A kind of antioxidant active whey and preparation thereof

Abstract

This invention aims to address the technical bottlenecks of existing milk curd products, such as weak functional activity, unclear process parameters, and unstable flavor and quality. It utilizes *Pediococcus pentosaceus* HM4, *Streptococcus thermophilus* HM6, *Lactococcus lactis* JY4, and *Lactobacillus plantarum* NC1, employing these four strains in an optimal ratio as a compound fermentation agent to achieve synergistic effects. Using milk and goji berries as raw materials, the fermentation process of milk curds and goji berry milk curds was systematically optimized through single-factor experiments, Plackett-Burman experiments, and response surface methodology. The physicochemical indicators and flavor characteristics of the products under different process conditions were analyzed. The obtained milk curds achieved a DPPH free radical scavenging rate of 91.38%–94.15%, providing technical support for the high-value utilization of milk curds and enriching the variety of functional dairy products.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, specifically relating to the technical field of fermentation for making milk curds, and more specifically to the technical field of a milk curd with antioxidant activity and its preparation. Background Technology

[0002] Milk curds, a traditional fermented dairy product, are mainly produced in small workshops or at the family level. They are typically made from cow's or sheep's milk, using yogurt as a starter, and processed through traditional methods such as whey separation, shaping, and drying. Essentially, they are a high-protein, shelf-stable cheese-like food. Because milk curds rely on the traditional process of "natural fermentation and sun-drying," the microbial community structure, quality, and flavor are unstable, posing challenges for industrial production. In recent years, research on process optimization has focused on strategies of "strain domestication and process coupling." For example, Gulnur Tulaxi screened lactic acid bacteria from milk curds from different regions of Xinjiang to create a suitable starter culture for cheese; TIAN et al., using flavor strains isolated in the laboratory and combined with flavor omics methods, significantly increased the concentration and diversity of pleasant volatile compounds in Kazakh cheese, resulting in a characteristic flavor profile that blends nutty and milky aromas; SHEN et al. used... Lactobacillus paracasei and Limosilactobacillus reuteri Mixed fermentation significantly enhances the flavor of Kazakh cheese; Peng Bin improved the shortcomings of traditional cheese curds, such as being too hard, too sour, and having poor palatability, by screening lactic acid bacteria and optimizing the process.

[0003] Free radicals are highly reactive molecules that, in excess, trigger oxidative stress, damaging DNA, proteins, and lipids. They are closely associated with various chronic diseases such as cancer, cardiovascular disease, neurodegenerative diseases, and diabetes. Antioxidants, by neutralizing reactive oxygen species and free radicals, reduce oxidative damage in organisms. Dietary intervention, consuming foods rich in antioxidants, can help extend lifespan and effectively alleviate oxidative stress, providing an economical, safe, and effective intervention strategy. Lactic acid bacteria, by producing their own antioxidants to scavenge free radicals and regulating host signaling pathways and gut microbiota, alleviate oxidative stress and thus have a positive impact on various health issues. Therefore, fermenting food ingredients with lactic acid bacteria can yield products rich in antioxidants. For example, fermentation with *Lactobacillus plantarum* for blueberry juice, *Lactobacillus plantarum* for dealcoholized apple juice, a combination of lactic acid bacteria and yeast for whole wheat flour, and lactic acid bacteria for goat milk has shown significantly increased antioxidant activity compared to the raw materials.

[0004] In preliminary experiments, Pediococcus pentosaceus was found to be... Pediococcus pentosaceus HM4, Streptococcus thermophilus ( Streptococcus thermophilus ) HM6, Lactococcus lactis ( Lactococcus lactisJY4 and Lactobacillus plantarum ( Lactobacillus plantarum NC1 bacteria exhibit good antioxidant activity, therefore, they will be used as fermentation agents. In the development of functional dairy products, research on the co-fermentation of wolfberry raw materials with lactic acid bacteria is still insufficient. How to effectively combine highly active fermentation strains with specialty raw materials, and improve the physicochemical quality and flavor characteristics of products through scientific process optimization, remains a pressing technical problem to be solved in the current dairy processing field. Summary of the Invention

[0005] Based on the existing technology, there is no information regarding the use of Pediococcus pentosaceus ( Pediococcus pentosaceus HM4, Streptococcus thermophilus ( Streptococcus thermophilus ) HM6, Lactococcus lactis ( Lactococcus lactis JY4 and Lactobacillus plantarum ( Lactobacillus plantarum This invention addresses the technical challenges of co-fermentation of milk curds with NC1 and wolfberry raw materials and lactic acid bacteria. It aims to solve the technical bottlenecks of existing milk curd products, such as weak functional activity, unclear process parameters, and unstable flavor and quality. The invention employs *Pediococcus pentosaceus* HM4, *Streptococcus thermophilus* HM6, *Lactococcus lactis* JY4, and *Lactobacillus plantarum* NC1, using these four strains in an optimal ratio as a compound fermentation agent to achieve synergistic effects. Using milk and wolfberry as raw materials, the fermentation process of milk curds and wolfberry milk curds was systematically optimized through single-factor experiments, Plackett-Burman experiments, and response surface methodology. The physicochemical indicators and flavor characteristics of the products under different process conditions were analyzed, providing technical support for the high-value utilization of milk curds and enriching the variety of functional dairy products.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a milk curd with antioxidant activity, made from whole milk and Pediococcus pentosaceus (Pediococcus pentosaceus). Pediococcus pentosaceus HM4, Streptococcus thermophilus ( Streptococcus thermophilus ) HM6, Lactococcus lactis ( Lactococcus lactis JY4 and Lactobacillus plantarum ( Lactobacillus plantarum ) Obtained by fermentation with NC1 compound microbial agent.

[0007] The above-mentioned milk curds with antioxidant activity can also be supplemented with goji berry powder.

[0008] Preferably, the compound microbial agent has a mass percentage of 0.5%-5%; Preferably, the percentage of goji berry powder by weight is 0.5%-3%.

