A method for synergistically preparing high-antioxidant biological bean curd by composite enzyme-probiotics-low-temperature plasma

Abstract

The application discloses a method for synergistically preparing high-antioxidant biological bean curd by using composite enzymes, probiotics and low-temperature plasma, and belongs to the technical field of deep processing of soybeans. The method comprises three key steps: composite biological enzymolysis pretreatment for optimizing antioxidant substances in soybean milk, probiotic synergistic fermentation for strengthening generation and transformation of antioxidant active ingredients, and low-temperature plasma treatment for activating antioxidant components in the bean curd. Experimental results show that the free radical scavenging capacity and oxidation stability of the high-antioxidant biological bean curd prepared by the method are significantly improved, and the nutritional quality is significantly optimized. The method has the advantages of mild production process, high retention rate of antioxidant components, and synergistic improvement of the nutritional value and functional characteristics of the product, and provides technical support for overcoming the defects of insufficient antioxidant activity, easy oxidation deterioration and single nutritional components of the bean curd.

Description

Technical Field

[0001] This invention relates to the field of soybean deep processing technology, and in particular to a method for the synergistic preparation of highly antioxidant bio-tofu using a compound enzyme-probiotic-low-temperature plasma. Background Technology

[0002] Soybeans are a high-quality and inexpensive plant protein source, with a dry-basis protein content of 35% to 40%, forming a distinctive traditional dietary system based on tofu. Tofu is low in calories and high in nutrient density, rich in active ingredients such as soy isoflavones and soy saponins, combining nutritional and health value, aligning with the global trend of upgrading to plant-based foods, and market demand continues to grow. However, tofu is prone to oxidative deterioration during processing and cold storage, which restricts product quality and shelf life. The unsaturated fatty acids and soy isoflavones in the tofu system are easily oxidized and depleted by free radicals, resulting in a significant decrease in antioxidant activity; the oxidation to form aldehydes and ketones can cause rancid odors, while the oxidative cross-linking of soy protein reduces the system's water retention and gel strength, resulting in a rough texture and hard consistency in the finished product, significantly reducing its marketability.

[0003] Traditional brine / gypsum coagulation and atmospheric pressure boiling processes are ineffective in inhibiting tofu oxidation. Existing antioxidant modification methods all have limitations: chemical antioxidants raise food safety concerns and do not meet the consumption needs of natural and healthy foods; single biological treatments have limited antioxidant capacity and are difficult to inhibit immediate oxidation during processing; physical sterilization treatments such as high pressure and microwaves can easily damage the spatial structure of soybean protein and instead promote the accumulation of oxidation products.

[0004] Based on the aforementioned deficiencies of existing technologies, this invention proposes to optimize and improve the antioxidant and storage preservation processes of tofu using a composite enzymatic hydrolysis-fermentation-low-temperature plasma synergistic technology, aiming to enhance the antioxidant activity of tofu, improve the texture and flavor of the product, enrich the nutritional components of tofu, and provide a feasible technical solution for improving the quality and efficiency of deep-processed soybean protein products. Summary of the Invention

[0005] The purpose of this invention is to provide a method for synergistic preparation of highly antioxidant bio-tofu using a combination of compound enzymes, probiotics, and low-temperature plasma, in order to solve the problems existing in the prior art. The method provided by this invention optimizes the antioxidant material basis of soy milk through compound enzymatic hydrolysis pretreatment, enhances the generation and transformation of active antioxidant components by combining probiotic fermentation, and then uses low-temperature plasma technology to activate the functional antioxidant components in tofu, ultimately achieving enhanced antioxidant performance, optimized nutritional quality, and expanded application value.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing antioxidant-rich bio-tofu, comprising the following steps: Soak soybeans, grind them, filter and collect the soy milk, sterilize and cool it, mix it with a compound enzyme preparation, carry out the first enzymatic hydrolysis reaction, add protease to carry out the second enzymatic hydrolysis reaction, sterilize it, and obtain enzymatically hydrolyzed soy milk. The compound bacterial solution is inoculated into the enzymatically hydrolyzed soybean milk, fermented, stirred, coagulant is added, and the mixture is kept warm to obtain curd. The curd is transferred to a mold and pressed to obtain compound bacteria fermented tofu. The antioxidant bio-tofu is obtained by subjecting the fermented tofu with the compound bacteria to low-temperature plasma treatment.

