Pediococcus pentosaceus SUN02 freeze-dried powder with high activity, no allergen and low sodium content and preparation method thereof

Abstract

The invention relates to the technical field of probiotic preparations, in particular to pediococcus pentosaceus SUN02 freeze-dried powder with high activity, no allergen and low sodium content and a preparation method of the pediococcus pentosaceus SUN02 freeze-dried powder. The method comprises the following steps: activating a strain and preparing a seed solution; the method comprises the following steps: carrying out fermentation culture on pediococcus pentosaceus SUN02 by adopting a fermentation culture medium which is free of active-source and plant-source proteins and is added with amino acid to supplement a nitrogen source, so as to prepare fermentation liquor; collecting bacterial sludge; mixing an allergen-free protective agent with the bacterial sludge, and pre-freezing the mixed bacterial liquid; and performing vacuum freeze drying on the pre-frozen product to obtain freeze-dried powder. The freeze-dried bacterial powder prepared by the preparation method has the advantages of no active-source or plant-source allergen, low sodium content and the like, has high viable count, and has wide and good application prospects.

Description

Technical Field

[0001] This application relates to the field of probiotic preparation technology, specifically to a highly active, allergen-free, low-sodium Pediococcus pentosaccharide SUN02 lyophilized powder and its preparation method. Background Technology

[0002] In the field of probiotic preparation technology, lactic acid bacteria, as an important probiotic, have great application potential in healthcare, food industry and animal husbandry due to their wide range of physiological functions and health benefits.

[0003] However, the current industrialization of probiotics faces two major challenges: how to achieve high-density cultivation and how to efficiently prepare highly active freeze-dried bacterial powder. The cultivation of lactic acid bacteria not only requires stringent nutritional conditions but is also susceptible to environmental factors. Therefore, it is necessary to screen culture media and optimize fermentation processes for different types of probiotics.

[0004] Compared to spray drying, bacterial agents prepared by vacuum freeze-drying have the advantages of high activity and good stability. However, lactic acid bacteria are subjected to low-temperature stress, dehydration stress, and oxidative damage during vacuum freeze-drying, leading to cell membrane damage and protein inactivation, which in turn significantly reduces the number of viable bacteria after freeze-drying.

[0005] Some existing technologies improve the survival rate of lactic acid bacteria during freeze-drying by adding protectants (such as trehalose, sucrose, skim milk powder, sorbitol, etc.) before freeze-drying, pre-treating the bacteria (such as heat stress, cold stress, alcohol stress), and optimizing freeze-drying process parameters. While these measures increase the number of viable bacteria to some extent, they still fall short of achieving the ideal high activity level, and low viable bacteria count remains a common problem.

[0006] Allergic reactions are abnormal physiological responses of the immune system to harmless substances, and are common in genetically susceptible individuals. Food allergens are components in food that can trigger abnormal immune system responses. The main components are proteins or glycoproteins with molecular weights ranging from 10,000 to 70,000 Daltons. Internationally, eight types of food, including milk, eggs, and peanuts, are listed as major allergens, accounting for more than 90% of allergic reactions.

[0007] Conventional fermentation media for lactic acid bacteria often require the addition of animal-derived proteins such as beef extract, beef bone extract, and fish peptone, or plant-derived proteins such as soybean peptone and pea peptone. These protein products can all cause allergic reactions. Furthermore, the animal or plant-derived proteins in the fermentation medium may remain unused during fermentation and end up in the lyophilized powder. Completely removing these proteins to avoid allergens often leads to restricted bacterial growth and a significant decrease in viable cell count.

[0008] Excessive sodium intake can lead to problems such as high blood pressure, cardiovascular disease, and increased burden on the kidneys. It can also cause osteoporosis and edema. Reducing sodium intake is one of the most cost-effective measures to improve health and reduce the burden of non-communicable diseases.

[0009] Traditional lactic acid bacteria fermentation processes often employ alkali supplementation to control the pH of the culture medium and increase the bacterial cell density. This alkali supplementation process typically uses sodium hydroxide as the alkali solution, and a certain amount of sodium salt is also added to the initial components of the culture medium to maintain its initial osmotic pressure. However, this method increases the introduction of sodium.

[0010] In summary, the current development of lactic acid bacteria faces the following prominent contradictions: First, it is difficult to simultaneously achieve both a high number of viable bacteria and the requirement of no allergens.

[0011] The animal or plant-derived proteins commonly added to lactic acid bacteria fermentation media, such as beef extract, fish peptone, and soy peptone, provide good nitrogen sources and promote bacterial growth. However, their residual proteins or glycoproteins may become allergens, posing a health risk to sensitive individuals. Completely removing these proteins to avoid allergens often results in restricted bacterial growth and a significant decrease in viable cell count. Furthermore, commonly used freeze-drying protectants (such as skim milk powder) may also introduce additional allergens. Therefore, current processes, while pursuing high viable cell counts, often struggle to guarantee the allergen-free nature of the product.

[0012] Second, there is a conflict between the regulation of the fermentation process and the demand for low-sodium products.

[0013] During lactic acid bacteria fermentation, alkali solutions are typically added to neutralize the acidic substances produced by bacterial metabolism, maintain a suitable pH, and thus increase cell density. Currently, sodium hydroxide solution is commonly used for alkali supplementation, which directly leads to an increase in the sodium content of the final product. Simultaneously, sodium salts such as sodium chloride and sodium acetate are often added to the culture medium to adjust osmotic pressure, further increasing sodium intake. Excessive sodium intake is associated with health risks such as hypertension, and the market demand for low-sodium health products is growing. However, existing fermentation processes struggle to effectively reduce the sodium content of products without affecting bacterial growth and viable cell count.

[0014] In summary, existing lactic acid bacteria preparation technologies struggle to simultaneously achieve the three key objectives of being allergen-free, having low sodium content, and possessing high viable bacteria counts. Developing a lactic acid bacteria preparation process that can achieve all these properties is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0015] To address the aforementioned problems in existing technologies, this application provides a method for preparing highly active, allergen-free, and low-sodium Pediococcus pentosacchari SUN02 lyophilized powder. This method effectively increases the viable count of Pediococcus pentosacchari SUN02 fermentation broth and lyophilized powder, and the entire process is free from the introduction of animal or plant-derived allergens. It primarily utilizes amino acids to optimize and replace allergen components in the culture medium. The resulting lyophilized powder is allergen-free, has low sodium content, and features low production costs, short fermentation time, and simple operation.

[0016] This application provides a highly active, allergen-free, low-sodium Pediococcus pentosaceus. (Pediococcus) pentosaceus) The preparation method of SUN02 lyophilized powder includes the following steps: S1. Activation of microorganisms and preparation of seed culture; S2. Fermentation culture: Using a fermentation medium free of animal and plant proteins and supplemented with amino acids to supplement nitrogen source, Pediococcus pentosaccharides SUN02 was fermented to obtain fermentation broth. During the fermentation process, ammonia water is used as a pH adjuster throughout to control the pH value of the fermentation system to 5.9-6.1. The fermentation medium comprises the following components: carbon source, yeast extract, phosphate, ammonium salt, magnesium sulfate, manganese sulfate, nonionic surfactant, and an amino acid composition consisting of glycine, threonine, L-cysteine ​​hydrochloride, tryptophan, serine, glutamic acid, arginine, and lysine; no sodium-containing pH buffering components are added to the fermentation medium, and ammonia is used as a pH adjuster to adjust the pH value to 5.9-6.1; S3. Collection of microbial sludge: The fermentation broth is separated into solid and liquid components, and the microbial sludge is collected. S4. Pre-freezing: The allergen-free preservative is mixed with the bacterial sludge, and the mixed bacterial solution is pre-frozen to obtain the pre-frozen product; S5. Vacuum freeze-drying: The pre-frozen product is vacuum freeze-dried to obtain the freeze-dried powder.

