A freeze-dried mare's milk powder and its preparation method

Abstract

This invention relates to a freeze-dried fermented mare's milk powder and its preparation method, belonging to the field of food processing technology. The preparation method of the freeze-dried fermented mare's milk powder includes the following steps: filtering, sterilizing, and cooling fresh mare's milk to obtain pretreated fresh mare's milk; adding a compound microbial agent to the pretreated fresh mare's milk for fermentation to obtain fermented mare's milk broth; concentrating the fermented mare's milk broth to obtain fermented mare's milk paste; adding a freeze-drying protectant to the fermented mare's milk paste; pre-cooling and freeze-drying to obtain the freeze-dried fermented mare's milk powder. This invention, through the compounding of trehalose, tremella polysaccharide, mannitol, glycine, grape seed polyphenols, and metal ions, results in a high survival rate of microorganisms in the freeze-dried fermented mare's milk powder.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a freeze-dried mare's milk powder and its preparation method. Background Technology

[0002] Fermented mare's milk is a traditional dairy beverage made from fresh mare's milk through natural fermentation by microorganisms such as lactic acid bacteria and yeast. Mongolian medical texts record its effects of dispelling cold, relaxing muscles and tendons, promoting blood circulation, and aiding digestion. Modern research has confirmed that fermented mare's milk is rich in probiotics, post-biotics, vitamins, and bioactive peptides, which have significant effects on regulating intestinal health, improving lipid metabolism, and enhancing the body's immunity. It is a unique dairy product that combines nutritional and health benefits.

[0003] However, traditional liquid fermented mare's milk suffers from inherent drawbacks such as short shelf life, stringent storage conditions, and limited transportation radius, severely hindering its industrialization and clinical application. To address these issues, existing technologies attempt to convert fermented mare's milk into solid powder. The preparation of this powder primarily relies on drying techniques, such as freeze-drying, spray-drying, and microwave drying. Spray-drying is widely used, but this method requires high-temperature air intake of 140-160°C, which can lead to the inactivation of probiotics, degradation of active peptides, and destruction of vitamins in the fermented mare's milk, significantly reducing the product's health benefits. Freeze-drying technology can preserve the activity of active ingredients to the greatest extent, but it still has many shortcomings: insufficient protection measures for active ingredients during freeze-drying, a lack of targeted process optimization and protective agent formulations, resulting in low viable bacteria counts in the freeze-dried powder. Therefore, developing a method for preparing freeze-dried mare's milk powder that efficiently preserves active ingredients, has stable processing, and delivers excellent overall quality has become a key requirement for the transformation and upgrading of the mare's milk industry. Summary of the Invention

[0004] The first objective of this invention is to provide a method for preparing lyophilized mare's milk powder to solve the technical problems of unreasonable process and low viable bacteria count in existing yogurt lyophilized powder.

[0005] The second objective of this invention is to provide a freeze-dried powder of fermented mare's milk.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing lyophilized mare's milk powder includes the following steps: S1: Fresh mare's milk is filtered, sterilized and cooled to obtain pretreated fresh mare's milk. Pretreated fresh mare's milk is added to compound microbial agents for fermentation to obtain fermented mare's milk broth. S2: After the fermented mare's milk liquid is concentrated, mare's milk paste is obtained. A freeze-drying protectant is added to the mare's milk paste, and after pre-cooling, it is freeze-dried to obtain mare's milk freeze-dried powder. The freeze-drying protectant comprises basic components and metal ions; the basic components, by mass, include: 5-8 parts trehalose, 6-10 parts tremella polysaccharide, 4-8 parts mannitol, 1-3 parts glycine, 2-6 parts grape seed polyphenols, and 100 parts water; the metal ions include potassium ions, calcium ions, and magnesium ions; the final concentration of potassium ions is 4-8 mmol / L, the final concentration of calcium ions is 0.7-0.72 mmol / L, and the final concentration of magnesium ions is 0.8-1.2 mmol / L.

[0007] Furthermore, the mass ratio of the sour mare's milk paste to the freeze-drying protectant in S2 is 1:2 to 4.