[0007] Furthermore, the compound enzyme preparation comprises superoxide dismutase, catalase, and glutathione peroxidase in a mass ratio of 0.02:1:0.33.

[0008] Furthermore, the amount of the compound enzyme preparation added is 0.2% by mass fraction; The amount of protease added is 0.15%.

[0009] Furthermore, the temperature of the first enzymatic hydrolysis reaction and the second enzymatic hydrolysis reaction are 40°C, the stirring rate is 500 rpm, and the time is 15 min.

[0010] Furthermore, the method for preparing the composite bacterial solution includes the following steps: Lactobacillus plantarum ( Lactobacillus plantarum ) and Lactobacillus helveticus ( Lactobacillus Swiss The bacteria were activated, passaged, mixed, and cultured at a constant temperature to obtain the mixed bacterial solution.

[0011] Furthermore, in the compound bacterial solution, the ratio of the number of viable Lactobacillus plantarum to the number of viable Lactobacillus helveticus is 1:1.

[0012] Furthermore, the inoculation amount of the compound bacterial solution is 4% by volume fraction.

[0013] Furthermore, the fermentation temperature is 37°C and the time is 5 hours.

[0014] Furthermore, the low-temperature plasma treatment has a power of 60 W, a discharge gap of 5 mm, and a time of 2 min.

[0015] The present invention also provides an antioxidant bio-tofu obtained according to the above preparation method.

[0016] The present invention discloses the following technical effects: This invention employs a combination of enzymatic hydrolysis, probiotic synergistic fermentation, and low-temperature plasma technology to enhance the antioxidant properties of tofu. Using soybean milk as the raw material, a complex of enzymes (superoxide dismutase, catalase, and glutathione peroxidase-protease) is used to pre-treat the soy milk through enzymatic hydrolysis to release antioxidant active substances. Then, probiotic synergistic fermentation further enhances the conversion and accumulation of antioxidant components. Finally, magnesium sulfate is used as a coagulant to prepare tofu coagulated through enzymatic hydrolysis and fermentation, which is then subjected to low-temperature plasma treatment to improve its antioxidant stability. Experimental results show that the high-antioxidant bio-tofu prepared by this invention exhibits significantly improved free radical scavenging ability and oxidative stability, is rich in probiotics, and has significantly optimized nutritional quality. The method provided by this invention has the advantages of a mild production process, high retention rate of antioxidant components, and synergistic enhancement of product nutritional value and functional characteristics. It provides technical support for overcoming the shortcomings of insufficient antioxidant activity and susceptibility to oxidative deterioration in tofu, and also provides a theoretical basis and practical reference for the application of complex enzyme preparations, probiotics, and low-temperature plasma technology in the antioxidant processing of soybean products. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Given the lack of effective methods to improve the antioxidant properties of tofu in this field, the innovative technical approach of combining compound bio-enzymatic hydrolysis, probiotic synergistic fermentation, and low-temperature plasma post-treatment has emerged, providing a completely new approach to solving the problem of tofu oxidation.

[0023] This innovative combination of technologies not only enhances the antioxidant activity of tofu from the source but also improves its flavor and texture: the organic acids produced by probiotic fermentation neutralize the bitterness of soy milk, improving its flavor acceptability; the small-molecule peptides produced by enzymatic hydrolysis are easily digested and absorbed, enhancing their nutritional value. Furthermore, low-temperature plasma treatment reduces processing energy consumption, aligning with the trend of green processing. However, research on the systematic integration of these three technologies in tofu processing is still in its early stages, and a mature system of process parameters has not yet been established; the relevant mechanisms of action require further clarification. Therefore, in-depth research on this technology has significant theoretical and practical value for improving the antioxidant activity of tofu, enhancing product quality, and promoting the development of the soybean protein deep processing industry.