[0009] Furthermore, this application also provides a method for preparing the aforementioned milk curd with antioxidant activity, which is prepared by the following steps: S1, strain Pediococcus pentosaceus ( Pediococcus pentosaceus HM4, Streptococcus thermophilus ( Streptococcus thermophilus ) HM6, Lactococcus lactis ( Lactococcus lactis JY4 and Lactobacillus plantarum ( Lactobacillus plantarum NC1 was inoculated into MRS broth medium and cultured at 37 ℃ for 24 h. The viable counts of the four lactic acid bacteria suspensions were determined. The viable counts of the four lactic acid bacteria suspensions were then serially diluted to ensure that they were of the same gradient for later use. S2. Aseptic whole milk is sterilized at 85°C for 20 minutes. S3. The bacterial suspension obtained from activation in step S1 is mixed according to the volume ratio of *Lactococcus lactis* (…). Lactococcus milk JY4 and Streptococcus thermophilus ( Streptococcus thermophilus HM6 was mixed in a ratio of (15-1):(1-15); Lactobacillus plantarum ( Lactobacillus plantarum NC1 and Pediococcus pentosaceus ( Pediococcus pentosaceous HM4 is mixed in a ratio of (15-1):(1-15) and added to the sterilized whole milk in S2 at a mass percentage of 0.5%-5%. S4. Ferment the whole milk inoculated with the bacterial agent in step S1 for 12 h-48 h; fermentation temperature is 35℃-43℃. S5. Pour the fermented product obtained in step S4 into a 300-mesh gauze, hang it up, and drain the whey for 12 hours. S6. Dry the filter material obtained from the gauze in step S5 at 35 ℃ for 2 h-10 h to obtain milk curds with antioxidant activity.

[0010] Furthermore, 0.5%-3% by weight of goji berry powder can be added in step S3 or step S5.

[0011] Lactococcus lactis ( Lactococcus lactis JY4 and Streptococcus thermophilus ( Streptococcus thermophilic HM6 was mixed in a 3:1 ratio; Lactobacillus plantarum ( Lactobacillus plantarum NC1 and Pediococcus pentosaceus ( Pediococcus pentosaceus HM4 is mixed in a 1:1 ratio; Preferably, the inoculation amount is 3%; Preferably, the fermentation time is 24 hours or 30 hours; Preferably, the fermentation temperature is 37℃ or 39℃; Preferably, the amount of goji berries added is 1.5%; Preferably, the drying time is 4 hours or 8 hours; Furthermore, this application also provides the application of the above-mentioned method for preparing milk curds with antioxidant activity in the preparation of milk curds with antioxidant activity.

[0012] Compared with the prior art, the present invention has the following features: This invention provides a type of milk curd with antioxidant activity, which is achieved by optimizing Lactococcus lactis (Lactococcus lactis). Lactococcus lactis JY4 and Streptococcus thermophilus ( Streptococcus thermophilus HM6 and Lactobacillus plantarum ( Lactobacillus plantarum NC1 and Pediococcus pentosaceus ( Pediococcus pentosaceus The optimal mixing ratio of HM4 strains fully leverages the synergistic effect among the four strains, achieving significant results in viable cell count, DPPH free radical scavenging rate, and superoxide anion scavenging rate. This provides technical support for the high-value utilization of milk curds and enriches the variety of functional dairy products.

[0013] (2) The technical solution provided by the company, based on the optimal results of the response surface methodology, shows that by optimizing the inoculum quantity, fermentation time, drying time, and amount of wolfberry added, the DPPH free radical scavenging rate of milk curds under each process condition reached 91.38%-94.15% - the error between the DPPH free radical scavenging rate of each process and the predicted value is less than 5%, proving the accuracy of the model. Furthermore, the protein, fat content, and total sugar characteristics are significantly improved.

[0014] (3) The milk curds prepared using the technical solution provided in this application all showed certain response values ​​at W1W, W5S, W2W, W2S, and W1S, indicating that the samples contained a large amount of sulfur-containing compounds, nitrogen oxides, and aromatic substances. The response values ​​of the prepared milk curds were all higher than those of the commercial control (SY). Principal component analysis (PCA) further verified the above results. The cumulative contribution rate of PC1 and PC2 reached 98.4%, which can fully reflect the volatility characteristics of the samples. The prepared milk curds and the commercial milk curds partially overlapped in the PCA plot, indicating that the odor composition of the three was relatively similar; while sensors such as W1W and W5S made significant contributions to PC1, consistent with the response pattern of the radar plot. Attached Figure Description

[0015] Figure 1 The diagram shows the selection of fermentation strains in the two substrates.

[0016] Figure A shows milk; Figure B shows 1% goji berry powder + milk.

[0017] Figure 2 The diagram shows the inoculation ratio of milk curds produced using different processes.

[0018] Figure A shows the original flavor milk curds; Figure B shows the mixed fermented goji berry milk curds.

[0019] Figure 3 The image shows the bacterial inoculation levels of milk curds produced using different processes.

[0020] Figure A shows the original flavor milk curds; Figure B shows the mixed fermented goji berry milk curds.

[0021] Figure 4 The image shows the fermentation time of milk curds produced using different processes.

[0022] Figure A shows the original flavor milk curds; Figure B shows the mixed fermented goji berry milk curds.

[0023] Figure 5 The diagram shows the fermentation temperature of milk curds produced using different processes.

[0024] Figure A shows the original flavor milk curds; Figure B shows the mixed fermented goji berry milk curds.

[0025] Figure 6 The image shows the amount of goji berries added to milk curds made using different processing methods.

[0026] Figure A shows the goji berry milk curd with added ingredients; Figure B shows the mixed fermented goji berry milk curd.

[0027] Figure 7 The image shows the drying time of goji berries for milk curds made using different processes.

[0028] Figure A shows the original flavor milk curd; Figure B shows the milk curd with added goji berries; and Figure C shows the mixed fermented goji berry milk curd.

[0029] Figure 8 The figure shows the effect of the interaction between various factors on the DPPH free radical scavenging rate in plain milk curds.

[0030] Figure A shows the contour plot of the interaction between inoculum amount and fermentation time on the DPPH free radical scavenging rate; Figure B shows the contour plot of the interaction between inoculum amount and drying time on the DPPH free radical scavenging rate; Figure C shows the contour plot of the interaction between fermentation time and drying time on the DPPH free radical scavenging rate; Figure D shows the response surface plot of the contour plot of the interaction between inoculum amount and fermentation time on the DPPH free radical scavenging rate; Figure E shows the response surface plot of the interaction between inoculum amount and drying time on the DPPH free radical scavenging rate; Figure F shows the response surface plot of the interaction between fermentation time and drying time on the DPPH free radical scavenging rate.

[0031] Figure 9 The figure shows the effect of the interaction between various factors on the DPPH free radical scavenging rate in added wolfberry milk curds. Figure A shows the contour plot of the interaction between inoculum amount and fermentation time on the DPPH free radical scavenging rate; Figure B shows the contour plot of the interaction between inoculum amount and wolfberry dosage on the DPPH free radical scavenging rate; Figure C shows the contour plot of the interaction between fermentation time and wolfberry dosage on the DPPH free radical scavenging rate; Figure D shows the response surface plot of the interaction between inoculum amount and fermentation time on the DPPH free radical scavenging rate; Figure E shows the response surface plot of the interaction between inoculum amount and wolfberry dosage on the DPPH free radical scavenging rate; Figure F shows the response surface plot of the interaction between fermentation time and wolfberry dosage on the DPPH free radical scavenging rate.

