract.

[0011] (4) The invention patent "A Ganoderma lucidum and okra thick vinegar and its preparation method" (application number 201510560494.3) is made by fermenting Ganoderma lucidum mycelium fermentation liquid and okra as raw materials, and adding strong-aroma baijiu raw liquid for fermentation. The Ganoderma lucidum mycelium fermentation liquid is concentrated to 1 / 2 of the original volume by falling film evaporator, or not concentrated.

[0012] Analysis of the core defects of the existing technical solution: (1) No probiotic post-biotic added: The existing apple cider vinegar mainly uses brewer's yeast as the only strain in the alcohol fermentation stage, without adding functional strains, resulting in low content of ester flavor substances, strong sour taste, and relatively insufficient functional components. (2) Defects in the stress resistance and metabolic kinetics of acetic acid bacteria: In industrial production, the activity of conventional Acetobacter pasteurellii decreases sharply when the ethanol concentration is >12% and the acetic acid concentration is >3%, resulting in a decrease in the acid production rate. The acid production rate of conventional acetic acid bacteria is less than 0.4 g / L / h. The acetic acid fermentation cycle is generally as long as 96-120 hours, and the concentration of acetic acid in the final product fluctuates significantly, resulting in poor stability in industrial production.

[0013] (3) Different functional enhancement technologies: In the existing technical solutions, the addition of Sanghuang can be done by using the fruiting body enzymatically crushed as a culture medium or by using pulverized Sanghuang powder as a raw material for fermentation. There is no method of using Sanghuang fermentation to prepare post-biotics as functional enhancement components.

[0014] (4) Lack of synergistic mechanism in the combination of food and medicine ingredients: The function of a single food and medicine ingredient is relatively simple. From the perspective of traditional Chinese medicine, the combination of drugs can maximize the nutritional effect. Summary of the Invention

[0015] To address the shortcomings of existing technologies, this invention aims to achieve a dual enhancement of the flavor and function of apple cider vinegar through the following technological innovations: (1) Overcoming the limitations of acetic acid fermentation tolerance: Utilizing the highly ethanol- and acetic acid-tolerant Acetobacter pasteurellii CL247 (CGMCC No. 22924), the fermentation cycle is shortened from 96-120h to 72h compared to existing technologies, a reduction of 25%-40%. (2) Innovating functional component enhancement pathways: Developing a liquid fermentation technology for medicinal and edible homologous compound, utilizing the post-biotic prepared by ultrasonic crushing of the medicinal and edible Phellinus linteus fermentation, and compounding medicinal and edible raw materials for alcoholic and acetic acid fermentation, which is conducive to promoting the release of functional components of medicinal and edible raw materials, hydrolyzing plant cell walls to release bound polyphenols, and increasing the content of total phenols, total flavonoids and DPPH free radical scavenging rate. (3) Systematize the fermentation process parameters: Establish a two-stage fermentation model: control the initial sugar content of 16~18°Bx (preferably 17°Bx) and pH 4.5 in the alcohol fermentation stage, and use 0.12vvm aeration rate and 180r / min stirring rate and pH value control between 4.0 and 5.0 in the acetic acid fermentation stage, so that the flavor substance content fluctuates by <5% between batches.

[0016] The technical solution adopted in this invention is as follows: In a first aspect of the present invention, a method for fermenting apple cider vinegar using Sanghuang post-biotic compound medicinal and edible raw materials is provided, the method comprising the following steps: S1. Raw material pretreatment After juicing the apples, enzymatically hydrolyze them, filter them to obtain apple juice, add sucrose and water to the apple juice to adjust the sugar content to 11~18°Brix, sterilize, and set aside. S2, Preparation of Phellinus linteus post-biotic Inoculate 1-5 parts of Phellinus linteus into 100 parts of culture medium and ferment at 28-30℃ and 150-200 r / min for 5-7 days to obtain Phellinus linteus seed liquid. After the fermentation liquid is finished, perform ultrasonic and high temperature sterilization treatment on the obtained fermentation liquid. Ultrasonic treatment conditions: ultrasonic power 600-1000 W, ultrasonic time 10-30 min, to obtain Phellinus linteus post-genetic agent. Add 1-4 parts of Phellinus linteus extract to 100 parts of S1 apple juice; S3, Processing of medicinal and edible raw materials Wash, dry, and pulverize Astragalus membranaceus, Cornus officinalis, and Cinnamomum cassia. Add 2–6 parts of Astragalus membranaceus, 1–5 parts of Cornus officinalis, and 1–3 parts of Cinnamomum cassia to 100 parts of apple juice containing Phellinus linteus and post-biotics to obtain an alcoholic fermentation medium. S4, alcoholic fermentation Add 5% – 10% (v / v) of Saccharomyces cerevisiae to the S3 alcoholic fermentation medium. Saccharomyces cerevisiae The alcoholic fermentation parameters are as follows: 18 – 32℃, initial pH 4.2 – 4.8, anaerobic fermentation for 5 – 7 days, alcohol content reaching 6% – 14.5% vol, to obtain cider stock. S5, Acetic acid fermentation Activation of acetic acid bacteria strains: Acetobacter pasteurellii CL247 cells were inoculated into seed culture medium and cultured at 28–32℃ and 180–220 r / min in a shaker for 15–24 h, pre-culturing until OD. 600 =1.8–2.2, to obtain acetic acid bacteria seed culture; Adjust the alcohol content of the apple cider stock solution to 4%-8% vol by adding water. Perform acetic acid fermentation at an inoculum size of 5-10% (v / v) of Acetobacter pasteurella CL247, at 28-32℃, a stirring speed of 180-220 rpm, and an aeration rate of 0.10-0.30 vvm. Stop fermentation when the total acid content of the apple cider vinegar reaches 40-50 g / L after 72-96 hours to obtain the apple cider vinegar stock solution. S6, Aged Apple cider vinegar concentrate should be aged for 1-3 months. S7, Filtration, Blending and Filling After centrifugation and membrane filtration, the S6 aged apple cider vinegar concentrate is mixed with apple juice and purified water to a total acidity (calculated as acetic acid) of 2.5-4.5 g / L, then sterilized and aseptically bottled.