[0024] In the complex enzymatic hydrolysis stage, this invention utilizes a complex enzyme preparation composed of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) to directly scavenge free radicals in soy milk. Subsequently, protease is added to hydrolyze soy protein to produce small molecule peptides with antioxidant activity. The two work synergistically to enhance the antioxidant potential of tofu. Following this, probiotics are introduced for co-fermentation. *Lactobacillus plantarum* and *Lactobacillus helveticus* not only co-ferment to produce lactic acid and other organic acids, lowering the pH of the system and inhibiting oxidase activity, but also enrich functional components such as γ-aminobutyric acid (GABA) and soy isoflavone aglycones through metabolic transformation. Finally, the formed tofu is subjected to antioxidant activation treatment using low-temperature plasma technology. This technology, as an emerging non-thermal processing technology, activates antioxidant groups such as phenolic hydroxyl groups and thiol groups in soy protein in tofu by generating reactive oxygen species (ROS) and reactive nitrogen species (RNS), while simultaneously improving the hydrophobicity of the protein surface and enhancing its binding ability with antioxidant components, further strengthening the antioxidant effect.

[0025] In this embodiment of the invention, the method for determining the DPPH free radical scavenging rate is as follows: Accurately weigh 4 g of tofu, homogenize and crush it, then add 10 mL of deionized water and mix well. Centrifuge at 8000 r / min for 15 min, and collect the supernatant for later use. Take 2 mL of the supernatant and add 2 mL of DPPH ethanol solution (0.2 mmol / L), mix, and react at 37℃ in the dark for 30 min. Measure the absorbance of the reaction solution at 517 nm. Vitamin C (VC) was used as a positive control. The formula for calculating the DPPH free radical scavenging rate is as follows: DPPH free radical scavenging rate (%) = (A0-A1+A2) / A0×100; Where: A0 is the absorbance of an equal volume of anhydrous ethanol replacing the sample, A1 is the absorbance of the sample, and A2 is the absorbance of an equal volume of anhydrous ethanol replacing DPPH.

[0026] Determination of ABTS + The methods for free radical scavenging are as follows: Dissolve ABTS (0.384 g) and potassium persulfate (0.066 g) in distilled water (100 mL) and stir in the dark for 12–16 h. Dilute the reaction solution with phosphate buffer (0.1 mol / L) to an absorbance of 0.7 to obtain the ABTS working solution.

[0027] The supernatant was treated under the same conditions as DPPH. 2 mL of the supernatant was added to 1 mL of ABTS working solution, and the reaction was carried out at 37°C in the dark for 30 min. The absorbance of the reaction solution was measured at 734 nm. Vitamin C was used as a positive control. ABTS + The formula for calculating free radical scavenging rate is as follows: ABTS + Free radical scavenging rate (%) = (A3 - A4 + A5) / A3 × 100; Where: A3 is the absorbance of an equal volume of anhydrous ethanol replacing the sample, A4 is the absorbance of the sample, and A5 is the absorbance of an equal volume of anhydrous ethanol replacing ABTS.

[0028] The method for determining iron-reducing antioxidant capacity (FRAP) is as follows: The FRAP working solution was prepared by mixing 0.3 mol / L, pH 3.6 acetate buffer, and tripyridine triazine (TPTZ) solution (10 mmol / L, dissolved in 40 mmol / L hydrochloric acid) in a volume ratio of 10:1:1.

[0029] Take 0.5 mL of the supernatant (under the same conditions as the DPPH method), add the prepared FRAP working solution, and incubate in the dark for 30 min. Measure the absorbance of the mixture at 593 nm using a multi-mode microplate reader. Ferrous sulfate (FeSO4) was used as the standard, and the final result is expressed as FeSO4 equivalent.

