Biological fermentation casein powder and preparation method thereof

Abstract

The invention relates to the field of milk powder processing, in particular to biological fermentation casein powder and a preparation method thereof. Various probiotics including streptococcus thermophilus, lactobacillus plantarum and lactobacillus bulgaricus are adopted, casein is subjected to staged fermentation, and natural plant extracts such as mulberry juice and functional components such as trehalose, inulin and fructo-oligosaccharide are added, so that the health-care food is prepared. And the biological fermentation casein powder with high nutritional value, good solubility and oxidation resistance is prepared. The method not only improves the biological activity and absorbability of casein, but also endows the product with more health benefits, such as immune regulation and intestinal health promotion, and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of milk powder processing, in particular to a biological fermentation casein powder and a preparation method thereof. Background Art

[0002] Casein is a major protein in mammalian milk, comprising 70%-80% of total milk protein. It possesses excellent emulsifying properties, stability, and nutritional value, making it a high-quality protein source. In the food industry, casein is widely used in the production of dairy products, beverages, baked goods, and other products to improve their texture, taste, and nutritional value.

[0003] However, despite the aforementioned excellent properties of casein, its bioavailability and functionality are still limited in certain applications. For example, unprocessed casein is difficult to be directly absorbed by the human body and has low antioxidant and bioactivity. In addition, existing fermentation methods often fail to effectively improve the decomposition efficiency of casein, resulting in low absorption rate in the intestine.

[0004] Therefore, there is an urgent need to develop a method that can significantly improve the bioavailability and functionality of casein. By improving the preparation process of casein, making it easier for the human body to absorb, and enhancing its antioxidant properties and bioactivity, it will help enhance its application value in the food and nutrition fields. Summary of the Invention

[0005] In view of this, the present invention aims to provide a bio-fermented casein powder and a method for preparing the same. By fermenting a casein solution with various plant powders and probiotics in steps, and then adding mulberry juice and other ingredients, a bio-fermented casein powder rich in small molecule peptides and bioactive substances is ultimately obtained. Based on the above objectives, the present invention provides a bio-fermented casein powder and a method for preparing the same.

[0006] A method for preparing bio-fermented casein powder comprises the following steps:

[0007] S1: fresh sterilized milk is centrifuged at 2°C-6°C and 5000r / min-6000r / min for 20 minutes for defatting, and the supernatant after removing the upper layer of fat is the defatted sterilized milk; the pH of the sterilized milk is adjusted to 4.0-4.5, and the milk is allowed to stand at room temperature until precipitation occurs to obtain a casein precipitate, sodium hydroxide solution is added dropwise to the casein precipitate, PBS buffer is added, and the mixture is stirred at 90°C-100°C for 15-20 minutes. After cooling, deionized water is added, and the mixture is stirred evenly to obtain a casein solution; fresh mulberry fruits are pulped, filtered to remove residue, and mulberry juice is obtained, and the pH of the mulberry juice is adjusted to 5.5-6.0 with sodium citrate to obtain mulberry juice.

[0008] S2: uniformly mixing casein solution, soybean meal powder, Atractylodes macrocephala powder, Astragalus root powder, licorice powder, ammonium sulfate, and potassium dihydrogen phosphate, and sterilizing at high temperature to obtain a casein culture medium, inoculating activated Streptococcus thermophilus into the casein culture medium, and fermenting anaerobically at 42°C-45°C for 9-12 hours to obtain culture medium A, inoculating activated Lactobacillus plantarum into culture medium A, and fermenting anaerobically at 30°C-40°C for 8-10 hours to obtain culture medium B, inoculating activated Lactobacillus bulgaricus into culture medium B, adding mulberry juice, and fermenting anaerobically at 42°C-45°C for 8-11 hours to obtain a casein fermentation liquid;

[0009] S3: Add trehalose, whey protein and gelatin to the casein fermentation broth, stir for 20-40 minutes at a stirring speed of 600-800 rpm, continue to add inulin, oligofructose and lecithin, stir for 30-40 minutes at a stirring speed of 800 rpm-1000 rpm, and after stirring, spray dry to obtain bio-fermented casein powder.

[0010] Preferably, the sterilized milk is any one of pasteurized cow's milk, goat's milk, yak's milk and buffalo's milk.