[0008] Furthermore, the freeze-drying step described in S2 includes: S1: Pre-cooling stage: Cool to -35 to -40 ℃ at a rate of 1 to 3 ℃ / min and hold for 2 to 3 h; S2: First sublimation drying: Under a vacuum of 4.0 to 4.3 Pa, heat to -15 to -20 °C and dry for 18 to 20 h, then heat to 20 to 25 °C and hold for 1 to 1.5 h; S3: Second analytical drying: Maintain vacuum at 4.0-4.3 Pa, raise temperature to 30-32 ℃ and dry for 10-15 hours.

[0009] Furthermore, the concentration conditions in S2 are as follows: temperature 25–35 °C, vacuum degree 0.08–0.09 MPa, stirring rate 50–80 r / min; the water content of the sour mare's milk paste is 3–5 wt%; the pre-cooling temperature for freeze drying after pre-cooling is 4–5 °C, and the time is 15–30 min.

[0010] Furthermore, the sterilization temperature in S1 is 45–50 °C, and the sterilization time is 15–20 min; the temperature for pre-treating the fresh mare's milk is 25–30 °C.

[0011] Furthermore, the amount of compound microbial agent added in S1 is 0.02 to 0.05 wt% of the pretreated fresh mare's milk.

[0012] Furthermore, the compound microbial agent mentioned in S1 includes Bacillus natto powder, Lactobacillus plantarum powder, and Saccharomyces cerevisiae powder, with the mass ratio of Saccharomyces cerevisiae powder to Lactobacillus plantarum powder being 1:1.5-3; and the total mass ratio of Bacillus natto powder to Lactobacillus plantarum powder and Saccharomyces cerevisiae powder being 0.2-0.55:1.

[0013] Furthermore, the viable count of the Bacillus natto powder described in S1 is 3 × 10⁻⁶. 11 ~8×10 11CFU / g; the viable count of the *Lactobacillus plantarum* powder is 3 × 10⁻⁶. 11 ~5×10 12 CFU / g; the viable count of the brewer's yeast powder is 1×10⁻⁶. 11 ~4×10 11 CFU / g.

[0014] Furthermore, the fermentation temperature described in S1 is 28–37 °C, and the fermentation time is 24–48 h.

[0015] A type of freeze-dried mare's milk powder is prepared using the above-mentioned method for preparing freeze-dried mare's milk powder.

[0016] The beneficial effects of this invention are: In this invention, trehalose, a freeze-drying protectant, can replace water molecules bound to the surface of proteins through hydroxyl groups, maintaining the structural stability of proteins and cell membranes. Tremella fuciformis polysaccharide can reduce damage to bacteria during freeze-drying by inhibiting cell membrane damage and maintaining membrane fluidity. Glycine, a small-molecule amino acid, can reduce cell dehydration damage through osmotic pressure regulation. Grape seed polyphenols have antioxidant functions, preventing oxidative stress damage to cells during freeze-drying. Simultaneously, polyphenol molecules can bind to the polar heads of cell membrane phospholipids and membrane proteins through hydrogen bonds and hydrophobic interactions, alleviating the damage caused by dehydration to bacteria. Metal ions can improve the bacteria's tolerance to osmotic pressure and dehydration during freeze-drying, increasing the survival rate of the bacteria. This invention, through the combination of trehalose, Tremella fuciformis polysaccharide, mannitol, glycine, grape seed polyphenols, and metal ions, results in a high survival rate of bacteria in the freeze-dried mare's milk powder.