[0030] The survival rate of probiotics in the final product was determined according to the method described in GB 4789.35-2023.

[0031] Untreated tofu (control group 1) → enzymatic hydrolysis without fermentation (experimental group 1) → enzymatic hydrolysis + single-strain Lactobacillus plantarum fermentation (control group 2) → enzymatic hydrolysis + single-strain Lactobacillus helveticus fermentation (control group 3) → enzymatic hydrolysis + compound bacteria fermentation (experimental group 2) → enzymatic hydrolysis + compound bacteria fermentation + low-temperature plasma treatment (experimental group 3).

[0032] Example 1 1. Preparation of untreated tofu Select plump soybeans and soak them in distilled water (the mass of distilled water is 3 times the mass of soybeans) for 12 hours. Mix them according to the mass ratio of 20% soybeans and 80% water, grind them for 1 minute at a speed of 10000 r / min, filter them through a 100-mesh sieve, and collect the raw soybean milk.

[0033] The raw soy milk is sterilized at 115℃ for 10 minutes, then cooled to 45℃, and then cooled to 37℃ for later use.

[0034] Add 2.0 mol / L magnesium sulfate (2% by volume) to the prepared soy milk and incubate at 85℃ for 20 min to obtain curd. Transfer the curd to a molding mold lined with filter cloth and measuring 10.0 cm × 10.0 cm × 8.0 cm, and heat at 20 g / cm³. 2 Tofu is obtained by pressing under pressure for 1 hour.

[0035] The untreated tofu prepared above was used as control group 1.

[0036] 2. Preparation of enzymatically hydrolyzed tofu Superoxide dismutase (SOD) (product number: S10116-50 mg, manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., derived from porcine blood, enzyme activity 2.5 × 10⁻⁶) was selected. 6 ~7×10 6 U / g), catalase (CAT) (product number: S25070-5g, manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., source: Aspergillus niger, enzyme activity 1×10⁻⁶ ... 5 U / g) and glutathione peroxidase (GPx) (Catalog No.: S10152-100UN, Manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., Biotechnology Grade, Yeast Source, Enzyme Activity 3×10 5 The enzymes (U / g) were compounded at a mass ratio of 0.02:1:0.33 to prepare a compound enzyme preparation, which was then sealed and stored for later use.

[0037] The above-mentioned compound enzyme preparation was added to the sterilized and cooled soy milk in "1. Preparation of Untreated Tofu" at a dosage of 0.2%. The reaction temperature was controlled at 40℃, the stirring speed at 500 rpm, and the enzymatic hydrolysis was carried out at a constant temperature for 15 min. The pH was 6.6-7.2 to pre-clear the native free radicals in the soy milk system. Then, neutral protease (product number: V32439-50ml, manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., enzyme activity ≥2.4 U / g) was added to the system at a dosage of 0.15% of the mass of soy milk. The temperature was maintained at 40℃, 500 rpm, and pH 6.6-7.2, and the enzymatic hydrolysis was continued at a constant temperature for 15 min, for a total enzymatic hydrolysis time of 30 min. After the enzymatic hydrolysis was completed, the enzymatically hydrolyzed soy milk was sterilized again at 115℃ for 5 min, and then cooled for later use.

[0038] Add 2.0 mol / L magnesium sulfate (2% by volume) to the enzymatically hydrolyzed soybean milk and incubate at 85℃ for 20 min to obtain curd. Transfer the curd to a molding mold lined with filter cloth and measuring 10.0 cm × 10.0 cm × 8.0 cm, and heat at 20 g / cm³. 2 Tofu was obtained by pressing under pressure for 1 hour. The enzymatically hydrolyzed tofu described above was used as experimental group 1.