[0011] Preferably, the soybean meal powder, Atractylodes macrocephala powder, Astragalus root powder and Licorice root powder are obtained by grinding soybean meal, Atractylodes macrocephala, Astragalus root and Licorice root separately and passing through a 100-mesh sieve.

[0012] Preferably, the sodium hydroxide solution is a 0.5 mol / L sodium hydroxide solution, and the PBS buffer is a 0.01 mol / L PBS buffer.

[0013] Preferably, the usage ratio of the casein precipitate, sodium hydroxide solution, PBS buffer and deionized water is 10 g: 8-12 ml: 16-20 ml: 58-66 ml.

[0014] Preferably, the viable count of the thermophilic Streptococcus after activation reaches 8.2×10 8 CFU / mL-8.5×10 8 CFU / mL, the number of viable Lactobacillus plantarum inoculation after activation reached 8.2×10 8 CFU / mL-8.5×10 8 CFU / mL, the number of viable Lactobacillus bulgaricus after activation reached 8.5×10 8 CFU / mL-8.8×10 8 CFU / mL.

[0015] Preferably, the inoculum amount of thermophilus streptococcus is 3% of the volume of the casein culture medium, and the volume ratio of the inoculum amount of thermophilus streptococcus, the inoculum amount of Lactobacillus plantarum and the inoculum amount of Lactobacillus bulgaricus is 3:2:1.

[0016] Preferably, the usage ratio of the casein solution, soybean meal powder, Atractylodes macrocephala powder, Astragalus root powder, licorice powder, ammonium sulfate, potassium dihydrogen phosphate, and mulberry juice is 80 mL: 3-5 g: 1.2-1.8 g: 1.2-1.8 g: 0.4-0.6 g: 0.5-0.8 g: 0.2-0.3 g: 20-30 mL.

[0017] Preferably, the mass ratio of the casein fermentation broth, inulin, oligofructose, trehalose, whey protein, gelatin and lecithin is 90:10-20:8-15 parts:5-10 parts:6-8 parts:5-8 parts:4-6 parts.

[0018] The traditional Chinese medicine pharmacology of the Atractylodes macrocephala, Astragalus membranaceus and Licorice is as follows:

[0019] Atractylodes macrocephala: bitter, sweet, warm in nature, enters the spleen and stomach meridians. It has the effects of invigorating the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and calming the fetus.

[0020] Astragalus: Sweet in taste, slightly warm in nature, it enters the spleen and lung meridians. It has the effects of replenishing qi and raising yang, strengthening the body's defense system, promoting diuresis and reducing swelling, and expelling toxins and promoting tissue regeneration.

[0021] Licorice: sweet in taste, neutral in nature, enters the heart, lung, spleen, and stomach meridians. It has the effects of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, relieving pain, and harmonizing various medicines.

[0022] Beneficial effects of the present invention:

[0023] 1. The present invention effectively decomposes casein into a large number of small molecular peptides through the fermentation process, which is conducive to intestinal absorption.

[0024] 2. The mulberry juice added during the fermentation process of the present invention increases the content of phenolic and flavonoid compounds in the bio-fermented casein powder, thereby improving its antioxidant performance.

[0025] 3. The bio-fermented casein powder prepared by the present invention can effectively improve the absorption of essential amino acids in rats. Atractylodes macrocephala, Astragalus membranaceus and Licorice root promote protein absorption by regulating spleen and stomach function, while probiotics such as Streptococcus thermophilus, Lactobacillus plantarum and Lactobacillus bulgaricus can decompose casein to generate small molecule peptides that are more easily absorbed by the intestine. DETAILED DESCRIPTION

[0026] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments.

[0027] Source of strain:

[0028] The Streptococcus thermophilus in the following examples was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC NO.22560.

[0029] The Lactobacillus plantarum in the following examples is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No. 27469.

[0030] The Lactobacillus bulgaricus in the following examples is Lactobacillus delbrueckii subsp. bulgaricus, which is deposited in the China Industrial Culture Collection Center of Microorganisms with a deposit number of CCTCCNO: M 2022316.