[0017] Bacillus natto can secrete proteases to break down proteins into peptides and amino acids, providing abundant substrates for Lactobacillus plantarum and Saccharomyces cerevisiae. This allows Lactobacillus plantarum and Saccharomyces cerevisiae to grow to the exponential growth phase more quickly. As can be seen from Comparative Example 1 and Example 1, the fermentation time of Example 1 was significantly shorter than that of Comparative Example 1 when the fermentation endpoint was reached. Simultaneously, Bacillus natto is an aerobic bacterium, and its rapid oxygen consumption in the early stages of fermentation provides an anaerobic environment for the fermentation of Lactobacillus plantarum. Furthermore, because fresh mare's milk has a higher lactose content, traditional fermentation of fresh mare's milk results in a higher lactic acid content and an overly sour taste. However, Bacillus natto produces amines during fermentation, which can neutralize the lactic acid in sour mare's milk, reducing its sourness. After entering the intestines, Bacillus natto can rapidly develop into vegetative cells and temporarily colonize, inhibiting pathogenic bacteria, promoting the growth of beneficial bacteria, and helping to maintain the balance of intestinal flora. Detailed Implementation

[0018] The present invention will be further described below with reference to embodiments thereof.

[0019] Lactobacillus plantarum was identified as Lactobacillus plantarum P-8; Saccharomyces cerevisiae was identified as Saccharomyces cerevisiae QH2-2; and Bacillus natto was identified as Bacillus natto FMN-1, with the preservation number CGMCC No. 20659. Lactobacillus plantarum P-8, Saccharomyces cerevisiae QH2-2, and Bacillus natto FMN-1 are all commercially available strains.

[0020] Example 1 The preparation method of the lyophilized mare's milk powder in Example 1 includes the following steps: S1: Fresh mare's milk from healthy Abaga black horses was selected, filtered through a 100-mesh filter to remove impurities, and then sterilized at 45 ℃ for 15 min. After cooling to 28 ℃, 1 kg of pretreated fresh mare's milk was obtained. 0.2 g of a compound microbial agent was added to the pretreated fresh mare's milk, and fermentation was carried out at 30 ℃ for 36 h. The final pH of the fermentation was controlled to be 4.2, thus obtaining the fermented mare's milk broth. The compound microbial agent included 0.034 g of Bacillus natto powder, 0.056 g of Saccharomyces cerevisiae powder, and 0.11 g of Lactobacillus plantarum powder. The viable count of the Bacillus natto powder was 3 × 10⁻⁶. 11 The viable count of *Lactobacillus plantarum* powder is 5 × 10⁻⁶ CFU / g. 12 CFU / g, the viable count of the brewer's yeast powder is 2×10⁻⁶. 11 CFU / g.

[0021] S2: The fermented mare's milk broth was placed in a vacuum distillation apparatus and concentrated at 25 ℃ and 0.08 MPa. During the concentration process, the mixture was continuously stirred at a stirring rate of 60 r / min to avoid local overheating. After concentration, a mare's milk paste with a water content of 4 wt% was obtained.

[0022] S3: Add the lyophilization protectant to the fermented mare's milk paste at a mass ratio of 1:3. After stirring evenly, pre-cool at 4 ℃ for 30 min to obtain pre-cooled fermented mare's milk. After freeze-drying, the pre-cooled fermented mare's milk is aseptically pulverized, passed through an 80-mesh sieve, and packaged using nitrogen-filled encapsulation. It is then stored at -20 ℃ in the dark to obtain lyophilized mare's milk powder. The lyophilization protectant includes basic components and metal ions. The basic components, by mass, include: 8 parts trehalose, 6 parts tremella polysaccharide, 7 parts mannitol, 3 parts glycine, 5 parts grape seed polyphenols, and 100 parts water. The metal ions include potassium, calcium, and magnesium ions. The final concentration of potassium ions in the lyophilization protectant is 5 mmol / L, the final concentration of calcium ions is 0.7 mmol / L, and the final concentration of magnesium ions is 0.8 mmol / L. Potassium ions are provided by potassium chloride, calcium ions by calcium chloride, and magnesium ions by magnesium chloride.

[0023] The steps of freeze drying are as follows: S1: Place the pre-cooled fermented mare's milk in a freeze dryer and cool it to -40°C at a rate of 1°C / min. Keep it at this temperature for 2 hours to ensure that the freeze-dried material is completely frozen below the eutectic point. S2: First sublimation drying: Under a vacuum of 4.0 Pa, the temperature is raised to -20 ℃ and dried for 20 h, then the temperature is naturally raised to 25 ℃ and held for 1 h to allow the internal moisture of the fermented mare's milk to fully migrate to the surface; S3: Second analytical drying: Under a vacuum of 4.0 Pa, the temperature is raised to 30 °C and dried for 10 h.