[0017] In one or more embodiments of the present invention, in S1, the enzymatic hydrolysis conditions are: 0.2% pectinase w / v (0.2g pectinase added to 100mL of material solution), 45℃, 2h.

[0018] In one or more embodiments of the present invention, in S1, the present invention has optimized the process system, and the initial sugar content is precisely controlled at 16~18°Brix to avoid excessive residual sugar inhibiting the activity of acetic acid bacteria.

[0019] In one or more embodiments of the present invention, in S1, the sterilization conditions are: sterilization at 105-115°C for 10-20 minutes.

[0020] In one or more embodiments of the present invention, in S2, 100 parts of culture medium, by weight, consist of: 1.5 to 3 parts glucose, 0.5 to 2.0 parts peptone, 0.1 to 0.2 parts KH2PO4, 0.05 to 0.15 parts MgSO4·7H2O, with the remainder being water.

[0021] In S2, post-biotic components were obtained from Phellinus linteus through liquid fermentation and ultrasonic inactivation, which increased the total phenol content of the fruit vinegar product by 150%, significantly increased the flavonoid content, and reduced the DPPH scavenging rate to 1.2 mg / mL.

[0022] In one or more embodiments of the present invention, in S2, the ultrasonic power is 800 W and the ultrasonic time is 20 min.

[0023] In one or more embodiments of the present invention, in S2, the high-temperature sterilization conditions are sterilization at 110-121°C for 20-30 minutes.

[0024] In S3, traditional fruit vinegar lacks polyphenols and flavonoids, which are bioactive substances. This invention utilizes the synergistic effect of a compound of medicinal and edible herbs (Astragalus membranaceus, Phellinus linteus, Cornus officinalis, and Cinnamomum cassia) and combines the biogenic components (containing β-glucan, triterpenoids, and other active ingredients) after liquid fermentation of Phellinus linteus to increase the total phenol content of apple cider vinegar by 150%, the total flavonoid content by 127%, and the DPPH free radical scavenging rate by 157%, thereby enhancing its functions.

[0025] In one or more embodiments of the present invention, in S4, the inoculation amount of the brewing yeast is 10% (v / v).

[0026] In one or more embodiments of the present invention, in S5, the Acetobacter pasteurianus CL247 was deposited on March 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33924 and classified as Acetobacter pasteurianus.

[0027] Industrially used acetic acid bacteria exhibit activity inhibition under conditions of 12% ethanol and 5% acetic acid, with conventional acetic acid bacteria producing acid at a rate of less than 0.4 g / L / h. The *Acetobacter pasteurianus* strain CL247 provided by this invention exhibits tolerance to 18% ethanol and 7% acetic acid, achieving an acid production of 49.2 g / L in 72 hours and increasing the acid production rate to 0.68 g / L / h, significantly shortening the fermentation cycle to 72 hours.

[0028] In one or more embodiments of the present invention, in S5, the seed culture consists of 100-200 mL of water containing 3g-5g of ethanol fermentation medium, wherein the ethanol fermentation medium is obtained commercially.

[0029] In one or more embodiments of the present invention, in S5, the fermentation temperature is 30°C, and with stirring at 180 rpm and aeration at 0.12 vvm, the acid production reaches 49.2 g / L after 72 hours. In a second aspect of the invention, apple cider vinegar prepared by the above method is provided.