[0031] Example 1:

[0032] A method for preparing bio-fermented casein powder comprises the following steps:

[0033] S1: fresh sterilized milk is centrifuged at 4°C and 5200 r / min for 20 min for defatting, and the supernatant after removing the upper layer of fat is the defatted sterilized milk; the pH of the sterilized milk is adjusted to 4.0, and the milk is allowed to stand at room temperature until precipitation occurs to obtain a casein precipitate; 1000 g of the casein precipitate is added dropwise with 860 mL of sodium hydroxide solution, 1700 mL of PBS buffer is added, and the mixture is stirred at 95°C for 16 min. After cooling, 6000 mL of deionized water is added, and the mixture is stirred evenly to obtain a casein solution; fresh mulberry fruits are pulped, filtered to remove residue, and mulberry juice is obtained; the pH of the mulberry juice is adjusted to 5.8 with sodium citrate to obtain mulberry juice.

[0034] S2: 8000 mL of casein solution, 450 g of soybean meal powder, 120 g of Atractylodes macrocephala powder, 140 g of Astragalus membranaceus powder, 46 g of licorice powder, 60 g of ammonium sulfate, and 30 g of potassium dihydrogen phosphate were mixed evenly and sterilized at high temperature to obtain casein culture medium. The inoculum amount of thermophilic Streptococcus was 3% of the volume of casein culture medium, and the volume ratio of thermophilic Streptococcus inoculum, Lactobacillus plantarum inoculum, and Lactobacillus bulgaricus inoculum was 3:2:1. The number of viable bacteria after activation reached 8.5 × 10 8 CFU / mL of thermophilic Streptococcus was inoculated into casein medium and fermented anaerobically at 42℃-45℃ for 10h to obtain medium A. The number of viable bacteria after activation reached 8.2×10 8CFU / mL of plant lactobacillus was inoculated into medium A and fermented anaerobically at 30℃-40℃ for 9h to obtain medium B. The number of viable bacteria after activation reached 8.6×10 8 CFU / mL of Lactobacillus bulgaricus was inoculated into culture medium B, 280 mL of mulberry juice was added, and the culture medium was anaerobically fermented at 42°C-45°C for 10 h to obtain casein fermentation liquid;

[0035] S3: 9000 g of casein fermentation liquid, 80 g of trehalose, 65 g of whey protein and 60 g of gelatin were stirred for 35 min at a stirring speed of 700 rpm, and then 150 g of inulin, 120 g of oligofructose and 50 g of lecithin were added and stirred for 32 min at a stirring speed of 900 rpm. After stirring, the mixture was spray-dried to obtain bio-fermented casein powder.

[0036] Example 2:

[0037] A method for preparing bio-fermented casein powder comprises the following steps:

[0038] S1: fresh sterilized milk is centrifuged at 3°C and 5200 rpm for 20 min for defatting, and the supernatant after removing the upper layer of fat is the defatted sterilized milk; the pH of the sterilized milk is adjusted to 4.5, and the milk is allowed to stand at room temperature until precipitation occurs to obtain a casein precipitate; 1000 g of the casein precipitate is dropwise added with 800 mL of sodium hydroxide solution, 1600 mL of PBS buffer is added, and the mixture is stirred at 90°C for 20 min. After cooling, 5800 mL of deionized water is added, and the mixture is stirred evenly to obtain a casein solution; fresh mulberry fruits are pulped, filtered to remove residue, and mulberry juice is obtained; the pH of the mulberry juice is adjusted to 5.5 with sodium citrate to obtain mulberry juice.

[0039] S2: 8000 mL of casein solution, 300 g of soybean meal powder, 120 g of Atractylodes macrocephala powder, 120 g of Astragalus membranaceus powder, 40 g of licorice powder, 50 g of ammonium sulfate, and 30 g of potassium dihydrogen phosphate were mixed evenly and sterilized at high temperature to obtain casein culture medium. The inoculum amount of thermophilic Streptococcus was 3% of the volume of casein culture medium, and the volume ratio of thermophilic Streptococcus inoculum, Lactobacillus plantarum inoculum, and Lactobacillus bulgaricus inoculum was 3:2:1. The number of viable bacteria after activation reached 8.2 × 10 8 CFU / mL of thermophilic Streptococcus was inoculated into casein medium and fermented anaerobically at 42℃-45℃ for 9h to obtain medium A. The number of viable bacteria after activation reached 8.2×10 8 CFU / mL of plant lactobacillus was inoculated into medium A and fermented anaerobically at 30℃-40℃ for 8h to obtain medium B. The number of viable bacteria after activation reached 8.5×10 8CFU / mL of Lactobacillus bulgaricus was inoculated into culture medium B, 200 mL of mulberry juice was added, and the culture medium was anaerobically fermented at 42°C-45°C for 8 h to obtain casein fermentation liquid;