[0032] Figure 10 The figure shows the effect of the interaction between various factors on the DPPH free radical scavenging rate in mixed fermented wolfberry milk curds.

[0033] Figure A shows the contour plot of the interaction between fermentation time and wolfberry dosage on the DPPH free radical scavenging rate; Figure B shows the contour plot of the interaction between fermentation time and drying time on the DPPH free radical scavenging rate; Figure C shows the contour plot of the interaction between wolfberry dosage and drying time on the DPPH free radical scavenging rate; Figure D shows the response surface plot of the interaction between fermentation time and wolfberry dosage on the DPPH free radical scavenging rate; Figure E shows the response surface plot of the interaction between fermentation time and drying time on the DPPH free radical scavenging rate; Figure F shows the response surface plot of the interaction between wolfberry dosage and drying time on the DPPH free radical scavenging rate.

[0034] Figure 11 The image shown is a radar image of the electronic nose and a PCA result image of the electronic nose data.

[0035] Figure A shows the radar image of the electronic nose, and Figure B shows the PCA result of the electronic nose data. Detailed Implementation

[0036] The following examples are provided to further illustrate the content of this invention, but should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and essence of the invention are within the scope of this invention.

[0037] The reagents used in this application are as follows: MRS agar medium and MRS broth medium, Qingdao Haibo Biotechnology Co., Ltd.; ox bile salts (choleic acid content >75%) and DPPH (2,2-biphenyl-1-picrylhydrazine), Shanghai Maclean Biochemical Technology Co., Ltd.; 1 M Tris-HCl solution (pH = 6.8), Lanjieke Technology Co., Ltd.; pyrogallol, Tianjin Beilian Fine Chemicals Development Co., Ltd.; 1 M NaOH standard titration solution, Beijing Zhongke Ruipu Biotechnology Co., Ltd.; and total sugar content reagent kit (catalog number JC0409-M), Nanjing Jice Biotechnology Co., Ltd. Aseptic whole milk, wolfberry powder, and commercial milk curds (SY) are all commercially available and can be purchased by the general public from supermarkets. The instruments and equipment used in this application are: SW-CJ-2D double-person clean bench, Zhejiang Fuxia Medical Technology Co., Ltd.; pHS-25 pH meter, Shanghai Leici Instrument Factory; 2-16R benchtop high-speed refrigerated centrifuge, Hunan Hengnuo Instrument Equipment Co., Ltd.; UV-1200 ultraviolet / visible spectrophotometer, Shanghai Meixi Instrument Co., Ltd.; HPX-9272MBE electric thermostatic incubator, Shanghai Boxun Industrial Co., Ltd.; A1204 electronic analytical balance, Mettler Toledo Group, Switzerland; Epoch microplate reader, Porton Instruments, USA; DU-6GW magnetic stirring water bath, Shanghai Shaying Scientific Instruments Co., Ltd.

[0038] The strains used in this invention, including Pediococcus pentosaceus (HM4), Streptococcus thermophilus (HM6), Lactococcus lactis (JY4), and Lactobacillus plantarum (NC1), were all isolated from fermented foods in Xinjiang. These strains have been disclosed in "Isolation and Screening of Lactic Acid Bacteria in Traditional Fermented Foods in Xinjiang and Their Non-Target Metabolomics Analysis after Fermentation of Milk Curds," and can be obtained from the Institute of Microbiology, Xinjiang Academy of Agricultural Sciences.

[0039] In this application, the terms “Pediococcus pentosaceus (HM4), Streptococcus thermophilus (HM6), Lactococcus lactis (JY4), and Lactobacillus plantarum (NC1)” are abbreviated as “HM4”, “HM6”, “JY4”, and “NC1” in the following embodiments.

[0040] Data processing and significance analysis (Duncan's method) in this application were performed using SPSS 27, and differences between experimental groups are represented by lowercase letters. P <0.05), the graph was plotted using Origin 2022, and all tests in this experiment were repeated 3 times.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] Example 1: A milk curd with antioxidant activity This invention provides a milk curd with antioxidant activity, made from whole milk and Pediococcus pentosaceus (Pediococcus pentosaceus). Pediococcus pentosaceus HM4, Streptococcus thermophilus ( Streptococcus thermophilus ) HM6, Lactococcus lactis ( Lactococcus lactis JY4 and Lactobacillus plantarum ( Lactobacillus plantarum ) Obtained by fermentation with NC1 compound microbial agent.

[0043] The above-mentioned milk curds with antioxidant activity can also be supplemented with goji berry powder.

[0044] Preferably, the compound microbial agent has a mass percentage of 0.5%-5%; Preferably, the percentage of goji berry powder by weight is 0.5%-3%.

[0045] Example 2: A method for preparing milk curds with antioxidant activity This application also provides a method for preparing the aforementioned milk curd with antioxidant activity, which is prepared by the following steps: S1, strain Pediococcus pentosaceus ( Pediococcus pentosaceus HM4, Streptococcus thermophilus ( Streptococcus thermophilus ) HM6, Lactococcus lactis ( Lactococcus lactis JY4 and Lactobacillus plantarum ( Lactobacillus plantarum NC1 was inoculated into MRS broth medium and cultured at 37 ℃ for 24 h. The viable counts of the four lactic acid bacteria suspensions were determined. The viable counts of the four lactic acid bacteria suspensions were then serially diluted to ensure that they were of the same gradient for later use.

[0046] S2. Aseptic whole milk is sterilized at 85°C for 20 minutes. S3. The bacterial suspension obtained from activation in step S1 is mixed according to the volume ratio of *Lactococcus lactis* (…). Lactococcus milk JY4 and Streptococcus thermophilus ( Streptococcus thermophilus HM6 was mixed in a ratio of (15-1):(1-15); Lactobacillus plantarum ( Lactobacillus plantarum NC1 and Pediococcus pentosaceus ( Pediococcus pentosaceous HM4 is mixed in a ratio of (15-1):(1-15) and added to the sterilized whole milk in S2 at a mass percentage of 0.5%-5%. S4. Ferment the whole milk inoculated with the bacterial agent in step S1 for 12 h-48 h; fermentation temperature is 35℃-43℃. S5. Pour the fermented product obtained in step S4 into a 300-mesh gauze, hang it up, and drain the whey for 12 hours. S6. Dry the filter material obtained from the gauze in step S5 at 35 ℃ for 2 h-10 h to obtain milk curds with antioxidant activity.

[0047] Furthermore, 0.5%-3% by weight of goji berry powder can be added in step S3 or step S5.