[0024] The lyophilized mare's milk powder of Example 1 was prepared according to the preparation method of the lyophilized mare's milk powder of Example 1.

[0025] Example 2 The preparation method of the lyophilized mare's milk powder in Example 2 includes the following steps: S1: Fresh mare's milk from healthy Abaga black horses was selected, filtered through a 100-mesh filter to remove impurities, and then sterilized at 50 ℃ for 15 min. After cooling to 25 ℃, 1 kg of pretreated fresh mare's milk was obtained. 0.4 g of a compound microbial agent was added to the pretreated fresh mare's milk, and fermentation was carried out at 28 ℃ for 48 h. The final pH of the fermentation was controlled to be 4.2, yielding the fermented mare's milk broth. The compound microbial agent consisted of 0.12 g of Bacillus natto powder, 0.07 g of Saccharomyces cerevisiae powder, and 0.21 g of Lactobacillus plantarum powder. The viable count of the Bacillus natto powder was 6 × 10⁻⁶. 11 The viable count of *Lactobacillus plantarum* powder is 3 × 10⁻⁶ CFU / g. 11 The CFU / g of the brewer's yeast powder is 4 × 10⁻⁶. 11 CFU / g.

[0026] S2: Place the fermented mare's milk broth in a vacuum distillation apparatus and concentrate it at 35 ℃ and 0.09 MPa. During the concentration process, stir continuously at a stirring rate of 50 r / min to avoid local overheating. After concentration, a mare's milk paste with a water content of 5 wt% is obtained.

[0027] S3: Add the lyophilization protectant to the fermented mare's milk paste at a mass ratio of 1:4. After stirring evenly, pre-cool at 5 ℃ for 20 min to obtain pre-cooled fermented mare's milk. After freeze-drying, the pre-cooled fermented mare's milk is aseptically pulverized, passed through an 80-mesh sieve, and packaged using nitrogen-filled encapsulation. It is then stored at -20 ℃ in the dark to obtain lyophilized mare's milk powder. The lyophilization protectant includes basic components and metal ions. The basic components, by mass, include: 5 parts trehalose, 8 parts tremella polysaccharide, 4 parts mannitol, 2 parts glycine, 6 parts grape seed polyphenols, and 100 parts water. The metal ions include potassium, calcium, and magnesium ions. The final concentration of potassium ions in the lyophilization protectant is 4 mmol / L, the final concentration of calcium ions is 0.72 mmol / L, and the final concentration of magnesium ions is 1 mmol / L. Potassium ions are provided by potassium chloride, calcium ions by calcium chloride, and magnesium ions by magnesium chloride.

[0028] The steps of freeze drying are as follows: S1: Place the pre-cooled fermented mare's milk in a freeze dryer and cool it to -35°C at a rate of 2°C / min. Keep it at this temperature for 3 hours to ensure that the freeze-dried material is completely frozen below the eutectic point. S2: First sublimation drying: Under a vacuum of 4.2 Pa, the temperature is raised to -18 ℃ and dried for 18 h, then the temperature is naturally raised to 22 ℃ and held for 1 h to allow the internal moisture of the fermented mare's milk to fully migrate to the surface; S3: Second analytical drying: Under a vacuum of 4.2 Pa, the temperature is raised to 30 °C and dried for 12 h.

[0029] The lyophilized mare's milk powder of Example 2 was prepared according to the preparation method of the lyophilized mare's milk powder of Example 1.

[0030] Example 3 The preparation method of the lyophilized mare's milk powder in Example 3 includes the following steps: S1: Fresh mare's milk from healthy Abaga black horses was selected, filtered through a 100-mesh filter to remove impurities, and then sterilized at 45 ℃ for 20 min. After cooling to 30 ℃, 1 kg of pretreated fresh mare's milk was obtained. 0.5 g of a compound microbial agent was added to the pretreated fresh mare's milk, and fermentation was carried out at 37 ℃ for 24 h. The final pH of the fermentation was controlled to be 4.2, thus obtaining the fermented mare's milk broth. The compound microbial agent included 0.17 g of Bacillus natto powder, 0.11 g of Saccharomyces cerevisiae powder, and 0.22 g of Lactobacillus plantarum powder. The viable count of the Bacillus natto powder was 8 × 10⁻⁶. 11 The CFU / g count of Lactobacillus plantarum powder is 8 × 10⁻⁶. 11 CFU / g, the viable count of the brewer's yeast powder is 1×10⁻⁶. 11 CFU / g.