[0040] S3: Add 50 g of trehalose, 60 g of whey protein and 50 g of gelatin to 9000 g of casein fermentation liquid, stir for 25 min at a stirring speed of 800 pm, continue to add 140 g of inulin, 100 g of oligofructose and 40 g of lecithin, stir for 40 min at a stirring speed of 800 rpm, and after stirring, spray dry to obtain bio-fermented casein powder.

[0041] Example 3:

[0042] A method for preparing bio-fermented casein powder comprises the following steps:

[0043] S1: fresh sterilized milk is centrifuged at 4°C and 5500 rpm for 20 min for defatting, and the supernatant after removing the upper layer of fat is the defatted sterilized milk; the pH of the sterilized milk is adjusted to 4.5, and the milk is allowed to stand at room temperature until precipitation occurs to obtain a casein precipitate; 1000 g of the casein precipitate is dropwise added with 990 mL of sodium hydroxide solution, 1700 mL of PBS buffer is added, and the mixture is stirred at 95°C for 20 min. After cooling, 6200 mL of deionized water is added, and the mixture is stirred evenly to obtain a casein solution; fresh mulberry fruits are pulped, filtered to remove residue, and mulberry juice is obtained; the pH of the mulberry juice is adjusted to 5.5 with sodium citrate to obtain mulberry juice.

[0044] S2: 8000 mL of casein solution, 410 g of soybean meal powder, 150 g of Atractylodes macrocephala powder, 140 g of Astragalus membranaceus powder, 42 g of liquorice powder, 75 g of ammonium sulfate, and 25 g of potassium dihydrogen phosphate were mixed evenly and sterilized at high temperature to obtain casein culture medium. The inoculum amount of thermophilic Streptococcus was 3% of the volume of casein culture medium, and the volume ratio of thermophilic Streptococcus inoculum, Lactobacillus plantarum inoculum, and Lactobacillus bulgaricus inoculum was 3:2:1. The number of viable bacteria after activation reached 8.4 × 10 8 CFU / mL of thermophilic Streptococcus was inoculated into casein medium and fermented anaerobically at 42℃-45℃ for 11h to obtain medium A. The number of viable bacteria after activation reached 8.5×10 8 CFU / mL of plant lactobacillus was inoculated into medium A and fermented anaerobically at 30℃-40℃ for 9.5h to obtain medium B. The number of viable bacteria after activation reached 8.6×10 8 CFU / mL of Lactobacillus bulgaricus was inoculated into culture medium B, 250 mL of mulberry juice was added, and the culture medium was anaerobically fermented at 42°C-45°C for 10 h to obtain casein fermentation liquid;

[0045] S3: Add 70 g of trehalose, 69 g of whey protein and 70 g of gelatin to 9000 g of casein fermentation liquid, stir for 31 min at a stirring speed of 700 pm, continue to add 165 g of inulin, 105 g of oligofructose and 52 g of lecithin, stir for 36 min at a stirring speed of 900 rpm, and after stirring, spray dry to obtain bio-fermented casein powder.

[0046] Example 4:

[0047] A method for preparing bio-fermented casein powder comprises the following steps:

[0048] S1: fresh sterilized milk is centrifuged at 4°C and 6000 rpm for 20 min for defatting, and the supernatant after removing the upper layer of fat is the defatted sterilized milk; the pH of the sterilized milk is adjusted to 4.5, and the milk is allowed to stand at room temperature until precipitation occurs to obtain a casein precipitate; 1000 g of the casein precipitate is dropwise added with 990 mL of sodium hydroxide solution, 1700 mL of PBS buffer is added, and the mixture is stirred at 98°C for 20 min. After cooling, 6200 mL of deionized water is added, and the mixture is stirred evenly to obtain a casein solution; fresh mulberry fruits are pulped, filtered to remove residue, and mulberry juice is obtained; the pH of the mulberry juice is adjusted to 6.0 with sodium citrate to obtain mulberry juice.

