Fermentation product with antioxidant activity as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fermentation products, and particularly relates to a fermentation product with antioxidant activity and a preparation method and application thereof. The fermented product with antioxidant activity is prepared by the following steps: preparing raw materials into a water-containing mixed solution, then adding an enzyme preparation for enzymolysis, and then adding a leavening agent for fermenting the obtained enzymatic hydrolysate, the raw materials comprise 8-22 parts of radix puerariae, 3-12 parts of purple sweet potatoes and 0-10 parts of oat; wherein fermentation is carried out by adopting a leavening agent, and the leavening agent comprises streptococcus salivarius and kluyveromyces marxianus; relative to the total mass of the water-containing mixed solution of the raw materials, the addition amount of the enzyme preparation is 1%-1.5%. The prepared fermented product has more excellent antioxidant activity, namely, the content of exopolysaccharides is higher, the scavenging rate of DPPH free radicals is higher, the activity of SOD and GSH-Px is higher, and the fermented product has more excellent effects in the aspects of shape, color, taste and smell.

Description

Technical Field

[0001] This invention belongs to the field of fermentation product technology, specifically relating to a fermentation product with antioxidant activity, its preparation method, and its application. Background Technology

[0002] Oxidation is a necessary metabolic process in the body, but when too many free radicals are produced, exceeding the body's ability to eliminate them, oxidative stress occurs. This is an imbalance between the generation and elimination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the body, leading to their excessive accumulation and subsequent damage to cells and tissues. This damage may trigger inflammatory responses, accelerate tissue and organ aging, and increase the risk of chronic diseases. Antioxidants can neutralize free radicals and help maintain the balance between oxidation and antioxidation; therefore, developing highly effective and stable antioxidant products is of great significance.

[0003] However, traditional fermented antioxidant products generally suffer from long production cycles and low efficiency. For example, the solid-state fermentation cycle of traditional citrus enzymes can take as long as 3-6 months. At the same time, the fermentation process is often difficult to control precisely, which can easily lead to contamination by miscellaneous bacteria, the production of undesirable byproducts, low dissolution rates of target active ingredients, and unstable product quality, thus limiting their practical application effectiveness.

[0004] Therefore, developing an antioxidant product that, compared to traditional fermented antioxidant products and other antioxidant fermented products, can significantly shorten fermentation time, improve production efficiency, enhance palatability, retain nutrients, enrich antioxidant substances, and enhance antioxidant activity is of great research significance. Summary of the Invention

[0005] The technical problem solved by this invention is that the antioxidant active products disclosed in the prior art have long fermentation cycles, low content of antioxidant substances, and their antioxidant activity needs to be further improved.

[0006] To address the aforementioned technical problems, this invention provides a fermented product with antioxidant activity, its preparation method, and its application.

[0007] Specifically, the present invention provides the following technical solution: In a first aspect, the present invention provides a fermented product with antioxidant activity, which is prepared by preparing an aqueous mixture of raw materials, adding an enzyme preparation for enzymatic hydrolysis, and then adding a fermenting agent to ferment the resulting hydrolysate. The raw materials, by weight, include 8-22 parts kudzu root, 3-12 parts purple sweet potato, and 0-10 parts oats; the enzyme preparation accounts for 1%-1.5% of the total mass of the aqueous mixture of the raw materials; the fermenting agent includes Streptococcus salivarius and Kluyveromyces martensii.

[0008] Preferably, the raw material comprises 10-20 parts by weight of kudzu root; more preferably, the raw material comprises 16-19 parts by weight of kudzu root.

[0009] And / or, preferably, the raw material comprises 5-10 parts purple sweet potato by weight; more preferably, the raw material comprises 7-9 parts purple sweet potato.

[0010] And / or, preferably, the raw material comprises 5 to 10 parts by weight of oats; more preferably, the raw material comprises 7 to 9 parts by weight of oats.

[0011] And / or, preferably, *Kluyveromyces martensii* is added to the enzymatic hydrolysate obtained by enzymatic hydrolysis for a first fermentation to obtain a preliminary fermentation broth; then *Streptococcus salivarius* is added for a second fermentation to obtain a fermentation broth; more preferably, the mass percentage of *Kluyveromyces martensii* added is 2%-6% relative to the total mass of the supernatant of the enzymatic hydrolysate; and / or, the mass percentage of *Streptococcus salivarius* added is 0.05%-0.2% relative to the total mass of the preliminary fermentation broth.

[0012] And / or, preferably, the *Streptococcus salivarius* includes a DNA sequence with antioxidant activity, as shown in SEQ ID NO. 1 and / or SEQ ID NO. 2.

[0013] And / or, preferably, the *Streptococcus salivarius* includes an amino acid sequence with antioxidant activity, as shown in SEQ ID NO. 3 and / or SEQ ID NO. 4.

[0014] And / or, preferably, the Streptococcus salivarius is Streptococcus thermophilus subspecies Jiacha.ST-685 strain, with accession number CCTCC NO: M20231495.

[0015] And / or, preferably, the Kluyveromyces martensii strain is Kluyveromyces martensii Linzhi. KM-D3, with accession number CCTCC NO: M 20231575.

[0016] And / or, preferably, the content of extracellular polysaccharides in the fermented product is 500-1100 mg / L; more preferably, the content of extracellular polysaccharides in the fermented product is 800-1100 mg / L; and even more preferably, the content of extracellular polysaccharides in the fermented product is 900-1100 mg / L.

[0017] Secondly, the present invention provides a method for preparing the fermented product with antioxidant activity, comprising the following steps: Step 1: Mix the raw materials with water to obtain an aqueous mixture; Step 2: Add enzyme preparation to the mixture to carry out enzymatic hydrolysis, and obtain the enzymatic hydrolysate; Step 3: Add a starter culture to the enzyme hydrolysate to ferment and obtain the fermentation broth; Step 4: Separate the solid and liquid components of the fermentation broth. The resulting liquid is the fermentation product with antioxidant activity.

[0018] Preferably, in step 1, the mixture of raw materials and water to obtain an aqueous mixture is also called a fermentation base liquid; more preferably, the raw materials are dried and pulverized, and then mixed with water to obtain a fermentation base liquid; even more preferably, the moisture content of the dried raw materials is 0-10%; and / or, even more preferably, the particle size of the dried raw materials after pulverization is 100-150 mesh.

[0019] And / or, preferably, in step 1, the mass ratio of the raw material to the volume ratio of water is 1:8-15, wherein the mass is in g and the volume is in mL; more preferably, the mass ratio of the raw material to the volume ratio of water is 1:9-11, wherein the mass is in g and the volume is in mL.

[0020] And / or, preferably, in step 2, the enzyme preparation accounts for 1.1%-1.3% of the total mass of the fermentation base liquid. More preferably, the enzyme preparation comprises α-amylase and pectinase; even more preferably, the mass ratio of α-amylase to pectinase is 4-8:0.5-4; even more preferably, the mass ratio of α-amylase to pectinase is 5-8:1-4; and still more preferably, the mass ratio of α-amylase to pectinase is 6-8:2-4.

[0021] And / or, more preferably, the enzyme activity of α-amylase is 240,000-280,000 u / mL; and / or, the enzyme activity of pectinase is 50,000-70,000 u / g; And / or, preferably, in step 2, the enzymatic hydrolysis temperature is 45-65°C; more preferably, the enzymatic hydrolysis temperature is 50-60°C.

[0022] And / or, preferably, in step 2, the enzymatic hydrolysis time is 2-5 hours; more preferably, the enzymatic hydrolysis time is 3-4 hours; And / or, preferably, in step 2, the pH of the enzymatic hydrolysis is 5.0-6.0; more preferably, the pH of the enzymatic hydrolysis is 5.3-6.7.

[0023] And / or, preferably, in step 2, after enzymatic hydrolysis, solid-liquid separation is performed, the supernatant is collected, sterilized, and then subjected to subsequent fermentation treatment; more preferably, solid-liquid separation is centrifugal separation; even more preferably, the centrifugation speed is 4000-6000 rpm, and / or, the centrifugation time is 5-10 min; and / or, more preferably, the sterilization temperature is 75-80℃, and / or, the sterilization time is 15-30 min.

[0024] And / or, preferably, in step 3, *Kluyveromyces martensii* is first added to the enzymatic hydrolysate obtained in step 2 for a first fermentation to obtain a preliminary fermentation broth; then *Streptococcus salivarius* is added for a second fermentation to obtain a fermentation broth.