[0031] S2: Place the fermented mare's milk broth in a vacuum distillation apparatus and concentrate it at 30 ℃ and 0.08 MPa. During the concentration process, stir continuously at a stirring rate of 80 r / min to avoid local overheating. After concentration, a mare's milk paste with a water content of 3 wt% is obtained.

[0032] S3: Add the lyophilization protectant to the fermented mare's milk paste at a mass ratio of 1:2. After stirring evenly, pre-cool at 5 ℃ for 15 min to obtain pre-cooled fermented mare's milk. After freeze-drying, the pre-cooled fermented mare's milk is aseptically pulverized, passed through an 80-mesh sieve, and packaged using nitrogen-filled encapsulation. It is then stored at -20 ℃ in the dark to obtain lyophilized mare's milk powder. The lyophilization protectant includes basic components and metal ions. The basic components, by mass, include: 7 parts trehalose, 10 parts tremella polysaccharide, 8 parts mannitol, 1 part glycine, 2 parts grape seed polyphenols, and 100 parts water. The metal ions include potassium, calcium, and magnesium ions. The final concentration of potassium ions in the lyophilization protectant is 8 mmol / L, the final concentration of calcium ions is 0.71 mmol / L, and the final concentration of magnesium ions is 1.2 mmol / L. Potassium ions are provided by potassium chloride, calcium ions by calcium chloride, and magnesium ions by magnesium chloride.

[0033] The steps of freeze drying are as follows: S1: Place the pre-cooled fermented mare's milk in a freeze dryer and cool it down to -40°C at a rate of 3°C / min. Keep it for 2 hours to ensure that the freeze-dried material is completely frozen below the eutectic point. S2: First sublimation drying: Under a vacuum of 4.3 Pa, the temperature is raised to -15 ℃ and dried for 20 h, then the temperature is naturally raised to 20 ℃ and held for 1.5 h to allow the internal moisture of the fermented mare's milk to fully migrate to the surface; S3: Second analytical drying: Under a vacuum of 4.3 Pa, the temperature is raised to 32 ℃ and dried for 10 h.

[0034] The lyophilized mare's milk powder of Example 3 was prepared according to the preparation method of the lyophilized mare's milk powder of Example 1.

[0035] Comparative Example 1 The preparation method of the freeze-dried mare's milk powder in Comparative Example 1 is roughly the same as that in Example 1. The difference between the preparation method of the freeze-dried mare's milk powder in Comparative Example 1 and Example 1 is that the compound microbial agent in Comparative Example 1 includes 0.067 g of Saccharomyces cerevisiae powder and 0.133 g of Lactobacillus plantarum powder, and the time to reach the fermentation endpoint pH 4.2 is 54 h.

[0036] Comparative Example 2 The preparation method of the freeze-dried mare's milk powder in Comparative Example 2 is roughly the same as that in Example 1. The difference between the preparation method of the freeze-dried mare's milk powder in Comparative Example 2 and Example 1 is that the freeze-drying protectant in Comparative Example 2 lacks trehalose, and the mass is made up according to the proportion of each substance in Example 1.

[0037] Comparative Example 3 The preparation method of the freeze-dried mare's milk powder in Comparative Example 3 is roughly the same as that in Example 1. The difference between the preparation method of the freeze-dried mare's milk powder in Comparative Example 3 and Example 1 is that the freeze-drying protectant in Comparative Example 3 lacks Tremella polysaccharide, and the mass is made up according to the proportion of each substance in Example 1.

