High-activity staphylococcus xylosus freeze-dried powder and preparation method thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to high-activity staphylococcus xylosus freeze-dried powder and a preparation method thereof.The preparation method comprises the following steps that a staphylococcus xylosus working seed solution is inoculated into a fermentation culture medium, shake cultivation is conducted at 37 DEG C, then centrifugation is conducted at 4 DEG C, bacterial sludge is collected, and staphylococcus xylosus freeze-dried powder is obtained; mixing the bacterial sludge with a freeze-drying protective agent, and performing vacuum freeze-drying to obtain freeze-dried powder; the inoculation amount of the staphylococcus xylosus working seed solution is 2-4% of the volume of the fermentation system; the fermentation culture medium is an improved BHI culture medium; the weight volume ratio of the bacterial sludge to the freeze-drying protective agent is 1: (1-2). The staphylococcus xylosus freeze-dried powder product is outstanding in performance index, the freeze-drying survival rate exceeds 90%, the viable count is high, and the staphylococcus xylosus freeze-dried powder product has remarkable advantages in industrial application.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a highly active Staphylococcus xylose freeze-dried powder and its preparation method. Background Technology

[0002] Staphylococcus xylose possesses excellent fermentation characteristics and flavor-forming ability, and has broad application prospects in functional foods, fermented meat products, and microecological preparations. Staphylococcus xylose (… Staphylococcus xylosus TG022 is a food-grade microorganism with significant application value. Its outstanding antioxidant activity has been verified in the patent application number 2023106329280. This strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2023516. This strain can effectively scavenge free radicals and reduce oxidative stress damage.

[0003] However, the production of Staphylococcus xylose inoculants currently faces bottlenecks such as low viable cell counts, low freeze-drying survival rates, and poor shelf-life stability. For example, the cultivation of Staphylococcus xylose mainly uses universal culture media (such as BHI and MRS), which suffer from low cell yield and insufficient cultivation efficiency, resulting in low cell density and failing to meet the high cell density requirements of industrial production. In the freeze-drying process, vacuum freeze-drying is mainly used. During this process, the formation of ice crystals and changes in osmotic pressure can cause cell membrane damage, severely affecting the maintenance of cell survival. At the same time, traditional protective agents have poor cell membrane protection effects, leading to rapid decline in viable cells during storage, which seriously affects product quality stability and directly restricts its industrial application.

[0004] Therefore, developing a process for preparing highly active and stable Staphylococcus xylose powder is of great significance for promoting its application in the food and health fields. Summary of the Invention

[0005] To address the above-mentioned deficiencies or improvement needs of existing technologies, this invention provides a highly active Staphylococcus xylose freeze-dried powder and its preparation method. This Staphylococcus xylose freeze-dried powder has outstanding performance indicators, with a freeze-drying survival rate of over 90% and a high number of viable bacteria, giving it significant advantages in industrial applications.

[0006] To achieve the above objectives, the present invention provides a method for preparing highly active Staphylococcus xylose lyophilized powder, comprising the following steps: (1) The working seed culture of Staphylococcus xylose was inoculated into the fermentation medium and cultured in a shaker at 37°C to obtain the culture medium; (2) Centrifuge the culture medium at 4°C and collect the bacterial sludge; (3) Mix the bacterial sludge with the freeze-drying protectant and freeze-dry it under vacuum to obtain freeze-dried powder; The inoculum volume of the Staphylococcus xylose working seed culture is 2-4% of the fermentation system volume; The fermentation medium is a modified BHI medium; The weight-to-volume ratio of the bacterial sludge to the freeze-drying protectant is 1:1-2, preferably 1:1.

[0007] This invention uses a modified BHI medium as the fermentation medium. The optimized medium composition provides suitable environmental conditions for the growth of the bacterial strain, achieving efficient accumulation of bacterial biomass, high cultivation efficiency, and high strain density, providing high-quality bacterial raw materials for subsequent freeze-drying processes. By selecting a novel freeze-drying protectant and controlling its ratio with the bacterial sludge, the integrity of the cell membrane can be effectively maintained, ice crystal damage can be reduced, and the survival rate and stability of the bacterial powder can be improved.

[0008] Furthermore, in the above technical solution, the carbon source in the modified BHI culture medium is galactooligosaccharide, with a content of 0-4 g / L; the nitrogen source is yeast extract, with a content of 5-15 g / L; and the inorganic salt is a combination of disodium hydrogen phosphate and sodium chloride, with a content of 4.5-7.5 g / L.

[0009] Furthermore, in the above technical solution, the mass ratio of disodium hydrogen phosphate to sodium chloride is 1:2.

