A probiotic preservation composition and its preparation method

Abstract

The present invention provides a probiotic fresh-keeping composition and a preparation method thereof, belonging to the technical field of probiotics. Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium acidipropionici FPHC0659 are inoculated into a culture medium for fermentation culture. The bacterial sludge, antibacterial peptides with a molecular weight of 2-10 kDa, and fermentation products are collected respectively. The antibacterial peptide-chitosan condensate is prepared by reacting the antibacterial peptides with chitosan, and the sustained-release microspheres are prepared by co-embedding the bacterial sludge with sodium alginate. The obtained product is mixed uniformly with the fermentation product, lysozyme, and vitamin C to obtain the probiotic fresh-keeping composition. The probiotic fresh-keeping composition prepared by the present invention has the advantages of strong antibacterial property, high safety, clean label, long fresh-keeping and anti-corrosion period, good biocompatibility, etc., and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and particularly relates to a probiotic preservation composition and a preparation method thereof. Background Art

[0002] The meaning of food anti-corrosion and preservation is to maintain the inherent color, aroma, taste, shape and nutritional components of food during storage. The chemicals used for this purpose are called anti-corrosion and preservation agents. The toxicity of natural anti-corrosion and preservation agents is far lower than that of synthetic ones. Therefore, people tend to choose natural food anti-corrosion and preservation agents.

[0003] Biological anti-corrosion and preservation agents are natural anti-corrosion and preservation agents that "control bacteria with bacteria". Generally, there are three mechanisms of action for biological preservation agents: 1. Microorganisms secrete extracellular polysaccharides and other film-forming substances to form a dense film on the outside of food, isolating oxygen and preventing water evaporation. 2. The preservation microorganisms can compete with pathogenic bacteria for nutrients such as sugars and living space in food, thereby inhibiting the growth of harmful microorganisms. 3. Microorganisms mainly inhibit or kill harmful microorganisms in food through antagonistic effects, so as to achieve the purpose of anti-corrosion and preservation. Compared with chemical anti-corrosion and preservation, biological preservation has the characteristics of being odorless, non-toxic and safe; compared with physical anti-corrosion and preservation, it has the characteristics of low technical requirements, low equipment requirements and low cost. Compared with other natural anti-corrosion and preservation agents prepared from Chinese herbal medicine extracts, biological anti-corrosion and preservation agents have the advantages of being able to effectively block oxygen and microorganisms, maintain moisture, protect color and antioxidant. At present, only nisin and natamycin are two kinds of biological anti-corrosion and preservation agents approved for use.

[0004] However, when using nisin as a biological anti-corrosion and preservation agent, if nisin is infected, it may cause inflammations such as erythema and papules, which may cause certain harm to the human body; when using natamycin as a biological anti-corrosion and preservation agent, due to the poor absorption of the human digestive tract and the low resistance of microorganisms to natamycin, long-term and large intake may increase some burdens on the digestive system. Therefore, it is urgent to find a biological anti-corrosion and preservation agent with good anti-corrosion and preservation effects and higher safety for the human body. Summary of the Invention

[0005] The purpose of the present invention is to provide a probiotic preservation composition and a preparation method thereof, which have the advantages of strong antibacterial property, high safety, clean label, long preservation and anti-corrosion period, good biocompatibility, etc., and have broad application prospects.

[0006] The technical solution of the present invention is realized as follows:

[0007] The present invention provides a method for preparing a probiotic preservation composition. Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium propionicum FPHC0659 are inoculated into a culture medium, fermented and cultured, and the bacterial sludge, antibacterial peptides with a molecular weight of 2-10 kDa, and fermentation products are collected respectively. The antibacterial peptides react with chitosan to obtain an antibacterial peptide-chitosan condensate, which is co-embedded with sodium alginate to prepare a sustained-release microsphere. The sustained-release microsphere is mixed evenly with the fermentation product, lysozyme, and vitamin C to prepare the probiotic preservation composition.