[0038] Comparative Example 4 The preparation method of the freeze-dried mare's milk powder in Comparative Example 4 is roughly the same as that in Example 1. The difference between the preparation method of the freeze-dried mare's milk powder in Comparative Example 4 and Example 1 is that the freeze-drying protectant in Comparative Example 4 lacks mannitol, and the mass is made up according to the proportion of each substance in Example 1.

[0039] Comparative Example 5 The preparation method of the lyophilized mare's milk powder of Comparative Example 5 is roughly the same as that of Example 1. The difference between the preparation method of the lyophilized mare's milk powder of Comparative Example 5 and Example 1 is that the lyophilization protectant in Comparative Example 5 lacks glycine, and the mass is made up according to the proportion of each substance in Example 1.

[0040] Comparative Example 6 The preparation method of the freeze-dried mare's milk powder of Comparative Example 6 is roughly the same as that of Example 1. The difference between the preparation method of the freeze-dried mare's milk powder of Comparative Example 6 and Example 1 is that the freeze-drying protectant in Comparative Example 6 lacks grape seed polyphenols, and the mass is made up according to the proportion of each substance in Example 1.

[0041] Comparative Example 7 The preparation method of the lyophilized mare's milk powder in Comparative Example 7 is roughly the same as that in Example 1. The difference between the preparation method of the lyophilized mare's milk powder in Comparative Example 7 and Example 1 is that the freeze-drying protectant in Comparative Example 7 lacks potassium ions.

[0042] Comparative Example 8 The preparation method of the freeze-dried mare's milk powder of Comparative Example 8 is roughly the same as that of Example 1. The difference between the preparation method of the freeze-dried mare's milk powder of Comparative Example 8 and Example 1 is that the freeze-drying protectant in Comparative Example 8 lacks calcium ions.

[0043] Comparative Example 9 The preparation method of the freeze-dried mare's milk powder of Comparative Example 9 is roughly the same as that of Example 1. The difference between the preparation method of the freeze-dried mare's milk powder of Comparative Example 9 and Example 1 is that the freeze-drying protectant in Comparative Example 9 lacks magnesium ions.

[0044] Comparative Example 10 The preparation method of the freeze-dried mare's milk powder of Comparative Example 10 is roughly the same as that of Example 1. The difference between the preparation method of the freeze-dried mare's milk powder of Comparative Example 10 and Example 1 is that the freeze-drying protectant in Comparative Example 1 only has basic components and no metal ions.

[0045] Comparative Example 11 The preparation method of the lyophilized mare's milk powder in Comparative Example 11 is roughly the same as that in Example 1. The difference between the preparation method of the lyophilized mare's milk powder in Comparative Example 11 and Example 1 is that no freeze-drying protectant was added to the lyophilized mare's milk paste in Comparative Example 11.

[0046] Experimental Example 1 Blending characteristics and organizational characteristics Table 1. Combination state and reconstitution characteristics of freeze-dried mare's milk powder

[0047] As can be seen from Table 1, the lyophilized mare's milk powder prepared by this invention has uniform particles without clumping, and can form a stable suspension after adding water, with good texture and reconstitution properties.

[0048] Experimental Example 2 viability test 1. Detection of Lactobacillus plantarum: The lyophilized mare's milk powder of Examples 1-3 and Comparative Examples 2-11 was dissolved in sterile physiological saline to obtain a suspension, and then serially diluted 10 times. 1 mL of the suspension with an appropriate dilution was inoculated into MRS agar medium and anaerobically cultured at 37 ℃ for 48 h. The results were observed and calculated, and the average value was taken.

[0049] 2. Detection of Saccharomyces cerevisiae: The freeze-dried mare's milk powder of Examples 1-3 and Comparative Examples 2-11 was dissolved in sterile physiological saline to obtain a suspension, and then serially diluted 10 times. 1 mL of the suspension with the appropriate dilution was inoculated into YPD medium and cultured aerobically at 28 ℃ for 48 h. The results were observed and calculated, and the average value was taken.