[0049] S2: 8000 mL of casein solution, 500 g of soybean meal powder, 170 g of Atractylodes macrocephala powder, 180 g of Astragalus membranaceus powder, 50 g of liquorice powder, 65 g of ammonium sulfate, and 30 g of potassium dihydrogen phosphate were mixed evenly and sterilized at high temperature to obtain casein culture medium. The inoculum amount of thermophilic Streptococcus was 3% of the volume of casein culture medium, and the volume ratio of thermophilic Streptococcus inoculum, Lactobacillus plantarum inoculum, and Lactobacillus bulgaricus inoculum was 3:2:1. The number of viable bacteria after activation reached 8.5 × 10 8 CFU / mL of thermophilic Streptococcus was inoculated into casein medium and fermented anaerobically at 42℃-45℃ for 12h to obtain medium A. The number of viable bacteria after activation reached 8.4×10 8 CFU / mL of plant lactobacillus was inoculated into medium A and fermented anaerobically at 30℃-40℃ for 10h to obtain medium B. The number of viable bacteria after activation reached 8.8×10 8 CFU / mL of Lactobacillus bulgaricus was inoculated into culture medium B, 300 mL of mulberry juice was added, and the culture medium was anaerobically fermented at 42°C-45°C for 11 h to obtain casein fermentation liquid;

[0050] S3: Add 95 g of trehalose, 80 g of whey protein and 75 g of gelatin to 9000 g of casein fermentation liquid, stir for 40 min at a stirring speed of 800 pm, continue to add 180 g of inulin, 150 g of oligofructose and 60 g of lecithin, stir for 40 min at a stirring speed of 900 rpm, and after stirring, spray dry to obtain bio-fermented casein powder.

[0051] Comparative Example 1:

[0052] Compared with Example 1, this comparative example did not add mulberry juice during the preparation process, and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, bio-fermented casein powder was obtained.

[0053] Comparative Example 2:

[0054] A method for preparing bio-fermented casein powder comprises the following steps:

[0055] S1: fresh sterilized milk is centrifuged at 4°C and 5200 rpm for 20 min for defatting, and the supernatant after removing the upper layer of fat is the defatted sterilized milk; the pH of the sterilized milk is adjusted to 4.0, and the milk is allowed to stand at room temperature until precipitation occurs to obtain a casein precipitate; 1000 g of the casein precipitate is added to 2560 mL of deionized water, and the mixture is stirred at 95°C for 16 min until uniformly stirred to obtain a casein suspension; fresh mulberry fruits are pulped, filtered to remove residue, and mulberry juice is obtained; the pH of the mulberry juice is adjusted to 5.8 with sodium citrate to obtain mulberry juice.

[0056] S2: 8000 mL of casein suspension, 450 g of soybean meal powder, 120 g of Atractylodes macrocephala powder, 140 g of Astragalus membranaceus powder, 46 g of licorice powder, 60 g of ammonium sulfate, and 30 g of potassium dihydrogen phosphate were mixed evenly and sterilized at high temperature to obtain casein culture medium. The inoculum amount of thermophilic Streptococcus was 3% of the volume of casein culture medium, and the volume ratio of thermophilic Streptococcus inoculum, Lactobacillus plantarum inoculum, and Lactobacillus bulgaricus inoculum was 3:2:1. The number of viable bacteria after activation reached 8.5 × 10 8 CFU / mL of thermophilic Streptococcus was inoculated into casein medium and fermented anaerobically at 42℃-45℃ for 10h to obtain medium A. The number of viable bacteria after activation reached 8.2×10 8 CFU / mL of plant lactobacillus was inoculated into medium A and fermented anaerobically at 30℃-40℃ for 9h to obtain medium B. The number of viable bacteria after activation reached 8.6×10 8 CFU / mL of Lactobacillus bulgaricus was inoculated into culture medium B, 300 mL of mulberry juice was added, and the mixture was fermented anaerobically at 42-45°C for 10 h to obtain casein fermentation liquid;

[0057] S3: 9000 g of casein fermentation liquid, 80 g of trehalose, 65 g of whey protein and 60 g of gelatin were stirred for 35 min at a stirring speed of 700 rpm, and then 150 g of inulin, 120 g of oligofructose and 50 g of lecithin were added and stirred for 32 min at a stirring speed of 900 rpm. After stirring, the mixture was spray-dried to obtain bio-fermented casein powder.

