Biological fermentation whey powder and preparation method thereof

Abstract

The invention relates to the technical field of biological fermentation, in particular to biological fermentation whey powder and a preparation method thereof, and solves the problems of low efficiency, single bacteriostasis, poor stability and the like of traditional whey treatment through multi-strain synergistic fermentation, auxiliary component synergism, precise process control and freeze-drying process optimization. A lactobacillus fermentum CECT5716, propionibacterium and saccharomyces cerevisiae MX2 triple bacteria fermentation system is adopted, high-density proliferation of thalli is achieved through metabolism complementation, lactobacillus fermentum produces organic acid to inhibit mold, propionibacterium produces propionic acid to achieve synergistic bacteriostasis, saccharomyces cerevisiae consumes glucose to reduce substrate inhibition, and the bacteriostatic activity and functional stability of the fermented whey powder can be ensured; a plurality of bacteria are fermented to generate a plurality of antibacterial substances, and auxiliary components are combined to form a composite antibacterial system, so that the problem of insufficient antibacterial effect of the traditional single bacteria is solved, and the method has a wide application prospect.

Description

Technical Field

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

[0002] Whey, a byproduct of cheese production, is rich in lactose, protein, and minerals. However, its direct discharge can cause serious environmental pollution. Currently, whey utilization is primarily focused on the production of feed and food additives, but the development of its bioactive components remains underdeveloped. Traditional whey processing methods suffer from low efficiency and limited added value. There is an urgent need for efficient, environmentally friendly, and value-added whey processing technologies.

[0003] Microbial fermentation is an effective way to increase the value of whey. For example, fermentation of whey with lactic acid bacteria produces metabolites with antibacterial activity, such as organic acids and bacteriocins. These products can be used as natural preservatives in the food industry. While advances have been made in the application of fermented whey powder, there is still room for improvement. For example, when using whey powder for ethanol production, Kluyveromyces has low tolerance to lactose and ethanol, resulting in suboptimal ethanol fermentation yields and productivity. While Saccharomyces cerevisiae offers many advantages, its wild form cannot directly utilize lactose, requiring the exogenous expression of β-galactosidase or the hydrolysis of lactose prior to fermentation. Furthermore, the substrates are often low in semi-synthetic culture media with low lactose concentrations, resulting in low ethanol yields and increased distillation costs. Fungal contamination is a serious problem in yogurt production. Traditional chemical preservatives no longer meet consumer demand for natural and safe products. While fermented whey powder has mold inhibition properties, it suffers from low fermentation efficiency and limited functionality.

[0004] Therefore, according to the relevant technologies mentioned above, there is an urgent need to develop a biological fermentation whey powder and a preparation method thereof. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a bio-fermented whey powder and a preparation method thereof, so as to provide a bio-fermented whey powder with multiple functions, thereby efficiently utilizing whey.

[0006] Based on the above objectives, the present invention provides a bio-fermented whey powder and a preparation method thereof.

[0007] A biologically fermented whey powder is produced by fermenting the following raw materials:

[0008] Whey powder, culture medium, glucose, yeast extract and auxiliary ingredients;

[0009] The bacterial species include Lactobacillus fermentum, Propionibacterium and Saccharomyces cerevisiae;

[0010] The auxiliary ingredients have specific functions, and include nutritional factors that promote fermentation and natural extracts that enhance antibacterial effects;

[0011] The nutritional factor is a mixture of B vitamins and amino acids, which can provide more sufficient nutrition for the growth of lactobacillus and propionibacterium and promote the fermentation process;

[0012] The natural extracts are rosemary extract and tea polyphenols, which not only have antioxidant effects but also can synergistically enhance the inhibitory effect of fermented whey powder on mold.

[0013] A method for preparing biological fermentation whey powder comprises the following steps:

[0014] Step S1, bacterial activation;

[0015] Step S101, activating Lactobacillus fermentum;

[0016] Step S102, activation of Propionibacterium;

[0017] Step S103: Activate the Saccharomyces cerevisiae strain.

[0018] Step S2, culture medium preparation:

[0019] Step S201, preparing seed culture medium;

[0020] Step S202: preparing fermentation medium;

[0021] Step S3, mixed fermentation:

[0022] Step S301, primary fermentation;

[0023] Step S302, secondary fermentation;

[0024] Step S4, centrifugal separation:

[0025] Step S5, preparation and mixing of a lyoprotectant;

[0026] Step S6: vacuum freeze-drying.

[0027] Preferably, the process of activating Lactobacillus fermentum in step S101 is as follows:

[0028] The fermented Lactobacillus strain was inoculated into MRS solid medium and cultured anaerobically at 37°C for 36 h. A single colony was picked for second-generation streak purification. The purified single colony was inoculated into 2 mL of liquid MRS medium and cultured anaerobically at 37°C for 24 h. The inoculation volume was then transferred to 5 mL of liquid MRS medium at 2% and cultured under the same conditions for 12 h until the number of viable bacteria was ≥1×10 8 CFU / mL, activation is completed, and activated Lactobacillus fermentum is obtained.

[0029] Preferably, the process of activating the Propionibacterium in step S102 is as follows:

[0030] Inoculate Propionibacterium into Propionibacterium culture medium (such as containing glucose and yeast extract), culture under anaerobic conditions at 30-32°C for 48-72 hours, and wait until the number of viable bacteria reaches 10 8 -10 9 CFU / mL, and activated Propionibacterium was obtained.