[0048] Lactococcus lactis ( Lactococcus lactis JY4 and Streptococcus thermophilus ( Streptococcus thermophilus HM6 was mixed in a 3:1 ratio; Lactobacillus plantarum ( Lactobacillus plantarum NC1 and Pediococcus pentosaceus ( Pediococcus pentosaceus HM4 is mixed in a 1:1 ratio; Preferably, the inoculation amount is 3%; Preferably, the fermentation time is 24 hours or 30 hours; Preferably, the fermentation temperature is 37℃ or 39℃; Preferably, the amount of goji berries added is 1.5%; Preferably, the drying time is 4 hours or 8 hours; Furthermore, this application also provides the application of the above-mentioned method for preparing milk curds with antioxidant activity in the preparation of milk curds with antioxidant activity.

[0049] Example 3: Selection of Fermentation Strains in Two Substrates Strains JY4, HM4, HM6 and their pairwise combinations (JH4, JH6, H46) were used to select strains for use in a milk matrix. Strains NC1, HM4, HM6 and their pairwise combinations (N14, N16, H46) were used to select strains for use in a 1% wolfberry powder + milk matrix. Viable cell count and antioxidant activity were used as indicators.

[0050] (1) Determination of viable lactic acid bacteria count The determination of viable bacteria count was performed in accordance with GB 4789.35-2023, "National Food Safety Standard: Microbiological Examination of Food - Lactic Acid Bacteria Examination".

[0051] (2) Determination of antioxidant activity The fermented milk after inoculation and fermentation is prepared as follows: Take 5 g of fermented milk into a 10 mL centrifuge tube, centrifuge at 4 ℃ and 10000 rpm for 10 min, collect the supernatant, filter the supernatant with qualitative filter paper, and use the resulting whey for later use. 1 mg / mL VC is used as a positive control.

[0052] The superoxide anion radical scavenging capacity was determined using the pyrogallol auto-oxidation method. The specific processing steps are as follows: A0 (blank control): 0.1 mL pure water + 4.5 mL Tris-HCl buffer + 0.4 mL pyrogallol solution + 0.1 mL HCl solution; A1 (sample to be tested): 0.1 mL bacterial culture + 4.5 mL Tris-HCl buffer + 0.4 mL pyrogallic acid solution + 0.1 mL HCl solution; A2 (Sample Control): 0.1 mL bacterial culture + 4.5 mL Tris-HCl buffer + 0.4 mL pure water + 0.1 mL HCl solution. Measure the absorbance of the solution at 320 nm wavelength, repeating three times.

[0053] DPPH free radical scavenging rate (refer to Zhao et al.)

[19] The method is as follows: The specific processing is as follows: A0 (blank control): 0.1 mL anhydrous ethanol + 0.1 mL DPPH ethanol solution; A1 (sample to be tested): 0.1 mL bacterial suspension + 0.1 mL DPPH ethanol solution; A2 (sample control): 0.1 mL bacterial culture + 0.1 mL anhydrous ethanol. Measure the absorbance at 517 nm wavelength, repeat three times.

[0054] The formula for calculating the free radical scavenging rate is as follows: Results in milk matrix as follows Figure 1 As shown in Figure A. In the viable cell count determination, at 24 h, JY4 and JH6 (JY4 and HM6) had higher viable cell counts, but the difference was not statistically significant (P < 0.05). In DPPH free radical scavenging ability, JH6 had the highest scavenging rate, with no significant difference compared to VC, while H46 (HM4 and HM6) had the lowest. In superoxide anion free radical scavenging rate, VC had the highest scavenging rate, followed by JH6, but there was no significant difference between JH6 and other groups. The results for the 1% wolfberry powder + milk matrix are shown below. Figure 1 As shown in Figure B, there was no significant difference in viable cell count between mixed-culture and single-culture fermentation. Regarding free radical scavenging rates, mixed-culture fermentation consistently showed higher rates than single-culture fermentation. N14 (NC1 and HM4) exhibited the highest DPPH and superoxide anion free radical scavenging rates, but these were not significantly different from other mixed-culture combinations. Considering all factors, JH6 was selected for the development of plain and additive-type milk curds, while N14 was selected for the development of mixed-fermentation milk curds.

[0055] Example 4: Optimization of Milk Curd Process I. Experimental Methods In the single-factor experiments, except for drying time, which was measured by moisture content and antioxidant activity, all other single-factors were measured by viable cell count and antioxidant activity. Among them, the single-factor experiments for the additive-type milk curds used some of the single-factors of the original flavor milk curds (inoculation ratio, inoculation amount, fermentation time, and fermentation temperature), and based on this, the single-factor experiments for the amount of goji berries added and drying time were conducted.

[0056] The method for determining the viable bacterial count and the method for determining the antioxidant activity were described in Example 3. The dried milk curds were processed as follows: 10 g of milk curds were placed in a 50 mL centrifuge tube, 15 mL of anhydrous ethanol was added, and the mixture was thoroughly mixed. The mixture was then centrifuged at 4 °C and 10,000 rpm for 10 min. The supernatant was collected, and the whey was filtered through qualitative filter paper. The water content was determined according to GB 5009.3-2016, "National Food Safety Standard - Determination of Moisture in Food".

[0057] (1) Single-factor experiment setup The inoculation ratios (JY4:HM6 and NC1:HM4) were 15:1, 10:1, 15:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:10, and 1:15; the inoculum amounts were 0.5%, 1%, 2%, 3%, 4%, and 5%; the fermentation times were 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, and 48 h; the fermentation temperatures were 35 ℃, 37 ℃, 39 ℃, 41 ℃, and 43 ℃; the amount of wolfberry added was 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, and 3%; and the drying times were 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h.

[0058] II. Experimental Results (1) Vaccination rate In the experiment with plain milk curds, see Appendix Figure 2 As shown in Figure A, the inoculation ratio of 3:1 showed the best performance in terms of viable bacteria count and DPPH free radical scavenging ability, but there was no significant difference compared with other ratios. In terms of DPPH free radical scavenging ability, the ratio of 3:1 was the highest, with no significant difference compared with other ratios or vitamin C. In terms of superoxide anion free radical scavenging rate, vitamin C was the highest, while the ratio of 3:1 was the second best. See Appendix for details on mixed fermented wolfberry milk curds. Figure 2 As shown in Figure B, the overall viable cell count and free radical scavenging rate increased with increasing inoculation ratio, but the DPPH free radical scavenging ability was strongest at a 1:1 ratio; while the superoxide anion scavenging rate was lowest at a 1:5 ratio, with no significant differences among the other ratios. Taking all factors into consideration, JY4 and HM6 at a 3:1 ratio and NC1 and HM4 at a 1:1 ratio were selected for subsequent experiments.