[0025] More preferably, the mass percentage of added Kluyveromyces martensii relative to the total mass of the supernatant of the enzymatic hydrolysate is 2%-6%; more preferably, the mass percentage of added Kluyveromyces martensii is 3%-5%; and even more preferably, the mass percentage of added Kluyveromyces martensii is 3%-4%.

[0026] And / or, more preferably, the temperature of the first fermentation is 28-32°C.

[0027] And / or, more preferably, the first fermentation time is 24-72 hours; even more preferably, the first fermentation time is 24-48 hours.

[0028] And / or, more preferably, after the first fermentation is completed, the initial fermentation broth is subjected to ultrasonic inactivation treatment; even more preferably, the temperature of ultrasonic inactivation treatment is 50-60°C; and / or, the time of ultrasonic inactivation treatment is 20-30 min.

[0029] And / or, preferably, in step 3, the mass percentage of added Streptococcus salivarius relative to the total mass of the initial fermentation broth is 0.05%-0.2%; more preferably, the mass percentage of added Streptococcus salivarius is 0.05%-0.15%.

[0030] And / or, preferably, in step 3, the temperature of the second fermentation is 35-39°C.

[0031] And / or, preferably, in step 3, the second fermentation time is 24-72 hours; more preferably, the second fermentation time is 24-48 hours.

[0032] And / or, preferably, in step 4, the solid-liquid separation is centrifugal separation; more preferably, the centrifugation speed is 5000-8000 rpm; and / or, more preferably, the centrifugation time is 10-15 min; And / or, preferably, in step 4, the supernatant obtained after solid-liquid separation is concentrated to obtain a fermented product; more preferably, the supernatant obtained after solid-liquid separation is concentrated to 30%-70% of its original volume; and / or, more preferably, the concentration temperature is 50-60°C.

[0033] Thirdly, the present invention provides a fermented product with antioxidant activity, which is prepared by the method for preparing the fermented product with antioxidant activity.

[0034] Fourthly, the present invention provides an application of the aforementioned fermented product with antioxidant activity in the preparation of food.

[0035] Fifthly, the present invention provides a food product comprising the aforementioned fermented product with antioxidant activity.

[0036] Beneficial effects of the present invention (1) The present invention involves enzymatic hydrolysis and fermentation of raw materials including kudzu root and purple sweet potato (and may also contain oats), and the fermented products prepared have antioxidant activity.

[0037] (2) By controlling the ratio of raw materials, the type of enzyme and the enzymatic hydrolysis conditions, the type of fermenting agent and the fermentation conditions, and the synergistic interaction between each component and each step, the fermented product obtained in the present invention has better antioxidant activity, that is, higher content of extracellular polysaccharides, higher DPPH free radical scavenging rate, higher activity of SOD and GSH-Px, and the fermented product has better effects in terms of morphology, color, taste and smell.

[0038] (3) The present invention uses an enzyme preparation containing α-amylase and pectinase to enzymatically hydrolyze the raw materials. The fermented products prepared by the present invention have stronger antioxidant activity and better sensory evaluation results compared with fermented products prepared by other enzyme preparations.

[0039] (4) The present invention employs two fermentations. Preferably, the enzyme hydrolysate is first fermented with Kluyveromyces martensii and then fermented with Streptococcus salivarius. The fermented product prepared has stronger antioxidant activity and better sensory evaluation results compared to the fermented product prepared by one fermentation.

[0040] (5) In this invention, Kluyveromyces marxi and Streptococcus salivarius are used together for the fermentation of the enzymatic hydrolysate. More preferably, the thermophilic subspecies of Streptococcus salivarius, Jiacha.ST-685 strain, has unique functional genes related to antioxidants, such as glycosyltransferase and dextran sucrase, which can synergistically interact with other components to make the fermented product more antioxidant and have better sensory evaluation results.

[0041] (6) The fermented products with antioxidant activity prepared by the present invention are safe and natural, have good palatability, can improve the taste defects of plant-based products, and solve the problem of unfriendly flavor of traditional food and medicine homologous fermented products.

[0042] (7) The present invention uses specific enzymatic hydrolysis and fermentation processes to carry out directional fermentation of raw materials, replacing traditional fermentation. On the one hand, it shortens the fermentation time and improves the preparation efficiency, while retaining the original active substances in the raw materials.

[0043] (8) The fermented products of the present invention can effectively remove oxygen free radicals in the body and outside the body, play an antioxidant role and slow down aging. When applied to food, they can enhance nutritional value and play a health function.

[0044] Strain Preservation Information Streptococcus salivarius subsp. thermophilus Jiacha. ST-685 ( Streptococcus salivarius subsp. therm ophilus Jiacha (ST-685), deposited on August 17, 2023, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20231495, address: Wuhan University, Wuhan, China, postcode: 430072; telephone: (027) 68754052. This strain has been described in invention patent application number 202411020209.4.

[0045] Kluyveromycin Linzhi. KM-D3 ( Kluyveromyces marxious The strain Linzhi. KM-D3 was deposited at the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with accession number CCTCC NO: M20231575. The depositary address is: Wuhan University, Wuhan, China, Postcode: 430072; Telephone: (027) 68754052. This strain has been described in the invention patent application No. 202411560647.X. Detailed Implementation

[0046] As described above, the purpose of this invention is to provide a fermented product with antioxidant activity, its preparation method, and its application.

[0047] In this invention, kudzu root, rich in isoflavones, particularly puerarin, daidzein, and daidzeinogen, primarily exerts its effects by directly scavenging free radicals and enhancing the body's own antioxidant enzyme system. Purple sweet potato, containing anthocyanins, possesses extremely strong antioxidant capabilities, effectively combating various free radicals. Oats, containing arnthramides, as well as other polyphenols and β-glucan, are characterized by their ability to effectively inhibit the oxidation of low-density lipoprotein ("bad cholesterol"). This application combines kudzu root and purple sweet potato, more preferably further combining them with oats. The components within these ingredients synergistically, and combined with specific enzymatic hydrolysis and fermentation processes, the resulting fermented product exhibits superior antioxidant properties and better sensory evaluation.

[0048] This invention intentionally adds a compound enzyme preparation containing α-amylase and pectinase to the selected raw materials for enzymatic hydrolysis, which can improve the fluidity and stability of the slurry, making it more conducive to the next fermentation step, and resulting in a fermented product with better taste and antioxidant properties.

[0049] Specifically, the present invention provides the following technical solution: Technical Solution 1: A fermented product with antioxidant activity is prepared by preparing raw materials into an aqueous mixture, then adding an enzyme preparation for enzymatic hydrolysis, and then adding a fermenting agent to ferment the resulting hydrolysate. The raw materials, by weight, include 8-22 parts kudzu root, 3-12 parts purple sweet potato, and 0-10 parts oats; the added enzyme preparation accounts for 1%-1.5% of the total mass of the aqueous mixture of the raw materials; the fermenting agent includes Streptococcus salivarius and Kluyveromyces martensii. For example, in some embodiments, the raw materials may include 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 parts of kudzu root, or contain kudzu root within a numerical range defined by any two of the above specific values ​​as endpoints; and / or, In some embodiments, the raw materials may include 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 parts of purple sweet potato, or contain purple sweet potato within a numerical range defined by any two of the above specific values ​​as endpoints; and / or, In some embodiments, the raw materials may include 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts of oats, or oats falling within a numerical range defined by any two of the above specific values ​​as endpoints; and / or, In some embodiments, the mass percentage of the enzyme preparation added during the enzymatic hydrolysis process can be 1%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, or 1.5%, or the content of enzymes within the numerical range formed by any two of the above specific values ​​as endpoints.

[0050] Technical Solution 2: The fermented product with antioxidant activity according to Technical Solution 1, wherein, by weight, the raw material includes 10-20 parts of kudzu root; preferably, the raw material includes 16-19 parts of kudzu root; And / or, by weight, the raw material comprises 5-10 parts purple sweet potato; preferably, the raw material comprises 7-9 parts purple sweet potato; And / or, by weight, the raw materials comprise 5 to 10 parts of oats; preferably, the raw materials comprise 7 to 9 parts of oats.

[0051] Technical Solution 3: A fermented product with antioxidant activity according to Technical Solution 1 or 2, wherein the Streptococcus salivarius includes a DNA sequence with antioxidant activity, as shown in SEQ ID NO. 1 and / or SEQ ID NO. 2; And / or, the *Streptococcus salivarius* includes an amino acid sequence with antioxidant activity, as shown in SEQ ID NO. 3 and / or SEQ ID NO. 4; And / or, the Streptococcus salivarius is strain Jiacha.ST-685 of Streptococcus salivarius thermophilus, with accession number CCTCC NO: M20231495.