[0017] In some embodiments, the preservation number of Pediococcus pentosaceus SUN02 is CGMCC No. 29705.

[0018] In some embodiments, the amount of the amino acid composition added to the fermentation medium is: glycine 0.95-1.05 g / L, threonine 0.95-1.05 g / L, L-cysteine ​​hydrochloride 0.95-1.05 g / L, tryptophan 0.45-0.55 g / L, serine 0.45-0.55 g / L, glutamic acid 0.08-0.12 g / L, arginine 0.04-0.06 g / L, and lysine 0.009-0.011 g / L.

[0019] In some embodiments, the preparation process of the fermentation medium is as follows: the raw material components of the fermentation medium are mixed and sterilized at 105-107℃ for 30-32 min, and then the pH is adjusted to 5.9-6.1 with ammonia water of concentration of 9.8-10.2 mol / L; wherein, the fermentation medium contains the following raw material components: glucose 39.5-40.5 g / L, Angel yeast extract FM985 19.5-20.5 g / L, dipotassium hydrogen phosphate 1.9-2.1 g / L, ammonium citrate 1.9-2.1 g / L, magnesium sulfate 0.095-0.15 g / L, manganese sulfate 0.048-0.052 g / L, Tween-80 0.95-1.05 g / L, glycine 0.95-1.05 g / L, threonine 0.95-1.05 g / L, L-cysteine ​​hydrochloride 0.95-1.05 g / L, tryptophan 0.45-0.55 g / L, serine 0.45-0.55 g / L, glutamic acid 0.08-0.12 g / L, arginine 0.04-0.06 g / L, lysine 0.009-0.011 g / L.

[0020] In some embodiments, in step S2, *Pediococcus pentosaceus* SUN02 is inoculated into a fermentation medium, and the *Pediococcus pentosaceus* SUN02 seed culture is fermented to obtain a fermentation broth; wherein the fermentation temperature is 36-38℃, the fermentation pressure is 0.03-0.05MPa, and the fermentation time is 12-15h.

[0021] In some embodiments, the components of the allergen-free lyophilized protectant include trehalose, maltodextrin, glycine, threonine, and sodium ascorbate, and do not contain skim milk powder, whey protein, or other animal-derived protectants.

[0022] In some embodiments, the raw material components of the allergen-free lyophilization protectant are mixed and sterilized at 105-107°C for 15-17 min to obtain the allergen-free lyophilization protectant; wherein the components of the allergen-free lyophilization protectant include: 14.5-15.5 g / L trehalose, 4.5-5.5 g / L maltodextrin, 1.95-2.05 g / L glycine, 1.95-2.05 g / L threonine, and 0.95-1.05 g / L sodium ascorbate.

[0023] In some embodiments, *Pediococcus pentosaceus* SUN02 is inoculated into a primary seed culture medium and cultured at 36.5-37.5°C under anaerobic conditions for 19-21 hours for primary expansion culture; then the culture is inoculated into a secondary seed culture medium and cultured at 36.5-37.5°C under anaerobic conditions for 7-8 hours for secondary expansion culture to obtain seed culture.

[0024] In some embodiments, the primary seed culture medium comprises: glucose 19.5-20.5 g / L, Angel yeast extract FM503 14.5-15.5 g / L, Angel yeast peptone FP103 9.8-10.2 g / L, sodium acetate 4.9-5.1 g / L, dipotassium hydrogen phosphate 1.9-2.1 g / L, ammonium citrate 1.9-2.1 g / L, magnesium sulfate 0.09-0.11 g / L, manganese sulfate 0.048-0.052 g / L, and Tween-80 0.95-1.05 g / L; the pH is adjusted to 6.4-6.6 using a 4.8-5.0 mol / L sodium hydroxide solution, and sterilization is performed at 121-123°C for 20-22 min. In some embodiments, the secondary seed culture medium comprises: glucose 14.5-15.5 g / L, Angel yeast extract FM503 7.8-8.2 g / L, Angel yeast peptone FP103 4.8-5.2 g / L, sodium acetate 4.9-5.1 g / L, dipotassium hydrogen phosphate 1.9-2.1 g / L, ammonium citrate 1.9-2.1 g / L, magnesium sulfate 0.09-0.11 g / L, manganese sulfate 0.048-0.052 g / L, and Tween-80 0.95-1.05 g / L; the pH is adjusted to 6.4-6.6 using a 4.8-5.0 mol / L sodium hydroxide solution, and sterilization is performed at 121-123℃ for 20-22 min.

[0025] In some embodiments, in step S4, the bacterial sludge and the allergen-free protective agent are mixed at a mass ratio of (0.9-1):1 to obtain a mixed bacterial solution. After mixing, the mixed bacterial solution is pre-frozen at -40-41°C for 14-15 hours.

[0026] In some embodiments, during step S5, the freezing temperature in the freeze-drying stage is -50°C to -70°C, the vacuum degree is 10-11 Pa, the drying time is 36-38 hours, and the moisture content of the freeze-dried powder after freeze-drying is controlled to be below 3%.

[0027] This application also provides a highly active, allergen-free, low-sodium Pediococcus pentosaceus SUN02 lyophilized powder, which is prepared by the method described above.

[0028] Compared with the prior art, this application has the following advantages and effects: The proposed method can effectively increase the viable count of Pediococcus pentosaceus SUN02 fermentation broth and freeze-dried powder, and the entire process is free from the introduction of animal or plant-derived allergens. It focuses on using specific amino acids to optimize and replace the allergen components contained in the culture medium. The resulting freeze-dried bacterial powder is free of allergens, has low sodium content, and has low production cost and simple operation. Attached Figure Description

[0029] To make the purpose, technical solution, and advantages of the verification experiments in this application clearer, the technical solutions in the verification experiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described verification experiments are only a part of the verification experiments in this application, not all of them. Based on the verification experiments in this application, all other verification experiments obtained by those skilled in the art without inventive effort are within the scope of protection of this application.

[0030] Figure 1 The viable cell counts of *Pediococcus pentosaceus* SUN02 in the fermentation broth under different fermentation culture conditions in Comparative Examples 1-4 and Example 2 of this application are shown.

[0031] Figure 2 The number of viable cells in the freeze-dried powder of Pediococcus pentosaceus SUN02 under different fermentation culture conditions in Comparative Examples 1-3, Comparative Example 5, and Example 2 of this application is the number of viable cells in the freeze-dried powder of Pediococcus pentosaceus SUN02.

[0032] Figure 3 This is a growth curve of Pediococcus pentosaceus SUN02 in Example 2 of this application.

[0033] Figure 4 This is a growth curve of Pediococcus pentosaceus SUN02 in Comparative Example 1 of this application.