[0010] Furthermore, in the above technical solution, the modified BHI medium contains 0 g / L of galactooligosaccharides, 10 g / L of yeast extract, and 6 g / L of the combination of disodium hydrogen phosphate and sodium chloride. This invention, through systematic optimization of carbon sources, nitrogen sources, and inorganic salt components, ultimately determined that a modified medium containing yeast extract, disodium hydrogen phosphate, and sodium chloride, but without galactooligosaccharides, can effectively increase the viable cell count of Staphylococcus xylose TG022 and shorten the culture cycle. This medium has a simple formulation, low cost, and significant application value.

[0011] Furthermore, in step (2) of the above technical solution, the centrifugation speed is 6000 rpm and the time is 10 min. After centrifugation, the centrifuge is washed 2-3 times with sterile physiological saline.

[0012] Furthermore, in the above technical solution, the freeze-drying protectant is a mixture containing 2-10% maltitol, 0.5-2% silk protein peptides, and 5-10% lactose. This invention creatively uses silk protein peptides + lactose + maltitol as a freeze-drying protectant system. Lactose protects cell structure by forming a glassy matrix and replacing water molecules; silk protein peptides reduce ice crystal damage through molecular chaperone action and physical barriers, and contain thiol amino acids (such as cysteine) to scavenge free radicals and reduce oxidative stress damage to cells; maltitol improves storage stability due to its anti-crystallization properties and low hygroscopicity. The combined use of these three components provides systemic protection: lactose provides basic protection, silk protein peptides enhance membrane stability, and maltitol optimizes physical properties, forming a complete protective system. The resulting highly active bacterial powder has good resolubility and low water content.

[0013] Furthermore, in the above technical solution, the preparation method of the freeze-drying protectant is as follows: maltitol, silk protein peptide and lactose are added to purified water to dissolve and mix well, then sterilized at high temperature and high pressure, taken out and cooled at room temperature, and then stored in a refrigerator at 4°C.

[0014] Furthermore, in step (3) of the above technical solution, the mixture is pre-frozen at -80℃ for 2 hours before vacuum freeze-drying; the procedure for vacuum freeze-drying is shown in the table below:

[0015] The present invention also provides a Staphylococcus xylose lyophilized powder prepared by the above preparation method, wherein the initial viable count of the Staphylococcus xylose lyophilized powder is 5 × 10⁻⁶. 11 CFU / g-10×10 11 CFU / g.

[0016] This invention also provides the application of the above-mentioned Staphylococcus xylose freeze-dried powder in the preparation of meat product preservation as a natural antioxidant, in the preparation of traditional fermented food production as a flavor enhancer, and in the preparation of probiotic preparations.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The improved BHI culture medium formulation of this invention uses an optimized combination of yeast extract as a nitrogen source, disodium hydrogen phosphate and sodium chloride as inorganic salts, and is completely free of galactooligosaccharides, achieving a viable cell count of 2.03 × 10⁻⁶. 9 The CFU / mL concentration is 5.07 times that of traditional BHI medium, significantly improving the efficiency of bacterial culture.

[0018] The freeze-drying protectant system of this invention innovatively uses silk protein peptides as probiotic protectant components, combined with lactose and maltitol to form a synergistic protection mechanism. With a 1:1 ratio of bacterial sludge to protectant, it significantly improves the freeze-drying survival rate and has excellent stability.

[0019] The product of this invention has outstanding performance indicators, with a freeze-drying survival rate exceeding 90% and a viable bacterial count as high as 9.47 × 10⁻⁶. 11 The CFU / g count was significantly better than the 1.02 × 10⁻⁶ viable bacteria count obtained by the traditional method. 11 CFU / g. Detailed Implementation

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the raw materials used in the following examples are all commercially available products and can be purchased from the market.

[0021] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.

[0022] The raw materials involved in the various embodiments of the present invention are either existing commercially available products or can be prepared according to existing methods.

[0023] BHI medium consists of: 10.0 g / L tryptone, 5.0 g / L dehydrated bovine heart extract, 5.0 g / L sodium chloride, 2.0 g / L glucose, 2.5 g / L disodium hydrogen phosphate, and pH 7.4 ± 0.2.

[0024] The vacuum freeze-drying procedures in each embodiment are shown in Table 1.

[0025] Table 1. Vacuum freeze-drying procedure

[0026] Example 1 A method for preparing a highly active Staphylococcus xylose mycelium slurry includes the following steps: (1) Cultivation of working seed culture of Staphylococcus xylose: Staphylococcus xylose stored frozen in a -80℃ freezer was thawed at room temperature and inoculated into liquid fermentation medium and cultured at a constant temperature of 37℃ for 12 h to obtain working seed culture. The formula of liquid fermentation medium is: yeast extract 10 g / L, disodium hydrogen phosphate 2 g / L, sodium chloride 4 g / L, and the remainder is water.