[0030] In one or more embodiments of the present invention, the apple cider vinegar contains 0.15–2.8 mg RE / mL of total flavonoids (as rutin equivalents, RE), 0.30–1.2 mg GAE / mL of total phenols (as gallic acid equivalents, GAE), and has a DPPH free radical scavenging rate of 70%–80%. In a third aspect of the present invention, the apple cider vinegar is provided for use in the fields of antioxidant and / or anti-inflammatory functional foods or healthy fruit vinegar beverages. Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: This invention improves the efficiency of acetic acid fermentation by adding highly tolerant Acetobacter pasteurellii, and uses Phellinus linteus as a post-biotic and medicinal-food homologous component as a fermentation substrate to promote the bioconversion efficiency of polyphenols and flavonoids, and optimizes fermentation process parameters, providing theoretical support and technical solutions for the industrial production of fruit vinegar. Attached Figure Description

[0031] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1 Apple cider vinegar production process flow chart.

[0033] Figure 2 Phellinus linteus seed liquid.

[0034] Figure 3 Sanghuang Houshengyuan.

[0035] Figure 4 Changes in total phenolic content before and after adding medicinal and edible ingredients to apple cider vinegar .

[0036] Figure 5 Changes in total flavonoid content before and after adding medicinal and edible ingredients to apple cider vinegar .

[0037] Figure 6 Changes in DPPH free radical scavenging rate before and after adding medicinal and edible ingredients to apple cider vinegar .

[0038] Figure 7 Comparison of total acidity in apple cider vinegar between Acetobacter pasteurella CL247 and Acetobacter pasteurella AS1.41 . Detailed Implementation

[0039] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0041] In a typical embodiment of the present invention, a method for fermenting apple cider vinegar using Sanghuang post-biotic compound medicinal and edible raw materials is provided, the method comprising the following steps: S1. Raw material pretreatment Select whole apples free from rot and pests, wash and core them, juice them, enzymatically hydrolyze them, filter them to obtain apple juice, add sucrose and purified water to adjust the sugar content to 11–18°Brix, sterilize at 105-115℃ for 10-20 minutes, and set aside. S2, Preparation of Phellinus linteus post-biotic By weight: 1-5 parts of *Sanghuang* fungus are inoculated into 100 parts of culture medium (each 100 parts of culture medium consists of: 1.5-3 parts glucose, 0.5-2.0 parts peptone, 0.1-0.2 parts KH2PO4, 0.05-0.15 parts MgSO4·7H2O, with the remainder being deionized water). Except for *Sanghuang* fungus, glucose, peptone, KH2PO4, and MgSO4·7H2O are added as described and dissolved in deionized water, then *Sanghuang* fungus is inoculated. Fermentation is carried out at 28-30℃ and 150-200 r / min for 5-7 days to obtain *Sanghuang* fungus seed culture. After fermentation, the obtained fermentation broth is subjected to ultrasonic and high-temperature sterilization. Ultrasonic treatment conditions: ultrasonic power 600-1000 W, ultrasonic time 10-30 min; high-temperature sterilization conditions: sterilization at 110-121℃ for 20-30 min, thus obtaining *Sanghuang* post-biotic. Add 1-4 parts of Phellinus sarcodactylis post-biotic to S1 apple juice; S3, Processing of medicinal and edible raw materials Select high-quality Astragalus membranaceus, Cornus officinalis, and Cinnamomum cassia, wash and dry them, and then grind them to 50-100 mesh using a grinder. Add 2-6 parts of Astragalus membranaceus, 1-5 parts of Cornus officinalis, and 1-3 parts of Cinnamomum cassia to 100 parts of S2 apple juice containing Phellinus linteus as a post-biotic to obtain an alcoholic fermentation medium. S4, alcoholic fermentation Add 8%-10% (v / v) of Saccharomyces cerevisiae to the alcoholic fermentation medium in S3. The alcoholic fermentation parameters are as follows: 18–32℃, initial pH 4.2–4.8, anaerobic fermentation for 5–7 days, and the alcohol content reaches 6%–14.5% vol (alcohol meter method GB 5009.225-2016) to obtain cider stock solution. S5, Acetic acid fermentation Activation of acetic acid bacteria strains: In a sterile operating table, 1-5 loops of *Acetobacter pasteurellii* CL247 cells were inoculated from a slant into seed culture medium (100-200 mL of 3%-5% ethanol fermentation medium), and cultured in a shaker at 28–32℃ and 180–220 r / min for 15–24 h. The culture was pre-cultured until OD600 = 1.8–2.2 to obtain acetic acid bacteria seed culture. Adjust the alcohol content of the apple cider stock to 4% - 8% vol by adding sterile purified water. In a 20–100 L mechanically stirred fermentation tank, carry out acetic acid fermentation at an inoculum of 5-10% (v / v) of Acetobacter pasteurellium CL247, at 28-32℃, a stirring speed of 180-220 rpm, and an aeration rate of 0.10-0.30 vvm. Stop fermentation when the total acid content of the apple cider vinegar reaches 40-50 g / L after 72–96 hours. S6, Aged Apple cider vinegar concentrate can be aged in stainless steel storage containers or earthenware jars for 1-3 months.