[0034] Figure 5 This is a growth curve of Pediococcus pentosaceus SUN02 in Comparative Example 2 of this application.

[0035] Figure 6 This is a growth curve of Pediococcus pentosaceus SUN02 in Comparative Example 3 of this application.

[0036] Figure 7 This is a growth curve of Pediococcus pentosaceus SUN02 in Comparative Example 4 of this application. Detailed Implementation

[0037] To make the purpose, technical solution, and advantages of the verification experiments in this application clearer, the technical solutions in the verification experiments of this application are described clearly and completely below. Obviously, the verification experiments described are only a part of the verification experiments in this application, not all of them. The technical features designed in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other. Based on the verification experiments in this application, all other verification experiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0038] In the description of this application, it should be noted that all terms used in this application (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and should not be construed as limiting this application; it should be further understood that the terms used in this application should be understood to have the same meaning as those in the context of this specification and the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this application.

[0039] The present application will be described in further detail below with reference to the embodiments, but the implementation of the present application is not limited thereto.

[0040] The Pediococcus pentosaceus strain SUN02 used in the following examples was deposited on January 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29705.

[0041] The bacterial strain is a publicly available strain, preserved and viable in a collection center, and can be obtained by technicians from the collection center based on the accession number. Therefore, this application does not involve the preservation of the bacterial strain and does not require proof of its viability.

[0042] The detection methods for viable lactic acid bacteria mentioned below shall be performed in accordance with the national standards GB4789.35-2023 Food Safety Standard for Microbiological Examination of Food - Lactic Acid Bacteria Examination and GB4789.2-2022 Food Safety Standard for Microbiological Examination of Food - Determination of Total Colony Count.

[0043] Example 1: Shake-flask experiment on amino acid compatibility of Pediococcus pentosaceus SUN02 Example 1 was used to verify the adaptability of Pediococcus pentosaceus SUN02 to different amino acids during its growth. The process is as follows: (1) Culture medium preparation Preparation of MRS liquid culture medium: Weigh the following components according to the specified ratio to prepare the culture medium: glucose 20.0 g / L, Angel yeast extract FM503 15 g / L, Angel yeast peptone FP103 10 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Prepare 200 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a primary seed culture medium.

[0044] Preparation of MRS liquid culture medium: Weigh the following components according to the specified ratio to prepare the culture medium: 20.0 g / L glucose, 15 g / L Angel yeast extract FM503, 10 g / L Angel yeast peptone FP103, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 g / L Tween-80. Prepare 400 ml of the medium and dilute to volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 minutes. This culture medium is a shake flask culture medium.

[0045] (2) Activation of strains and preparation of seed culture The glycerol tubes of Pediococcus pentosacchari SUN02 stored at low temperature were placed at room temperature to thaw. The thawed Pediococcus pentosacchari SUN02 was inoculated into 200 ml of primary seed culture medium at an inoculation rate of 0.028%. The culture was then incubated at 37°C under anaerobic conditions for 20 h to obtain the primary seed culture. The primary seed culture was then inoculated into 400 ml shake flask culture medium at an inoculation rate of 2%, and different amino acids were added according to the amino acid types in Table 1. The mixture was then statically cultured at 37°C under anaerobic conditions for 12 h to obtain the shake flask fermentation broth, which was then subjected to OD analysis. 600 And live bacteria count detection.

[0046] Table 1 shows the test data from the shake flask experiment: Table 1. Shake-flask experiment on amino acid compatibility of Pediococcus pentosaceus SUN02

[0047] Example 2: High-density fermentation stage of Pediococcus pentosaceus SUN02 (1) Preparation of MRS liquid culture medium: Weigh the following components according to the specified ratio to prepare the culture medium: glucose 20.0 g / L, Angel yeast extract FM503 15 g / L, Angel yeast peptone FP103 10 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Prepare 200 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a primary seed culture medium. Weigh the following components according to the specified ratio to prepare the culture medium: 15.0 g / L glucose, 8 g / L Angel yeast extract FM503, 5 g / L Angel yeast peptone FP103, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 g / L Tween-80. Prepare 400 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a secondary seed culture medium. (2) Activation of strains and preparation of seed culture: The glycerol tubes of Pediococcus pentosacchari SUN02 stored at low temperature were placed at room temperature to thaw. The thawed Pediococcus pentosacchari SUN02 was inoculated into 200 ml of primary seed culture medium at an inoculation rate of 0.028%. The culture was then incubated at 37°C under anaerobic conditions for 20 h to obtain the primary seed culture. The primary seed culture was then inoculated into 400 ml of secondary seed culture medium at an inoculation rate of 2%, and cultured statically at 37°C under anaerobic conditions for 8 hours to obtain the secondary seed culture.

[0048] (3) High-density culture: Fermentation medium preparation: Weigh the following components according to the specified ratio to prepare the culture medium: glucose 40 g / L, Angel yeast extract FM985 20 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, glycine 1 g / L, threonine 1 g / L, L-cysteine ​​hydrochloride 1 g / L, tryptophan 0.5 g / L, serine 0.5 g / L, glutamic acid 0.1 g / L, magnesium sulfate 0.1 g / L, arginine 0.05 g / L, manganese sulfate 0.05 g / L, lysine 0.01 g / L, Tween-80 1 g / L. Prepare 8 L of the medium and transfer it to a 10 L fermenter for sterilization at 105 °C for 30 min. After sterilization, adjust the pH to 5.9-6.1 with 10.0 mol / L ammonia solution.

[0049] After the culture medium temperature cools to 37℃, inoculate the Pediococcus pentosacchari SUN02 seed culture into the fermenter at a ratio of 5%. Control the fermentation conditions as follows: temperature 36-38℃, fermentation pressure 0.03-0.05MPa, and rotation speed 100rpm. During the process, use a pH electrode meter to detect the pH and control the pH to 5.9-6.1 by adding 10.0mol / L ammonia water. Ferment for 12-15 hours until the alkali addition stops increasing, which is the fermentation endpoint. Immediately start cooling to 4℃ to obtain the Pediococcus pentosacchari SUN02 fermentation broth. The growth curve of Pediococcus pentosaceus SUN02 during fermentation culture in Example 2 is shown below. Figure 3 As shown in the figure. The OD value in the figure refers to the OD of the fermentation broth at this point. 600 The measured value, or alkali replenishment amount, refers to the amount of ammonia water with a concentration of 10.0 mol / L that is added, in ml.

[0050] (4) Collection of fungal sludge: The fermentation broth of Pediococcus pentosaceus SUN02 was centrifuged at 4℃ and 6000rpm for 20min, and the bacterial sludge was collected. (5) Vacuum freeze drying: The bacterial sludge and the protective agent were mixed in a 1:1 mass ratio. After mixing, the mixed bacterial solution was pre-frozen at -40℃ for 14 hours. After pre-freezing at -40℃ for 14 hours, vacuum freeze-drying was carried out. The vacuum freeze-drying parameters were: temperature -50 to -70℃, vacuum degree 10Pa, drying time 36 hours. The moisture content of the freeze-dried bacterial powder was controlled below 3%.

[0051] The protective agent formulation used was 15.0 g / L trehalose, 5.0 g / L maltodextrin, 2.0 g / L glycine, 2.0 g / L threonine, and 1.0 g / L sodium ascorbate. The sterilization conditions were 105℃ for 15 min.