[0027] (2) Fermentation culture: The working seed liquid of Staphylococcus xylose was inoculated into the modified BHI medium at 2% of the fermentation system volume and cultured in a shaker at 37℃ for 12h to obtain the culture medium; the culture medium was centrifuged at 6000rpm for 10min at 4℃, washed 2-3 times with sterile physiological saline, and the bacterial sludge was collected. The modified BHI culture medium consisted of: 10.0 g / L yeast extract, 0 g / L galactooligosaccharides, 4.0 g / L sodium chloride, 2 g / L disodium hydrogen phosphate, and a pH of 7.4 ± 0.2.

[0028] Example 2 A method for preparing a highly active Staphylococcus xylose mycelium slurry includes the following steps: (1) Cultivation of working seed culture of Staphylococcus xylose: Staphylococcus xylose stored frozen in a -80℃ freezer was thawed at room temperature and inoculated into liquid fermentation medium and cultured at a constant temperature of 37℃ for 12 h to obtain working seed culture. The formula of liquid fermentation medium is: yeast extract 10 g / L, disodium hydrogen phosphate 2 g / L, sodium chloride 4 g / L, and the remainder is water.

[0029] (2) Fermentation culture: The working seed liquid of Staphylococcus xylose was inoculated into the modified BHI medium at 3% of the fermentation system volume and cultured in a shaker at 37℃ for 12h to obtain the culture medium; the culture medium was centrifuged at 6000rpm for 10min at 4℃, washed 2-3 times with sterile physiological saline, and the bacterial sludge was collected. The modified BHI culture medium consisted of: 10.0 g / L yeast extract, 2 g / L galactooligosaccharides, 3 g / L sodium chloride, 1.5 g / L disodium hydrogen phosphate, and a pH of 7.4 ± 0.2.

[0030] Example 3 A method for preparing a highly active Staphylococcus xylose mycelium slurry includes the following steps: (1) Cultivation of working seed culture of Staphylococcus xylose: Staphylococcus xylose stored frozen in a -80℃ freezer was thawed at room temperature and inoculated into liquid fermentation medium and cultured at a constant temperature of 37℃ for 12 h to obtain working seed culture. The formula of liquid fermentation medium is: yeast extract 10 g / L, disodium hydrogen phosphate 2 g / L, sodium chloride 4 g / L, and the remainder is water.

[0031] (2) Fermentation culture: The working seed liquid of Staphylococcus xylose was inoculated into the modified BHI medium at 4% of the fermentation system volume and cultured in a shaker at 37℃ for 12h to obtain the culture medium; the culture medium was centrifuged at 6000rpm for 10min at 4℃, washed 2-3 times with sterile physiological saline, and the bacterial sludge was collected. The modified BHI culture medium consisted of: 15.0 g / L yeast extract, 0 g / L galactooligosaccharides, 4.0 g / L sodium chloride, 2 g / L disodium hydrogen phosphate, and a pH of 7.4 ± 0.2.

[0032] Example 4 A method for preparing highly active Staphylococcus xylose freeze-dried powder includes the following steps: mixing the bacterial sludge from Example 1 with a freeze-drying protectant at a weight-volume ratio of 1:1, pre-freezing the mixture at -80℃ for 2 hours, and then freeze-drying it according to the procedure in Table 1 to obtain freeze-dried powder. The method for preparing freeze-dried protection is as follows: maltitol, silk protein peptides and lactose are added to purified water to dissolve and mix well to prepare a mixture with 6% maltitol, 2% silk protein peptides and 8% lactose. Then, it is sterilized by high temperature and high pressure, and then used after being taken out and cooled at room temperature.

[0033] Example 5 A method for preparing highly active Staphylococcus xylose freeze-dried powder includes the following steps: mixing the bacterial sludge from Example 2 with a freeze-drying protectant at a weight-volume ratio of 1:1, pre-freezing the mixture at -80℃ for 2 hours, and then freeze-drying it according to the procedure in Table 1 to obtain freeze-dried powder. The method for preparing freeze-dried protection is as follows: maltitol, silk protein peptides and lactose are added to purified water to dissolve and mix well to prepare a mixture with 8% maltitol, 1% silk protein peptides and 6% lactose. Then, it is sterilized by high temperature and high pressure, taken out and cooled at room temperature before use.