[0008] As a further improvement of the present invention, it includes the following steps:

[0009] S1. Preparation of the culture medium: Desalted whey powder, glucose, yeast extract powder, and anhydrous sodium acetate are added to water and sterilized to obtain the culture medium.

[0010] S2. Activation of probiotics: Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium propionicum FPHC0659 are respectively inoculated into MRS broth medium for activation culture to obtain bacterial strain seed solutions.

[0011] S3. Fermentation: The bacterial strain seed solutions of Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium propionicum FPHC0659 prepared in step S2 are inoculated into the culture medium prepared in step S1, fermented and cultured, filtered, and the bacterial sludge is collected. The filtrate is ultrafiltered to collect a solution with a molecular weight of 2-10 kDa, freeze-dried to obtain antibacterial peptides, and the remaining liquid is freeze-dried to obtain fermentation products.

[0012] S4. Preparation of the antibacterial peptide-chitosan condensate: The antibacterial peptides prepared in step S3 are activated by N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide and reacted with chitosan to obtain the antibacterial peptide-chitosan condensate.

[0013] S5. Preparation of the sustained-release microsphere: The antibacterial peptide-chitosan condensate, sodium alginate, and lecithin prepared in step S4 are added to water, the bacterial sludge prepared in step S3 is added, and the mixture is dispersed evenly. Then it is dropped into fish oil for emulsification, and a calcium chloride solution is dropped for curing at room temperature. After centrifugation, washing, and drying, the sustained-release microsphere is obtained.

[0014] S6. Natural preservative composition: Lysozyme and vitamin C are mixed evenly to obtain the natural preservative composition.

[0015] S7. Preparation of the probiotic preservation composition: The fermentation product prepared in step S3, the sustained-release microsphere prepared in step S5, and the natural preservative composition prepared in step S6 are mixed evenly to obtain the probiotic preservation composition.

[0016] As a further improvement of the present invention, the mass ratio of the desalted whey powder, glucose, yeast extract powder, sodium acetate anhydrous and water in step S1 is 12-15:10-15:3-5:2-4:200-400.

[0017] As a further improvement of the present invention, the conditions for the activation culture in step S2 are anaerobic conditions, 36-38 °C, and cultured for 18-24 h. The bacterial content of the bacterial strain seed liquid is 10 8 -10 9 cfu / mL.

[0018] As a further improvement of the present invention, the inoculation amounts of the Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium acidipropionici FPHC0659 bacterial strain seed liquids in step S3 are 1-3 v / v%, 2-3 v / v%, and 3-5 v / v% respectively. The conditions for the fermentation culture are anaerobic conditions, 36-38 °C, and fermented for 48-52 h. The filtration is carried out by filtering with a 0.22 μm microporous membrane.

[0019] As a further improvement of the present invention, the mass ratio of the antimicrobial peptide, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and chitosan in step S4 is 10-15:11-12:14-16:17-22. The temperature of the activation reaction is 0-4 °C and the time is 30-40 min. The temperature of the reaction is 35-40 °C and the time is 18-24 h.

[0020] As a further improvement of the present invention, the mass ratio of the antimicrobial peptide-chitosan condensate, sodium alginate, lecithin, water, bacterial sludge, and fish oil in step S5 is 10-15:7-12:1-2:200-300:25-30:500.

[0021] As a further improvement of the present invention, the mass ratio of the lysozyme and vitamin C in step S6 is 7-10:2-4; the mass ratio of the fermentation product, sustained-release microspheres, and natural preservation composition in step S7 is 12-15:7-10:2-3.