[0039] 3. Antioxidant capacity testing Untreated tofu and enzymatically hydrolyzed tofu were prepared according to the above method, and the process was repeated three times. DPPH free radical scavenging rate and ABTS were measured. + The free radical scavenging rate and ferric ion reducing power (FRAP) were averaged, and the results are shown in Table 1.

[0040] Table 1. Antioxidant activity of the soy milk experimental group and the control group. As can be seen from Table 1: After compound enzymatic hydrolysis treatment, the ABTS of the enzymatically hydrolyzed tofu in experimental group 1 was... + The free radical scavenging rate increased from 33.12% in control group 1 to 49.21%, the DPPH free radical scavenging rate increased from 32.98% to 47.45%, and the FRAP rate increased from 29.51% to 45.25%. All three antioxidant indicators were significantly improved compared to control group 1 without enzymatic hydrolysis. These results indicate that the compound enzyme preparation of this invention can efficiently remove reactive free radicals such as superoxide anions and hydrogen peroxide in the soy milk system, reduce the oxidative loss of endogenous active substances such as small molecule antioxidant peptides and free isoflavones, and simultaneously improve the free radical scavenging capacity and iron ion reducing capacity of the sample, significantly enhancing the basic antioxidant level of soy milk and demonstrating a significant antioxidant improvement effect.

[0041] Example 2 Select Lactobacillus plantarum ( Lactobacillus plantarum) and Lactobacillus helveticus ( Lactobacillus Swiss (as a compound strain)

[0042] The *Lactobacillus plantarum* strain of this invention is *Lactobacillus plantarum* LL257, isolated from traditional dairy products in Tongliao, Inner Mongolia. The isolation process involved serially diluting traditional yogurt samples and inoculating them into MRS medium. Physiological and biochemical methods, including colony morphology observation, Gram staining microscopy, and catalase assays, were performed, initially identifying it as a lactic acid bacterium.

[0043] Further molecular biological methods, including 16S rDNA RFLP-PCR and 16S-23S rDNA RFLP-PCR fingerprinting combined with 16S rDNA sequencing, were used. After gene sequencing identification and comparison with NCBI, it was found to have 100% homology with *Lactobacillus plantarum*, confirming it as *Lactobacillus plantarum*, and named it *Lactobacillus plantarum*. Lactobacillus plantarum LL257. The finally isolated Lactobacillus plantarum was biopreserved and stored in the laboratory of the College of Food Science and Technology, Northeast Agricultural University.

[0044] The depositary institution for Lactobacillus plantarum LL257 is the China General Microbiological Culture Collection Center (CGMCC); the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC No. 35067; and the deposit date is July 1, 2025.

[0045] The *Lactobacillus helveticus* of this invention has been disclosed in a paper entitled "Immunomodulatory potential of *Lactobacillus helveticus* KLDS 1.8701 postbiotics: By regulating the Th17 / Treg balance".

[0046] Prepare the MRS liquid culture medium for activating the compound strains and autoclave it for later use.

[0047] Take the freeze-dried Lactobacillus plantarum and Lactobacillus helveticus powders that have been frozen and stored, and activate them by subculturing twice at 37°C using MRS liquid medium. After detecting the number of viable bacteria, mix them in equal volumes at a 1:1 ratio to obtain a compound bacterial solution. Then, inoculate the solution with 4% (w / w) of the total inoculum into sterile MRS liquid medium and incubate at 37°C for 12 h to obtain a stable activated compound bacterial solution for later use.

[0048] Equal amounts of freeze-dried *Lactobacillus plantarum* powder were activated and subcultured twice at 37°C using MRS liquid medium to obtain *Lactobacillus plantarum* single-cell suspensions. Equal amounts of freeze-dried *Lactobacillus helveticus* powder were also activated and subcultured twice at 37°C using MRS liquid medium to obtain *Lactobacillus helveticus* single-cell suspensions. Both single-cell suspensions were inoculated at a 4% (w / w) inoculum into sterile MRS liquid medium and incubated at 37°C for 12 h to obtain stable activated *Lactobacillus plantarum* and *Lactobacillus helveticus* single-cell suspensions, respectively, for later use.