[0058] Comparative Example 3:

[0059] Comparative Example 1 is the casein culture medium prepared in Example 1.

[0060] For the bio-fermented casein powder prepared in Examples 1-4 and Comparative Example 2, the molecular weight distribution of the fermented casein and its decomposed small molecule peptides was determined by gel chromatography. The chromatographic column used was a tandem TSK-GEL gel chromatography column, the mobile phase was acetonitrile + 0.06% trifluoroacetic acid solution, and cytochrome C (12.4 kDa), aprotinin (6.5 kDa), insulin (5.8 kDa), glutathione (0.307 kDa), bovine serum albumin (BSA, 66 kDa), and thyroglobulin (669 kDa) were used as standards. The results are shown in Table 1:

[0061] Table 1

[0062]

[0063] The biological fermentation casein powder prepared in Examples 1-4 and Comparative Examples 1-3 and the casein culture medium prepared in the comparative examples were tested. 10 g of the biological fermentation casein powder prepared in Examples 1-4 and Comparative Examples 1-3 and the casein culture medium prepared in the comparative examples were weighed and placed in a 50 mL centrifuge tube. 15 mL of acidified methanol (methanol: hydrochloric acid = 299: 1) was added. After 0.5 h of ultrasonic extraction at 300 W, the mixture was allowed to stand at -20 ° C for 1 h to allow the protein to fully precipitate. Centrifuged at 4 ° C and 7000 rmp / min for 10 min, the supernatant was taken, filtered with a 0.22 μm organic phase filter membrane, and the volume was adjusted to 25 mL to obtain an extract. The content of total phenols and total flavonoids in the extract, as well as the free radical scavenging rate, ABTS and FRAP were detected. The detailed results are shown in Table 2.

[0064] Total phenol content was determined using the Folin-Scholl colorimetric method. 100 μL of the extract was placed in a test tube, 0.4 mL of distilled water was added, and 1 mL of 0.25 M Folin reagent (Folin-phenol:water = 1:8, v / v) was pipetted in. The mixture was thoroughly mixed and allowed to stand at room temperature for 5 minutes. Then, 1 mL of 15% sodium carbonate solution was added, the mixture was vortexed for 30 seconds, and the mixture was allowed to stand in the dark for 0.5 hour. The absorbance of each sample was measured at λ = 760 nm, with acidified methanol solution used as a blank for zero adjustment. A standard curve was constructed using gallic acid as the standard. Results are expressed as gallic acid equivalents (mg / 10 g of extract).

[0065] The total flavonoid content was determined by spectrocolorimetry. 1 mL of the extract was transferred to a colorimetric tube, 0.3 mL of a 5% NaNO₂ solution was added, vortexed to mix, and allowed to stand for 6 minutes. 0.3 mL of a 10% Al(NO₃)₃ solution was then added, vortexed again to mix, and allowed to stand for 6 minutes. Finally, 2 mL of a 1 mol / L NaOH solution was added, and each sample was made up to 5 mL with 85% methanol. The sample was shaken and placed in a dark room for 15 minutes. The absorbance was immediately read at 510 nm. A standard curve was constructed using rutin as the standard, and the results were expressed as rutin equivalents (mg / 10 g of extract).

[0066] The DPPH free radical assay method involves placing 40 μL of sample solution and 360 μL of DPPH working solution in a 2 mL centrifuge tube and mixing thoroughly. An equal volume of anhydrous ethanol replaces the DPPH working solution as a blank control (A0) and an equal volume of anhydrous ethanol solution replaces the sample solution as a color control (Aj). After incubation at room temperature in the dark for 0.5 h, 200 μL of each test solution is transferred to a 96-well plate and the absorbance value, Ai, of each test solution at 515 nm is measured using a microplate reader. Formula:

[0067] ABTS and FRAP assays were performed using the detection kits.

[0068] Table 2

[0069]

[0070] Verify the absorbability of bio-fermented casein powder:

[0071] Experimental Animals: Healthy adult Sprague-Dawley rats weighing 180-220 g were randomly divided into five groups, each containing 10 mice (5 males and 5 females), housed in separate cages. Mice were acclimated for one week in a barrier environment with free access to food and water. Bedding was changed daily, and the mice were maintained on a standard light cycle (12 h light / 12 h dark).