[0050] 3. Detection of Bacillus natto: The lyophilized mare's milk powder of Examples 1-3 and Comparative Examples 2-11 was dissolved in sterile physiological saline to obtain a suspension, and then serially diluted 10 times. 1 mL of the suspension with an appropriate dilution was inoculated into nutrient agar medium and cultured aerobically at 37°C for 48 h. The results were observed and calculated, and the average value was taken.

[0051] Survival rate (%) = (number of viable bacteria in freeze-dried mare's milk powder / initial number of viable bacteria) × 100%.

[0052] Table 2 Survival rate of bacterial culture in freeze-dried mare's milk

[0053] As can be seen from Table 2, the bacterial survival rate of the freeze-dried mare's milk in Examples 1-3 was the highest. When one of the substances was missing, the survival rate of the bacterial strain decreased to varying degrees. The freeze-drying protectant of the present invention has a good function of improving the survival rate of the bacterial strain.

[0054] The viable bacterial counts of the lyophilized mare's milk powder from Examples 1-3 after nitrogen filling and packaging and storage at -20 °C for 12 months are shown in Table 3.

[0055] Table 3. Viable bacteria count in freeze-dried mare's milk powder

[0056] Experimental Example 3 Prepare a sufficient number of 10 mL centrifuge tubes and weigh them, marking the weight on the tubes. Inoculate the culture medium with the bacterial strain at a rate of 3%, and incubate for 18 hours. After incubation, centrifuge, wash, and collect the bacterial sludge. Prepare a freeze-drying protectant, then mix the collected bacterial sludge with the freeze-drying protectant at a mass ratio of 1:2. Perform freeze-drying according to the procedure described in Example 1. Weigh the centrifuge tubes and the bacterial powder to calculate the mass of the bacterial powder. Add sterile water to the bacterial powder and bring the volume to 10 mL. Calculate the viability using dilution plates and algorithms. MRS agar was used for *Lactobacillus plantarum*, YPD agar for *Saccharomyces cerevisiae*, and nutrient agar for *Bacillus natto*.

[0057] 1. Effect of different mass fractions of trehalose on viable bacterial count Table 4. Effect of different mass fractions of trehalose on viable bacterial count

[0058] As shown in Table 4, when the amount of trehalose added was 5-8 parts by weight, the survival rate of Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus natto was the highest.

[0059] 2. Effects of different mass fractions of Tremella polysaccharide on viable cell rate Table 5. Effects of different mass fractions of Tremella polysaccharide on viable cell rate

[0060] As shown in Table 5, when the amount of Tremella polysaccharide added was 6-10 parts by weight, the survival rate of Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus natto was the highest.

[0061] 3. Effect of different mass fractions of mannitol on viable bacterial count Table 6. Effect of different mass fractions of mannitol on viable bacterial count

[0062] As shown in Table 6, the survival rates of Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus natto were highest when the amount of mannitol added was 4–8 parts by weight.

[0063] 4. Effect of different mass fractions of glycine on viable bacterial count Table 7. Effect of different mass fractions of glycine on viable bacterial count

[0064] As can be seen from Table 7, when the glycine content is 1-3 parts by mass, Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus natto all have high survival rates.

[0065] 5. Effect of different mass fractions of grape seed polyphenols on viable cell rate Table 8. Effect of different mass fractions of grape seed polyphenols on viable cell rate

[0066] As shown in Table 8, when the amount of grape seed polyphenols added is 2 to 6 parts by weight, Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus natto all have high survival rates.

[0067] 6. Effect of different potassium ion concentrations on viable cell rate Table 9. Effects of different potassium ion concentrations on viable cell rate

[0068] 7. Effect of different calcium ion concentrations on viable bacterial count Table 10 Effect of different calcium ion concentrations on viable bacterial count

[0069] 8. Effects of different magnesium ions on viable bacterial count Table 11 Effects of different magnesium ions on viable bacterial count

[0070] As can be seen from Tables 9-11, the survival rates of *Lactobacillus plantarum*, *Saccharomyces cerevisiae*, and *Bacillus natto* were relatively high when the final concentrations of potassium ions were 4-8 mmol / L, calcium ions were 0.7-0.72 mmol / L, and magnesium ions were 0.8-1.2 mmol / L.