[0049] The activated *Lactobacillus plantarum*, *Lactobacillus helveticus*, and *Lactobacillus plantarum*-*Lactobacillus helveticus* complex cultures were inoculated into the sterilized enzymatically hydrolyzed soy milk prepared in Example 1 at an inoculation rate of 4% (v / v). Fermentation was carried out at 37°C for 5 h. The fermented soy milk was then stirred at 75°C and 750 rpm for 1 min, and 2.0 mol / L magnesium sulfate (2% by volume) was added. The mixture was kept at this temperature for 20 min to obtain curd. The curd was transferred to a molding mold lined with filter cloth and measuring 10.0 cm × 10.0 cm × 8.0 cm. The curd was then heated at 20 g / cm³. 2 Pressed under pressure for 1 h, tofu fermented with enzymatic hydrolysis + Lactobacillus plantarum (as control group 2), tofu fermented with enzymatic hydrolysis + Lactobacillus helveticus (as control group 3), and tofu fermented with enzymatic hydrolysis + compound bacteria (as experimental group 2) were obtained respectively.

[0050] Table 2 Results of the Enzymatic Hydrolysis + Fermentation Tofu Experiment As can be seen from Table 2: Pre-treatment of soy milk with a combination of antioxidant enzymes and proteases releases more small-molecule active peptides and polyphenolic antioxidants. Following this, single fermentation with *Lactobacillus plantarum* and *Lactobacillus helveticus* further enhances the ABTS and DPPH free radical scavenging capacity, FRAP total reducing power, and probiotic survival rate in the final product. Compared to the two single-strain fermentation treatments, the *Lactobacillus plantarum*-*Lactobacillus helveticus* complex fermentation group achieved optimal levels in all antioxidant indicators and probiotic retention. The metabolites of the two lactic acid bacteria complement each other, forming a synergistic antioxidant system. This indicates a significant synergistic effect between the combined enzymatic pretreatment and the dual-strain fermentation, which further strengthens the antioxidant activity of tofu and significantly increases the survival rate of probiotics in the finished product.

[0051] Example 3 The enzymatic hydrolysis + compound bacteria fermented tofu (experimental group 2) prepared in Example 2 was placed in an atmospheric pressure cold plasma device for low-temperature plasma treatment. The treatment power was controlled at 60 W, the discharge gap at 5 mm, and the treatment time at 2 min. After treatment, the enzymatic hydrolysis + compound bacteria fermented + low-temperature plasma treated tofu was obtained as experimental group 3.

[0052] The low-temperature plasma used in this embodiment is magnetized arc plasma, generated by a magnetized arc plasma generator. This magnetized arc plasma generator consists of two parts: a mercury arc plasma generator and an induced electromagnetic field. Mercury arc plasma generator: tube length 25 cm, inner diameter 2.5 cm, vacuum degree 10-20 Pa, discharge power 400 W, maximum discharge power 1.6 kW; Induced electromagnetic field: It consists of 7 interconnected magnetic field systems formed by 8 sets of induction coils connected in opposite directions. The spatial variation range of the magnetic field in the longitudinal direction is 0-500 Gauss, and the spatial variation range of the magnetic field in the radial direction is 0-175 Gauss.

[0053] The magnetic field coil is powered by an AC power supply with a voltage of 300 V and a continuously adjustable current ranging from 0 to 5 A.

[0054] The experiment was repeated three times according to the treatment methods for control groups 1-3 and experimental groups 1-3. Tofu was made from the tofu, and the DPPH and ABTS of each group of tofu were measured. + The results of free radical scavenging rate, FRAP, and probiotic final product survival rate are shown in Table 3.

[0055] Table 3. Results of the control group and Examples 1-3 As can be seen from the results in Table 3, the composite treatment process of the present invention can significantly improve the in vitro antioxidant activity of tofu and maintain a high survival rate of probiotics.