[0072] Feed configuration and experimental period: The experimental group was provided with 90% basal feed + 10% bio-fermented casein powder prepared in Example 1; control group 1 was provided with 90% basal feed + 10% bio-fermented casein powder prepared in Comparative Example 1; control group 2 was provided with 90% basal feed + 10% bio-fermented casein powder prepared in Comparative Example 2; control group 3 was provided with 90% basal feed + 10% fermented casein culture medium prepared in Comparative Example 3; and the blank group was provided with 100% basal feed. The experimental period was 2 weeks.

[0073] Data collection and recording:

[0074] The rats' initial body weight before the experiment and final body weight after the experiment were recorded.

[0075] Detailed results are shown in Table 3. Sampling and Testing: At the end of the experiment, blood was collected after a 12-hour fast. The concentrations of essential amino acids in rat plasma were determined by high-performance liquid chromatography (HPLC). Chromatographic conditions were as follows: Hypersiliods C18 column (4.6 mm × 150 mm, 5 μm); column temperature 40°C; detection wavelength 338 nm (262 nm for proline); mobile phase A: 27.6 mmol / L sodium acetate-triethylamine-tetrahydrofuran solution (v:v:v = 500:0.11:2.5, pH = 7.2); mobile phase B: 80.9 mmol / L sodium acetate-methanol-acetonitrile (v:v:v = 1:2:2, pH = 7.2); flow rate 1.0 mL / min. Amino acid content was quantified using the external standard method. Detailed results are shown in Table 4.

[0076] Table 3

[0077] Group Initial body weight (g) Final body weight (g) Weight gain rate (%) Experimental group 195±8 240±10 23.1±2.6 Control group 1 198±7 230±9 16.2±2.1 Control group 2 197±6 225±8 14.2±.9 Control group 3 196±7 220±7 12.2±1.8 Blank group 200±9 215±6 7.5±1.5

[0078] Table 4

[0079] Amino acid types Example 1 Control group 3 Blank group Leu (μmol / mL) 0.28 0.19 0.13 Phe+Tyr (μmol / mL) 0.38 0.25 0.18 Lys (μmol / mL) 0.22 0.16 0.10 Val (μmol / mL) 0.20 0.14 0.09 Ile (μmol / mL) 0.18 0.12 0.07 Thr (μmol / mL) 0.25 0.17 0.11 Met+Cys (μmol / mL) 0.15 0.10 0.06 His (μmol / mL) 0.11 0.08 0.05 Trp (μmol / mL) 0.07 0.05 0.03

[0080] Data Analysis:

[0081] As can be seen from Table 1, in the bio-fermented casein powder of Examples 1-4, small molecule peptides with a molecular weight in the range of 1000-5000 accounted for 83.06%-83.71%, and small molecule peptides with a molecular weight less than 1000 accounted for 7.05%-7.82%, indicating that the fermentation process effectively decomposed casein and generated a large number of small molecule peptides, which are conducive to intestinal absorption. In Comparative Example 2, small molecule peptides with a molecular weight in the range of 1000-5000 accounted for 76.29%, and small molecule peptides with a molecular weight of 1000 accounted for 4.01, which is lower than that of the examples, indicating that preparing casein solution by precipitating casein is more conducive to the decomposition of casein.

[0082] As can be seen from Table 2, the total phenol and total flavonoid contents of Examples 1-4 are significantly higher than those of Comparative Example 1, indicating that the mulberry juice added during the fermentation process increases the content of phenolic and flavonoid compounds in the bio-fermented casein powder, and improves the antioxidant properties of the bio-fermented casein powder.

[0083] As can be seen from Table 3, the bio-fermented casein powder prepared by the present invention can effectively improve the absorption of essential amino acids in rats. Atractylodes macrocephala, Astragalus membranaceus and Licorice promote the absorption of protein by regulating the spleen and stomach function. The probiotics such as thermophilic Streptococcus, Lactobacillus plantarum and Lactobacillus bulgaricus used in the fermentation process can decompose casein to generate small molecule peptides and amino acids, which are more easily absorbed by the intestine. The addition of mulberry juice provides more antioxidant components and bioactive substances, which synergize with Atractylodes macrocephala, Astragalus membranaceus and Licorice to further improve the absorption efficiency of essential amino acids.