[0052] Technical Solution 4: A fermented product with antioxidant activity according to any one of Technical Solutions 1-3, wherein the Kluyveromyces martensii is Kluyveromyces martensii Linzhi. KM-D3, and its preservation number is CCTCC NO: M20231575.

[0053] Technical Solution 5: A fermented product with antioxidant activity according to any one of Technical Solutions 1-4, wherein the content of extracellular polysaccharides in the fermented product is 500-1100 mg / L; for example, in some embodiments, the content of extracellular polysaccharides in the fermented product can be 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or 1100 mg / L, or contain an extracellular polysaccharide content within the numerical range formed by any two of the above specific values ​​as endpoints; Preferably, the content of extracellular polysaccharides in the fermented product is 800-1100 mg / L; More preferably, the content of extracellular polysaccharides in the fermented product is 900-1100 mg / L.

[0054] Technical Solution 6: A method for preparing a fermented product with antioxidant activity as described in any one of Technical Solutions 1-5, characterized in that it includes the following steps: Step 1: Mix the raw materials with water to obtain an aqueous mixture; Step 2: Add enzyme preparation to the mixture to carry out enzymatic hydrolysis, and obtain the enzymatic hydrolysate; Step 3: Add a starter culture to the enzyme hydrolysate to ferment and obtain the fermentation broth; Step 4: Separate the solid and liquid components of the fermentation broth. The resulting liquid is the fermentation product with antioxidant activity.

[0055] Technical Solution 7: The method for preparing a fermented product with antioxidant activity according to Technical Solution 6, wherein in step 1, the raw materials are mixed with water to obtain an aqueous mixture, also known as a fermentation base liquid; preferably, the raw materials are dried and pulverized, and then mixed with water to obtain the fermentation base liquid; more preferably, the water content of the dried raw materials is 0-10%; and / or, even more preferably, the particle size of the dried raw materials after pulverization is 100-150 mesh; for example, in some embodiments, the particle size of the dried raw materials after pulverization can be 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 mesh, or contain particle sizes within the numerical range formed by any two of the above specific values ​​as endpoints; And / or, preferably, the mass-to-volume ratio of the raw material to the water is 1:8-15, wherein the mass is in g and the volume is in mL; more preferably, the mass-to-volume ratio of the raw material to the water is 1:9-11, wherein the mass is in g and the volume is in mL; for example, in some embodiments, in the mass-to-volume ratio of the raw material to the water, the mass of the raw material is 1, and the volume of the water can be 8, 9, 10, 11, 12, 13, 14 or 15, or a range of water proportions within the numerical range formed by any two of the above specific values ​​as endpoints.

[0056] Technical Solution 8: A method for preparing a fermented product with antioxidant activity according to Technical Solution 6 or 7, wherein, in step 2, the amount of enzyme preparation added is 1.1%-1.3% based on the mass of the fermentation base liquid; Preferably, the enzyme preparation includes α-amylase and pectinase; More preferably, the mass ratio of α-amylase to pectinase is 4-8:0.5-4; further preferably, the mass ratio of α-amylase to pectinase is 5-8:1-4; even more preferably, the mass ratio of α-amylase to pectinase is 6-8:2-4; for example, in some embodiments, in the mass ratio of α-amylase to pectinase, the mass of α-amylase is 4-8, and the mass of pectinase can be 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4, or a mass range of pectinase within the numerical range formed by any two of the above specific values ​​as endpoints; or, in some embodiments, in the mass ratio of α-amylase to pectinase, the mass of pectinase is 0.5-4, and the mass of α-amylase can be 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8, or a mass range of α-amylase within the numerical range formed by any two of the above specific values ​​as endpoints. And / or, more preferably, the activity of α-amylase is 240,000-280,000 u / mL; and / or, the activity of pectinase is 50,000-70,000 u / g.

[0057] Technical Solution 9: A method for preparing a fermented product with antioxidant activity according to any one of Technical Solutions 6-8, wherein, in step 2, the enzymatic hydrolysis temperature is 45-65℃; preferably, the enzymatic hydrolysis temperature is 50-60℃; for example, in some embodiments, the enzymatic hydrolysis temperature can be 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65℃, or the enzymatic hydrolysis temperature within the numerical range formed by any two of the above specific values ​​as endpoints; And / or, the enzymatic hydrolysis time is 2-5 hours; preferably, the enzymatic hydrolysis time is 3-4 hours; for example, in some embodiments, the enzymatic hydrolysis time can be 2, 2.5, 3, 3.5, 4, 4.5 or 5 hours, or the enzymatic hydrolysis time is within the numerical range formed by any two of the above specific values ​​as endpoints. And / or, the pH of the enzymatic hydrolysis is 5.0-6.0; preferably, the pH of the enzymatic hydrolysis is 5.3-6.7; for example, in some embodiments, the pH of the enzymatic hydrolysis can be 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6, or the pH of the enzymatic hydrolysis within the numerical range formed by any two of the above specific values ​​as endpoints.

[0058] Technical Solution 10: A method for preparing a fermented product with antioxidant activity according to any one of Technical Solutions 6-9, wherein in step 2, after enzymatic hydrolysis, solid-liquid separation is performed, the supernatant is taken, sterilized, and then subjected to subsequent fermentation treatment. Preferably, the solid-liquid separation is centrifugal separation; more preferably, the centrifugation speed is 4000-6000 rpm, and / or the centrifugation time is 5-10 min; And / or, the sterilization temperature is 75-80℃, and / or, the sterilization time is 15-30 min.

[0059] Technical Solution 11: A method for preparing a fermented product with antioxidant activity according to any one of technical solutions 6-10, wherein in step 3, *Kluyveromyces martensii* is first added to the enzymatic hydrolysate obtained in step 2 for a first fermentation to obtain a preliminary fermentation broth; then *Streptococcus salivarius* is added for a second fermentation to obtain a fermentation broth. Preferably, the mass percentage of *Kluyveromyces martensii* added relative to the total mass of the supernatant of the enzymatic hydrolysate is 2%-6%; more preferably, the mass percentage of *Kluyveromyces martensii* added is 3%-5%; more preferably, the mass percentage of *Kluyveromyces martensii* added is 3%-4%; for example, in some embodiments, the mass percentage of *Kluyveromyces martensii* added can be 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or 5%, or the amount of *Kluyveromyces martensii* added within the numerical range formed by any two of the above specific values ​​as endpoints; And / or, preferably, the temperature of the first fermentation is 28-32°C; for example, in some embodiments, the temperature of the first fermentation can be 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5 or 32°C, or a temperature within the range of any two of the above specific values ​​as endpoints. And / or, preferably, the first fermentation time is 24-72 hours; preferably, the first fermentation time is 24-48 hours; for example, in some embodiments, the first fermentation time is 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72 hours, or a time within the range of any two of the above specific values ​​as endpoints; And / or, preferably, after the first fermentation is completed, the initial fermentation broth is subjected to ultrasonic inactivation treatment; preferably, the temperature of ultrasonic inactivation treatment is 50-60℃; and / or, the time of ultrasonic inactivation treatment is 20-30 min.

[0060] Technical Solution 12: According to the preparation method of the fermented product with antioxidant activity described in Technical Solution 11, in step 3, the mass percentage of added Streptococcus salivarius relative to the total mass of the initial fermentation broth is 0.05%-0.2%; preferably, the mass percentage of added Streptococcus salivarius is 0.05%-0.15%; for example, in some embodiments, the mass percentage of added Streptococcus salivarius can be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% or 0.2%, or the amount of Streptococcus salivarius added within the numerical range formed by any two of the above specific values ​​as endpoints; And / or, the temperature of the second fermentation is 35-39°C; for example, in some embodiments, the temperature of the second fermentation can be 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5 or 39°C, or a temperature within the range of any two of the above specific values ​​as endpoints. And / or, the second fermentation time is 24-72 hours; preferably, the second fermentation time is 24-48 hours; for example, in some embodiments, the second fermentation time is 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72 hours, or a time within the range of any two of the above specific values ​​as endpoints.

[0061] Technical Solution 13: A method for preparing a fermented product with antioxidant activity according to any one of technical solutions 6-12, wherein, in step 4, the solid-liquid separation is centrifugal separation; preferably, the centrifugation speed is 5000-8000 rpm; and / or, preferably, the centrifugation time is 10-15 min; And / or, the supernatant obtained after solid-liquid separation is concentrated to obtain a fermented product; preferably, the supernatant obtained after solid-liquid separation is concentrated to 30%-70% of its original volume; and / or, preferably, the concentration temperature is 50-60℃.