[0031] Preferably, the preparation process of the Propionibacterium culture medium is as follows:

[0032] 20.0 g of glucose, 20.0 g of lactose, 10.0 g of yeast extract, 1.0 g of glycerol, 1.0 g of Na2HPO4, and 0.4 g of MgSO4·7H2O were placed in a conical flask. 1.0 L of distilled water was measured using a graduated cylinder and added to the conical flask. The conical flask was placed on a magnetic stirrer and stirred to fully dissolve the ingredients. 20.0 g of agar was added and stirring was continued. The pH of the culture medium was adjusted to 6.8-7.0. Finally, the culture medium was sterilized at 121°C for 15-20 min to obtain a Propionibacterium culture medium.

[0033] Preferably, the process of activating the Saccharomyces cerevisiae strain in step S103 is as follows:

[0034] Take the yeast strain of brewer's yeast and inoculate it into YPD medium, and culture it at 30℃ and 200rpm in a shaking incubator for 12h. 600 When it reaches 1.2-1.5, it is in the logarithmic growth stage and is ready for use.

[0035] Preferably, the process of preparing the seed culture medium in step S201 is as follows:

[0036] Yeast extract, lactose, glucose, sodium acetate, diammonium hydrogen citrate, dipotassium hydrogen phosphate, MgSO4, MnSO4, Tween-80, and carrot juice were mixed in mass volume ratios of 4.5% (g / 100 mL), 0.7% (g / 100 mL), 1.3% (g / 100 mL), 0.5% (g / 100 mL), 0.2% (g / 100 mL), 0.2% (g / 100 mL), 0.02% (g / 100 mL), 0.005% (g / 100 mL), 0.05% (g / 100 mL), and 4% (g / 100 mL), respectively, the pH was adjusted to 5.8-6.2, and the mixture was sterilized at 115°C for 15 min;

[0037] Preferably, the fermentation medium preparation process in step S202 is as follows:

[0038] 1.5% (g / 100mL) of glucose, 0.5% (g / 100mL) of lactose, and 0.5% (g / 100mL) of sodium acetate were dissolved in the deprecipitated corn steep liquor powder solution (corn steep liquor powder concentration was 3% (g / 100mL), boiled in a boiling water bath for 10 minutes, adjusted to pH 5.8, and centrifuged at 8000rpm for 10 minutes to obtain the supernatant). 5% (g / 100mL) of whey powder, 2% (g / 100mL) of yeast extract, and 0.38% (g / 100mL) of auxiliary ingredients were added, the volume was adjusted to 1L, the pH was adjusted to 6.0, and the solution was sterilized at 115°C for 15 minutes.

[0039] Preferably, the auxiliary ingredients include 0.1% (g / 100 mL) of B vitamins, 0.2% (g / 100 mL) of amino acids, 0.05% (g / 100 mL) of rosemary extract, and 0.03% (g / 100 mL) of tea polyphenols.

[0040] Preferably, the preparation process of the auxiliary component is as follows:

[0041] First, dissolve B vitamins and amino acids in warm water, and then mix them with rosemary extract and tea polyphenols to obtain auxiliary ingredients.

[0042] Preferably, the process of the primary fermentation in step S301 is as follows:

[0043] The activated Lactobacillus fermentum was inoculated into the seed culture medium at a 3% inoculation rate, the pH was maintained at 5.8-6.2, and the culture was placed in an anaerobically static manner at 37°C for 16 h until the viable bacteria count reached 3.69×10 10 CFU / mL, and the first-level fermentation broth was obtained.

[0044] Preferably, the process of the secondary fermentation in step S302 is as follows:

[0045] The primary fermentation liquid was inoculated with a fermentation medium at a volume percentage of 10%, and was also inoculated with 5% volume percentage of activated saccharomyces cerevisiae and 3% volume percentage of activated Propionibacterium, and was placed in a fermentation tank containing whey powder, glucose, yeast extract and auxiliary components. During the fermentation process, the fermentation tank speed was controlled at 100 rpm, the initial temperature was 37°C, and a 1 mol / L Na2CO3 solution was added at a uniform rate to maintain the pH at 5.8-6.2. After culturing for 16 hours, 300 mL of a feed solution with a carbon-nitrogen ratio of 1:3 (with the same ingredients as the fermentation medium) was added, and the fermentation was continued for 38 hours, during which the temperature was maintained at 37°C and the pH was maintained at 5.8-6.2 to obtain a secondary fermentation liquid.

[0046] Preferably, the carbon-nitrogen ratio of the feed solution is calculated based on the mass ratio of the carbon source to the nitrogen source.

[0047] Preferably, the centrifugal separation process in step S4 is as follows:

[0048] The secondary fermentation broth was centrifuged at 8000 rpm and 4°C for 15 min, the supernatant was discarded, and the bacterial sludge was collected. The bacterial sludge was washed twice with sterile saline, each time centrifuged at 8000 rpm and 4°C for 10 min, the supernatant was discarded, and the bacterial sludge was collected.

[0049] Preferably, the preparation process of the lyoprotectant in step S5 is as follows:

[0050] 18% (g / 100 mL) of skim milk powder, 3% (g / 100 mL) of maltodextrin, and 7% (g / 100 mL) of trehalose were dissolved in sterile water to prepare a protective agent solution.

[0051] Preferably, the mixing process of the lyoprotectant in step S5 is as follows:

[0052] The bacterial sludge and the protective agent were evenly mixed in a volume ratio of 1:2, and the pH was adjusted to 6.4-6.6 to obtain a mixed solution.

[0053] Preferably, the vacuum freeze-drying process in step S6 is as follows:

[0054] The mixed liquid was pre-frozen at -80°C for 5 hours, and then transferred to a vacuum freeze dryer with a cold trap temperature of -55°C and a vacuum degree of ≤10Pa. The freeze-drying process was carried out in stages: the first stage was maintained at -55°C for 4 hours, the second stage was heated to -20°C at a rate of 5°C / h and maintained for 4 hours, and the third stage was heated to 20°C at a rate of 10°C / h and maintained for 4 hours. The total freeze-drying time was 12 hours to obtain bio-fermented whey powder.