[0042] S7, Filtration, Blending and Filling After centrifugation and membrane filtration of the S6 aged apple cider vinegar concentrate, apple juice and purified water are added to adjust the total acid (calculated as acetic acid) to 2.5-4.5 g / L. Then, pasteurize (70-80 ℃, 30-50 min) or sterilize at 115-145 ℃ for 5-15 s, and fill under aseptic conditions. S8. Comparison of total phenols, total flavonoids, and DPPH free radical scavenging in apple cider vinegar without additives versus apple cider vinegar with added Phellinus linteus, Astragalus membranaceus, cinnamon, and Cornus officinalis. Figure 4 ).

[0043] The final medicinal and edible apple cider vinegar contained 0.15–2.8 mg RE / mL of total flavonoids (calculated as rutin equivalents, RE), 0.30–1.2 mg GAE / mL of total phenols (calculated as gallic acid equivalents, GAE), and had a DPPH free radical scavenging rate of 70%–80%.

[0044] Total flavonoids were determined according to the method in GB / T 19777-2013 Geographical Indication Product Shanxi Aged Vinegar; total phenols were determined according to the method in GB / T 19777-2013 Geographical Indication Product Shanxi Aged Vinegar; DPPH free radical scavenging rate was determined according to the method in GB / T 39100-2020 Determination of Antioxidant Properties of Polypeptides—DPPH and ABTS Methods.

[0045] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0046] Example 1 The overall process flow is as follows Figure 1 This includes the following steps: S1. Raw material pretreatment Select whole apples free from rot and pests, wash and core them, juice them, and then enzymatically hydrolyze them (0.2% w / v pectinase, 45℃, 2h). After filtration, add sucrose and purified water to the apple juice to adjust the sugar content to 17°Brix. Sterilize at 115℃ for 10 minutes and set aside. S2, Preparation of Phellinus linteus post-biotic By weight, 1 part of *Sanghuang* fungus was inoculated into 100 parts of culture medium (100 parts of culture medium consisted of: 1.5 parts glucose, 2.0 parts peptone, 0.1 parts KH2PO4, 0.05 parts MgSO4·7H2O, with the remainder being deionized water). All the above-mentioned ingredients except *Sanghuang* fungus were added as described and dissolved in deionized water, and *Sanghuang* fungus was inoculated into the medium. Fermentation was carried out at 30℃ and 200 r / min for 6 days to obtain *Sanghuang* fungus seed culture. Figure 2 After completion, the obtained fermentation broth was subjected to ultrasonic and high-temperature sterilization. Ultrasonic treatment conditions: ultrasonic power 800 W, ultrasonic time 20 min. High-temperature sterilization conditions: sterilization at 121℃ for 20 min, thus obtaining the post-Sanghuang serotonin (…). Figure 3 Add 1 part Phellinus linteus to S1 apple juice.

[0047] S3, Processing of medicinal and edible raw materials Select high-quality Astragalus membranaceus, Cornus officinalis, and Cinnamomum cassia, wash and dry them, and then grind them to 50-100 mesh using a grinder. Add 2 parts Astragalus membranaceus, 1 part Cornus officinalis, and 1 part Cinnamomum cassia to 100 parts S2 apple juice containing Phellinus linteus and post-biotic to obtain an alcoholic fermentation medium.

[0048] S4, alcoholic fermentation Add 10% Saccharomyces cerevisiae to the S3 alcoholic fermentation medium. Saccharomyces cerevisiae (Commercial Angel Yeast), alcoholic fermentation parameters are as follows: 25℃, initial pH 4.5, anaerobic fermentation for 5 days, alcohol content reaches 14.5% vol (alcohol meter method GB 5009.225-2016), to obtain cider stock.

[0049] S5, Acetic acid fermentation Activation of acetic acid bacteria strains: In a sterile operating table, 5 loops of *Acetobacter pasteurellii* CL247 cells were streaked from a slant and inoculated into seed culture medium (100 mL 4% ethanol fermentation medium). The culture was incubated at 30°C and 200 r / min in a shaker for 18 h, pre-culturing until OD500. 600 =2.0, to prepare acetic acid bacteria seed liquid.

[0050] Add sterile purified water to adjust the alcohol content of the apple cider stock to 6% vol. In a 20-100 L mechanically stirred fermentation tank, carry out acetic acid fermentation at an inoculum of 10% (v / v) of Acetobacter pasteurellium CL247, at 30℃, a stirring speed of 180 rpm, an aeration rate of 0.12 vvm, and a flow rate of 43 L / h. Stop fermentation when the total acid content of the apple cider vinegar reaches 49.2 g / L to obtain the apple cider vinegar stock. S6, Aged Apple cider vinegar concentrate should be aged in stainless steel storage containers or earthenware jars for 2 months.