[0052] The fermentation broth and lyophilized powder of *Pediococcus pentosaceus* SUN02 prepared by the above method were tested, and the viable count in the fermentation broth reached 1.0 x 10⁻⁶. 10 The CFU / ml lyophilized powder can contain up to 1.1 x 10⁻⁶ live bacteria. 12 CFU / g.

[0053] Thanks to the osmotic adaptability of Pediococcus pentosaceus SUN02 as a marine probiotic, removing sodium acetate from the initial culture medium had no significant effect on the viable count of the fermentation broth and freeze-dried powder.

[0054] Example 3: High-density fermentation stage of Pediococcus pentosaceus SUN02 (1) Preparation of MRS liquid culture medium: Weigh the following components according to the specified ratio to prepare the culture medium: glucose 20.2 g / L, Angel yeast extract FM503 15.3 g / L, Angel yeast peptone FP103 10.1 g / L, sodium acetate 5.1 g / L, dipotassium hydrogen phosphate 2.1 g / L, ammonium citrate 1.9 g / L, magnesium sulfate 0.11 g / L, manganese sulfate 0.048 g / L, Tween-80 1 g / L. Prepare 200 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 122℃ for 21 min. This culture medium is a primary seed culture medium. Weigh the following components according to the specified ratio to prepare the culture medium: glucose 15.2 g / L, Angel yeast extract FM503 8.1 g / L, Angel yeast peptone FP103 5.1 g / L, sodium acetate 5.1 g / L, dipotassium hydrogen phosphate 2.1 g / L, ammonium citrate 2.1 g / L, magnesium sulfate 0.11 g / L, manganese sulfate 0.052 g / L, Tween-80 1.02 g / L. Prepare 400 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 22 min. This culture medium is a secondary seed culture medium. (2) Activation of strains and preparation of seed culture: The glycerol tubes of Pediococcus pentosacchari SUN02 stored at low temperature were thawed at room temperature. The thawed Pediococcus pentosacchari SUN02 was inoculated into 200 ml of primary seed culture medium at an inoculation rate of 0.028%. The culture was then incubated at 37.1 ℃ under anaerobic conditions for 14 h to obtain the primary seed culture. The primary seed culture was then inoculated into 400 ml of secondary seed culture medium at an inoculation rate of 2%, and cultured statically at 36.9 ℃ under anaerobic conditions for 7.5 h to obtain the secondary seed culture.

[0055] (3) High-density culture: Fermentation medium preparation: Weigh the following components according to the specified ratio to prepare the culture medium: glucose 40.2 g / L, Angel yeast extract FM985 20.1 g / L, dipotassium hydrogen phosphate 1.98 g / L, ammonium citrate 2.07 g / L, glycine 1.02 g / L, threonine 1.01 g / L, L-cysteine ​​hydrochloride 0.99 g / L, tryptophan 0.47 g / L, serine 0.51 g / L, glutamic acid 0.10 g / L, magnesium sulfate 0.12 g / L, arginine 0.04 g / L, manganese sulfate 0.049 g / L, lysine 0.011 g / L, Tween-80 1.02 g / L. Prepare 8 L of the medium and transfer it to a 10 L fermenter for sterilization at 106 °C for 31 min. After sterilization, adjust the pH to 5.9-6.1 with 10.0 mol / L ammonia solution.

[0056] Once the culture medium temperature has cooled to 37.1℃, inoculate the Pediococcus pentosacchari SUN02 seed culture into the fermenter at a ratio of 5%. Control the fermentation conditions as follows: temperature 36-38℃, fermentation pressure 0.03-0.05MPa, and rotation speed 100rpm. During the process, use a pH electrode meter to detect the pH and control the pH to 5.9-6.1 by adding 10.0mol / L ammonia water. Ferment for 12-15 hours until the alkali addition stops increasing, which is the fermentation endpoint. Immediately begin cooling to 4℃ to obtain the Pediococcus pentosacchari SUN02 fermentation broth. (4) Collection of fungal sludge: The fermentation broth of Pediococcus pentosaceus SUN02 was centrifuged at 4℃ and 6000rpm for 20min, and the bacterial sludge was collected. (5) Vacuum freeze drying: The bacterial sludge and the protective agent were mixed at a mass ratio of 1:1.02. After mixing, the mixed bacterial solution was pre-frozen at -41℃ for 14.5 hours. After pre-freezing at -41℃ for 14.5 hours, vacuum freeze-drying was carried out. The vacuum freeze-drying parameters were: temperature -50 to -70℃, vacuum degree 10Pa, drying time 37 hours. The moisture content of the freeze-dried bacterial powder was controlled below 3%.

[0057] The protective agent formulation used was 15.2 g / L trehalose, 5.2 g / L maltodextrin, 2.01 g / L glycine, 2.02 g / L threonine, and 1.02 g / L sodium ascorbate. The sterilization conditions were 106℃ for 16 min.

[0058] The fermentation broth and lyophilized powder of *Pediococcus pentosaceus* SUN02 prepared by the above method were tested, and the viable cell count in the fermentation broth reached 1.0 × 10¹. 0 The live bacteria count in the freeze-dried powder can reach 1.2 × 10¹² CFU / g.

[0059] Thanks to the osmotic adaptability of Pediococcus pentosaceus SUN02 as a marine probiotic, removing sodium acetate from the initial culture medium had no significant effect on the viable count of the fermentation broth and freeze-dried powder.

[0060] Comparative Example 1: High-density fermentation of Pediococcus pentosaceus SUN02 (1) Preparation of MRS liquid culture medium: Weigh the following components according to the specified ratio to prepare the culture medium: glucose 20.0 g / L, Angel yeast extract FM503 15 g / L, Angel yeast peptone FP103 10 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Prepare 200 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a primary seed culture medium.

[0061] Weigh the following components according to the specified ratio to prepare the culture medium: glucose 15.0 g / L, Angel yeast extract FM503 8 g / L, Angel yeast peptone FP103 5 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Prepare 400 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a secondary seed culture medium.

[0062] (2) Activation of strains and preparation of seed culture: The glycerol tubes of Pediococcus pentosacchari SUN02 stored at low temperature were placed at room temperature to thaw. The thawed Pediococcus pentosacchari SUN02 was inoculated into 200 ml of primary seed culture medium at an inoculation rate of 0.028%. The culture was then incubated at 37°C under anaerobic conditions for 20 h to obtain the primary seed culture. The primary seed culture was then inoculated into 400 ml of secondary seed culture medium at an inoculation rate of 2%, and cultured statically at 37°C under anaerobic conditions for 8 hours to obtain the secondary seed culture. (3) High-density culture: Weigh the following components according to the specified ratio to prepare the culture medium: 40 g / L glucose, 20 g / L Angel yeast extract FM985, 15 g / L beef extract, 5 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 g / L Tween-80. Prepare 8 L of the medium and transfer it to a 10 L fermenter for sterilization at 105 °C for 30 min. After sterilization, adjust the pH to 5.9-6.1 with 20% sodium hydroxide solution.