[0062] Technical Solution 14: A method for preparing a fermented product with antioxidant activity according to any one of technical solutions 11-13, wherein the Kluyveromyces martensii is Kluyveromyces martensii powder; preferably, the number of live bacteria in the Kluyveromyces martensii powder is 1.0×10^10~1.2×10^10 CFU / g; And / or, the Streptococcus salivarius is Streptococcus salivarius powder; preferably, the number of live bacteria in the Streptococcus salivarius powder is 5.0×10^10~8.0×10^10 CFU / g.

[0063] Technical Solution 15: The method for preparing fermented products with antioxidant activity according to Technical Solution 14, wherein the method for preparing Kluyveromyces martensii powder includes the following steps: Step 1: Activate the Kluyveromycin yeast; Step 2: Cultivate the activated Kluyveromyces martensii, centrifuge to obtain the cells, add a freeze-drying protectant, freeze-dry to obtain the bacterial powder; Preferably, in step 1, the activation conditions are 28-32°C; the time is 22-26 h; and / or, by weight, the culture medium comprises 8-12 parts yeast extract, 18-22 parts glucose, 18-22 parts peptone and 800-1200 parts water; And / or, in step 2, the culture conditions are: temperature 28-32℃; time 22-26h; inoculum size of the strain 1%-3%; and / or, the culture medium includes peptone 9-11g / L, beef extract 9-11g / L, yeast extract 4-6g / L, glucose 19-21g / L, Tween 80 0.5-1.5g / L, dipotassium hydrogen phosphate 1-3g / L, sodium acetate 4-6g / L, triammonium citrate 1-3g / L, magnesium sulfate 0.05-0.15g / L, and manganese sulfate 0.01-0.1g / L; And / or, in step 2, the freeze-drying protectant comprises, by weight, 15-30 parts skim milk, 5-15 parts sorbitol and 5-15 parts trehalose.

[0064] Technical Solution 16: The method for preparing a fermented product with antioxidant activity according to Technical Solution 14, wherein the preparation method of Streptococcus salivarius powder includes the following steps: Step 1: Activate the salicylic streptococci; Step 2: Cultivate the activated Streptococcus salivarius, centrifuge to obtain bacterial cells, add a freeze-drying protectant, freeze-dry to obtain bacterial powder.

[0065] Technical Solution 17. A method for preparing a fermented product with antioxidant activity according to Technical Solution 16, wherein, in step 1, the activation conditions are a temperature of 35-39°C and a time of 22-26 h; and / or, by weight, the culture medium comprises 8-12 parts yeast extract, 18-22 parts glucose, 18-22 parts peptone and 800-1200 parts water; And / or, in step 2, the culture conditions are: temperature 35-39℃; time 22-26h; inoculum size of the strain 1%-3%; and / or, the culture medium includes: temperature 28-32℃; time 22-26h; inoculum size of the strain 1%-3%; and / or, the culture medium includes: peptone 9-11g / L, beef extract 9-11g / L, yeast extract 4-6g / L, glucose 19-21g / L, Tween 80 0.5-1.5g / L, dipotassium hydrogen phosphate 1-3g / L, sodium acetate 4-6g / L, triammonium citrate 1-3g / L, magnesium sulfate 0.05-0.15g / L, and manganese sulfate 0.01-0.1g / L; And / or, in step 2, the freeze-drying protectant comprises, by weight, 15-30 parts skim milk, 5-15 parts mannitol, 5-15 parts trehalose and 1-5 parts monosodium glutamate.

[0066] Technical Solution 18: A fermented product with antioxidant activity, characterized in that it is prepared by any one of the preparation methods of the fermented product with antioxidant activity described in Technical Solutions 6-17.

[0067] Technical Solution 19: The application of a fermented product with antioxidant activity as described in any one of Technical Solutions 1-5 or the fermented product with antioxidant activity as described in Technical Solution 18 in the preparation of food.

[0068] Technical Solution 20: A food product, characterized in that it includes any one of the fermented products with antioxidant activity described in Technical Solutions 1-5 or the fermented product with antioxidant activity described in Technical Solution 18.

[0069] Unless otherwise stated, all reagents / instruments used in the embodiments and comparative examples of this invention are conventional commercially available products. Information on the experimental materials and instruments used in this invention is shown in the table below: Table 1 Experimental Materials / Instruments and Manufacturers

[0070]

[0071] This invention does not limit the source of peptone; any commercially available or prepared yeast extract can be used in this invention. Preferably, the inventors have found that as long as the total nitrogen content of the commercially available or prepared peptone is ≥12% and / or the amino nitrogen content is ≥2%, it can be used in this invention. More preferably, the total nitrogen content of the peptone is 12%~17%, and / or the amino nitrogen content is 2%~5%.

[0072] In some specific embodiments, the present invention does not limit the source of the yeast extract powder, and any commercially available or prepared yeast extract powder can be used in the present invention; preferably, the inventors have found through research that as long as the total nitrogen content of the commercially available or prepared yeast extract powder is ≥10.0% and / or the amino nitrogen content is ≥5.0%, it can be used in the present invention; more preferably, the total nitrogen content of the yeast extract powder is 10%~15%, and / or the amino nitrogen content is 5%~8%.

[0073] 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.

[0074] Example 1: Strain screening, identification, and unique gene analysis Streptococcus salivarius thermophilus subspecies is Streptococcus salivarius thermophilus subspecies ( Streptococcus salivarius subsp. thermophilus Jiacha. ST-685 strain was obtained by screening homemade yogurt from herders in Jiacha County, Shannan City, Tibet Autonomous Region, using the following method: (1) Take 25g of homemade yogurt from a herdsman's family in Jiacha County, Shannan City, Tibet Autonomous Region and mix it with 225mL of sterile physiological saline to obtain a homogeneous sample solution. Then, perform serial dilution of the sample solution and take 10g of each solution. -3 10 -4 10 -5 10 -6 The diluted solution was spread on MRS agar plates and cultured at 32°C for 48 hours. Colonies grew on the MRS agar.

[0075] (2) Initial screening of bacterial strains Based on the standard colony characteristics of Streptococcus salivarius subsp. thermophilus, single colonies were picked and isolated and purified on MRS solid medium and cultured at 37℃ for at least three consecutive times to obtain purified colonies.

[0076] (3) Acid production experiment Single colonies obtained from the initial screening were cultured on MRS solid medium plates containing 0.2% CaCO3. After incubation at 32°C for 48 hours, the formation of a clear zone around the colony was observed. Single colonies with strong acid-producing ability and large clear zones were selected for further isolation and purification. In MRS solid medium, the colonies typically grow as round or nearly round, milky white or grayish-white colonies, exhibiting a smooth surface, small cell size, and slightly irregular edges.

[0077] (4) Gram staining Gram staining was performed on the strains obtained from the secondary screening. Those showing typical Gram staining positivity were identified as the target strain. Microscopic observation revealed that the cells were spherical, approximately 0.5-1.0 μm in diameter, without flagella, and did not produce spores or exhibit motility.

[0078] (5) Identification of strains The isolated and purified strain was Gram-positive, H2O2 catalase-negative, acid-producing, and non-gas-producing. 16S rDNA sequencing was performed, and the results were compared with those in the NCBI GenBank database for homology analysis. The results showed that this strain is *Streptococcus salivarius* subsp. *thermophilus*, and it was named *Streptococcus salivarius* subsp. *thermophilus* Jiacha. ST-685. Streptococcus salivarius subsp. thermophilusJiacha. ST-685), this strain was deposited at the China Center for Type Culture Collection (CCTCC) on August 17, 2023, with accession number CCTCC NO: M 20231495. The deposit address is: Wuhan University, Wuhan, China, Postcode: 430072; Telephone: (027) 68754052.

[0079] (6) Screening analysis of unique genes of Streptococcus salivarius subsp. thermophilus Jiacha. ST-685 Gene structure annotation of *Streptococcus thermophilus* subspecies *thermophilus* (genus *Streptococcus* - species *thermophilus*) was performed using Prokka. The complete genome of *Streptococcus thermophilus* subspecies *thermophilus* was downloaded from NCBI. Comparative genomic analysis was conducted between *Streptococcus thermophilus* subspecies *thermophilus* and *Streptococcus thermophilus* subspecies *thermophilus* using Roary. Multiple sequence alignment of core genes was then performed using MAFFT. A total of 42 unique genes were identified in *Streptococcus thermophilus* subspecies *thermophilus* *Jiacha.ST-685*, two of which are related to antioxidant function, with gene IDs ST685_01915 and ST685_01010.