[0055] Beneficial effects of the present invention:

[0056] The present invention provides a bio-fermented whey powder and a preparation method thereof. The present invention solves the problems of low efficiency, single antibacterial property and poor stability of traditional whey processing through multi-strain synergistic fermentation, auxiliary ingredient synergy, precise process control and freeze-drying process optimization.

[0057] The present invention adopts a triple-bacteria fermentation system of Lactobacillus fermentum, Propionibacterium and Saccharomyces cerevisiae to achieve high-density bacterial proliferation through metabolic complementation. Lactobacillus fermentum produces organic acids (such as lactic acid) to inhibit mold, Propionibacterium produces propionic acid to synergistically inhibit bacteria, and Saccharomyces cerevisiae consumes glucose to reduce substrate inhibition, thereby ensuring the antibacterial activity and functional stability of fermented whey powder. Multi-species fermentation produces multiple antibacterial substances (such as lactic acid, propionic acid, and bacteriocins), which are combined with auxiliary ingredients (rosemary extract and tea polyphenols) to form a composite antibacterial system. The antibacterial rate is improved compared with that of a single strain, thereby solving the problem of insufficient antibacterial effect of a traditional single strain. The invention has broad application prospects. DETAILED DESCRIPTION

[0058] 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.

[0059] The Propionibacterium in the present invention is Propionibacterium janaphyseum, which is deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M2013071;

[0060] The brewer's yeast is Saccharomyces cerevisiae MX2, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC NO.20378.

[0061] Example 1: A method for preparing biological fermentation whey powder, comprising the following steps:

[0062] S1. Lactobacillus fermentum CECT5716 was inoculated into MRS solid medium and incubated anaerobically at 37°C for 36 h. A single colony was picked and streaked for purification for the second generation. The purified single colony was inoculated into 2 mL of liquid MRS medium and incubated anaerobically at 37°C for 24 h. The inoculum was then transferred to 5 mL of liquid MRS medium at a 2% inoculum volume and incubated under the same conditions for 12 h until the viable count was ≥1×10 8 CFU / mL, activation was completed to obtain activated Lactobacillus fermentum CECT5716;

[0063] S2. Place 20.0 g of glucose, 20.0 g of lactose, 10.0 g of yeast extract, 1.0 g of glycerol, 1.0 g of Na2HPO4, and 0.4 g of MgSO4·7H2O in a conical flask. Measure 1.0 L of distilled water using a graduated cylinder and add it to the conical flask. Place the conical flask on a magnetic stirrer and stir to fully dissolve the ingredients. Add 20.0 g of agar and continue stirring. Adjust the pH of the culture medium to 6.8. Finally, sterilize at 121°C for 15 min to obtain a Propionibacterium culture medium. Inoculate Propionibacterium into a Propionibacterium culture medium (e.g., containing glucose and yeast extract) and culture under anaerobic conditions at 30°C for 48 h. When the viable count reaches 10 8 -10 9 CFU / mL, to obtain activated Propionibacterium;

[0064] S3. Take the MX2 strain of Saccharomyces cerevisiae and inoculate it into YPD medium. Incubate it at 30℃ and 200rpm for 12h. 600 When the value reaches 1.2-1.5, it is in the logarithmic growth phase and is set aside to obtain the activated MX2 strain of Saccharomyces cerevisiae;

[0065] S4. Yeast extract, lactose, glucose, sodium acetate, diammonium hydrogen citrate, dipotassium hydrogen phosphate, MgSO4, MnSO4, Tween-80, and carrot juice were mixed in a mass volume ratio of 4.5% (g / 100 mL), 0.7% (g / 100 mL), 1.3% (g / 100 mL), 0.5% (g / 100 mL), 0.2% (g / 100 mL), 0.2% (g / 100 mL), 0.02% (g / 100 mL), 0.005% (g / 100 mL), 0.05% (g / 100 mL), and 4% (g / 100 mL), respectively, the pH was adjusted to 5.8-6.2, and the mixture was sterilized at 115°C for 15 min to obtain a seed culture medium;

[0066] S5. First, dissolve B vitamins and amino acids in warm water, and then mix them with rosemary extract and tea polyphenols to obtain auxiliary ingredients, wherein the proportions of each component are 0.1% (g / 100mL) of B vitamins, 0.2% (g / 100mL) of amino acids, 0.05% (g / 100mL) of rosemary extract, and 0.03% (g / 100mL) of tea polyphenols. Dissolve 1.5% (g / 100mL) of glucose, 0.5% (g / 100mL) of lactose, and 0.5% (g / 100mL) of sodium acetate in the deprecipitated corn steep liquor powder solution (corn steep liquor powder concentration 3% (g / 100mL), boil in water bath for 10min, adjust the pH to 5.8, and simmer at 8000rpm. The mixture was centrifuged for 10 minutes and the supernatant was collected. 5% (g / 100 mL) of whey powder, 2% (g / 100 mL) of yeast extract, and 0.38% (g / 100 mL) of auxiliary ingredients were added, the volume was adjusted to 1 L, the pH was adjusted to 6.0, and the mixture was sterilized at 115° C. for 15 minutes to obtain a fermentation medium. The auxiliary ingredients have specific functions, including nutritional factors that promote fermentation and natural extracts that enhance antibacterial effects. A mixture of B vitamins and amino acids, as nutritional factors, can provide more sufficient nutrition for the growth of lactobacilli and propionibacteria, thereby promoting the fermentation process. Rosemary extract and tea polyphenols, as natural extracts, not only have antioxidant effects but also synergistically enhance the inhibitory effect of fermented whey powder on mold.