[0063] After the temperature cools to 37℃, inoculate 400ml of secondary seed culture at a 5% inoculation ratio. Control the fermentation conditions as follows: temperature 36-38℃, fermentation pressure 0.03-0.05MPa, and rotation speed 100rpm. During the process, use a pH electrode meter to detect the pH and control the pH to 5.9-6.1 by adding 20% ​​sodium hydroxide. Ferment for 12-15 hours. When the amount of alkali added stops increasing, it is the end point of fermentation. Immediately start cooling to 4℃ to obtain the Pediococcus pentosaceus SUN02 fermentation broth. Among them, the growth curve of Pediococcus pentosaceus SUN02 in Comparative Example 1 during fermentation culture is as follows: Figure 4 As shown in the figure. The OD value in the figure refers to the OD of the fermentation broth at this point. 600 The measured value, or alkali replenishment amount, refers to the amount of 20% sodium hydroxide solution added, measured in ml.

[0064] (4) Collection of fungal sludge: The fermentation broth of Pediococcus pentosaceus SUN02 was centrifuged at 4℃ and 6000rpm for 20min, and the bacterial sludge was collected. (5) Vacuum freeze drying: The bacterial sludge and the protective agent were mixed in a 1:1 mass ratio. After mixing, the mixed bacterial solution was pre-frozen at -40℃ for 14 hours. After pre-freezing at -40℃ for 14 hours, vacuum freeze-drying was carried out. The vacuum freeze-drying parameters were: temperature -50 to -70℃, vacuum degree 10Pa, drying time 36 hours. The moisture content of the freeze-dried bacterial powder was controlled below 3%.

[0065] The protective agent formulation used was 15.0 g / L trehalose, 5.0 g / L maltodextrin, 2.0 g / L glycine, 2.0 g / L threonine, and 1.0 g / L sodium ascorbate. The sterilization conditions were 105℃ for 15 min.

[0066] The fermentation broth and lyophilized powder of *Pediococcus pentosaceus* SUN02 prepared by the above method were tested, and the viable cell count in the fermentation broth reached 7.6 x 10⁻⁶. 9 The CFU / ml lyophilized powder can reach a viable bacteria count of 8.9 x 10⁻⁶. 11 CFU / g.

[0067] Comparative Example 2: High-density fermentation of Pediococcus pentosaceus SUN02 with a small amount of amino acids (1) Preparation of MRS liquid culture medium: Weigh the following components according to the specified ratio to prepare the culture medium: glucose 20.0 g / L, Angel yeast extract FM503 15 g / L, Angel yeast peptone FP103 10 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Prepare 200 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a primary seed culture medium.

[0068] Weigh the following components according to the specified ratio to prepare the culture medium: glucose 15.0 g / L, Angel yeast extract FM503 8 g / L, Angel yeast peptone FP103 5 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Prepare 400 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a secondary seed culture medium.

[0069] (2) Activation of strains and preparation of seed culture: The glycerol tubes of Pediococcus pentosacchari SUN02 stored at low temperature were placed at room temperature to thaw. The thawed Pediococcus pentosacchari SUN02 was inoculated into 200 ml of primary seed culture medium at an inoculation rate of 0.028%. The culture was then incubated at 37°C under anaerobic conditions for 20 h to obtain the primary seed culture. The primary seed culture was then inoculated into 400 ml of secondary seed culture medium at an inoculation rate of 2%, and cultured statically at 37°C under anaerobic conditions for 8 hours to obtain the secondary seed culture.

[0070] (3) High-density culture: Weigh the following components according to the specified ratio to prepare the culture medium: 40 g / L glucose, 20 g / L Angel yeast extract FM985, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 0.5 g / L glycine, 0.5 g / L threonine, 1 g / L L-cysteine ​​hydrochloride, 0.25 g / L tryptophan, 0.25 g / L serine, 0.05 g / L glutamic acid, 0.1 g / L magnesium sulfate, 0.025 g / L arginine, 0.05 g / L manganese sulfate, 0.005 g / L lysine, and 1 g / L Tween-80. Prepare 8 L of the medium and transfer it to a 10 L fermenter for sterilization at 105 °C for 30 min. After sterilization, adjust the pH to 5.9-6.1 with 10.0 mol / L ammonia solution.

[0071] After the temperature cools to 37℃, inoculate 400ml of secondary seed culture at a 5% inoculation ratio. Control the fermentation conditions as follows: temperature 36-38℃, fermentation pressure 0.03-0.05MPa, and rotation speed 100rpm. During the process, use a pH electrode meter to detect the pH and control the pH to 5.9-6.1 by adding 10.0mol / L ammonia water. Ferment for 12-15h, and when the amount of alkali added stops increasing, it is the end point of fermentation. Immediately start cooling to 4℃ to obtain the Pediococcus pentosaceus SUN02 fermentation broth. Among them, the growth curve of Pediococcus pentosaceus SUN02 in Comparative Example 2 during fermentation culture is as follows: Figure 5 As shown in the figure. The OD value in the figure refers to the OD of the fermentation broth at this point. 600 The measured value, or alkali replenishment amount, refers to the amount of ammonia water with a concentration of 10.0 mol / L that is added, in ml.

[0072] (4) Collection of fungal sludge: The fermentation broth of Pediococcus pentosaceus SUN02 was centrifuged at 4℃ and 6000rpm for 20min, and the bacterial sludge was collected. (5) Vacuum freeze drying: The bacterial sludge and the preservative were mixed at a 1:1 mass ratio. After thorough mixing, the mixed bacterial solution was pre-frozen at -40℃ for 14 hours. Following this, it underwent vacuum freeze-drying. The vacuum freeze-drying parameters were: temperature -50 to -70℃, vacuum degree 10 Pa, and drying time 36 hours. The moisture content of the freeze-dried bacterial powder was controlled below 3%. The preservative formulation used was 15.0 g / L trehalose, 5.0 g / L maltodextrin, 2.0 g / L glycine, 2.0 g / L threonine, and 1.0 g / L sodium ascorbate. The sterilization conditions were 105℃ for 15 minutes.

[0073] The fermentation broth and lyophilized powder of *Pediococcus pentosaceus* SUN02 prepared by the above method were tested, and the viable cell count in the fermentation broth reached 5.2 x 10⁻⁶. 9 The CFU / ml lyophilized powder can contain up to 5.1 x 10⁻⁶ live bacteria. 11 CFU / g.

[0074] Comparative Example 3: High-density fermentation of Pediococcus pentosaceus SUN02 with sufficient amino acids (1) Preparation of MRS liquid culture medium: Weigh the following components according to the specified ratio to prepare the culture medium: glucose 20.0 g / L, Angel yeast extract FM503 15 g / L, Angel yeast peptone FP103 10 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Prepare 200 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a primary seed culture medium.

[0075] Weigh the following components according to the specified ratio to prepare the culture medium: glucose 15.0 g / L, Angel yeast extract FM503 8 g / L, Angel yeast peptone FP103 5 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Prepare 400 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a secondary seed culture medium.

[0076] (2) Activation of strains and preparation of seed culture: The glycerol tubes of Pediococcus pentosacchari SUN02 stored at low temperature were placed at room temperature to thaw. The thawed Pediococcus pentosacchari SUN02 was inoculated into 200 ml of primary seed culture medium at an inoculation rate of 0.028%. The culture was then incubated at 37°C under anaerobic conditions for 20 h to obtain the primary seed culture. The primary seed culture was then inoculated into 400 ml of secondary seed culture medium at an inoculation rate of 2%, and cultured statically at 37°C under anaerobic conditions for 8 hours to obtain the secondary seed culture.