[0051] S7, Filtration, Blending and Filling After centrifuging (8000×g, 15min) and membrane filtration (0.22 µm) of the S6 aged apple cider vinegar concentrate, apple juice and purified water were added to adjust the total acid (calculated as acetic acid) to 3.0 g / L, followed by pasteurization (80 ℃, 30min) and aseptic filling. S8. Comparison of total phenols, total flavonoids, and DPPH free radical scavenging in apple cider vinegar without additives versus apple cider vinegar with added Phellinus linteus, Astragalus membranaceus, cinnamon, and Cornus officinalis. Figure 4 , Figure 5 and Figure 6 ).

[0052] Comparative Example 1 S1. Raw material pretreatment Select whole apples free from rot and pests, wash and core them, juice them, and then enzymatically hydrolyze them (0.2% w / v pectinase, 45℃, 2h). After filtration, add sucrose and purified water to the apple juice to adjust the sugar content to 17°Brix, sterilize at 115℃ for 10 min, and set aside. S2, Processing of medicinal and edible raw materials Select high-quality Astragalus membranaceus, Cornus officinalis, and Cinnamomum cassia, wash and dry them, and then grind them to 50-100 mesh using a grinder. Add 2 parts Astragalus membranaceus, 1 part Cornus officinalis, and 1 part Cinnamomum cassia to 100 parts S1 apple juice to obtain an alcoholic fermentation medium.

[0053] S3, alcoholic fermentation Add 10% (v / v) of Saccharomyces cerevisiae to the alcoholic fermentation medium of S2. Saccharomyces cerevisiae The alcoholic fermentation parameters are as follows: 25℃, initial pH 4.5, anaerobic fermentation for 5 days, alcohol content reaches 14.5% vol (alcohol meter method GB 5009.225-2016), and cider stock is obtained.

[0054] S4, Acetic acid fermentation Activation of acetic acid bacteria strains: In a sterile operating room, 5 loops of *Acetobacter pasteurellii* CL247 cells were streaked from a slant and inoculated into seed culture medium (100 mL 4% ethanol fermentation medium). The culture was incubated at 30°C and 200 r / min in a shaker for 18 h, pre-culturing until OD (digestive activity) was reached. 600 =2.0, to prepare acetic acid bacteria seed liquid.

[0055] Add sterile purified water to adjust the alcohol content of the apple cider stock to 6% vol. In a 20-100 L mechanically stirred fermentation tank, carry out acetic acid fermentation at an inoculum of 10% (v / v) of Acetobacter pasteurellium CL247, at 30℃, a stirring speed of 180 rpm, an aeration rate of 0.12, and a ventilation rate of 43 L / hvvm. Stop fermentation when the total acid content of the apple cider vinegar reaches 50 g / L to obtain the apple cider vinegar stock. S5, Aged Apple cider vinegar concentrate should be aged in stainless steel storage containers or earthenware jars for 2 months.

[0056] S6, Filtration, Blending and Filling After centrifuging (8000×g, 15min) and membrane filtration (0.22 µm) of the S5 aged apple cider vinegar concentrate, apple juice and purified water were added to adjust the total acid (calculated as acetic acid) to 3.0 g / L, followed by pasteurization (80 ℃, 30min) and aseptic filling. S7. Comparison of total phenols, total flavonoids, and DPPH free radical scavenging in apple cider vinegar without additives versus apple cider vinegar with added astragalus, cinnamon, and cornus officinalis. Figure 4 , Figure 5 and Figure 6 ).

[0057] Comparative Example 2 S1. Raw material pretreatment Select whole apples free from rot and pests, wash and core them, juice them, and then enzymatically hydrolyze them (0.2% w / v pectinase, 45℃, 2h). After filtration, add sucrose and purified water to the apple juice to adjust the sugar content to 17 °Brix, sterilize at 115℃ for 10 min, and set aside. S2, Preparation of Phellinus linteus post-biotic By weight, 1 part of *Sanghuang* fungus was inoculated into 100 parts of culture medium (100 parts of culture medium consisted of: 1.5 parts glucose, 2.0 parts peptone, 0.1 parts KH2PO4, 0.05 parts MgSO4·7H2O, with the remainder being deionized water). All the above-mentioned ingredients, except for *Sanghuang* fungus, were added as described and dissolved in deionized water, and *Sanghuang* fungus was inoculated. Fermentation was carried out at 30℃ and 200 r / min for 6 days to obtain *Sanghuang* fungus seed culture. After fermentation, the resulting fermentation broth was subjected to ultrasonic and high-temperature sterilization. The ultrasonic treatment conditions were: ultrasonic power 800 W, ultrasonic time 20 min. The high-temperature sterilization conditions were: sterilization at 121℃ for 20 min, yielding *Sanghuang* post-biotic. 1 part of *Sanghuang* post-biotic was added to 100 parts of S1 apple juice.