[0067] S6. The activated Lactobacillus fermentum CECT5716 was inoculated into the seed culture medium at a 3% inoculum volume, the pH was maintained at 5.8, and the culture was anaerobically cultured at 37°C for 16 h until the viable cell count reached 3.69 × 10 10 CFU / mL, and the first-level fermentation broth was obtained;

[0068] S7. The primary fermentation broth was inoculated with 10% volume percentage of fermentation medium, and 5% volume percentage of activated Saccharomyces cerevisiae MX2 strains and 3% volume percentage of activated Propionibacterium were simultaneously accessed, and a fermenter containing whey powder, glucose, yeast extract and auxiliary ingredients was added. During the fermentation process, the fermentor speed was controlled at 100 rpm, the initial temperature was 37 ° C, and a 1 mol / L Na2CO3 solution was added at a constant rate to maintain the pH at 5.8. When the culture was continued for 16 h, 300 mL of a feed solution with a carbon-nitrogen ratio of 1:3 (compositions of the same fermentation medium) was added, and the fermentation was continued for 38 h, during which the temperature was maintained at 37 ° C and the pH was maintained at 5.8 to obtain a secondary fermentation broth, wherein the carbon-nitrogen ratio of the feed solution was calculated based on the mass ratio of the carbon source and the nitrogen source;

[0069] S8. Centrifuge the secondary fermentation broth at 8000 rpm and 4°C for 15 min, discard the supernatant, and collect the bacterial sludge. Wash the sludge twice with sterile saline, centrifuging each time at 8000 rpm and 4°C for 10 min, discard the supernatant, and collect the bacterial sludge.

[0070] S9. Dissolve 18% (g / 100 mL) of skim milk powder, 3% (g / 100 mL) of maltodextrin, and 7% (g / 100 mL) of trehalose in sterile water to prepare a protective agent solution. Mix the bacterial sludge and protective agent solution in a volume ratio of 1:2, and adjust the pH to 6.4 to obtain a mixed solution.

[0071] S10. The mixed liquid was placed in a -80°C pre-freeze state for 5 hours, and then transferred to a vacuum freeze dryer with a cold trap temperature of -55°C and a vacuum degree of ≤10Pa. The temperature was increased in stages during the freeze-drying process: in the first stage, the temperature was maintained at -55°C for 4 hours; in the second stage, the temperature was increased to -20°C at a rate of 5°C / h and maintained for 4 hours; in the third stage, the temperature was increased to 20°C at a rate of 10°C / h and maintained for 4 hours. The total freeze-drying time was 12 hours to obtain bio-fermented whey powder.

[0072] Example 2: A method for preparing biological fermentation whey powder, comprising the following steps:

[0073] S1. Lactobacillus fermentum CECT5716 was inoculated into MRS solid medium and incubated anaerobically at 37°C for 36 h. A single colony was picked and streaked for purification for the second generation. The purified single colony was inoculated into 2 mL of liquid MRS medium and incubated anaerobically at 37°C for 24 h. The inoculum was then transferred to 5 mL of liquid MRS medium at a 2% inoculum volume and incubated under the same conditions for 12 h until the viable count was ≥1×10 8 CFU / mL, activation was completed to obtain activated Lactobacillus fermentum CECT5716;

[0074] S2. Place 20.0 g of glucose, 20.0 g of lactose, 10.0 g of yeast extract, 1.0 g of glycerol, 1.0 g of Na2HPO4, and 0.4 g of MgSO4·7H2O in a conical flask. Measure 1.0 L of distilled water using a graduated cylinder and add it to the conical flask. Place the conical flask on a magnetic stirrer and stir to fully dissolve the ingredients. Add 20.0 g of agar and continue stirring. Adjust the pH of the culture medium to 6.9. Finally, sterilize at 121°C for 17 min to obtain a Propionibacterium culture medium. Inoculate Propionibacterium into a Propionibacterium culture medium (e.g., containing glucose and yeast extract) and culture under anaerobic conditions at 31°C for 60 h. When the viable count reaches 10 8 -10 9 CFU / mL, to obtain activated Propionibacterium;

[0075] S3. Take the MX2 strain of Saccharomyces cerevisiae and inoculate it into YPD medium. Incubate it at 30℃ and 200rpm for 12h. 600 When the value reaches 1.2-1.5, it is in the logarithmic growth phase and is set aside to obtain the activated MX2 strain of Saccharomyces cerevisiae;

[0076] S4. Yeast extract, lactose, glucose, sodium acetate, diammonium hydrogen citrate, dipotassium hydrogen phosphate, MgSO4, MnSO4, Tween-80, and carrot juice were mixed in mass volume ratios of 4.5% (g / 100 mL), 0.7% (g / 100 mL), 1.3% (g / 100 mL), 0.5% (g / 100 mL), 0.2% (g / 100 mL), 0.2% (g / 100 mL), 0.02% (g / 100 mL), 0.005% (g / 100 mL), 0.05% (g / 100 mL), and 4% (g / 100 mL), respectively. The pH was adjusted to 6.0, and the mixture was sterilized at 115°C for 15 min to obtain a seed culture medium.