[0022] As a further improvement of the present invention, it specifically includes the following steps:

[0023] S1. Preparation of the culture medium: Add 12-15 parts by weight of desalted whey powder, 10-15 parts by weight of glucose, 3-5 parts by weight of yeast extract powder, and 2-4 parts by weight of sodium acetate anhydrous to 200-400 parts by weight of water, sterilize, and obtain the culture medium;

[0024] S2. Activation of probiotics: Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium acidipropionici FPHC0659 were respectively inoculated into MRS broth medium. Under anaerobic conditions, at 36 - 38 °C, they were activated and cultured for 18 - 24 h to obtain a bacterial seed solution with a bacterial content of 10 8 -10 9 cfu / mL;

[0025] S3. Fermentation: The bacterial seed solutions of Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium acidipropionici FPHC0659 prepared in step S2 were inoculated into the medium prepared in step S1, and the inoculation amounts were 1 - 3 v / v%, 2 - 3 v / v%, and 3 - 5 v / v% respectively. Under anaerobic conditions, at 36 - 38 °C, they were fermented and cultured for 48 - 52 h, filtered through a 0.22 μm microporous membrane, the bacterial sludge was collected, the filtrate was ultrafiltered to collect a solution with a molecular weight of 2 - 10 KDa, freeze-dried to obtain antibacterial peptides, and the remaining liquid was freeze-dried to obtain fermentation products;

[0026] S4. Preparation of antibacterial peptide-chitosan condensate: 10 - 15 parts by weight of the antibacterial peptides prepared in step S3 were added to water, 11 - 12 parts by weight of N-hydroxysuccinimide and 14 - 16 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, activated at 0 - 4 °C for 30 - 40 min, 17 - 22 parts by weight of chitosan were added, and stirred and reacted at 35 - 40 °C for 18 - 24 h to obtain an antibacterial peptide-chitosan condensate;

[0027] S5. Preparation of sustained-release microspheres: 10 - 15 parts by weight of the antibacterial peptide-chitosan condensate prepared in step S4, 7 - 12 parts by weight of sodium alginate, and 1 - 2 parts by weight of lecithin were added to 200 - 300 parts by weight of water, 25 - 30 parts by weight of the bacterial sludge prepared in step S3 were added, dispersed evenly, dropped into 500 parts by weight of fish oil, emulsified, 20 - 30 parts by weight of a 1 - 3 wt% calcium chloride solution were dropped, solidified at room temperature for 20 - 30 min, centrifuged, washed, and dried to obtain sustained-release microspheres;

[0028] S6. Natural preservative composition: 7 - 10 parts by weight of lysozyme and 2 - 4 parts by weight of vitamin C were mixed evenly to obtain a natural preservative composition;

[0029] S7. Preparation of probiotic preservative composition: 12 - 15 parts by weight of the fermentation products prepared in step S3, 7 - 10 parts by weight of the sustained-release microspheres prepared in step S5, and 2 - 3 parts by weight of the natural preservative composition prepared in step S6 were mixed evenly to obtain a probiotic preservative composition.

[0030] The present invention further protects a probiotic preservative composition prepared by the above preparation method.

[0031] The present invention has the following beneficial effects:

[0032] The probiotic preservation composition of the present invention contains slow-release microspheres, which encapsulate probiotics through embedding, improving the stability, temperature resistance and humidity resistance of probiotics. When added to food, the probiotics can naturally produce a variety of antibacterial active substances through fermentation. Among them, the bacteriocin produced by Lactobacillus plantarum JLA-9 has broad-spectrum antibacterial activity and strong antibacterial effects against a variety of bacteria and fungi, especially spore-forming bacteria; Lactobacillus rhamnosus LR05 can produce a variety of organic acids, such as lactic acid, etc., which have good inhibitory effects on bacteria and yeasts; Propionibacterium acidipropionici FPHC0659 mainly produces propionic acid during fermentation, which has good inhibitory effects on a variety of molds.

[0033] The present invention uses desalted whey powder, glucose, yeast extract powder, and anhydrous sodium acetate as raw materials for the medium for fermentation. After fermentation by composite probiotics (Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, Propionibacterium acidipropionici FPHC0659), the fermentation product contains abundant antibacterial peptides such as bacteriocin, as well as antibacterial active substances such as short-chain fatty acids. Antibacterial peptides with a molecular weight of 2-10 kDa are separated by ultrafiltration, and the remaining active product is freeze-dried to obtain a fermentation product, which also has good antibacterial activity.