[0059] 2. Inoculation volume See Appendix for the original flavor milk curd experiment. Figure 3 As shown in Figure A, an inoculum size of 3% exhibited the best overall performance: the highest viable cell count at 24 hours (8.14 log CFU / mL), and while the DPPH free radical scavenging rate was slightly lower than the VC control group, it was still the highest (95.75%), with no significant difference compared to partial inoculum sizes; the superoxide anion free radical scavenging rate showed no significant difference among 0.5%, 1%, and 3% inoculum sizes, but was highest at 3% (22.10%). See Appendix for the mixed fermentation experiment of wolfberry milk curds. Figure 3 As shown in Figure B, there was no significant difference in the number of viable bacteria among the groups after 24 hours, which may be related to the fact that the strains entered the stationary phase of growth. The antioxidant capacity generally showed a trend of first increasing and then decreasing. This is because if the inoculum amount is too low, the bacterial growth is insufficient, and the production of metabolites (such as antioxidants) is limited; if the inoculum amount is too high, the nutrients in the culture medium are consumed too quickly, which is not conducive to fermentation and may also cause changes in metabolic pathways, reducing the yield of antioxidant products. Among the inoculum amounts, 4% showed the highest DPPH free radical scavenging rate, but there was no significant difference compared to 0.5% and 3%; while 3% showed the highest superoxide anion free radical scavenging rate. Taking all factors into consideration, a 3% inoculum amount was selected for both the original flavor milk curd and the mixed fermented wolfberry milk curd in subsequent experiments.

[0060] 3. Fermentation time See the appendix for the results of the fermentation time test. Figure 4 As shown, the free radical scavenging rates of both plain and mixed-fermentation milk curds initially increased and then decreased with prolonged fermentation time. This is because insufficient accumulation of antioxidants occurs with too short a fermentation time, while excessively long fermentation may lead to degradation of active ingredients. Regarding viable cell counts, the trends differed: plain milk curds decreased with prolonged fermentation time, likely due to excessively high total acid content in the solution inhibiting bacterial growth; mixed-fermentation milk curds, however, gradually stabilized with time, possibly due to the addition of goji berries increasing nutrient content.

[0061] For details, see the appendix for original flavor milk curds. Figure 4 As shown in Figure A, the DPPH free radical scavenging rate was highest at 30 h of fermentation, showing no significant difference compared to the VC control group. The superoxide anion scavenging rate showed no significant difference at multiple time points, but was highest at 36 h, followed by 30 h. Considering the antioxidant performance, 30 h was selected as the fermentation time for the original flavor milk curds in subsequent experiments. For the mixed fermentation type of goji berry milk curds, please refer to the appendix. Figure 4 As shown in Figure B, the scavenging rates of both free radicals reached their highest levels at 24 h, while the viable cell count showed a trend of first increasing and then stabilizing. Therefore, 24 h was determined to be the optimal fermentation time for subsequent experiments.

[0062] 4. Fermentation temperature Based on the results of the fermentation temperature test Figure 5The viable bacteria counts of both the original flavor milk curds and the mixed fermented goji berry milk curds showed a trend of first increasing and then decreasing with rising temperature. This is because higher fermentation temperatures accelerate lactic acid production, leading to a faster drop in pH, which is detrimental to the survival of the bacteria. The free radical scavenging rates of both products also showed a similar overall trend, generally increasing first and then decreasing. This is because when the temperature rises above a certain threshold, excessively high temperatures can damage the structure of antioxidant substances, weakening their antioxidant capacity.

[0063] Specifically, see the appendix for original flavor milk curds. Figure 5 As shown in Figure A, the highest viable bacterial count was observed at 39 °C, but the DPPH free radical scavenging rate was not significantly affected by temperature, meaning that fermentation temperature had no significant impact on the DPPH free radical scavenging rate during fermentation. The superoxide anion scavenging rate, however, reached its highest level at 37 °C. Considering overall antioxidant capacity, 37 °C was chosen as the subsequent fermentation temperature for the original flavor milk curds. For the mixed fermentation type of goji berry milk curds, please refer to the appendix. Figure 5 As shown in Figure B, the number of viable bacteria was highest at 39 °C, but the scavenging rates of both free radicals reached their maximum values ​​at 41 °C. Therefore, 41 °C was ultimately determined as the fermentation temperature used in subsequent experiments.

[0064] 5. Amount of goji berries added In the experiment on the amount of goji berries added, the goji berry-added milk curds were observed (see appendix). Figure 6 The viable bacteria count shown in Figure A did not differ significantly among different addition amounts. The DPPH free radical scavenging rate increased with increasing goji berry addition, stabilizing after reaching 1.5%. The superoxide anion scavenging rate continued to increase, reaching its peak at 3%. However, excessive goji berry addition can affect product color and other aspects. See Appendix for mixed fermented goji berry milk curds. Figure 6 As shown in Figure B, the viable bacterial count, DPPH, and superoxide anion scavenging rate all showed a trend of first increasing and then decreasing with increasing wolfberry addition. The viable bacterial count after adding wolfberry was significantly higher than that in the unadded group, indicating that the sugars, amino acids, and other nutrients in wolfberry juice can meet the needs of bacterial growth. The free radical scavenging rate showed a trend of first increasing and then decreasing, possibly because lower wolfberry content contains wolfberry polysaccharides and has a higher anthocyanin content, and the uronic acid in the polysaccharides can effectively inhibit free radicals. The decrease may be due to the increased polysaccharide content in the substrate with increasing wolfberry addition, and the changes in branched structure caused by fermentation affecting the efficiency of polysaccharides in scavenging free radicals. At an addition of 1.5%, both the viable bacterial count and the two free radical scavenging rates reached their highest levels. Considering all factors, both the added and mixed fermentation wolfberry milk curds will use a 1.5% wolfberry addition level for subsequent studies.

[0065] 6. Drying time See Appendix for drying time test. Figure 7 As shown, the moisture content of all types of milk curds decreased significantly with increasing drying time. See Appendix. Figure 7 As shown in Figure A, the DPPH free radical scavenging rate of the plain milk curds showed a trend of first decreasing and then increasing, which is due to the post-ripening of the cheese at this temperature. However, there was no significant difference in the DPPH free radical scavenging rate between drying times of 8 h and 10 h. The superoxide anion scavenging rate was not significantly affected by the drying time. After comprehensive consideration, the subsequent drying time for the plain milk curds was determined to be 8 h.

[0066] The free radical scavenging rates of both additive-type and mixed-fermentation-type goji berry milk curds showed a change from initial increase to subsequent decrease with prolonged drying time. This may be related to the release of antioxidants from goji berry cells during the initial heating phase and the subsequent oxidative losses over time. (See appendix) Figure 7 As shown in Figure B, there was no significant difference in DPPH scavenging rate between 4 h and 6 h for the added goji berry milk curds, and no significant difference in superoxide anion scavenging rate between 2 h and 4 h. Therefore, a drying time of 4 h was ultimately selected. (See Appendix) Figure 7 As shown in Figure C, there was no significant difference in the DPPH scavenging rate of the mixed fermented wolfberry milk curds at 4 h and 6 h, but the superoxide anion scavenging rate reached its highest level at 6 h. Therefore, the subsequent drying time was determined to be 6 h.