[0077] (3) High-density culture: Weigh the following components according to the specified ratio to prepare the culture medium: 40 g / L glucose, 20 g / L Angel yeast extract FM985, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 2 g / L glycine, 2 g / L threonine, 2 g / L L-cysteine ​​hydrochloride, 1 g / L tryptophan, 1 g / L serine, 0.2 g / L glutamic acid, 0.1 g / L magnesium sulfate, 0.1 g / L arginine, 0.05 g / L manganese sulfate, 0.02 g / L lysine, and 1 g / L Tween-80. Prepare 8 L of the medium and transfer it to a 10 L fermenter for sterilization at 105 °C for 30 min. After sterilization, adjust the pH to 5.9-6.1 with 10.0 mol / L ammonia solution.

[0078] After the temperature cools to 37℃, inoculate 400ml of secondary seed culture at a 5% inoculation ratio. Control the fermentation conditions as follows: temperature 36-38℃, fermentation pressure 0.03-0.05MPa, and rotation speed 100rpm. During the process, use a pH electrode meter to detect the pH and control the pH to 5.9-6.1 by adding 10.0mol / L ammonia water. Ferment for 12-15h, and when the amount of alkali added stops increasing, it is the end point of fermentation. Immediately start cooling to 4℃ to obtain the Pediococcus pentosaceus SUN02 fermentation broth. Among them, the growth curve of Pediococcus pentosaceus SUN02 in Comparative Example 3 during fermentation culture is as follows: Figure 6 As shown in the figure. The OD value in the figure refers to the OD of the fermentation broth at this point. 600 The measured value, or alkali replenishment amount, refers to the amount of ammonia water with a concentration of 10.0 mol / L that is added, in ml.

[0079] (4) Collection of fungal sludge: The fermentation broth of Pediococcus pentosaceus SUN02 was centrifuged at 4℃ and 6000rpm for 20min, and the bacterial sludge was collected. (5) Vacuum freeze drying: The bacterial sludge and the preservative were mixed at a 1:1 mass ratio. After thorough mixing, the mixed bacterial solution was pre-frozen at -40℃ for 14 hours. Following this, it underwent vacuum freeze-drying. The vacuum freeze-drying parameters were: temperature -50 to -70℃, vacuum degree 10 Pa, and drying time 36 hours. The moisture content of the freeze-dried bacterial powder was controlled below 3%. The preservative formulation used was 15.0 g / L trehalose, 5.0 g / L maltodextrin, 2.0 g / L glycine, 2.0 g / L threonine, and 1.0 g / L sodium ascorbate. The sterilization conditions were 105℃ for 15 minutes.

[0080] The fermentation broth and lyophilized powder of *Pediococcus pentosaceus* SUN02 prepared by the above method were tested, and the viable cell count in the fermentation broth reached 8.9 x 10⁻⁶. 9 The CFU / ml lyophilized powder can reach a viable bacterial count of 9.8 x 10⁻⁶. 11 CFU / g.

[0081] Comparative Example 4: High-density fermentation of Pediococcus pentosaceus SUN02 with appropriate amounts of amino acids and tyrosine. (1) Preparation of MRS liquid culture medium: Weigh the following components according to the specified ratio to prepare the culture medium: glucose 20.0 g / L, Angel yeast extract FM503 15 g / L, Angel yeast peptone FP103 10 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Prepare 200 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a primary seed culture medium.

[0082] Weigh the following components according to the specified ratio to prepare the culture medium: glucose 15.0 g / L, Angel yeast extract FM503 8 g / L, Angel yeast peptone FP103 5 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Prepare 400 ml of the medium and bring it to a final volume with sterile water. After preparation, adjust the pH to 6.4-6.6 with a 5.0 mol / L sodium hydroxide solution. Sterilize at 121℃ for 20 min. This culture medium is a secondary seed culture medium.

[0083] (2) Activation of strains and preparation of seed culture: The glycerol tubes of Pediococcus pentosacchari SUN02 stored at low temperature were placed at room temperature to thaw. The thawed Pediococcus pentosacchari SUN02 was inoculated into 200 ml of primary seed culture medium at an inoculation rate of 0.028%. The culture was then incubated at 37°C under anaerobic conditions for 20 h to obtain the primary seed culture. The primary seed culture was then inoculated into 400 ml of secondary seed culture medium at an inoculation rate of 2%, and cultured statically at 37°C under anaerobic conditions for 8 hours to obtain the secondary seed culture.

[0084] (3) High-density culture: Weigh the following components according to the specified ratio to prepare the culture medium: 40 g / L glucose, 20 g / L Angel yeast extract FM985, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 1 g / L tyrosine, 1 g / L threonine, 1 g / L L-cysteine ​​hydrochloride, 0.5 g / L tryptophan, 0.5 g / L serine, 0.1 g / L glutamic acid, 0.1 g / L magnesium sulfate, 0.05 g / L arginine, 0.05 g / L manganese sulfate, 0.01 g / L lysine, and 1 g / L Tween-80. Prepare 8 L of the medium and transfer it to a 10 L fermenter for sterilization at 105 °C for 30 min. After sterilization, adjust the pH to 5.9-6.1 with 10.0 mol / L ammonia solution.

[0085] After the temperature cools to 37℃, inoculate 400ml of secondary seed culture at a 5% inoculation ratio. Control the fermentation conditions as follows: temperature 36-38℃, fermentation pressure 0.03-0.05MPa, and rotation speed 100rpm. During the process, use a pH electrode meter to detect the pH and control the pH to 5.9-6.1 by adding 10.0mol / L ammonia water. Ferment for 12-15h, and when the amount of alkali added stops increasing, it is the end point of fermentation. Immediately start cooling to 4℃ to obtain the Pediococcus pentosaceus SUN02 fermentation broth. Among them, the growth curve of Pediococcus pentosaceus SUN02 in Comparative Example 4 during fermentation culture is as follows: Figure 7 As shown in the figure. The OD value refers to the OD600 value of the fermentation broth at this time, and the amount of alkali added refers to the amount of 10.0 mol / L ammonia water added, in ml.

[0086] (4) Collection of fungal sludge: The fermentation broth of Pediococcus pentosaceus SUN02 was centrifuged at 4℃ and 6000rpm for 20min, and the bacterial sludge was collected. (5) Vacuum freeze drying: The bacterial sludge and the protective agent were mixed in a 1:1 mass ratio. After mixing, the mixed bacterial solution was pre-frozen at -40℃ for 14 hours. After pre-freezing at -40℃ for 14 hours, vacuum freeze-drying was carried out. The vacuum freeze-drying parameters were: temperature -50 to -70℃, vacuum degree 10Pa, drying time 36 hours. The moisture content of the freeze-dried bacterial powder was controlled below 3%.

[0087] The protective agent formulation used was 15.0 g / L trehalose, 5.0 g / L maltodextrin, 2.0 g / L glycine, 2.0 g / L threonine, and 1.0 g / L sodium ascorbate. The sterilization conditions were 105℃ for 15 min.