[0034] Chitosan is also a natural antibacterial active substance, which can insert into the bacterial cell wall and destroy its structure by attracting the negative charges on the bacterial cell wall. This destructive effect causes the bacterial cell wall to increase in permeability, expand, become brittle, and ultimately cause the death of bacterial cells. However, due to its easy crystallization and poor solubility, the application of chitosan is greatly limited. The present invention carries out a condensation reaction between the prepared antibacterial peptide and chitosan. The prepared antibacterial peptide-chitosan condensate not only has good film-forming properties, but also has significantly improved antibacterial and bacteriostatic properties. At the same time, it has good biocompatibility and can be mixed with sodium alginate to form a better shell layer to encapsulate probiotics. At the same time, it degrades continuously in the environment, slowly releases probiotics, and at the same time serves as a prebiotic to provide nutrition for probiotics, thereby exerting the synergistic preservation effect of antibacterial peptides, chitosan, and probiotics and prolonging the preservation and anti-corrosion time.

[0035] In addition, the present invention also adds natural preservative compositions such as lysozyme and vitamin C. Through synergistic antioxidant and antibacterial effects, it destroys the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, decomposes the insoluble mucopolysaccharide in the cell wall into soluble glycopeptides, causes the cell wall to rupture and the contents to escape, resulting in the dissolution of bacteria, and improves the preservation and antibacterial effects.

[0036] The probiotic preservation composition prepared by the present invention has the following advantages:

[0037] 1. Strong antibacterial property: It has excellent inhibitory effects on foodborne bacteria (such as Escherichia coli, Staphylococcus aureus, etc.) and various common food molds.

[0038] 2. High safety: Bacteriocins and antimicrobial peptides produced by probiotics can be decomposed into essential amino acids by pepsin and trypsin in the human body, without residual risk.

[0039] 3. Clean label: As consumers' health awareness gradually increases, the ingredient list and nutrition label of food have received more and more attention. Yixiansu can help products achieve natural without additives, simple ingredients, and have the concept of probiotics. Detailed implementation methods

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0041] Lactobacillus plantarum JLA-9, taxonomically named Lactobacillus plantarum, deposit number CGMCC NO.10686, deposit date April 2, 2015, depositary institution China General Microbiological Culture Collection Center, address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101.

[0042] Lactobacillus rhamnosus LR05, taxonomically named Lactobacillus rhamnosus, deposit number CGMCC NO.22773, deposit date June 24, 2021, depositary institution China General Microbiological Culture Collection Center, address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101.

[0043] Propionibacterium acidipropionici FPHC0659, taxonomically named Acidipropionibacterium acidipropionici, deposit number CGMCC NO.29345, deposit date December 18, 2023, depositary institution China General Microbiological Culture Collection Center, address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101.

[0044] Example 1

[0045] This embodiment provides a method for preparing a probiotic preservation composition, which specifically includes the following steps:

[0046] S1. Preparation of the culture medium: Add 12 parts by weight of desalted whey powder, 10 parts by weight of glucose, 3 parts by weight of yeast extract powder, and 2 parts by weight of anhydrous sodium acetate to 200 parts by weight of water, sterilize, and obtain the culture medium;

[0047] S2. Activation of probiotics: Inoculate Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium acidipropionici FPHC0659 into MRS broth medium respectively. Under anaerobic conditions, at 36 °C, activate and culture for 18 h to obtain a strain seed solution with a bacterial content of 10 8 -10 9 cfu / mL;

[0048] S3. Fermentation: Inoculate the strain seed solutions of Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium acidipropionici FPHC0659 prepared in step S2 into the culture medium prepared in step S1, with inoculation amounts of 1 v / v%, 2 v / v%, and 3 v / v% respectively. Under anaerobic conditions, at 36 °C, ferment and culture for 48 h, filter with a 0.22 μm microporous membrane, collect the bacterial sludge, ultrafilter the filtrate to collect the solution with a molecular weight of 2-10 KDa, freeze-dry to obtain the antibacterial peptide, and freeze-dry the remaining liquid to obtain the fermentation product;