[0056] Comparing experimental group 1 (enzymatic hydrolysis only), control groups 2-3 (single / compound probiotic fermentation), and experimental group 2 with control group 1, it can be seen that enzymatic hydrolysis can initially improve antioxidant levels, and compound probiotic fermentation can further significantly improve ABTS. + It has DPPH free radical scavenging rate and FRAP iron ion reducing ability, while giving the product probiotic activity, and the fermentation effect of compound bacteria is better than that of single strain fermentation.

[0057] Comparing the plasma-treated tofu product in experimental group 3 with that in experimental group 2, it can be seen that low-temperature plasma treatment can further improve all antioxidant indicators, and the antioxidant capacity is close to that of the VC positive control. The survival rate of probiotics is slightly reduced but still at a high level, indicating that low-temperature plasma treatment will not excessively destroy the activity of probiotics and can also significantly enhance the antioxidant effect.

[0058] The above results indicate that the combined use of enzymatic hydrolysis, probiotic fermentation, and low-temperature plasma physical treatment significantly enhances the free radical scavenging ability and iron ion reducing power of tofu through multiple pathways. This is achieved by using compound enzyme preparations to remove native free radicals in soy milk, using proteases to hydrolyze soy protein to generate small molecule antioxidant peptides, using probiotic fermentation to enrich phenolic active substances through biotransformation, and using low-temperature plasma physical modification to further expose antioxidant active groups. At the same time, it can maintain a high survival level of probiotics and has a significant synergistic effect.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing antioxidant-rich bio-tofu, characterized in that, Includes the following steps: Soak soybeans, grind them, filter and collect the soy milk, sterilize and cool it, mix it with a compound enzyme preparation, carry out the first enzymatic hydrolysis reaction, add protease to carry out the second enzymatic hydrolysis reaction, sterilize it, and obtain enzymatically hydrolyzed soy milk. The compound bacterial solution is inoculated into the enzymatically hydrolyzed soybean milk, fermented, stirred, coagulant is added, and the mixture is kept warm to obtain curd. The curd is transferred to a mold and pressed to obtain compound bacteria fermented tofu. The antioxidant bio-tofu is obtained by subjecting the fermented tofu with the compound bacteria to low-temperature plasma treatment.

2. The preparation method according to claim 1, characterized in that, The compound enzyme preparation comprises superoxide dismutase, catalase, and glutathione peroxidase in a mass ratio of 0.02:1:0.

33.

3. The preparation method according to claim 2, characterized in that, The amount of the compound enzyme preparation added is 0.2% by mass fraction; The amount of protease added is 0.15%.

4. The preparation method according to claim 1, characterized in that, The temperature for the first enzymatic hydrolysis reaction and the temperature for the second enzymatic hydrolysis reaction were 40°C, the stirring rate was 500 rpm, and the time was 15 min.

5. The preparation method according to claim 1, characterized in that, The method for preparing the composite bacterial solution includes the following steps: Lactobacillus plantarum ( Lactobacillus plantarum ) and Lactobacillus helveticus ( Lactobacillus helveticus The bacteria were activated, passaged, mixed, and cultured at a constant temperature to obtain the mixed bacterial solution.

6. The preparation method according to claim 5, characterized in that, In the compound bacterial solution, the ratio of viable Lactobacillus plantarum to viable Lactobacillus helveticus is 1:

1.

7. The preparation method according to claim 1, characterized in that, The inoculation amount of the compound bacterial solution is 4% by volume.

8. The preparation method according to claim 1, characterized in that, The fermentation was carried out at a temperature of 37°C for 5 hours.

9. The preparation method according to claim 1, characterized in that, The low-temperature plasma treatment has a power of 60W, a discharge gap of 5 mm, and a time of 2 min.

10. An antioxidant bio-tofu obtained by the preparation method according to any one of claims 1-9.

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