[0080] The DNA sequence of gene ST685_01915 is shown in SEQ ID NO. 1, and the amino acid sequence it encodes is shown in SEQ ID NO. 3. The DNA sequence of gene ST685_01010 is shown in SEQ ID NO. 2, and the amino acid sequence it encodes is shown in SEQ ID NO. 4.

[0081] The gene ST685_01915 is responsible for encoding glycosyltransferase family 92 (GT92). Enzymes like GT92 participate in a variety of biological processes in organisms, including cell wall synthesis, glycoprotein modification, and signal transduction. They are particularly important in microorganisms because they can help bacteria synthesize polysaccharides. Extracellular polysaccharides of lactic acid bacteria can enhance antioxidant capacity through multiple mechanisms such as free radical scavenging, metal ion chelation, cell protection, and immune regulation.

[0082] The gene ST685_01010 encodes dextran sucrase activity. Dextran sucrase is a glucose transferase that primarily catalyzes the breakdown of sucrose into dextran and fructose. Dextran mainly affects antioxidant capacity through its molecular structure, immune regulation, and free radical scavenging ability. Low molecular weight dextran can activate the Nrf2 signaling pathway, promote the expression of HO-1 (heme oxygenase-1), and enhance the cell's resistance to oxidative damage. Dextran can reduce the activation of NF-κB (nuclear factor κB) and decrease the expression of pro-inflammatory cytokines (TNF-α, IL-6), thereby alleviating inflammation-related oxidative stress. Fructose is mainly involved in the regulation of metabolic pathways, inflammatory responses, and oxidative stress. Fructose metabolism can increase the supply of NADPH (Nicotinamide Adenine Dinucleotide Phosphate), promote GSH (Glutathione) regeneration, and enhance the antioxidant capacity of cells.

[0083] Example 2: Preparation of antioxidant-active fermented products A method for preparing a fermented product with antioxidant activity, comprising the following steps: Step 1: Preparation of fermentation base liquid Take 10 parts by weight of kudzu root, 10 parts by weight of purple sweet potato, and 5 parts by weight of oats, wash them, dry them (moisture content after drying is 8%), and pulverize them (particle size after pulverization is 120 mesh) to obtain a solid mixture. Add the solid mixture to drinking water at a material-to-liquid ratio of 1:10 (g:mL) to obtain the fermentation base liquid; Step 2: Enzymatic hydrolysis Relative to the total weight of the fermentation base liquid, 1.2 wt% of a compound enzyme preparation, comprising α-amylase and pectinase in a weight ratio of 7:3, was added to the fermentation base liquid. Enzymatic hydrolysis was performed for 4 hours at pH 5.5 and temperature 50℃. Separation was carried out for 10 minutes at a rotation speed of 5000 r / min, and the supernatant was retained. Sterilization was performed using pasteurization at a temperature of 75℃ for 15 minutes. Step 3: Fermentation Primary fermentation: Add 3 wt% (relative to the total weight of the supernatant retained after enzymatic hydrolysis) of Kluyveromyces martensii Linzhi. KM-D3 (preservation number CCTCC NO: M 20231575) bacterial powder (viable count of 1.0×10^10~1.2×10^10 CFU / g) to the supernatant retained after centrifugation, mix thoroughly, and let ferment statically at 30℃ for 48 h to obtain the preliminary fermentation broth; sonicate the preliminary fermentation broth at 60℃ for 25 min. The preparation method of Kluyveromyces martensii Linzhi. KM-D3 powder includes the following steps: (1) Kluyveromyces martensii Linzhi. KM-D3 was activated at a temperature of 30℃ for 24 hours. The medium used for activation was YPD medium. The preparation method of YPD liquid medium includes the following steps: 10g of yeast extract, 20g of glucose, 20g of peptone and water were mixed and the volume was adjusted to 1000 mL. The mixture was sterilized at 115℃ for 20 min to obtain YPD liquid medium.

[0084] (2) The activated Kluyveromyces martensii Linzhi. KM-D3 was inoculated into MRS liquid medium at an inoculation rate of 2% and cultured at 30℃ for 24 h. The bacterial sludge was collected by centrifugation (4℃, 8000 r / min, 10 min), washed three times with sterile physiological saline, and then freeze-dried with freeze-drying agent at a ratio of bacterial cells to freeze-drying agent of 1:3 (w / w) to obtain bacterial powder. The freeze-drying agent formulation included 20 parts skim milk, 10 parts sorbitol and 10 parts trehalose by weight.

[0085] Secondary fermentation: 0.1 wt% (relative to the total weight of the primary fermentation broth) of Streptococcus salivarius subsp. thermophilus Jiacha. ST-685 (preservation number: CCTCC NO: M 20231495) bacterial powder (viable count of 5.0×10^10~8.0×10^10 CFU / g) was added to the primary fermentation broth, and the mixture was sealed and fermented at 37℃ for 48 hours to obtain the fermentation broth; The preparation method of Streptococcus salivarius thermophilus subspecies Jiacha. ST-685 bacterial powder includes the following steps: (1) Activate Streptococcus salivarius subsp. thermophilicus Jiacha. ST-685. The activation conditions are: temperature 37℃, time 24h. The activation medium is YPD medium (seller: Guangdong Huankai Microbial Technology Co., Ltd.). The preparation method of YPD liquid medium includes the following steps: take 10g of yeast extract powder, 20g of glucose, 20g of peptone and water, mix them and make up to 1000 mL. Sterilize at 115℃ for 20 min to prepare YPD liquid medium.

[0086] (2) Activated Streptococcus salivarius subsp. thermophilus Jiacha. ST-685 was inoculated into MRS medium at an inoculum rate of 2% and cultured at 37°C for 24 h. The bacterial sludge was collected by centrifugation (4°C, 8000 r / min, 10 min), washed three times with sterile physiological saline, and then freeze-dried with lyophilization protectant at a ratio of bacterial cells to lyophilization protectant of 1:4 (w / w) to obtain bacterial powder. The lyophilization protectant formulation, by weight, included 20 parts skim milk, 10 parts mannitol, 10 parts trehalose, and 3 parts monosodium glutamate.

[0087] Step 4: Separation and Concentration The fermentation broth was centrifuged at 5000 rpm for 10 min, and the supernatant was retained. The supernatant was concentrated to 50% of its original volume at 55℃ to obtain a fermented product with antioxidant properties.

[0088] Example 3: Preparation of antioxidant-active fermented products A method for preparing a fermented product with antioxidant activity, comprising the following steps: Step 1: Preparation of fermentation base liquid Take 20 parts by weight of kudzu root, 5 parts by weight of purple sweet potato, and 5 parts by weight of oats, wash them, dry them (moisture content after drying is 8%), and pulverize them (particle size after pulverization is 120 mesh) to obtain a solid mixture. Add the solid mixture to drinking water at a material-to-liquid ratio of 1:10 (g:mL) to obtain the fermentation base liquid; Steps 2-4 are the same as in Example 2.

[0089] Example 4: Preparation of antioxidant-active fermented products A method for preparing a fermented product with antioxidant activity, comprising the following steps: Step 1: Preparation of fermentation base liquid Take 18 parts by weight of kudzu root, 8 parts by weight of purple sweet potato, and 8 parts by weight of oats, wash them, dry them (moisture content after drying is 8%), and pulverize them (particle size after pulverization is 120 mesh) to obtain a solid mixture. Add the solid mixture to drinking water at a material-to-liquid ratio of 1:10 (g:mL) to obtain the fermentation base liquid; Steps 2-4 are the same as in Example 2.

[0090] Example 5: Preparation of antioxidant-active fermented products A method for preparing a fermented product with antioxidant activity, comprising the following steps: Step 1: Preparation of fermentation base liquid Take 18 parts by weight of kudzu root, 8 parts by weight of purple sweet potato, and 8 parts by weight of oats, wash them, dry them (moisture content after drying is 8%), and pulverize them (particle size after pulverization is 120 mesh) to obtain a solid mixture. Add the solid mixture to drinking water at a material-to-liquid ratio of 1:10 (g:mL) to obtain the fermentation base liquid; Step 2: Enzymatic hydrolysis Add 1.2 wt% (relative to the total weight of the fermentation base liquid) of a compound enzyme preparation, which includes α-amylase and pectinase in a weight ratio of 5:1, to the fermentation base liquid. Enzymatic hydrolysis was carried out for 3 hours at pH 5.5 and temperature 60℃. Separation was performed for 10 minutes at a speed of 5000 r / min, and the supernatant was retained. Sterilization was carried out by pasteurization at a temperature of 75℃ for 15 minutes. Steps 3-4 are the same as in Example 2.