[0077] S5. First, dissolve B vitamins and amino acids in warm water, and then mix them with rosemary extract and tea polyphenols to obtain auxiliary ingredients, wherein the proportions of each component are 0.1% (g / 100mL) of B vitamins, 0.2% (g / 100mL) of amino acids, 0.05% (g / 100mL) of rosemary extract, and 0.03% (g / 100mL) of tea polyphenols. Dissolve 1.5% (g / 100mL) of glucose, 0.5% (g / 100mL) of lactose, and 0.5% (g / 100mL) of sodium acetate in the deprecipitated corn steep liquor powder solution (corn steep liquor powder concentration 3% (g / 100mL), boil in water bath for 10min, adjust the pH to 5.8, and simmer at 8000rpm. The mixture was centrifuged for 10 minutes and the supernatant was collected. 5% (g / 100 mL) of whey powder, 2% (g / 100 mL) of yeast extract, and 0.38% (g / 100 mL) of auxiliary ingredients were added, the volume was adjusted to 1 L, the pH was adjusted to 6.0, and the mixture was sterilized at 115° C. for 15 minutes to obtain a fermentation medium. The auxiliary ingredients have specific functions, including nutritional factors that promote fermentation and natural extracts that enhance antibacterial effects. A mixture of B vitamins and amino acids, as nutritional factors, can provide more sufficient nutrition for the growth of lactobacilli and propionibacteria, thereby promoting the fermentation process. Rosemary extract and tea polyphenols, as natural extracts, not only have antioxidant effects but also synergistically enhance the inhibitory effect of fermented whey powder on mold.

[0078] S6. The activated Lactobacillus fermentum CECT5716 was inoculated into the seed culture medium at a 3% inoculum volume, the pH was maintained at 6.0, and the culture was anaerobically cultured at 37°C for 16 h until the viable cell count reached 3.69 × 10 10 CFU / mL, and the first-level fermentation broth was obtained;

[0079] S7. The primary fermentation broth was inoculated with 10% volume percentage of fermentation medium, and 5% volume percentage of activated Saccharomyces cerevisiae MX2 strains and 3% volume percentage of activated Propionibacterium were simultaneously accessed, and a fermenter containing whey powder, glucose, yeast extract and auxiliary ingredients was added. During the fermentation process, the fermentor speed was controlled at 100 rpm, the initial temperature was 37 ° C, and a 1 mol / L Na2CO3 solution was added at a constant rate to maintain the pH at 6.0. When the culture was continued for 16 h, 300 mL of a feed solution with a carbon-nitrogen ratio of 1:3 (compositions of the same fermentation medium) was added, and the fermentation was continued for 38 h, during which the temperature was maintained at 37 ° C and the pH was maintained at 6.0 to obtain a secondary fermentation broth, wherein the carbon-nitrogen ratio of the feed solution was calculated based on the mass ratio of the carbon source and the nitrogen source;

[0080] S8. Centrifuge the secondary fermentation broth at 8000 rpm and 4°C for 15 min, discard the supernatant, and collect the bacterial sludge. Wash the sludge twice with sterile saline, centrifuging each time at 8000 rpm and 4°C for 10 min, discard the supernatant, and collect the bacterial sludge.

[0081] S9. Dissolve 18% (g / 100 mL) of skim milk powder, 3% (g / 100 mL) of maltodextrin, and 7% (g / 100 mL) of trehalose in sterile water to prepare a protective agent solution. Mix the bacterial sludge and protective agent solution in a volume ratio of 1:2, and adjust the pH to 6.5 to obtain a mixed solution.

[0082] S10. The mixed liquid was placed in a -80°C pre-freeze state for 5 hours, and then transferred to a vacuum freeze dryer with a cold trap temperature of -55°C and a vacuum degree of ≤10Pa. The temperature was increased in stages during the freeze-drying process: in the first stage, the temperature was maintained at -55°C for 4 hours; in the second stage, the temperature was increased to -20°C at a rate of 5°C / h and maintained for 4 hours; in the third stage, the temperature was increased to 20°C at a rate of 10°C / h and maintained for 4 hours. The total freeze-drying time was 12 hours to obtain bio-fermented whey powder.

[0083] Example 3: A method for preparing biological fermentation whey powder, comprising the following steps:

[0084] S1. Lactobacillus fermentum CECT5716 was inoculated into MRS solid medium and incubated anaerobically at 37°C for 36 h. A single colony was picked and streaked for purification for the second generation. The purified single colony was inoculated into 2 mL of liquid MRS medium and incubated anaerobically at 37°C for 24 h. The inoculum was then transferred to 5 mL of liquid MRS medium at a 2% inoculum volume and incubated under the same conditions for 12 h until the viable count was ≥1×10 8 CFU / mL, activation was completed to obtain activated Lactobacillus fermentum CECT5716;

[0085] S2. Place 20.0 g of glucose, 20.0 g of lactose, 10.0 g of yeast extract, 1.0 g of glycerol, 1.0 g of Na2HPO4, and 0.4 g of MgSO4·7H2O in a conical flask. Measure 1.0 L of distilled water using a graduated cylinder and add it to the conical flask. Place the conical flask on a magnetic stirrer and stir to fully dissolve the ingredients. Add 20.0 g of agar and continue stirring. Adjust the pH of the culture medium to 7.0. Finally, sterilize at 121°C for 20 min to obtain a Propionibacterium culture medium. Inoculate Propionibacterium into a Propionibacterium culture medium (e.g., containing glucose and yeast extract) and culture under anaerobic conditions at 32°C for 72 h. Wait until the viable count reaches 10 8 -10 9 CFU / mL, to obtain activated Propionibacterium;

[0086] S3. Take the MX2 strain of Saccharomyces cerevisiae and inoculate it into YPD medium. Incubate it at 30℃ and 200rpm for 12h. 600 When the value reaches 1.2-1.5, it is in the logarithmic growth phase and is set aside to obtain the activated MX2 strain of Saccharomyces cerevisiae;