[0084] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0085] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing biological fermentation casein powder, characterized in that, The method comprises the following preparation steps: Mixing casein solution, soybean meal powder, Atractylodes macrocephala powder, Astragalus root powder, licorice powder, ammonium sulfate, and potassium dihydrogen phosphate uniformly, sterilizing at high temperature to obtain a casein culture medium, inoculating activated Streptococcus thermophilus into the casein culture medium, and fermenting at 42°C-45°C for 9-12 hours to obtain culture medium A, inoculating activated Lactobacillus plantarum into culture medium A, and fermenting at 30°C-40°C for 8-10 hours to obtain culture medium B, inoculating activated Lactobacillus bulgaricus into culture medium B, adding mulberry juice, and fermenting at 42°C-45°C for 8-11 hours to obtain a casein fermentation liquid; Trehalose, whey protein and gelatin are added to the casein fermentation broth, and the mixture is stirred for 20-40 minutes at a stirring speed of 600-800 rpm. Inulin, oligofructose and lecithin are then added, and the mixture is stirred for 30-40 minutes at a stirring speed of 800-1000 rpm. After the stirring is completed, the mixture is spray-dried to obtain bio-fermented casein powder.

2. The method for preparing biological fermentation casein powder according to claim 1, wherein: Fresh sterilized milk is centrifuged at 2°C-6°C and 5000r / min-6000r / min for 20 minutes for defatting, and the clear liquid after removing the upper layer of fat is the defatted sterilized milk; the pH of the sterilized milk is adjusted to 4.0-4.5, and the milk is allowed to stand at room temperature until precipitation is obtained to obtain a casein precipitate, sodium hydroxide solution is added dropwise to the casein precipitate, PBS buffer is added, and the mixture is stirred at 90°C-100°C for 15-20 minutes. After cooling, deionized water is added, and the mixture is stirred evenly to obtain a casein solution.

3. The method for preparing biological fermentation casein powder according to claim 2, wherein: The sodium hydroxide solution is a 0.5 mol / L sodium hydroxide solution, and the PBS buffer is a 0.01 mol / L PBS buffer.

4. The method for preparing biological fermentation casein powder according to claim 1, wherein: The usage ratio of the casein precipitate, sodium hydroxide solution, PBS buffer solution and deionized water is 10g: 8-12ml: 16-20ml: 58-66ml.

5. The method for preparing biological fermentation casein powder according to claim 1, wherein The preparation method of the mulberry juice comprises the following steps: beating fresh mulberry fruits, filtering and removing residues to obtain mulberry juice, and adjusting the pH of the mulberry juice to 5.5-6.0 with sodium citrate to obtain the mulberry juice.

6. The method for preparing biological fermentation casein powder according to claim 1, wherein: The number of viable bacteria of Streptococcus thermophilus after activation reached 8.2×10 8 CFU / mL-8.5×10 8 CFU / mL, the number of viable Lactobacillus plantarum inoculation after activation reached 8.0×10 8 CFU / mL-8.5×10 8 CFU / mL, the number of viable Lactobacillus bulgaricus after activation reached 8.5×10 8 CFU / mL-9.0×10 8 CFU / mL.

7. The method for preparing biological fermentation casein powder according to claim 1, wherein: The thermophilic streptococcus inoculation amount is 3% of the volume of the casein culture medium, and the volume ratio of the thermophilic streptococcus inoculation amount, the plantarum lactobacillus inoculation amount, and the bulgaricus lactobacillus inoculation amount is 3:2:

1.

8. The method for preparing biological fermentation casein powder according to claim 1, wherein: The dosage ratio of the casein solution, soybean meal powder, Atractylodes macrocephala powder, Astragalus root powder, Licorice root powder, ammonium sulfate, potassium dihydrogen phosphate, and mulberry juice is 80 mL: 3-5 g: 1.2-1.8 g: 1.2-1.8 g: 1.2-1.8 g: 0.5-0.8 g: 0.2-0.3 g: 20-30 mL.

9. The method for preparing biological fermentation casein powder according to claim 1, wherein: The mass ratio of the casein fermentation liquid, inulin, oligofructose, trehalose, whey protein, gelatin and lecithin is 90:10-20:8-15 parts:5-10 parts:6-8 parts:5-8 parts:4-6 parts.

10. A biological fermentation casein powder, characterized in that: The method for preparing the bio-fermented casein powder according to claims 1-9 is adopted.

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