[0091] Example 6: Preparation of antioxidant-active fermented products A method for preparing a fermented product with antioxidant activity, comprising the following steps: Step 1: Preparation of fermentation base liquid Take 18 parts by weight of kudzu root, 8 parts by weight of purple sweet potato, and 8 parts by weight of oats, wash them, dry them (moisture content after drying is 8%), and pulverize them (particle size after pulverization is 120 mesh) to obtain a solid mixture. Add the solid mixture to drinking water at a material-to-liquid ratio of 1:10 (g:mL) to obtain the fermentation base liquid; Step 2: Enzymatic hydrolysis Add 1.2 wt% (relative to the total weight of the fermentation base liquid) of a compound enzyme preparation, which includes α-amylase and pectinase in a weight ratio of 7:3, to the fermentation base liquid. Enzymatic hydrolysis was carried out for 3 hours at pH 5.5 and temperature 55℃. Separation was performed for 10 minutes at a speed of 5000 r / min, and the supernatant was retained. Sterilization was carried out by pasteurization at a temperature of 75℃ for 15 minutes. Steps 3-4 are the same as in Example 2.

[0092] Example 7: Preparation of antioxidant-active fermented products A method for preparing a fermented product with antioxidant activity, comprising the following steps: Steps 1-2 are the same as in Example 2.

[0093] Step 3: Fermentation Primary fermentation: Add 5 wt% (relative to the total weight of the supernatant retained after enzymatic hydrolysis and centrifugation) of Kluyveromyces martensii Linzhi. KM-D3 (preservation number CCTCC NO: M 20231575) mycelial powder (the preparation steps of the mycelial powder are the same as in Example 2) to the supernatant retained after enzymatic hydrolysis and centrifugation, mix thoroughly, and let it ferment statically at 28°C for 24 h to obtain a preliminary fermentation broth; sonicate the preliminary fermentation broth at 60°C for 25 min; Secondary fermentation: Add 0.05wt% (relative to the total weight of the primary fermentation broth) of Streptococcus thermophilus subsp. Jiacha.ST-685 (CCTCC NO: M 20231495) bacterial powder (the preparation steps of the bacterial powder are the same as in Example 2) to the primary fermentation broth, and seal and ferment at 35°C for 48 hours to obtain the fermentation broth; Step 4 is the same as in Example 2.

[0094] Example 8: Preparation of antioxidant-active fermented products A method for preparing a fermented product with antioxidant activity, comprising the following steps: Step 1: Preparation of fermentation base liquid: Take 10 parts by weight of kudzu root and 10 parts by weight of purple sweet potato, wash them, dry them (moisture content after drying is 8%), and pulverize them (particle size after pulverization is 120 mesh) to obtain a solid mixture. Add the solid mixture to drinking water at a material-to-liquid ratio of 1:10 (g:mL) to obtain the fermentation base liquid; Steps 2-4 are the same as in Example 2.

[0095] Comparative Example 1: Preparation of Fermented Products with Antioxidant Activity A method for preparing a fermented product with antioxidant activity, comprising the following steps: Step 1: Preparation of fermentation base liquid: Take 10 parts by weight of kudzu root, 10 parts by weight of purple sweet potato, and 5 parts by weight of oats, wash them, dry them (moisture content after drying is 8%), and pulverize them (particle size after pulverization is 120 mesh) to obtain a solid mixture. Add the solid mixture to drinking water at a material-to-liquid ratio of 1:10 (g:mL) to obtain the fermentation base liquid; Step 2: Enzymatic hydrolysis Add 1.2 wt% (relative to the total weight of the fermentation base liquid) of a compound enzyme preparation, which includes α-amylase and pectinase in a weight ratio of 7:3, to the fermentation base liquid. Enzymatic hydrolysis was carried out at pH 5.5 and 60°C for 4 hours. Separation was performed at 5000 r / min for 10 minutes, and the supernatant was retained. Sterilization was carried out by pasteurization at 75°C for 15 minutes. Step 3: Fermentation Primary fermentation: 0.1 wt% (relative to the total weight of the supernatant retained after enzymatic hydrolysis and centrifugation) of Streptococcus salivarius subsp. thermophilus Jiacha.ST-685 (preservation number: CCTCC NO: M20231495) bacterial powder (preparation method is the same as in Example 2) was added to the supernatant retained after enzymatic hydrolysis and centrifugation. The mixture was then sealed and fermented at 37°C for 36 h to obtain the preliminary fermentation broth. Step 4 is the same as in Example 2.

[0096] Comparative Example 2: Preparation of Fermented Products with Antioxidant Activity A method for preparing a fermented product with antioxidant activity, comprising the following steps: Step 1: Preparation of fermentation base liquid: Take 10 parts by weight of kudzu root, 10 parts by weight of purple sweet potato, and 5 parts by weight of oats, wash them, dry them (moisture content after drying is 8%), and pulverize them (particle size after pulverization is 120 mesh) to obtain a solid mixture. Add the solid mixture to drinking water at a material-to-liquid ratio of 1:10 (g:mL) to obtain the fermentation base liquid; Step 2: Enzymatic hydrolysis Add 1.2 wt% (relative to the total weight of the fermentation base liquid) of a compound enzyme preparation, which includes α-amylase and pectinase in a weight ratio of 7:3, to the fermentation base liquid. Enzymatic hydrolysis was carried out at pH 5.0 and 60°C for 4 hours. Separation was performed at 5000 r / min for 10 minutes, and the supernatant was retained. Sterilization was carried out by pasteurization at 75°C for 15 minutes. Step 3: Fermentation Primary fermentation: Add 3 wt% (relative to the total weight of the supernatant retained after enzymatic hydrolysis and centrifugation) of Kluyveromyces martensii Linzhi. KM-D3 (preservation number CCTCC NO: M 20231575) to the supernatant retained after enzymatic hydrolysis and centrifugation, mix thoroughly, and ferment at 28℃ for 72 h to obtain the preliminary fermentation broth; sonicate the preliminary fermentation broth at 55℃ for 20 min. Step 4 is the same as in Example 2.

[0097] Comparative Example 3: Preparation of Fermented Products with Antioxidant Activity A method for preparing a fermented product with antioxidant activity, comprising the following steps: Step 1: Preparation of fermentation base liquid Take 10 parts by weight of kudzu root, 10 parts by weight of purple sweet potato, and 5 parts by weight of oats, wash them, dry them (moisture content after drying is 8%), and pulverize them (particle size after pulverization is 120 mesh) to obtain a solid mixture. Add the solid mixture to drinking water at a material-to-liquid ratio of 1:10 (g:mL) to obtain the fermentation base liquid; Step 2: Enzymatic hydrolysis: Add 2 wt% (relative to the total weight of the fermentation base liquid) of a compound enzyme preparation, which includes α-amylase and pectinase in a weight ratio of 7:3, and enzymatically hydrolyze for 4 h at pH 5.5 and 60 °C; separate for 10 min at 5000 r / min, and retain the supernatant; sterilize by pasteurization at 75 °C for 15 min. Steps 3-4 are the same as in Example 2.

[0098] Comparative Example 4: Preparation of Fermented Products with Antioxidant Activity A method for preparing a fermented product with antioxidant activity, comprising the following steps: Steps 1-2 are the same as in Example 2.

[0099] Step 3: Fermentation Primary fermentation: Add 3 wt% (relative to the total weight of the supernatant retained after enzymatic hydrolysis and centrifugation) of Kluyveromyces martensii Linzhi. KM-D3 (preservation number CCTCC NO: M20231575) mycelial powder (the preparation steps of the mycelial powder are the same as in Example 2) to the supernatant retained after enzymatic hydrolysis and centrifugation, mix thoroughly, and ferment at 32°C for 24 h to obtain a preliminary fermentation broth; sonicate the preliminary fermentation broth at 55°C for 25 min; Secondary fermentation: Add 0.1wt% (relative to the total weight of the primary fermentation broth) of Streptococcus thermophilus CICC 6038 bacterial powder to the primary fermentation broth, and seal and ferment at 39℃ for 48 hours to obtain the fermentation broth; Step 4 is the same as in Example 2.

[0100] Comparative Example 5: Preparation of Fermented Products with Antioxidant Activity A method for preparing a fermented product with antioxidant activity, comprising the following steps: Steps 1-2 are the same as in Example 2.