[0049] S4. Preparation of antibacterial peptide-chitosan condensate: Add 10 parts by weight of the antibacterial peptide prepared in step S3 to water, add 11 parts by weight of N-hydroxysuccinimide and 14 parts by weight of 1-ethyl-(3-dimethylaminopropyl) carbodiimide, activate at 0 °C for 30 min, add 17 parts by weight of chitosan, and stir and react at 35 °C for 18 h to obtain the antibacterial peptide-chitosan condensate;

[0050] S5. Preparation of sustained-release microspheres: Add 10 parts by weight of the antibacterial peptide-chitosan condensate prepared in step S4, 7 parts by weight of sodium alginate, and 1 part by weight of lecithin to 200 parts by weight of water, add 25 parts by weight of the bacterial sludge prepared in step S3, stir and mix for 20 min, drop into 500 parts by weight of fish oil, emulsify at 7000 r / mmin for 15 min, drop 20 parts by weight of 1 wt% calcium chloride solution, cure at room temperature for 20 min, centrifuge, wash, and dry to obtain the sustained-release microspheres;

[0051] S6. Natural preservative composition: Stir and mix 7 parts by weight of lysozyme and 2 parts by weight of vitamin C for 10 min to obtain the natural preservative composition;

[0052] S7. Preparation of probiotic fresh-keeping composition: Stir and mix 12 parts by weight of the fermentation product obtained in step S3, 7 parts by weight of the slow-release microspheres obtained in step S5, and 2 parts by weight of the natural fresh-keeping composition obtained in step S6 for 15 min to obtain the probiotic fresh-keeping composition.

[0053] Example 2

[0054] This example provides a preparation method of a probiotic fresh-keeping composition, which specifically includes the following steps:

[0055] S1. Preparation of culture medium: Add 15 parts by weight of desalted whey powder, 15 parts by weight of glucose, 5 parts by weight of yeast extract powder, and 4 parts by weight of anhydrous sodium acetate to 400 parts by weight of water, sterilize to obtain the culture medium;

[0056] S2. Activation of probiotics: Inoculate Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium propionicum FPHC0659 into MRS broth culture medium respectively. Under anaerobic conditions, at 38 °C, activate and culture for 24 h to obtain a strain seed solution with a bacterial content of 10 8 -10 9 cfu / mL;

[0057] S3. Fermentation: Inoculate the strain seed solutions of Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium propionicum FPHC0659 obtained in step S2 into the culture medium obtained in step S1, with the inoculation amounts being 3 v / v%, 3 v / v%, and 5 v / v% respectively. Under anaerobic conditions, at 38 °C, ferment and culture for 52 h, filter through a 0.22 μm microporous filter membrane, collect the bacterial sludge, ultrafilter the filtrate to collect the solution with a molecular weight of 2 - 10 KDa, freeze-dry to obtain the antibacterial peptide, and freeze-dry the remaining liquid to obtain the fermentation product;

[0058] S4. Preparation of antibacterial peptide-chitosan condensate: Add 15 parts by weight of the antibacterial peptide obtained in step S3 to water, add 12 parts by weight of N-hydroxysuccinimide and 16 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, activate at 4 °C for 40 min, add 22 parts by weight of chitosan, and stir and react at 40 °C for 24 h to obtain the antibacterial peptide-chitosan condensate;

[0059] S5. Preparation of slow-release microspheres: Add 15 parts by weight of the antibacterial peptide-chitosan condensate obtained in step S4, 12 parts by weight of sodium alginate, and 2 parts by weight of lecithin to 300 parts by weight of water, add 30 parts by weight of the bacterial sludge obtained in step S3, stir and mix for 20 min, dropwise add to 500 parts by weight of fish oil, emulsify at 7000 r / mmin for 15 min, dropwise add 30 parts by weight of 3 wt% calcium chloride solution, cure at room temperature for 30 min, centrifuge, wash, and dry to obtain the slow-release microspheres;