[0088] The fermentation broth and lyophilized powder of *Pediococcus pentosaceus* SUN02 prepared by the above method were tested, and the viable cell count in the fermentation broth reached 9.6 x 10⁻⁶. 9 The viable bacteria count in the CFU / ml lyophilized powder can reach 1.0 x 10⁻⁶. 12 CFU / g.

[0089] Comparative Example 5: Pediococcus pentosaceus SUN02 Protectant Control Comparative Example 5 serves as a control to verify the protective effect of the protective agent. In Example 2, after collecting the bacterial mud in step (4), a portion of the bacterial mud was taken and the process of Example 2 was carried out. The bacterial mud and the protective agent were mixed in a mass ratio of 1:1. After mixing, the mixed bacterial solution was pre-frozen in a -40℃ refrigerator for 14 hours. After pre-freezing in a -40℃ refrigerator for 14 hours, vacuum freeze-drying was carried out. The vacuum freeze-drying parameters were: temperature -50 to -70℃, vacuum degree 10Pa, drying time 36 hours. The moisture content of the freeze-dried bacterial powder was controlled to be below 3%.

[0090] The protective agent formula used is 20.0 g / L skim milk powder, 10.0 g / L trehalose, and 1.0 g / L sodium ascorbate. The sterilization conditions are 105℃ for 15 min.

[0091] The test results after freeze-drying showed that the viable bacteria count in the freeze-dried powder was 1.1 x 10⁻⁶. 12 The CFU / g result demonstrates that the protective effect of the allergen-free protectant in this application is not significantly different from that of the protectant containing the allergen protectant during the freeze-drying process. The allergen-free protectant in this application effectively reduces the allergen content while maintaining good protective efficacy.

[0092] The above embodiments and comparative test data are summarized as follows: Table 2

[0093] In summary, based on the above analysis, we can conclude that: Based on the development objective of preparing allergen-free, low-sodium, high-viable-count, and highly stable marine probiotic Pediococcus pentosaccharide SUN02 freeze-dried powder, this application proposes a process for preparing allergen-free, low-sodium, high-viable-count, and highly stable marine probiotic Pediococcus pentosaccharide SUN02 freeze-dried powder by replacing allergen components with amino acids, replacing sodium hydroxide with ammonia, removing sodium salts from the initial culture medium, and using an allergen-free protective agent formulation.

[0094] The freeze-dried Pediococcus pentosaceus SUN02 prepared by this method has significant advantages over traditional freeze-dried bacterial powder preparation methods, including the absence of animal or plant-derived allergens and low sodium content. Furthermore, it exhibits a high viable count, reaching up to 1.1 x 10⁻⁶ viable bacteria in the freeze-dried powder. 12 Up to 1.2x10 12 CFU / g has broad and promising application prospects.

[0095] In summary, the preparation method provided in this application has at least the following design concept and technical effects: 1. Conventional fermentation media for lactic acid bacteria often contain animal-derived proteins such as beef extract, beef bone extract, and fish peptone, or plant-derived proteins such as soybean peptone and pea peptone. These protein products can all cause allergic reactions. Furthermore, the animal or plant-derived proteins in the fermentation medium containing these products may remain unused during fermentation and end up in the lyophilized powder.

[0096] For the preparation of Pediococcus pentosaceus SUN02 (a type of lactic acid bacteria), the conventional fermentation culture medium for lactic acid bacteria mentioned above is commonly used in the market. However, it's important to note that while the animal and plant-derived protein components added to these conventional lactic acid bacteria fermentation media provide a good nitrogen source and promote bacterial growth, the residual proteins or glycoproteins may become allergens, posing a health risk to sensitive individuals. Completely removing these protein components to avoid allergens often leads to restricted bacterial growth, resulting in a significant decrease in the number of viable bacteria. Furthermore, commonly used freeze-drying protectants (such as skim milk powder) may also introduce additional allergens.

[0097] This application relates to the preparation of Pediococcus pentosaceus SUN02 (a type of lactic acid bacteria) formulations. It employs a method of removing all animal or plant-derived protein components from the culture medium while supplementing it with a specific amino acid combination. This ensures the absence of animal or plant-derived allergens and maintains the integrity of the amino acid composition and total amount in the culture medium after the removal of animal or plant-derived proteins. This ensures that the lactic acid bacteria can grow normally during fermentation culture while preparing a freeze-dried powder free of animal or plant-derived allergens. The prepared freeze-dried bacterial powder still maintains high viable cell counts and high stability.

[0098] In addition, the selection of freeze-drying protectants removes commonly used allergens such as skim milk powder, eliminating the possibility of introducing allergens during the freeze-drying process, ensuring that the final freeze-dried powder product is free of allergens throughout the entire process, and ultimately producing an allergen-free freeze-dried powder product.

[0099] In summary, this application uses a specific amino acid combination to supplement the culture medium components, and the entire process is free of animal or plant-derived allergens. While increasing the viable cell count in the fermentation broth and freeze-dried powder, it avoids the introduction of animal or plant-derived allergens, effectively preventing allergic reactions caused by residual animal or plant-derived allergens in the freeze-dried powder. At the same time, it removes allergens from the freeze-drying protectant, ensuring that no allergens are added throughout the entire process.

[0100] 2. Traditional lactic acid bacteria fermentation processes often employ alkali supplementation to control the pH of the culture medium, thereby increasing the bacterial density. This alkali control process typically uses sodium hydroxide as the alkali solution. Furthermore, a certain amount of sodium salt is added to the initial components of the culture medium to maintain its initial osmotic pressure, leading to an increased sodium content in the final product. Excessive sodium intake is associated with health risks such as hypertension, and the market demand for low-sodium health products is growing. However, existing fermentation processes struggle to effectively reduce the sodium content of the product without affecting bacterial growth and the number of viable bacteria.

[0101] This application uses ammonia water instead of sodium hydroxide to control the pH of the culture medium during fermentation, which can effectively reduce the introduction of sodium salt. At the same time, as a marine probiotic, Pediococcus pentosaceus SUN02 has better adaptability to different osmotic pressures and growth characteristics, so it can remove the initial sodium salt in the culture medium and effectively reduce the introduction of sodium salt. Through these two methods, a low-sodium freeze-dried bacterial powder with significantly lower sodium content can be prepared.

[0102] In summary, this application uses ammonia water to replenish alkali and removes the initial sodium salt components in the culture medium, effectively reducing the sodium content of the freeze-dried powder. This provides a freeze-dried powder with low sodium intake for daily consumption by the general population and those who need to reduce sodium intake.

[0103] 3. By screening and optimizing the composition and dosage of the culture medium, the viable cell count in both the fermentation broth and the freeze-dried powder was increased, raising the viable cell count in the freeze-dried powder to 1.1 x 10⁻⁶. 12 Up to 1.2x10 12 The CFU / g level provides a certain experimental basis for the development and utilization of functional foods based on Pediococcus pentosaceus SUN02.

[0104] It should be noted that: (1) Definition: In this paper, “numerical-numerical” is used to represent the range of numerical values. The range of values ​​represented by this expression includes two endpoint values.

[0105] (2) The raw materials used in the implementation (nitrogen source, carbon source, bacteria, peptides, etc.) are all existing commercially available products that can be purchased and obtained by those skilled in the art.