[0101] Step 3: Fermentation Primary fermentation: Add 3 wt% (relative to the total weight of the supernatant retained after enzymatic hydrolysis and centrifugation) of Kluyveromyces martensii Linzhi. KM-D3 (preservation number CCTCC NO: M20231575) mycelial powder (the preparation steps of the mycelial powder are the same as in Example 2) to the supernatant retained after enzymatic hydrolysis and centrifugation, mix thoroughly, and ferment at 28°C for 48 h to obtain a preliminary fermentation broth; sonicate the preliminary fermentation broth at 60°C for 20 min; Secondary fermentation: Add 0.1wt% (relative to the total weight of the primary fermentation liquid) of Lactobacillus plantarum ATCC 8014 bacterial powder to the primary fermentation liquid, and seal and ferment at 40℃ for 48 hours to obtain fermentation liquid; Step 4 is the same as in Example 2.

[0102] Comparative Example 6 A method for preparing a fermented product with antioxidant activity, comprising the following steps: The preparation of the fermentation base liquid in step 1 is the same as in Example 2.

[0103] Step 2: Enzymatic hydrolysis Add 1.2 wt% (relative to the total weight of the fermentation base liquid) of a compound enzyme preparation, which includes cellulase and pectinase in a weight ratio of 7:3, and perform enzymatic hydrolysis for 4 h at pH 5.5 and temperature 50 °C; separate for 10 min at a rotation speed of 5000 r / min, and retain the supernatant; sterilize by pasteurization at a temperature of 75 °C for 15 min. Step 3: Fermentation Primary fermentation: Add 3 wt% (relative to the total weight of the supernatant retained after enzymatic hydrolysis and centrifugation) of Kluyveromyces martensii Linzhi. KM-D3 (preservation number CCTCC NO: M20231575) mycelial powder (the preparation steps of the mycelial powder are the same as in Example 2) to the supernatant retained after centrifugation and centrifugation, mix thoroughly, and ferment at 33°C for 24 h to obtain a preliminary fermentation broth; sonicate the preliminary fermentation broth at 50°C for 20 min; Secondary fermentation: 0.1 wt% (relative to the total weight of the primary fermentation broth) of Streptococcus salivarius thermophilic subsp. Jiacha. ST-685 (preservation number: CCTCC NO: M 20231495) bacterial powder was added to the primary fermentation broth (the preparation steps of the bacterial powder are the same as in Example 2), and the broth was sealed and fermented at 40°C for 24 hours to obtain the fermentation broth; Step 4, the separation and concentration, is the same as in Example 2.

[0104] Application Example 1: Determination of Polysaccharide Content (1) Extraction of crude polysaccharides The fermentation products prepared in the examples and comparative examples were heated in a 90°C water bath for 10 min to remove enzymes that might degrade polysaccharides, and then cooled to room temperature. 80% trichloroacetic acid (TCA) solution was added to the fermentation product until the concentration was 5% (m / v), and the mixture was stirred at room temperature for 2 h. The mixture was then centrifuged at 8000 rpm for 15 min at 4°C, and the supernatant was retained to remove any precipitates containing cells and proteins. Then, three times the volume of 95% ethanol solution was added to the supernatant, and the mixture was incubated overnight at 4°C. The ethanol-precipitated solution was centrifuged at 8000 rpm for 15 min at 4°C. Finally, the supernatant was placed in a dialysis bag (molecular weight cutoff 14000 Da) and dialyzed with ultrapure water at 4°C for 2 days, changing the water every 8 hours.

[0105] For the preparation of 80% trichloroacetic acid (TCA) solution: Accurately weigh 80.0 g of TCA solid into a beaker, slowly add approximately 50-60 mL of deionized water while stirring. After the solution cools to room temperature, transfer it entirely to a 100 mL volumetric flask, top up with deionized water to the mark, and mix well. Transfer to a reagent bottle and store at 4°C.

[0106] (2) Determination of polysaccharide content The content of EPS (extracellular polysaccharides) was determined by the phenol-sulfuric acid method, and a standard curve was prepared using glucose as a standard.

[0107] An appropriate amount of analytically pure glucose was placed in a forced-air drying oven and dried at 80℃ for 2 h. After cooling, 100 mg of glucose was accurately weighed into a 500 mL volumetric flask, and distilled water was added to the mark. The volumes of each solution in the reaction system were added according to the table below. First, the standard glucose solution was added to a stoppered graduated test tube, then 6% (v / v) phenol solution was added, and finally 10 mL of concentrated sulfuric acid was added, mixed well, and allowed to stand. After cooling, the absorbance at a wavelength of 490 nm was measured, with three replicates per group. A standard curve was plotted with glucose content (mg / L) on the x-axis and absorbance value (A490) on the y-axis. The absorbance of the dialysis-treated solutions prepared in each example and comparative example was measured at 490 nm using the same method, and the EPS yield was calculated using a regression equation.

[0108] Table 2 Data on extracellular polysaccharide content

[0109] As can be seen from the results in Table 2, the fermented products prepared in Examples 1-8 can significantly produce extracellular polysaccharides compared with the fermented products prepared in Comparative Examples 1-6. The extracellular polysaccharides of lactic acid bacteria can enhance antioxidant capacity through multiple mechanisms such as free radical scavenging, metal ion chelation, cell protection, and immune regulation.

[0110] Application Example 2: Cellular Experiments on Antioxidant Effects Assay of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine), SOD (superoxide dismutase), and GSH-Px (glutathione peroxidase) activities. Subculture normal human colonic epithelial cells (sold by Beijing Bio-Innovation Technology Co., Ltd.). Subculturing steps: Discard the old culture medium from the culture flask. Add an appropriate amount of preheated PBS buffer (sold by Beijing Solarbio Science & Technology Co., Ltd.), gently agitate to wash the cell surface and remove residual serum and dead cells. Discard the PBS buffer. Add an appropriate amount of preheated trypsin-EDTA digestion solution (sold by Beijing Solarbio Science & Technology Co., Ltd.), just enough to cover the cell layer. Gently agitate to evenly cover the cells, and immediately return the flask to a 37°C incubator for incubation (usually only 1-3 minutes). Observe closely under a microscope. When most cells become rounded, the edges shrink, the intercellular spaces increase, and a small number of cells begin to float, immediately stop digestion; do not over-digest. Termination of digestion: Quickly add an equal or twice the volume of complete culture medium (composition: DMEM high-glucose medium (Thermo Fisher Scientific), Hepes (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) 20 mmol / L, insulin 200 U / L, heparin sodium 100 mg / L, FBS (fetal bovine serum) 25 mL / L, EGF (epidermal growth factor) 10 ng / mL, bovine pituitary extract 5 mg / L, penicillin 10,000 units / mL, streptomycin 100 μg / mL, gentamicin 100 μg / mL, amphotericin B 3 μg / mL). Gently and repeatedly pipette the bottom of the culture flask to completely detach the cells, forming a single-cell suspension. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at a relatively low speed (e.g., 800-1200 rpm) for 5 minutes at room temperature to pellet the cells. Resuspension and Counting: After centrifugation, carefully aspirate the supernatant, being careful not to touch the cell clumps at the bottom of the tube. Add an appropriate amount of fresh complete culture medium and gently pipette (avoiding air bubbles) to redisperse the cell clumps into a uniform single-cell suspension. A small amount of the suspension can be used for cell counting to determine the subsequent seeding density. Seeding in Flasks: Based on the counting results and the required passage ratio (usually 1:2 to 1:4), add the cell suspension to new culture flasks in the correct ratio and replenish with sufficient fresh complete culture medium. Gently shake the culture flasks back and forth and side to side to distribute the cells evenly. Continued Culture: Label the culture flasks (cell name, passage number, date, operator). Return the culture flasks to a 37°C, 5% CO2 incubator for continued culture. Replace the culture medium with fresh complete medium every 2-3 days, depending on cell growth.

[0111] Cells from the third generation of passage culture were added to cell culture medium (i.e., complete medium), followed by fermentation products prepared in the examples or comparative studies at a concentration of 15 mg / L. Each treatment group was configured in triplicate. Culture conditions were: gas phase: 95% air and 5% carbon dioxide; temperature: 37℃; humidity: 70%-80%. Antioxidant enzyme activity was measured in cells cultured for 72 h: cells from each group were collected and sonicated. After centrifugation at 1000 rpm for 5 min, the supernatant was collected, and DPPH, SOD, and GSH-Px activities were measured (refer to the kit instructions). The kit for DPPH detection was catalog number BC4755, sold by Beijing Solarbio Science & Technology Co., Ltd.; the kit for SOD activity detection was catalog number BC0170, sold by Beijing Solarbio Science & Technology Co., Ltd.; and the kit for GSH-Px activity detection was catalog number SY6454, sold by Beijing Solarbio Science & Technology Co., Ltd. The results are shown in Table 3.