[0067] Example 5: Response Surface Optimization Based on the single-factor experimental results recorded in the above embodiments, a response surface optimization preparation process was carried out. I. Experimental Methods (1) Plackett-Burman test screening Based on the single-factor analysis, the DPPH radical scavenging rate was used as the response value to evaluate the main single factors under different processes. The optimal value range of each factor obtained from the single-factor experiment was analyzed. Each factor was set to two levels: the lowest (-1) and the highest (1). The Plackett-Burman experiment was designed using Design-Expert. The factors used in the experiment and their codes are shown in Table 1.

[0068] Table 1: Plackett-Burman Experimental Factor Coding Table (2) Box-Behnken response surface design Based on the Plackett-Burman experiment, three significant factors affecting milk curd formation under various process conditions were selected for the steepest ramp-up experiment. Furthermore, using DPPH radical scavenging rate as the response variable, a three-factor, three-level Box-Behnken response surface methodology was designed using Design-Expert to optimize the experiment. The experimental designs for each process are shown in Tables 2, 3, and 4.

[0069] Table 2: Box-Behnken Experimental Design for Original Flavor Milk Curds Table 3: Box-Behnken experimental design for milk curds with added goji berries Table 4: Box-Behnken experimental design for mixed-fermented wolfberry milk curds II. Experimental Results 1. Plackett-Burman assay to screen key factors affecting antioxidant activity The Plackett-Burman (PB) design is an experimental design method used to quickly screen out key influencing factors from multiple variables, particularly suitable for the initial experimental stage. Its main purpose is to identify significant factors from a large number of variables that may affect experimental results, so that these key factors can be focused on in subsequent optimization experiments, while ignoring or fixing insignificant variables to reduce the number of experiments and complexity.

[0070] (1) Experimental design and results of the original flavor milk curd PB experiment The design and results of the original flavor milk curd PB experiment are shown in Table 6, and the evaluation of the DPPH free radical scavenging rate of each factor is shown in Table 7.

[0071] Table 6: PB Experimental Design and Response Values ​​for Original Flavor Milk Curds Table 7: Experimental Design and Evaluation of Factors in the Original Flavor Milk Curd Experiment (PB) Note: * indicates P <0.05, ** indicates P <0.01 Therefore, three factors that significantly affect the DPPH free radical scavenging rate in plain milk curds can be identified, in descending order of influence: drying time, inoculum quantity, and fermentation time. P <0.05). Meanwhile, the regression equation is: (2) Experimental design and results of PB test for added wolfberry milk curds The experimental design and results of the PB test for the added wolfberry milk curd are shown in Table 8, and the evaluation of the DPPH free radical scavenging rate of each factor is shown in Table 9.

[0072] Table 8: Experimental Design and Response Values ​​of Added Goji Berry Milk Curds (PB Experiment) Table 9: Experimental Design and Evaluation of Factors in the PB Experiment for Added Goji Berry Milk Curds Note: * indicatesP <0.05, ** indicates P <0.01 This reveals three factors that significantly influence the DPPH free radical scavenging rate in added wolfberry milk curds, ranked from largest to smallest as follows: inoculum quantity, fermentation time, and wolfberry dosage. P <0.05). Meanwhile, the regression equation is: (3) Experimental design and results of mixed fermentation type milk curd PB experiment The experimental design and results of the mixed fermentation type wolfberry milk curd PB experiment are shown in Table 10, and the evaluation of the DPPH free radical scavenging rate of each factor is shown in Table 11.

[0073] Table 10: Experimental Design and Response Values ​​of Mixed Fermented Goji Berry Milk Curds (PB) Table 11: Experimental Design and Evaluation of Factors in Mixed Fermented Goji Berry Milk Curd Experiment Note: * indicates P <0.05, ** indicates P <0.01 This reveals three factors that significantly affect the DPPH free radical scavenging rate in mixed fermented wolfberry milk curds, ranked from largest to smallest as follows: wolfberry addition amount, drying time, and fermentation time. P <0.05). Meanwhile, the regression equation is: 2. Experimental Design and Results of the Steepest Climb Test (1) Design and results of the steepest climbing test of plain milk curds Based on the PB test results of the original flavor milk curds, drying time, inoculum quantity, and fermentation time are three factors that significantly affect the DPPH free radical scavenging rate. P <0.05). Regression equation analysis showed that B (inoculum quantity), C (fermentation time), and E (drying time) all had positive effects. Among them, the effect of drying time was the most significant, so it was used as the unit of steepest slope to conduct the steepest slope experiment.

[0074] Step sizes B and C were set to 0.5 and 3 respectively, based on actual conditions. The experimental design and results are shown in Table 12. Treatment 3 showed the highest DPPH radical scavenging rate, therefore, treatment 3 was used as the center point for response surface methodology optimization.

[0075] Table 12: Experimental Design and Results of Steepest Climbing Test for Original Flavor Milk Curds (2) Design and results of the steepest climbing test of the wolfberry-added milk curds According to the PB test results of the additive-type milk curd, the inoculum amount, the amount of wolfberry added, and the fermentation time are the three factors that have a significant impact on the DPPH free radical scavenging rate. P <0.05). The regression equation showed that B (inoculum quantity), C (fermentation time), and E (goji berry addition amount) all had positive effects. Since the inoculum quantity was the most significant factor, it was used as the unit of measurement for steepest inoculation.

[0076] The step sizes C and E were set to 4 and 0.5 respectively, based on actual conditions. The experimental design and results are shown in Table 13. Treatment 3 showed the highest DPPH radical scavenging rate, therefore, treatment 3 was used as the center point for response surface methodology optimization.

[0077] Table 13: Experimental Design and Results of Steepest Climbing Test for Added Goji Berry Milk Curds (3) Design and results of the steepest climbing test of mixed fermented wolfberry milk curds According to the PB test results of mixed fermented milk curds, fermentation time, wolfberry addition amount, and drying time are the three factors that have a significant impact on DPPH free radical scavenging rate. P <0.05). The regression equation showed that C (fermentation time) and E (goji berry addition amount) had positive effects, while F (drying time) had a negative effect. Since the amount of goji berries added was the most significant factor, it was used as the unit of measurement for the steepest climb experiment.

[0078] The step sizes C and F were set to 3 and 2 respectively based on actual conditions. The experimental design and results are shown in Table 14. Treatment 3 showed the highest DPPH radical scavenging rate, therefore, treatment 3 was used as the center point for response surface methodology optimization.