[0106] In summary, the specific parameters or some commonly used reagents or raw materials in the above verification experiments are specific verification experiments or preferred verification experiments under the concept of this application, and are not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of this application.

[0107] Furthermore, unless otherwise specified, the raw materials used may be commercially available products in the field or prepared by conventional methods in the field; that is, the reagents and instruments used in this verification experiment, without specifying the manufacturer, are all conventional products that can be purchased from the market. Finally, it should be noted that the above verification experiments are only used to illustrate the technical solutions of this application, and not to limit them; although this application has been described in detail with reference to the foregoing verification experiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing verification experiments, or make equivalent substitutions for some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the verification experiments of this application.

Claims

1. A highly active, allergen-free, low-sodium Pediococcus pentosaceus (Pediococcus pentosaceus) The method for preparing SUN02 lyophilized powder is characterized by, Includes the following steps: S1. Activation of microorganisms and preparation of seed culture; S2. Fermentation culture: Using a fermentation medium free of animal and plant proteins and supplemented with amino acids to supplement nitrogen source, Pediococcus pentosaccharides SUN02 was fermented to obtain fermentation broth. During the fermentation process, ammonia water is used as a pH adjuster throughout to control the pH value of the fermentation system to 5.9-6.

1. The fermentation medium comprises the following components: carbon source, yeast extract, phosphate, ammonium salt, magnesium sulfate, manganese sulfate, nonionic surfactant, and an amino acid composition consisting of glycine, threonine, L-cysteine ​​hydrochloride, tryptophan, serine, glutamic acid, arginine, and lysine; no sodium-containing pH buffering components are added to the fermentation medium, and ammonia is used as a pH adjuster to adjust the pH value to 5.9-6.1; S3. Collection of microbial sludge: The fermentation broth is separated into solid and liquid components, and the microbial sludge is collected. S4. Pre-freezing: The allergen-free preservative is mixed with the bacterial sludge, and the mixed bacterial solution is pre-frozen to obtain the pre-frozen product; S5. Vacuum freeze-drying: The pre-frozen product is vacuum freeze-dried to obtain the freeze-dried powder.

2. The preparation method according to claim 1, characterized in that, The preservation number of Pediococcus pentosaccharis SUN02 is CGMCC No. 29705.

3. The preparation method according to claim 1, characterized in that, The amount of the amino acid composition added to the fermentation medium is as follows: Glycine 0.95-1.05 g / L, threonine 0.95-1.05 g / L, L-cysteine ​​hydrochloride 0.95-1.05 g / L, tryptophan 0.45-0.55 g / L, serine 0.45-0.55 g / L, glutamic acid 0.08-0.12 g / L, arginine 0.04-0.06 g / L, lysine 0.009-0.011 g / L.

4. The preparation method according to claim 1, characterized in that, The preparation process of the fermentation medium is as follows: the raw material components of the fermentation medium are mixed and sterilized at 105-107℃ for 30-32 min, and then the pH is adjusted to 5.9-6.1 with ammonia water of 9.8-10.2 mol / L concentration. The fermentation medium comprises the following raw material components: glucose 39.5-40.5 g / L, Angel yeast extract FM985 19.5-20.5 g / L, dipotassium hydrogen phosphate 1.9-2.1 g / L, ammonium citrate 1.9-2.1 g / L, magnesium sulfate 0.095-0.15 g / L, manganese sulfate 0.048-0.052 g / L, and Tween-80. 0.95-1.05 g / L, glycine 0.95-1.05 g / L, threonine 0.95-1.05 g / L, L-cysteine ​​hydrochloride 0.95-1.05 g / L, tryptophan 0.45-0.55 g / L, serine 0.45-0.55 g / L, glutamic acid 0.08-0.12 g / L, arginine 0.04-0.06 g / L, lysine 0.009-0.011 g / L.

5. The preparation method according to claim 1, characterized in that, In step S2, Pediococcus pentosaceus SUN02 is inoculated into a fermentation medium, and the Pediococcus pentosaceus SUN02 seed culture is fermented to obtain a fermentation broth. The fermentation temperature is 36-38℃, the fermentation pressure is 0.03-0.05MPa, and the fermentation time is 12-15h.

6. The preparation method according to claim 1, characterized in that, The components of the allergen-free lyophilized protectant include trehalose, maltodextrin, glycine, threonine, and sodium ascorbate, and do not contain skim milk powder, whey protein, or other animal-derived protective ingredients.

7. The preparation method according to claim 6, characterized in that, The raw material components of the allergen-free lyophilization protectant are mixed and sterilized at 105-107℃ for 15-17 min to obtain the allergen-free lyophilization protectant. The components of the allergen-free lyophilized protectant include: 14.5-15.5 g / L trehalose, 4.5-5.5 g / L maltodextrin, 1.95-2.05 g / L glycine, 1.95-2.05 g / L threonine, and 0.95-1.05 g / L sodium ascorbate.

8. The preparation method according to claim 1, characterized in that, Pediococcus pentosaceus SUN02 was inoculated into a primary seed culture medium and cultured at 36.5-37.5℃ under anaerobic conditions for 19-21 hours for primary expansion. Then, the culture was inoculated into a secondary seed culture medium and cultured at 36.5-37.5℃ under anaerobic conditions for 7-8 hours for secondary expansion to obtain the seed culture. The primary seed culture medium comprises: glucose 19.5-20.5 g / L, Angel yeast extract FM503 14.5-15.5 g / L, Angel yeast peptone FP103 9.8-10.2 g / L, sodium acetate 4.9-5.1 g / L, dipotassium hydrogen phosphate 1.9-2.1 g / L, ammonium citrate 1.9-2.1 g / L, magnesium sulfate 0.09-0.11 g / L, manganese sulfate 0.048-0.052 g / L, and Tween-80 0.95-1.05 g / L; the pH is adjusted to 6.4-6.6 using a 4.8-5.0 mol / L sodium hydroxide solution, and sterilization is performed at 121-123℃ for 20-22 min. The secondary seed culture medium comprises: glucose 14.5-15.5 g / L, Angel yeast extract FM503 7.8-8.2 g / L, Angel yeast peptone FP103 4.8-5.2 g / L, sodium acetate 4.9-5.1 g / L, dipotassium hydrogen phosphate 1.9-2.1 g / L, ammonium citrate 1.9-2.1 g / L, magnesium sulfate 0.09-0.11 g / L, manganese sulfate 0.048-0.052 g / L, and Tween-80 0.95-1.05 g / L; the pH is adjusted to 6.4-6.6 using a 4.8-5.0 mol / L sodium hydroxide solution, and sterilization is performed at 121-123℃ for 20-22 min.

9. The preparation method according to claim 1, characterized in that: In step S4, the bacterial mud and the allergen-free protective agent are mixed at a mass ratio of (0.9-1):1 to obtain a mixed bacterial solution. After mixing, the mixed bacterial solution is pre-frozen at -40-41℃ for 14-15 hours. In step S5, the freezing temperature during the freeze-drying stage is -50°C to -70°C, the vacuum degree is 10-11 Pa, the drying time is 36-38 hours, and the moisture content of the freeze-dried powder after freeze-drying is controlled below 3%.

10. A highly active, allergen-free, low-sodium Pediococcus pentosaceus SUN02 lyophilized powder, characterized in that: It is prepared by the method described in any one of claims 1-9.

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