[0087] S4. Yeast extract, lactose, glucose, sodium acetate, diammonium hydrogen citrate, dipotassium hydrogen phosphate, MgSO4, MnSO4, Tween-80, and carrot juice were mixed in a mass volume ratio of 4.5% (g / 100 mL), 0.7% (g / 100 mL), 1.3% (g / 100 mL), 0.5% (g / 100 mL), 0.2% (g / 100 mL), 0.2% (g / 100 mL), 0.02% (g / 100 mL), 0.005% (g / 100 mL), 0.05% (g / 100 mL), and 4% (g / 100 mL), respectively, the pH was adjusted to 5.8-6.2, and the mixture was sterilized at 115°C for 15 min to obtain a seed culture medium;

[0088] S5. First, dissolve B vitamins and amino acids in warm water, and then mix them with rosemary extract and tea polyphenols to obtain auxiliary ingredients, wherein the proportions of each component are 0.1% (g / 100mL) of B vitamins, 0.2% (g / 100mL) of amino acids, 0.05% (g / 100mL) of rosemary extract, and 0.03% (g / 100mL) of tea polyphenols. Dissolve 1.5% (g / 100mL) of glucose, 0.5% (g / 100mL) of lactose, and 0.5% (g / 100mL) of sodium acetate in the deprecipitated corn steep liquor powder solution (corn steep liquor powder concentration 3% (g / 100mL), boil in water bath for 10min, adjust the pH to 5.8, and simmer at 8000rpm. The mixture was centrifuged for 10 minutes and the supernatant was collected. 5% (g / 100 mL) of whey powder, 2% (g / 100 mL) of yeast extract, and 0.38% (g / 100 mL) of auxiliary ingredients were added, the volume was adjusted to 1 L, the pH was adjusted to 6.0, and the mixture was sterilized at 115° C. for 15 minutes to obtain a fermentation medium. The auxiliary ingredients have specific functions, including nutritional factors that promote fermentation and natural extracts that enhance antibacterial effects. A mixture of B vitamins and amino acids, as nutritional factors, can provide more sufficient nutrition for the growth of lactobacilli and propionibacteria, thereby promoting the fermentation process. Rosemary extract and tea polyphenols, as natural extracts, not only have antioxidant effects but also synergistically enhance the inhibitory effect of fermented whey powder on mold.

[0089] S6. The activated Lactobacillus fermentum CECT5716 was inoculated into the seed culture medium at a 3% inoculum volume, the pH was maintained at 6.2, and the culture was anaerobically cultured at 37°C for 16 h until the viable cell count reached 3.69 × 10 10 CFU / mL, and the first-level fermentation broth was obtained;

[0090] S7. The primary fermentation broth was inoculated with 10% volume percentage of fermentation medium, and 5% volume percentage of activated Saccharomyces cerevisiae MX2 strains and 3% volume percentage of activated Propionibacterium were simultaneously accessed, and a fermenter containing whey powder, glucose, yeast extract and auxiliary ingredients was added. During the fermentation process, the fermentor speed was controlled at 100 rpm, the initial temperature was 37 ° C, and a 1 mol / L Na2CO3 solution was added at a constant rate to maintain the pH at 6.2. When the culture was continued for 16 h, 300 mL of a feed solution with a carbon-nitrogen ratio of 1:3 (compositions of the same fermentation medium) was added, and the fermentation was continued for 38 h, during which the temperature was maintained at 37 ° C and the pH was maintained at 6.2 to obtain a secondary fermentation broth, wherein the carbon-nitrogen ratio of the feed solution was calculated based on the mass ratio of the carbon source and the nitrogen source;

[0091] S8. Centrifuge the secondary fermentation broth at 8000 rpm and 4°C for 15 min, discard the supernatant, and collect the bacterial sludge. Wash the sludge twice with sterile saline, centrifuging each time at 8000 rpm and 4°C for 10 min, discard the supernatant, and collect the bacterial sludge.

[0092] S9. Dissolve 18% (g / 100 mL) of skim milk powder, 3% (g / 100 mL) of maltodextrin, and 7% (g / 100 mL) of trehalose in sterile water to prepare a protective agent solution. Mix the bacterial sludge and protective agent solution in a volume ratio of 1:2, and adjust the pH to 6.6 to obtain a mixed solution.

[0093] S10. The mixed liquid was placed in a -80°C pre-freeze state for 5 hours, and then transferred to a vacuum freeze dryer with a cold trap temperature of -55°C and a vacuum degree of ≤10Pa. The temperature was increased in stages during the freeze-drying process: in the first stage, the temperature was maintained at -55°C for 4 hours; in the second stage, the temperature was increased to -20°C at a rate of 5°C / h and maintained for 4 hours; in the third stage, the temperature was increased to 20°C at a rate of 10°C / h and maintained for 4 hours. The total freeze-drying time was 12 hours to obtain bio-fermented whey powder.

[0094] Comparative Example 1: Single strain fermentation:

[0095] Compared with Example 1, this comparative example only uses Lactobacillus fermentum for fermentation, and does not add saccharomyces cerevisiae and Propionibacterium. The remaining steps and parameters are the same and will not be repeated in this comparative example. Finally, bio-fermented whey powder is obtained.

[0096] Comparative Example 2: No auxiliary ingredients added:

[0097] Compared with Example 1, this comparative example did not add auxiliary components during the preparation of the fermentation medium, and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, bio-fermented whey powder was obtained.

[0098] Comparative Example 3: Changing culture conditions:

[0099] Compared with Example 1, in the secondary fermentation process, no Na2CO3 solution was added to maintain pH, and the fermentation was allowed to proceed naturally. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, bio-fermented whey powder was obtained.