[0071] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the claims.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. A method for preparing freeze-dried mare's milk powder, characterized in that, Includes the following steps: S1: Fresh mare's milk is filtered, sterilized and cooled to obtain pretreated fresh mare's milk. Pretreated fresh mare's milk is added to compound microbial agents for fermentation to obtain fermented mare's milk broth. S2: After the fermented mare's milk liquid is concentrated, mare's milk paste is obtained. A freeze-drying protectant is added to the mare's milk paste, and after pre-cooling, it is freeze-dried to obtain mare's milk freeze-dried powder. The freeze-drying protectant comprises basic components and metal ions; the basic components, by mass, include: 5-8 parts trehalose, 6-10 parts tremella polysaccharide, 4-8 parts mannitol, 1-3 parts glycine, 2-6 parts grape seed polyphenols, and 100 parts water; the metal ions include potassium ions, calcium ions, and magnesium ions; the final concentration of potassium ions is 4-8 mmol / L, the final concentration of calcium ions is 0.7-0.72 mmol / L, and the final concentration of magnesium ions is 0.8-1.2 mmol / L.

2. The method for preparing lyophilized mare's milk powder according to claim 1, characterized in that, The mass ratio of the sour mare's milk paste to the freeze-drying protectant in S2 is 1:2 to 4.

3. The method for preparing freeze-dried mare's milk powder according to claim 1, characterized in that, The freeze-drying step described in S2 includes: S1: Pre-cooling stage: Cool to -35 to -40 ℃ at a rate of 1 to 3 ℃ / min and hold for 2 to 3 h; S2: First sublimation drying: Under a vacuum of 4.0 to 4.3 Pa, heat to -15 to -20 °C and dry for 18 to 20 h, then heat to 20 to 25 °C and hold for 1 to 1.5 h; S3: Second analytical drying: Maintain vacuum at 4.0–4.3 Pa, raise temperature to 30–32 °C and dry for 10–15 h.

4. The method for preparing freeze-dried mare's milk powder according to claim 1, characterized in that, The concentration conditions in S2 are as follows: temperature 25–35 °C, vacuum degree 0.08–0.09 MPa, stirring rate 50–80 r / min; the water content of the fermented mare's milk paste is 3–5 wt%; the pre-cooling temperature for freeze drying after pre-cooling is 4–5 °C, and the time is 15–30 min.

5. The method for preparing freeze-dried mare's milk powder according to claim 1, characterized in that, The sterilization temperature in S1 is 45-50 ℃, and the sterilization time is 15-20 min; the temperature for pre-treating fresh mare's milk is 25-30 ℃.

6. The method for preparing freeze-dried mare's milk powder according to claim 1, characterized in that, The amount of compound microbial agent added in S1 is 0.02 to 0.05 wt% of the pretreated fresh mare's milk.

7. The method for preparing freeze-dried mare's milk powder according to claim 1, characterized in that, The compound microbial agent described in S1 includes Bacillus natto powder, Lactobacillus plantarum powder, and Saccharomyces cerevisiae powder. The mass ratio of Saccharomyces cerevisiae powder to Lactobacillus plantarum powder is 1:1.5-3. The total mass ratio of Bacillus natto powder to Lactobacillus plantarum powder and Saccharomyces cerevisiae powder is 0.2-0.55:

1.

8. The method for preparing freeze-dried mare's milk powder according to claim 7, characterized in that, The viable count of Bacillus natto powder described in S1 is 3 × 10⁻⁶. 11 ~8×10 11 CFU / g; the viable count of the *Lactobacillus plantarum* powder is 3 × 10⁻⁶. 11 ~5×10 12 CFU / g; the viable count of the brewer's yeast powder is 1×10⁻⁶. 11 ~4×10 11 CFU / g.

9. The method for preparing freeze-dried mare's milk powder according to claim 1, characterized in that, The fermentation temperature described in S1 is 28–37 °C, and the fermentation time is 24–48 h.

10. A freeze-dried powder of fermented mare's milk, characterized in that, It is prepared by the method for preparing lyophilized mare's milk according to any one of claims 1 to 9.

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