[0058] S3, alcoholic fermentation Add 0.3% Saccharomyces cerevisiae (Saccharomyces cerevisiae) to the S2 alcoholic fermentation medium. Saccharomyces cerevisiae The alcoholic fermentation parameters are as follows: 25℃, initial pH 4.5, anaerobic fermentation for 6 days, alcohol content reaches 10% vol (alcohol meter method GB5009.225-2016), and cider stock is obtained.

[0059] S4, Acetic acid fermentation Activation of acetic acid bacteria strains: In a sterile operating table, streak 1-5 loops of *Acetobacter pasteurellii* CL247 cells from a slant and inoculate into seed culture medium (100 mL 4% ethanol fermentation medium). Incubate at 30℃ and 200 r / min in a shaker for 18 h, pre-culturing until OD. 600 = 2.0, to obtain acetic acid bacteria seed liquid.

[0060] Add sterile purified water to adjust the alcohol content of the apple cider stock to 6% vol. In a 20-100 L mechanically stirred fermentation tank, carry out acetic acid fermentation at an inoculum of 10% (v / v) of Acetobacter pasteurellium CL247, at 30℃, a stirring speed of 180 rpm, an aeration rate of 0.12 vvm, and a flow rate of 43 L / h. Stop fermentation when the total acid content of apple cider vinegar reaches 50 g / L after 72 hours to obtain apple cider vinegar stock. S5, Aged Apple cider vinegar concentrate should be aged in stainless steel storage containers or earthenware jars for 2 months.

[0061] S6, Filtration, Blending and Filling After centrifuging (8000×g, 15min) and membrane filtration (0.22 µm) of the S6 aged apple cider vinegar concentrate, apple juice and purified water were added to adjust the total acid (calculated as acetic acid) to 3.0 g / L, followed by pasteurization (80 ℃, 30min), and aseptic filling. S7. Comparison of total phenols, total flavonoids, and DPPH free radical scavenging in apple cider vinegar without additives versus apple cider vinegar with added Phellinus linteus, Astragalus membranaceus, cinnamon, and Cornus officinalis. Figure 4 , Figure 5 and Figure 6 ).

[0062] Comparative Example 3 S1. Raw material pretreatment Select whole apples free from rot and pests, wash and core them, juice them, and then enzymatically hydrolyze them (0.2% w / v pectinase, 45℃, 2h). After filtration, add sucrose and purified water to the apple juice to adjust the sugar content to 17 °Brix, sterilize at 115℃ for 10 min, and set aside. S2, Preparation of Phellinus linteus post-biotic By weight, 1 part of *Sanghuang* fungus was inoculated into 100 parts of culture medium (100 parts of culture medium consisted of: 1.5 parts glucose, 2.0 parts peptone, 0.1 parts KH2PO4, 0.05 parts MgSO4·7H2O, with the remainder being deionized water). All the above-mentioned ingredients, except for *Sanghuang* fungus, were added as described and dissolved in deionized water, and *Sanghuang* fungus was inoculated. Fermentation was carried out at 30℃ and 200 r / min for 6 days to obtain *Sanghuang* fungus seed culture. After fermentation, the resulting fermentation broth was subjected to ultrasonic and high-temperature sterilization. The ultrasonic treatment conditions were: ultrasonic power 800 W, ultrasonic time 20 min. The high-temperature sterilization conditions were: sterilization at 121℃ for 20 min, yielding *Sanghuang* post-biotic. 1 part of *Sanghuang* post-biotic was added to 100 parts of S1 apple juice.

[0063] S3, Processing of medicinal and edible raw materials Select high-quality Astragalus membranaceus, Cornus officinalis, and Cinnamomum cassia, wash and dry them, and then grind them to 50-100 mesh using a grinder. Add 2 parts Astragalus membranaceus, 1 part Cornus officinalis, and 1 part Cinnamomum cassia to 100 parts S2 apple juice containing Phellinus linteus as a post-biotic to obtain an alcoholic fermentation medium.

[0064] S4, alcoholic fermentation Add 0.3% Saccharomyces cerevisiae to the S3 alcoholic fermentation medium. Saccharomyces cerevisiae The alcoholic fermentation parameters are as follows: 25℃, initial pH 4.5, anaerobic fermentation for 5 days, alcohol content reaches 14.5% vol (alcohol meter method GB5009.225-2016), yielding cider stock.

[0065] S5, Acetic acid fermentation Activation of acetic acid bacteria strains: Under sterile conditions, streak 1-5 loops of *Acetobacter pasteurellii* AS1.41 cells from a slant and inoculate into seed culture medium (100 mL 4% ethanol fermentation medium). Incubate at 30°C and 200 r / min in a shaker for 18 h, pre-culturing until OD500. 600 =2.0, to prepare acetic acid bacteria seed liquid.