[0112] Table 3. Results of DPPH, SOD and GSH-Px activity assays

[0113] As can be seen from the results in Table 3, the fermentation products prepared in Examples 1-8 significantly improved the DPPH free radical scavenging rate and significantly improved the activities of SOD and GSH-Px compared with the fermentation products prepared in Comparative Examples 1-6.

[0114] Application Example 3: Sensory Evaluation of Antioxidant Fermented Products The sensory evaluation panel consisted of 15 trained personnel with certain professional knowledge. They evaluated the form, color, taste and aroma of the fermented products according to the sensory scoring criteria, as shown in Table 4. The full score was 100 points, and the average value was taken as the final sensory score, as shown in Table 5.

[0115] Table 4 Sensory Evaluation Standards for Fermented Products

[0116] Table 5 Sensory Evaluation Scores for Antioxidant Fermented Products

[0117] As shown in Table 5, the antioxidant fermented products prepared in the embodiments of the present invention have good scores in terms of morphology, color, taste and aroma. They have a delicate and silky taste, a fresh aroma and a moderate sweet and sour taste.

[0118] Table 6 Sequence List

[0119]

[0120]

[0121]

[0122] While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A fermented product with antioxidant activity, characterized in that, It is prepared by preparing the raw materials into an aqueous mixture, then adding an enzyme preparation for enzymatic hydrolysis, and then adding a fermenting agent to ferment the resulting hydrolysate. The raw materials, by weight, include 8-22 parts kudzu root, 3-12 parts purple sweet potato, and 0-10 parts oats; the amount of enzyme preparation added is 1wt%-1.5wt% relative to the total mass of the aqueous mixture of the raw materials; the fermenting agent includes Streptococcus salivarius and Kluyveromyces martensii.

2. The fermented product with antioxidant activity according to claim 1, wherein, The raw materials comprise 10-20 parts by weight of kudzu root; preferably, the raw materials comprise 16-19 parts by weight of kudzu root. And / or, by weight, the raw material comprises 5-10 parts purple sweet potato; preferably, the raw material comprises 7-9 parts purple sweet potato; And / or, by weight, the raw materials comprise 5 to 10 parts of oats; preferably, the raw materials comprise 7 to 9 parts of oats.

3. The fermented product with antioxidant activity according to claim 1 or 2, wherein, The *Streptococcus salivarius* contains DNA sequences with antioxidant activity, as shown in SEQ ID NO. 1 and / or SEQ ID NO. 2; And / or, the *Streptococcus salivarius* includes an amino acid sequence with antioxidant activity, as shown in SEQ ID NO. 3 and / or SEQ ID NO. 4; And / or, the *Streptococcus salivarius* is strain Jiacha.ST-685, with accession number CCTCCNO: M20231495.

4. The fermented product with antioxidant activity according to any one of claims 1-3, wherein, The Kluyveromyces masculinus mentioned is Kluyveromyces masculinus Linzhi. KM-D3, with the accession number CCTCC NO: M 20231575.

5. The fermented product with antioxidant activity according to any one of claims 1-4, wherein, The content of extracellular polysaccharides in the fermented product is 500-1100 mg / L; Preferably, the content of extracellular polysaccharides in the fermented product is 800-1100 mg / L; More preferably, the content of extracellular polysaccharides in the fermented product is 900-1100 mg / L.

6. A method for preparing a fermented product with antioxidant activity as described in any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Mix the raw materials with water to obtain an aqueous mixture; Step 2: Add enzyme preparation to the mixture to carry out enzymatic hydrolysis, and obtain the enzymatic hydrolysate; Step 3: Add a starter culture to the enzyme hydrolysate to ferment and obtain the fermentation broth; Step 4: Separate the solid and liquid components of the fermentation broth. The resulting liquid is the fermentation product with antioxidant activity.

7. The method for preparing the fermented product with antioxidant activity according to claim 6, wherein, In step 1, the raw materials are mixed with water to obtain an aqueous mixture, also known as a fermentation base liquid; preferably, the raw materials are dried and pulverized, and then mixed with water to obtain the fermentation base liquid; more preferably, the moisture content of the dried raw materials is 0-10%; and / or, even more preferably, the particle size of the dried raw materials after pulverization is 100-150 mesh. And / or, preferably, the mass ratio of the raw material to the volume of water is 1:8-15, wherein the mass is in g and the volume is in mL; more preferably, the mass ratio of the raw material to the volume of water is 1:9-11, wherein the mass is in g and the volume is in mL.

8. The method for preparing the fermented product with antioxidant activity according to claim 6 or 7, wherein, In step 2, the enzyme preparation accounts for 1.1%-1.3% of the total mass of the fermentation base liquid. Preferably, the enzyme preparation includes α-amylase and pectinase; More preferably, the mass ratio of α-amylase to pectinase is 4-8:0.5-4; even more preferably, the mass ratio of α-amylase to pectinase is 5-8:1-4; and even more preferably, the mass ratio of α-amylase to pectinase is 6-8:2-4. And / or, more preferably, the activity of α-amylase is 240,000-280,000 u / mL; and / or, the activity of pectinase is 50,000-70,000 u / g.

9. A method for preparing a fermented product with antioxidant activity according to any one of claims 6-8, wherein, In step 2, the enzymatic hydrolysis temperature is 45-65℃; preferably, the enzymatic hydrolysis temperature is 50-60℃. And / or, the enzymatic hydrolysis time is 2-5 hours; preferably, the enzymatic hydrolysis time is 3-4 hours; And / or, the pH of the enzymatic hydrolysis is 5.0-6.0; preferably, the pH of the enzymatic hydrolysis is 5.3-6.

7.

10. A method for preparing a fermented product with antioxidant activity according to any one of claims 6-9, wherein, In step 2, after the enzymatic hydrolysis is completed, the solid and liquid are separated, the supernatant is taken, sterilized, and then subjected to subsequent fermentation treatment. Preferably, the solid-liquid separation is centrifugal separation; more preferably, the centrifugation speed is 4000-6000 rpm, and / or the centrifugation time is 5-10 min; And / or, the sterilization temperature is 75-80℃, and / or, the sterilization time is 15-30 min.

11. The method for preparing a fermented product with antioxidant activity according to any one of claims 6-10, wherein, In step 3, *Kluyveromyces martensii* is first added to the enzymatic hydrolysate obtained in step 2 for the first fermentation to obtain the preliminary fermentation broth; then *Streptococcus salivarius* is added for the second fermentation to obtain the fermentation broth. Preferably, the mass percentage of *Kluyveromyces martensii* relative to the total mass of the supernatant of the enzymatic hydrolysate is 2%-6%; more preferably, the mass percentage of *Kluyveromyces martensii* is 3%-5%; more preferably, the mass percentage of *Kluyveromyces martensii* is 3%-4%. And / or, preferably, the temperature for the first fermentation is 28-32°C; And / or, preferably, the first fermentation time is 24-72 hours; preferably, the first fermentation time is 24-48 hours; And / or, preferably, after the first fermentation is completed, the initial fermentation broth is subjected to ultrasonic inactivation treatment; preferably, the temperature of ultrasonic inactivation treatment is 50-60℃; and / or, the time of ultrasonic inactivation treatment is 20-30 min.

12. The method for preparing the fermented product with antioxidant activity according to claim 11, wherein, In step 3, the mass percentage of *Streptococcus salivarius* relative to the total mass of the initial fermentation broth is 0.05%-0.2%; preferably, the mass percentage of *Streptococcus salivarius* is 0.05%-0.15%. And / or, the temperature for the second fermentation is 35-39℃; And / or, the second fermentation time is 24-72 hours; preferably, the second fermentation time is 24-48 hours.

13. The method for preparing the fermented product with antioxidant activity according to any one of claims 6-12, wherein, In step 4, the solid-liquid separation is centrifugal separation; preferably, the centrifugation speed is 5000-8000 rpm; and / or, preferably, the centrifugation time is 10-15 min; And / or, the supernatant obtained after solid-liquid separation is concentrated to obtain a fermented product; preferably, the supernatant obtained after solid-liquid separation is concentrated to 30%-70% of its original volume; and / or, preferably, the concentration temperature is 50-60℃.

14. A fermented product with antioxidant activity, characterized in that, It is prepared by the method for preparing the fermented product with antioxidant activity as described in any one of claims 6-12.

15. The use of a fermented product with antioxidant activity as described in any one of claims 1-5 or the fermented product with antioxidant activity as described in claim 14 in the preparation of food.

16. A food product, characterized in that, It includes the fermented product with antioxidant activity as described in any one of claims 1-5 or the fermented product with antioxidant activity as described in claim 14.

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