[0034] Example 6 A method for preparing highly active Staphylococcus xylose freeze-dried powder includes the following steps: mixing the bacterial sludge from Example 3 with a freeze-drying protectant at a weight-to-volume ratio of 1:2, pre-freezing the mixture at -80℃ for 2 hours, and then freeze-drying it according to the procedure in Table 1 to obtain freeze-dried powder. The method for preparing freeze-dried protection is as follows: maltitol, silk protein peptides and lactose are added to purified water to dissolve and mix well to prepare a mixture with 6% maltitol, 2% silk protein peptides and 10% lactose. Then, it is sterilized by high temperature and high pressure, and then used after being taken out and cooled at room temperature.

[0035] Comparative Example 1 A method for preparing Staphylococcus xylose mycelium sludge differs from Example 2 in that the carbon source in the modified BHI medium is glucose.

[0036] Comparative Example 2 A method for preparing Staphylococcus xylitol mycelium sludge differs from Example 2 in that the carbon source in the modified BHI medium is fructooligosaccharide.

[0037] Comparative Example 3 A method for preparing Staphylococcus xylose mycelium sludge differs from Example 2 in that the nitrogen source in the modified BHI medium is tryptone.

[0038] Comparative Example 4 A method for preparing Staphylococcus xylitol mycelium sludge differs from Example 2 in that the nitrogen source in the modified BHI medium is beef extract powder.

[0039] Comparative Example 5 A method for preparing Staphylococcus xylose mycelium sludge differs from Example 2 in that the inorganic salt in the modified BHI medium is disodium hydrogen phosphate.

[0040] Comparative Example 6 A method for preparing highly active Staphylococcus xylose mycelium sludge differs from Example 2 in that the inorganic salt in the modified BHI medium is sodium chloride.

[0041] Comparative Example 7 A method for preparing Staphylococcus xylitol mycelium slurry includes the following steps: (1) Cultivation of working seed culture of Staphylococcus xylose: Staphylococcus xylose stored frozen in a -80℃ freezer was thawed at room temperature, inoculated into BHI liquid medium, and cultured at a constant temperature of 37℃ for 12 h to obtain working seed culture; (2) Fermentation culture: The working seed liquid of Staphylococcus xylose was inoculated into sterilized BHI medium at 3% of the fermentation system volume and cultured in a shaker at 37℃ for 12h to obtain the culture solution; the culture solution was centrifuged at 6000rpm for 10min at 4℃, and the bacterial sludge was washed 2-3 times with sterile physiological saline and collected.

[0042] Comparative Example 8 A method for preparing Staphylococcus xylose freeze-dried powder differs from Example 4 in that the ratio of mycelial sludge to freeze-drying protectant is 1:3.

[0043] Comparative Example 9 A method for preparing Staphylococcus xylitol freeze-dried powder differs from Example 4 in that the freeze-drying protectant is maltitol.

[0044] Comparative Example 10 A method for preparing Staphylococcus xylose freeze-dried powder differs from Example 4 in that the freeze-drying protectant is silk protein peptide.

[0045] Comparative Example 11 A method for preparing Staphylococcus xylose freeze-dried powder differs from Example 4 in that the freeze-drying protectant is lactose.

[0046] Comparative Example 12 A method for preparing Staphylococcus xylitol freeze-dried powder differs from Example 4 in that the freeze-drying protectant is maltitol and lactose.

[0047] Comparative Example 13 A method for preparing Staphylococcus xylose freeze-dried powder includes the following steps: mixing the bacterial sludge of Comparative Example 7 with a freeze-drying protectant at a weight-volume ratio of 1:1, loading the mixture into a freeze-drying pan for vacuum freeze-drying, and freeze-drying according to the procedure in Table 1 to obtain freeze-dried powder. The preparation method of the freeze-drying protectant is as follows: skim milk powder, inulin and maltodextrin are added to purified water to dissolve and mix well to make a mixture with skim milk powder content of 10%, inulin content of 5% and maltodextrin content of 5%. Then, it is sterilized by high temperature and high pressure, and then used after being taken out and cooled at room temperature.

[0048] Test case 1. The viable cell counts of the culture media obtained in Examples 1-3 and Comparative Examples 1-7 were detected (the viable cell count per unit was calculated using the plate count method), and the results are shown in Table 2.

[0049] Table 2 Viable Bacterial Count Results

[0050] As shown in Table 2, the viable count of Staphylococcus xylose cultured on the modified BHI medium of this invention was consistently above 18 × 10⁻⁶. 8 A concentration of CFU / mL or higher indicates that the modified BHI medium is more conducive to the growth of Staphylococcus xylose and has a higher culture efficiency.