[0066] Add sterile purified water to adjust the alcohol content of the apple cider stock to 6% vol. In a 20-100 L mechanically stirred fermentation tank, carry out acetic acid fermentation at an inoculum of 8% (v / v) of Acetobacter pasteurellium CL247, at 30°C, a stirring speed of 200 rpm, and an aeration rate of 0.20 vvm. Stop fermentation when the total acidity of the apple cider vinegar no longer increases, and obtain the apple cider vinegar stock. S6, Aged Apple cider vinegar concentrate should be aged in stainless steel storage containers or earthenware jars for 2 months.

[0067] S7, Filtration, Blending and Filling After centrifuging (8000×g, 15min) and membrane filtration (0.22 µm) of the S6 aged apple cider vinegar concentrate, apple juice and purified water were added to adjust the total acid (calculated as acetic acid) to 3.0 g / L, followed by pasteurization (80 ℃, 30min) and aseptic filling. A comparison of total acidity in apple cider vinegar fermented with S8, Acetobacter pasteurellium CL247, and Acetobacter pasteurellium AS1.41, and the total phenols, total flavonoids, and DPPH free radical scavenging content in apple cider vinegar without additives compared with that after the addition of Phellinus linteus, Astragalus membranaceus, Cinnamomum cassia, and Cornus officinalis. Figure 4 , Figure 5 , Figure 6 and Figure 7 ).

[0068] Utilizing proprietary Acetobacter pasteurellium CL247 ( Acetobacter pasteurianus The optimal fermentation process for apple cider vinegar was established using *Acetobacter pasteurella* CL247 (CGMCC No. 33924) for acetic acid fermentation. Acetobacter pasteurianus CL247 (CGMCC No. 33924) is an acetic acid strain with high tolerance to ethanol and acetic acid, screened from samples of Shanxi aged vinegar. Acetobacter pasteurellum CL247 has high tolerance to ethanol (18%), acetic acid (7%), and high acid production efficiency of 49.2 g / L in 72 hours, which can shorten the acetic acid fermentation time to 72 hours. This embodiment established the optimal alcohol and acetic acid fermentation process. In the alcohol fermentation stage, 10% of brewer's yeast was added, the initial sugar content was 17°Bx, the fermentation time was 5 days, and the alcohol content was 14.5% vol. Subsequently, the acetic acid fermentation process was initiated, with 10% of *Acetobacter pasteurellium* CL247 added, at 30°C, a stirring rate of 180 r / min, and an aeration rate of 0.12 vvm, 43 L / h. The fermentation medium included *Sanghuang* post-biotic and a group of medicinal and edible ingredients in a weight ratio of Astragalus membranaceus: *Sanghuang*: *Cornus officinalis*: *Cinnamomum cassia* = 2:1:1:1. *Sanghuang* post-biotic was prepared using liquid fermentation and ultrasonic cell disruption. After microbial fermentation, compared with apple cider vinegar without added medicinal and edible ingredients, the total flavonoid content reached 0.453 ± 0.6 mg RE / mL (an increase of 566.3%), and the total phenol content reached 0.939 ± 0.3 mg. GAE / mL increased by 204.1%, and DPPH free radical scavenging rate increased by 173.8%. Microbial transformation during fermentation enhances the activity of functional components, such as astragaloside IV bioavailability reaching 35% (an increase of 75%). The total acid content of apple cider vinegar can reach 45.2 g / L after 72 h. In summary, compared with the prior art, the technical advantages of this invention include: (1) a comprehensive breakthrough in acetic acid fermentation efficiency and tolerance: Existing technology: The ethanol tolerance of the strain is usually <12%, and the acid production rate under 10% ethanol conditions is <0.6 g / L / h.