[0079] Table 14: Experimental Design and Results of Steepest Climbing Test for Mixed Fermented Goji Berry Milk Curds 3. Results of Box-Behnken Design Experiments and Regression and Significance Analysis Based on the steepest climb experiment, the Box-Benhnken central composite design principle of Design-Expert 13 software was used to design milk curds with different processes using a 3-factor, 3-level response surface methodology, with DPPH free radical scavenging rate as the response value.

[0080] (1) Results of response surface methodology experiment and regression and significance analysis of original flavor milk curds Three factors (A: inoculum size, B: fermentation time, C: drying time) that significantly affected the DPPH free radical scavenging rate of plain milk curds were selected as independent variables, with the DPPH free radical scavenging rate as the response value. The experimental results are shown in Table 15 below: Table 15: Response Surface Optimization Experimental Design and Results for Original Flavor Milk Curds Multiple regression fitting was performed on the experimental data, and the regression equation model was obtained as follows: As can be seen from the analysis of variance (Table 16), the regression model is highly significant. P The main effects B and C, and the quadratic terms A², B², and C² are all significant (<0.000 1), while the interaction terms AC are significant, and AB and BC are not. The model's R² is 0.9832, Adj R² is 0.9616, and Pred R² is 0.8777. These three values ​​are close and relatively high, indicating excellent model fit and good predictive ability. In summary, this model can effectively explain changes in response values ​​and can be used for process optimization and parameter prediction.

[0081] Table 16: Analysis of Variance of the Regression Model for Original Flavor Milk Curds Note: * indicates P <0.05, ** indicates P <0.01 (2) Results of response surface methodology and regression and significance analysis of added wolfberry milk curds Three factors (A: inoculum size, B: fermentation time, C: goji berry dosage) that significantly affected the DPPH free radical scavenging rate of added goji berry milk curds were selected as independent variables, with the DPPH free radical scavenging rate as the response value. The experimental results are shown in Table 17 below: Table 17: Response Surface Optimization Experimental Design and Results of Added Goji Berry Milk Curds Multiple regression fitting was performed on the experimental data, and the regression equation model was obtained as follows: As can be seen from the analysis of variance (Table 18), the regression model is highly significant. PThe main effects A and B, and the quadratic terms A², B², and C² are all significant (<0.000 1), while the interaction terms AB are significant, and AC and BC are not. The model's R² = 0.9925, Adj R² = 0.982.8, and Pred R² = 0.9860. These three values ​​are close and relatively high, indicating excellent model fit and good predictive ability. In summary, this model can effectively explain changes in response values ​​and can be used for process optimization and parameter prediction.

[0082] Table 18: Analysis of Variance of Regression Model for Added Goji Berry Milk Curds Note: * indicates P <0.05, ** indicates P <0.01 (3) Results of response surface methodology and regression and significance analysis of mixed fermented wolfberry milk curds Three factors that significantly affect the DPPH free radical scavenging rate of mixed fermented milk curds (A: fermentation time, B: amount of wolfberry added, C: drying time) were selected as independent variables, and the DPPH free radical scavenging rate was the response value. The results are shown in Table 19.

[0083] Table 19: Response Surface Optimization Experimental Design and Results for Mixed Fermented Milk Curds Multiple regression fitting was performed on the experimental data, and the regression equation model was obtained as follows: As can be seen from the analysis of variance (Table 20), the regression model is highly significant. P The main effects A and C, and the quadratic terms A², B², and C² are all significant (<0.000 1), while the interaction terms AB and AC are significant, and BC is not. The model's R² is 0.9837, Adj R² is 0.9627, and Pred R² is 0.9436. These three values ​​are close and relatively high, indicating excellent model fit and good predictive ability. In summary, this model can effectively explain changes in response values ​​and can be used for process optimization and parameter prediction.

[0084] Table 20: Analysis of Variance for the Regression Model of Mixed Fermented Goji Berry Milk Curds Note: * indicates P <0.05, ** indicates P <0.01 According to the optimal results of the response surface methodology, the optimal process for YW was: inoculum size 2.969%, fermentation time 30.664 h, drying time 7.793 h, and DPPH radical scavenging rate 91.781%; for TJ, the optimal process was: inoculum size 3.063%, fermentation time 29.231 h, goji berry addition 1.478%, and DPPH radical scavenging rate 94.051%; and for HH, the optimal process was: fermentation time 25.172 h, goji berry addition 1.353%, drying time 4.098 h, and DPPH radical scavenging rate 93.711%. Based on actual conditions, the optimal inoculum size for YW was 3%, fermentation time 30.5 h, and drying time 7.75 h; for TJ, the optimal inoculum size was 3%, fermentation time 29.25 h, and goji berry addition 1.5%; and for HH, the optimal fermentation time was 25.25 h, goji berry addition 1.35%, and drying time 4 h. The final results are shown in Table 21. The DPPH free radical scavenging rates of milk curds under each process condition were 91.38%, 94.15%, and 93.61%, respectively. The error between the DPPH free radical scavenging rate of each process and the predicted value was less than 5%, which proved the accuracy of the model.

[0085] Example 6: Physicochemical Index Analysis The method for determining the viable bacteria count was the same as that described in Example 3 above; the method for determining the antioxidant content was the same as that described in Example 3 above; the method for determining the water content was the same as that described in GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food"; the method for determining the total sugar content was the same as that described in the kit method, the total sugar content kit (catalog number JC0409-M) (Nanjing Jice Biotechnology Co., Ltd.); the method for determining the total acid was the same as that described in GB 5009.239-2016 "National Food Safety Standard - Determination of Acidity in Food"; the method for determining the protein content was the same as that described in GB 5009.5-2025 "National Food Safety Standard - Determination of Protein in Food"; the method for determining the fat content was the same as that described in GB 5009.6-2016 "National Food Safety Standard - Determination of Fat in Food".