[0051] 2. The freeze-dried survival rate and shelf-life survival rate of the *Staphylococcus xylose* powders obtained in Examples 4-5 and Comparative Examples 8-13 were tested. The shelf-life survival rate was determined by packaging the *Staphylococcus xylose* powders in food-grade aluminum foil bags, sealing them, and placing them in a 37°C incubator for 7 days for accelerated shelf-life testing under high-temperature conditions. After 7 days, the samples were removed, and the viable cell count was determined using the dilution plating method to calculate the shelf-life survival rate. The shelf-life survival rate was calculated as follows: Shelf-life survival rate = Shelf-life viable cell count (CFU / g) / Initial viable cell count (CFU / g) × 100%. The results are shown in Table 3.

[0052] Table 3 Survival rate and shelf-life survival rate results

[0053] As can be seen from the results in Table 3, by adopting the freeze-drying protectant formulation of this invention and controlling its ratio with the bacterial sludge, the survival rate and stability of the strain can be effectively improved. The resulting freeze-dried powder has a high number of live bacteria and a survival rate of over 90%, resulting in good quality.

[0054] In summary, this invention improves culture and transformation efficiency by optimizing culture medium components; and by improving the formulation of the freeze-drying protectant and innovatively introducing silk fibroin peptides as the core protective component, a novel freeze-drying protection system is constructed, which effectively improves the survival rate and stability of the strains and has broad application prospects.

[0055] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing highly active Staphylococcus xylose lyophilized powder, characterized in that, Includes the following steps: (1) The working seed culture of Staphylococcus xylose was inoculated into the fermentation medium and cultured in a shaker at 37°C to obtain the culture medium; (2) Centrifuge the culture medium at 4°C and collect the bacterial sludge; (3) Mix the bacterial sludge with the freeze-drying protectant and freeze-dry it under vacuum to obtain freeze-dried powder; The inoculum volume of the Staphylococcus xylose working seed culture is 2-4% of the fermentation system volume; The fermentation medium is a modified BHI medium; The weight-to-volume ratio of the bacterial sludge to the freeze-drying protectant is 1:1-2.

2. The method for preparing a highly active Staphylococcus xylose lyophilized powder according to claim 1, characterized in that, In the modified BHI medium, the carbon source is galactooligosaccharide, with a content of 0-4 g / L; the nitrogen source is yeast extract, with a content of 5-15 g / L; and the inorganic salt is a combination of disodium hydrogen phosphate and sodium chloride, with a content of 4.5-7.5 g / L.

3. The method for preparing highly active Staphylococcus xylose lyophilized powder according to claim 2, characterized in that, The mass ratio of disodium hydrogen phosphate to sodium chloride is 1:

2.

4. The method for preparing a highly active Staphylococcus xylose lyophilized powder according to claim 2 or 3, characterized in that, In the modified BHI medium, the content of the oligogalactose is 0 g / L, the content of the yeast extract is 10 g / L, and the content of the combination of disodium hydrogen phosphate and sodium chloride is 6 g / L.

5. The method for preparing a highly active Staphylococcus xylose lyophilized powder according to claim 1, characterized in that, In step (2), the centrifugation speed is 6000 rpm and the time is 10 min. After centrifugation, the centrifuge is rinsed 2-3 times with sterile physiological saline.

6. The method for preparing a highly active Staphylococcus xylose lyophilized powder according to claim 1, characterized in that, The freeze-drying protectant is a mixture containing 2-10% maltitol, 0.5-2% silk protein peptides, and 5-10% lactose.

7. The method for preparing a highly active Staphylococcus xylose lyophilized powder according to claim 6, characterized in that, The method for preparing the freeze-drying protectant is as follows: maltitol, silk protein peptides and lactose are added to purified water to dissolve and mix well, then sterilized at high temperature and high pressure, taken out and cooled at room temperature, and then stored in a refrigerator at 4°C.

8. The method for preparing highly active Staphylococcus xylose lyophilized powder according to claim 1, characterized in that, In step (3), the mixture is pre-frozen at -80℃ for 2 hours before vacuum freeze-drying; the procedure for vacuum freeze-drying is shown in the table below: 。 9. The method for preparing a highly active Staphylococcus xylose lyophilized powder according to claim 1, characterized in that, The weight-to-volume ratio of the bacterial sludge to the freeze-drying protectant is 1:

1.

10. A freeze-dried Staphylococcus xylose powder prepared by the preparation method according to any one of claims 1-9, characterized in that, The initial viable count of the Staphylococcus xylose freeze-dried powder was 5 × 10⁻⁶. 11 CFU / g-10×10 11 CFU / g.

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