[0100] Comparative Example 4: Using traditional freeze-drying protective agent:

[0101] Compared with Example 1, in the preparation of the freeze-dried protective agent, this comparative example uses traditional glycerol as the protective agent, and does not use skim milk powder, maltodextrin and trehalose. The remaining steps and parameters are the same and will not be repeated in this comparative example. Finally, bio-fermented whey powder is obtained.

[0102] Comparative Example 5: Freeze-drying process does not use three-stage heating:

[0103] The freeze-drying process in this comparative example was heated in stages: in the first stage, -55°C was maintained for 4 hours, in the second stage, the temperature was increased to 20°C at 10°C / h and maintained for 8 hours, and the total freeze-drying time was 12 hours. The remaining steps and parameters were the same as in Example 1, and finally, bio-fermented whey powder was obtained.

[0104] Performance testing:

[0105] 1. Viable bacteria count and microbial detection: refer to GB 4789.35-2016 "National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria", use the plate count method, take 1g of sample, dilute it and inoculate it into MRS medium, incubate it anaerobically at 37℃ for 48h, and count the colony forming units (CFU / g). The specific method is to weigh 1g of sample, add 9mL of sterile saline, shake and mix to make a 1:10 dilution, and dilute it step by step to 10 -6 , 10 -7 , 10 -8 , 100 μL of each was inoculated into MRS solid medium, cultured anaerobically at 37°C for 48 h, and the plates with 30-300 colonies were selected for counting and CFU / g was calculated;

[0106] 2. Freeze-drying survival rate: Calculation method: The percentage of viable bacterial count before freeze-drying (CFU / mL) to the viable bacterial count after freeze-drying (CFU / g). The formula is: Survival rate (%) = viable bacterial count before freeze-drying / viable bacterial count after freeze-drying × 100%. The specific method is: Before freeze-drying, take 1 mL of fermentation broth, dilute and count the viable bacteria (N0, CFU / mL). After freeze-drying according to the patented method, weigh 1 g of freeze-dried powder, dissolve it in 1 mL of sterile water, dilute and count the viable bacteria (N, CFU / g), and calculate the survival rate:

[0107] 3. Inhibition rate (for Aspergillus versicolor): Referring to the patent examples and the standard "Food Microbiology Examination Mold and Yeast Count" (GB 4789.15-2016), use the agar diffusion method to add the fermented whey powder solution to PDA medium containing Aspergillus versicolor spores. Incubate at 28°C for 72 hours and measure the diameter of the inhibition zone or colony inhibition rate: The specific method is as follows: prepare Aspergillus versicolor spore suspension (10 6 CFU / mL), evenly spread on PDA medium, stick on filter paper containing 10 μL fermented whey powder solution (10 mg / mL), culture at 28°C for 72 h, measure the diameter of the inhibition zone (if any) or the colony area, and calculate the inhibition rate;

[0108] 4. Moisture content: Referring to GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food" (direct drying method), weigh 1g of sample, dry at 105°C to constant weight, and calculate the percentage of moisture loss. The specific method is as follows: weigh the dried constant weight Petri dish (m1), add 1g of sample (m2), dry in a 105°C oven for 4 hours, remove and cool, and weigh (m3). Calculate the moisture content:

[0109] 5. Rehydration time: Take 5 g of sample and add 50 mL of 40°C warm water, stir until completely dissolved, and record the dissolution time (min). The specific method is to take 5 g of sample and add 50 mL of 40°C warm water, stir at 200 rpm, and record the time for complete dissolution (no precipitation);

[0110] 6. pH stability (storage at 4°C): Referring to GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count", store the samples at 4°C for 5 months, regularly measure the pH value and viable cell count, and record the viable cell count retention rate (compared with the initial value). The specific method is to store the samples at 4°C, take samples at 0, 1, 2, 3, and 5 months, measure the pH value (precision pH meter) and viable cell count (same as step 1), and record the viable cell count retention rate (4°C, 5 months).

[0111] The results are shown in Table 1 below:

[0112] Table 1

[0113]

[0114]

[0115] Data Analysis:

[0116] As can be seen from Table 1, Examples 1 to 3 of the present invention have higher viable bacteria counts, higher antibacterial rates, and higher performance;

[0117] The present invention has the following advantages:

[0118] Necessity of multi-species synergistic fermentation: Advantages of the embodiment: Lactobacillus fermentum CECT5716 (producing organic acids to inhibit bacteria) forms metabolic complementarity with Propionibacterium (producing propionic acid to synergistically inhibit bacteria) and Saccharomyces cerevisiae (consuming glucose to reduce substrate inhibition). The number of viable bacteria increased by 117% compared with a single strain, and the inhibition rate increased by 44%. Comparative Example 1 only relies on a single strain and lacks metabolic product synergy, resulting in insufficient production of antibacterial substances and easy development of mold tolerance.

[0119] The core role of auxiliary ingredients: Nutritional factors (B vitamins + amino acids): promote the rapid entry of bacteria into the logarithmic growth phase, shorten the fermentation cycle by 4 hours, and increase the peak number of viable bacteria by 30%; Natural extracts (rosemary + tea polyphenols): Rosemary extract destroys the phospholipid bilayer of the mold cell membrane, and tea polyphenols inhibit the activity of the mold respiratory chain enzyme. The two synergistically increase the antibacterial rate by 15%-20% (compared to the group without auxiliary ingredients);

[0120] Importance of pH control and feeding strategy: In the embodiment, the pH is maintained at 5.8-6.2 by feeding Na2CO3 to avoid autolysis of bacteria caused by lactic acid accumulation. The number of viable bacteria is increased by 87.5% compared with natural fermentation (Comparative Example 3). The feeding solution (carbon-nitrogen ratio 1:3) supplements nutrients during the logarithmic growth phase, prolongs the peak fermentation period by 8 hours, and the ethanol production (implicit indicator) is increased by 25% compared with the non-fed group.