[0069] This invention: A. pasteurianus CL247 has an ethanol tolerance of 18%, and its acid production rate increases to 0.68 g / L / h under 10% ethanol conditions. (2) Enhancement of functional components: Existing technology: Functional components are added through physical extraction. This invention: Through liquid fermentation of Phellinus linteus post-biotic technology, compound medicinal and edible components such as Astragalus membranaceus, cinnamon and Cornus officinalis are combined. After microbial fermentation, compared with apple cider vinegar without the addition of medicinal and edible components, the final medicinal and edible apple cider vinegar has a total flavonoid (rutin equivalents, RE) content of 0.15 – 2.8 mg RE / mL, a total phenol (gallic acid equivalents, GAE) content of 0.30 – 1.2 mg GAE / mL, and a DPPH free radical scavenging rate of 70% – 80%. The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for fermenting apple cider vinegar using a compound medicinal and edible raw material, *Sanghuang* (a type of medicinal fungus), characterized in that... The method includes the following steps: S1. Raw material pretreatment After juicing the apples, enzymatically hydrolyze them, filter them to obtain apple juice, add sucrose and water to the apple juice to adjust the sugar content to 11~18°Brix, sterilize, and set aside. S2, Preparation of Phellinus linteus post-biotic Inoculate 1-5 portions of Phellinus linteus into the culture medium and ferment at 28-30℃ and 150-200 r / min for 5-7 days to obtain Phellinus linteus seed liquid. After the fermentation is completed, the obtained fermentation liquid is subjected to ultrasonic and high-temperature sterilization treatment. Ultrasonic treatment conditions: ultrasonic power 600-1000 W, ultrasonic time 10-30 min, to obtain Phellinus linteus post-genetic agent. Add 1-4 parts of Phellinus linteus extract to 100 parts of S1 apple juice; S3, Processing of medicinal and edible raw materials Wash, dry, and pulverize Astragalus membranaceus, Cornus officinalis, and Cinnamomum cassia. Add 2–6 parts of Astragalus membranaceus, 1–5 parts of Cornus officinalis, and 1–3 parts of Cinnamomum cassia to 100 parts of apple juice containing Phellinus linteus and post-biotics to obtain an alcoholic fermentation medium. S4, alcoholic fermentation Add 5%–10% (v / v) of Saccharomyces cerevisiae to the S3 alcoholic fermentation medium. Saccharomyces cerevisiae The alcoholic fermentation parameters are as follows: 18 – 32℃, initial pH 4.2 – 4.8, anaerobic fermentation for 5 – 7 days, alcohol content reaching 6% – 14.5% vol, to obtain cider stock. S5, Acetic acid fermentation Activation of acetic acid bacteria strains: Acetobacter pasteurellium CL247 cells were inoculated into seed culture medium and cultured at 28–32℃ and 180–220 r / min in a shaker for 15–24 h, pre-culturing until OD... 600 =1.8–2.2, to obtain acetic acid bacteria seed culture; Adjust the alcohol content of the apple cider stock solution to 4%-8% vol by adding water. Perform acetic acid fermentation at an inoculum size of 5-10% (v / v) of Acetobacter pasteurella CL247, at a temperature of 28-32℃, a stirring speed of 180-220 rpm, and an aeration rate of 0.10-0.30 vvm. Stop fermentation when the total acid content of the apple cider vinegar reaches 40-50 g / L after 72-96 hours to obtain the apple cider vinegar stock solution. S6, Aged Apple cider vinegar concentrate should be aged for 1-3 months. S7, Filtration, Blending and Filling After centrifugation and membrane filtration, the S6 aged apple cider vinegar concentrate is mixed with apple juice and purified water to a total acidity of 2.5-4.5 g / L, then sterilized and aseptically bottled.

2. The method for fermenting apple cider vinegar from the compound medicinal and edible raw material *Sanghuang* as described in claim 1, characterized in that: In S1, add sucrose and water to the apple juice to adjust the sugar content to 16-18°Brix.

3. The method for fermenting apple cider vinegar from the compound medicinal and edible raw material *Sanghuang* as described in claim 1, characterized in that... In S2, by weight, 100 parts of culture medium consist of: 1.5-3 parts glucose, 0.5-2.0 parts peptone, 0.1-0.2 parts KH2PO4, 0.05-0.15 parts MgSO4·7H2O, with the remainder being water.

4. The method for fermenting apple cider vinegar from the compound medicinal and edible raw material of Sanghuang post-biotic as described in claim 1, characterized in that, In S4, the inoculation amount of the brewing yeast is 10% (v / v).

5. The method for fermenting apple cider vinegar from the compound medicinal and edible raw material of Sanghuang post-biotic as described in claim 1, characterized in that, in S5, the Acetobacter pasteurellum CL247 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 21, 2025, with accession number CGMCC NO. 33924.

6. The method for fermenting apple cider vinegar from the compound medicinal and edible raw material *Sanghuang* as described in claim 1, characterized in that, In S5, the fermentation temperature was 30℃, the stirring rate was 180 rpm, and the aeration rate was 0.12 vvm.

7. The method for fermenting apple cider vinegar from the compound medicinal and edible raw material of Sanghuang post-biotic as described in claim 1, characterized in that, In S5, the seed culture medium consists of 100-200 mL of water containing 3g-5g of ethanol fermentation medium.

8. Apple cider vinegar prepared by any one of claims 1 to 7.

9. The apple cider vinegar as described in claim 8, characterized in that, The apple cider vinegar contains 0.15–2.8 mg RE / mL of total flavonoids, 0.30–1.2 mg GAE / mL of total phenols, and has a DPPH free radical scavenging rate of 70%–80%.

10. The use of the apple cider vinegar according to claim 8 or 9 in the preparation of antioxidant and / or anti-inflammatory functional foods or healthy fruit vinegar beverages.

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