[0086] Table 21: Quality Analysis of Milk Curds Processed by Different Methods The physicochemical properties of milk curds produced using different processes are shown in Table 21. All three samples had high viable bacterial counts (approximately 8.5-8.7 logCFU / g), indicating they are all foods rich in active probiotics. In terms of antioxidant activity, TJ showed the best performance, with a DPPH free radical scavenging rate of 94.15% and a superoxide anion scavenging rate of 27.84%, both higher than YW and HH. HH was second best, while YW was the weakest. This indicates that whether added directly or through mixed fermentation, goji berries can significantly improve antioxidant performance, possibly due to the presence of goji berry polysaccharides, flavonoids, and carotenoids. However, direct addition was more effective, possibly due to the reduction in antioxidants caused by fermentation. Compared to unfermented goji berry juice, fermented goji berry juice showed significantly lower total phenol and flavonoid content, but increased total sugar content. In terms of major nutritional components, HH had the lowest total acid content (103.10°T), while YW had the highest (138.83°T). Furthermore, HH had the highest moisture content (76.13%), while YW had the lowest (53.79%), which may contribute to HH's smoother texture. HH had the highest total sugar content (18.68 g / 100g), followed by TJ, with YW having the lowest. However, in terms of protein content, YW (38.08 g / 100g) had the highest, while TJ (32.94 g / 100g) and HH (34.10 g / 100g) were relatively low. This may be because the addition of goji berries diluted the overall protein concentration in the milk, or it could be due to a reaction between the polyphenols and polysaccharides in goji berries and the protein. The fat content showed a trend of YW (28.80%) > TJ (21.07%) > HH (18.21%), indicating that the addition of goji berries reduced the product's fat ratio.

[0087] In summary, milk curds made using different processes each have their own characteristics. YW has the highest protein and fat content, but lower carbohydrate content and some antioxidant indicators. The two products with added goji berries (TJ and HH) have significant advantages in antioxidant activity and carbohydrate nutrition, with HH having the highest total sugar content and the lowest fat content, while TJ shows the best performance in antioxidant indicators.

[0088] Example 7: Electronic Nose Analysis Following the method of Wang Jiao et al., the differences in volatile odors and aroma-producing components of three types of milk curds and commercial milk curd (SY) samples were analyzed using electronic nose technology. The sensitive substances corresponding to the sensors are shown in Table 5.

[0089] Table 5: Sensitive Substances Corresponding to Electronic Nose Sensors An electronic nose (E-nose) is a biomimetic olfactory system that uses a sensor array to detect volatile organic compounds released from food, thereby evaluating its flavor. The flavor analysis results of four milk curd samples based on the electronic nose are shown below. Figure 11 The radar charts showed that the overall outlines of the samples were similar, but the sensor response values ​​differed. All four samples showed certain response values ​​on sensors W1W, W5S, W2W, W2S, and W1S, indicating that the samples contained a significant amount of sulfur-containing compounds, nitrogen oxides, and aromatic substances. Among them, the response values ​​of the three experimental milk curds (YW, TJ, and HH) were all higher than the commercial control (SY), especially on sensors such as W1W and W5S. The additive-type milk curd (TJ) showed the strongest response on most sensors, presumably related to the direct addition of goji berry powder, which introduced more volatile components.

[0090] Principal component analysis (PCA) further validated the above results. The cumulative contribution of PC1 and PC2 reached 98.4%, which can fully reflect the volatility characteristics of the sample. SY, HH, and YW partially overlapped in the PCA plot, indicating that their odor compositions are relatively similar. Meanwhile, sensors such as W1W and W5S made significant contributions to PC1, consistent with the radar plot response pattern, jointly revealing the influence of different processes on the flavor characteristics of milk curds.

[0091] The above embodiments are merely examples to clearly illustrate the present invention and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A type of milk curd with antioxidant activity, characterized in that, The antioxidant-active milk curds mentioned above are made from whole milk and Pediococcus pentosaceus (Pentose Pediococcus). Pediococcus pentosaceus HM4, Streptococcus thermophilus ( Streptococcus thermophilus ) HM6, Lactococcus lactis ( Lactococcus lactis JY4 and Lactobacillus plantarum ( Lactobacillus plantarum ) Obtained by fermentation with NC1 compound microbial agent.

2. The milk curd with antioxidant activity as described in claim 1, characterized in that, The antioxidant-rich milk curds can also be made with goji berry powder.

3. The milk curd with antioxidant activity as described in claim 1, characterized in that, The compound microbial agent has a mass percentage of 0.5%-5%.

4. The milk curd with antioxidant activity as described in claim 2, characterized in that, The weight percentage of goji berry powder is 0.5%-3%.

5. A method for preparing an antioxidant-active milk curd as described in any one of claims 1 to 2, characterized in that, It is prepared by the following steps: S1, strain Pediococcus pentosaceus ( Pediococcus pentosaceus HM4, Streptococcus thermophilus ( Streptococcus thermophilus ) HM6, Lactococcus lactis ( Lactococcus lactis JY4 and Lactobacillus plantarum ( Lactobacillus plantarum NC1 was inoculated into MRS broth medium and cultured at 37 ℃ for 24 h. The viable counts of the four lactic acid bacteria suspensions were determined. The viable counts of the four lactic acid bacteria suspensions were then serially diluted to ensure that they were of the same gradient for later use. S2. Aseptic whole milk is sterilized at 85°C for 20 minutes. S3. The bacterial suspension obtained from activation in step S1 is mixed according to the volume ratio of *Lactococcus lactis* (…). Lactococcus lactis JY4 and Streptococcus thermophilus ( Streptococcus thermophilus HM6 is mixed in a ratio of (15-1):(1-15); Lactobacillus plantarum ( Lactobacillus plantarum NC1 and Pediococcus pentosaceus ( Pediococcus pentosaceus HM4 is mixed in a ratio of (15-1):(1-15) and added to the sterilized whole milk in S2 at a mass percentage of 0.5%-5%. S4. Ferment the whole milk inoculated with the bacterial agent in step S1 for 12 h-48 h; fermentation temperature is 35℃-43℃. S5. Pour the fermented product obtained in step S4 into a 300-mesh gauze, hang it up, and drain the whey for 12 hours. S6. Dry the filter material obtained from the gauze in step S5 at 35 ℃ for 2 h-10 h to obtain milk curds with antioxidant activity.

6. The method for preparing an antioxidant-active milk curd as described in claim 5, characterized in that, You can also add 0.5%-3% by weight of goji berry powder in step S3 or step S5.

7. The method for preparing an antioxidant-active milk curd as described in claim 5, characterized in that, Lactococcus lactis ( Lactococcus lactis JY4 and Streptococcus thermophilus ( Streptococcus thermophilus HM6 is mixed in a 3:1 ratio; Lactobacillus plantarum ( Lactobacillus plantarum NC1 and Pediococcus pentosaceus ( Pediococcus pentosaceus HM4 was mixed in a 1:1 ratio; the inoculation amount was 3%.

8. The method for preparing an antioxidant-active milk curd as described in claim 5, characterized in that, Fermentation time is 24 hours or 30 hours; fermentation temperature is 37°C or 39°C.

9. The method for preparing an antioxidant-active milk curd as described in claim 6, characterized in that, The amount of goji berries added is 1.5%; the drying time is 4 hours or 8 hours.

10. The application of the method for preparing an antioxidant-active milk curd as described in any one of claims 1 to 3 in the preparation of an antioxidant-active milk curd.

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