[0121] Optimization of freeze-drying protectants and processes: Protective agent formula: skim milk powder (18%) provides protein membrane protection, maltodextrin (3%) lowers the freezing point, and trehalose (7%) stabilizes intracellular macromolecules through hydrogen bonds. The combination of the three makes the freeze-drying survival rate exceed 90%, far exceeding the traditional single protectant (glycerol is only 55.7%). The staged heating process: the low-temperature stage (-55℃→-20℃) slowly sublimates free water to avoid ice crystal damage; the second stage (-20℃→20℃) removes bound water to ensure the integrity of the cell membrane during rehydration. The rehydration time is shortened by 40% compared with the traditional process.

[0122] Conclusion of the core advantages of this application process:

[0123] Multi-strain fermentation system: through the metabolic complementation of triple bacteria, high-density culture (viable bacteria count ≥ 5.9×10 11 CFU / g) and strong antibacterial effect (inhibition rate ≥88.5%), which is more than 50% higher than that of a single strain;

[0124] Innovation in auxiliary ingredients: B vitamins and amino acids promote bacterial proliferation, while rosemary and tea polyphenols synergistically inhibit bacteria, forming a "nutrition + antibacterial" dual-effect system to solve the problem of single antibacterial effect of traditional starter cultures;

[0125] Precise process control: Dynamic pH adjustment (Na2CO3 feeding) and feeding strategy extend the fermentation cycle, ensure stable bacterial metabolism, and increase the stability of viable bacterial count by 30%;

[0126] Freeze-drying process optimization: The combination of a composite protective agent formula and a staged heating process has resulted in a freeze-drying survival rate of 91.58%, a rehydration time shortened to 5.2 minutes, and a live bacteria retention rate of ≥89% after five months of storage, far exceeding similar products in the industry.

[0127] 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.

[0128] 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 biological fermentation whey powder, characterized in that, Made by fermentation of the following ingredients: Whey powder, culture medium, glucose, yeast extract and auxiliary ingredients; The bacterial species include Lactobacillus fermentum, Propionibacterium and Saccharomyces cerevisiae; The auxiliary ingredients include nutritional factors that promote fermentation and natural extracts that enhance antibacterial effects; The nutritional factor is a mixture of B vitamins and amino acids; The natural extracts are rosemary extract and tea polyphenols.

2. A method for preparing biological fermentation whey powder, characterized in that: The following steps are involved: Step S1, bacterial activation; Step S2, culture medium preparation; Step S3, mixed fermentation; Step S4, centrifugal separation; Step S5, preparation and mixing of a lyoprotectant; Step S6: vacuum freeze-drying.

3. The method for preparing biological fermentation whey powder according to claim 2, wherein The process of bacterial activation in step S1 is as follows: Step S101, activating Lactobacillus fermentum; Step S102, activation of Propionibacterium; Step S103: Activate the Saccharomyces cerevisiae strain.

4. The method for preparing biological fermentation whey powder according to claim 3, wherein The activation process of Lactobacillus fermentum in step S101 is carried out in MRS solid medium, and the number of viable bacteria after activation is ≥1×10 8 CFU / mL; The process of propionibacterium activation in step S102 is as follows: in the propionibacterium culture medium, the number of viable bacteria after activation reaches 10 8 -10 9 CFU / mL; The activation process of the cerevisiae yeast strain in step S103 is carried out in YPD medium. 600 When the growth rate reaches 1.2-1.5, it is in the logarithmic growth phase and an activated brewer's yeast strain is obtained.

5. The method for preparing biological fermentation whey powder according to claim 2, wherein The process of preparing the culture medium in step S2 is as follows: Step S201, preparing seed culture medium; Step S202: preparing fermentation medium.

6. The method for preparing biological fermentation whey powder according to claim 5, characterized in that: The seed culture medium in step S201 is prepared from yeast extract, lactose, glucose, sodium acetate, diammonium hydrogen citrate, dipotassium hydrogen phosphate, MgSO4, MnSO4, Tween-80, and carrot juice.

7. The method for preparing biological fermentation whey powder according to claim 5, characterized in that: The fermentation medium in step S202 is prepared from glucose, lactose, sodium acetate, corn steep liquor dry powder solution, whey powder, yeast extract and auxiliary components.

8. The method for preparing biological fermentation whey powder according to claim 2, characterized in that: The process of mixed fermentation in step S3 is as follows: Step S301, primary fermentation; Step S302: secondary fermentation.

9. The method for preparing biological fermentation whey powder according to claim 8, characterized in that: The primary fermentation in step S301 is to activate the fermentation of Lactobacillus fermentum in the seed culture medium until the viable bacteria count reaches 3.69×10 10 CFU / mL, and the first-level fermentation broth was obtained; The secondary fermentation in step S302 is performed by fermenting the primary fermentation liquid in a fermentation medium with an inoculum amount of 10% by volume, 5% by volume of activated Saccharomyces cerevisiae and 3% by volume of activated Propionibacterium to obtain a secondary fermentation liquid.

10. The method for preparing biological fermentation whey powder according to claim 2, characterized in that: The vacuum freeze drying in step S6 adopts staged heating, wherein the staged heating is as follows: the first stage is -55°C and maintained for 4 hours, the second stage is heated to -20°C at a rate of 5°C / h and maintained for 4 hours, and the third stage is heated to 20°C at a rate of 10°C / h and maintained for 4 hours.

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