[0060] S6. Natural preservative composition: 10 parts by weight of lysozyme and 4 parts by weight of vitamin C are stirred and mixed for 10 min to obtain the natural preservative composition;

[0061] S7. Preparation of probiotic preservative composition: 15 parts by weight of the fermentation product prepared in step S3, 10 parts by weight of the sustained-release microspheres prepared in step S5, and 3 parts by weight of the natural preservative composition prepared in step S6 are stirred and mixed for 15 min to obtain the probiotic preservative composition.

[0062] Example 3

[0063] This example provides a method for preparing a probiotic preservative composition, which specifically includes the following steps:

[0064] S1. Preparation of culture medium: 13 parts by weight of desalted whey powder, 12 parts by weight of glucose, 4 parts by weight of yeast extract powder, and 3 parts by weight of anhydrous sodium acetate are added to 300 parts by weight of water, sterilized, and the culture medium is obtained;

[0065] S2. Activation of probiotics: Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium propionicum FPHC0659 are respectively inoculated into MRS broth medium, and under anaerobic conditions, at 37 °C, activated and cultured for 21 h to obtain a bacterial strain seed solution with a bacterial content of 10 8 -10 9 cfu / mL;

[0066] S3. Fermentation: The bacterial strain seed solutions of Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium propionicum FPHC0659 prepared in step S2 are inoculated into the culture medium prepared in step S1, and the inoculation amounts are 2 v / v%, 2.5 v / v%, and 4 v / v% respectively. Under anaerobic conditions, at 37 °C, fermented and cultured for 50 h, filtered through a 0.22 μm microporous filter membrane, the bacterial sludge is collected, the filtrate is ultrafiltered to collect a solution with a molecular weight of 2-10 KDa, freeze-dried to obtain the antibacterial peptide, and the remaining liquid is freeze-dried to obtain the fermentation product;

[0067] S4. Preparation of antibacterial peptide-chitosan condensate: 12 parts by weight of the antibacterial peptide prepared in step S3 are added to water, 11.5 parts by weight of N-hydroxysuccinimide and 15 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added, activated at 2 °C for 35 min, 20 parts by weight of chitosan are added, and stirred and reacted at 37 °C for 21 h to obtain the antibacterial peptide-chitosan condensate;

[0068] S5. Preparation of sustained-release microspheres: Add 12 parts by weight of the antibacterial peptide-chitosan condensate prepared in step S4, 10 parts by weight of sodium alginate, and 1.5 parts by weight of lecithin to 250 parts by weight of water. Add 28 parts by weight of the bacterial sludge prepared in step S3, stir and mix for 20 min, then dropwise add it into 500 parts by weight of fish oil, emulsify at 7000 r / mmin for 15 min, dropwise add 25 parts by weight of a 2 wt% calcium chloride solution, cure at room temperature for 25 min, centrifuge, wash, and dry to obtain the sustained-release microspheres;

[0069] S6. Natural preservative composition: Stir and mix 8.5 parts by weight of lysozyme and 3 parts by weight of vitamin C for 10 min to obtain the natural preservative composition;

[0070] S7. Preparation of probiotic preservative composition: Stir and mix 13.5 parts by weight of the fermentation product prepared in step S3, 8.5 parts by weight of the sustained-release microspheres prepared in step S5, and 2.5 parts by weight of the natural preservative composition prepared in step S6 for 15 min to obtain the probiotic preservative composition.

[0071] Comparative Example 1

[0072] Compared with Example 3, the difference is that Lactobacillus plantarum JLA-9 was not inoculated in step S3.

[0073] Specifically as follows:

[0074] S3. Fermentation: Inoculate the seed solutions of Lactobacillus rhamnosus LR05 and Propionibacterium acidipropionici FPHC0659 prepared in step S2 into the culture medium prepared in step S1, with inoculation amounts of 4.5 v / v% and 4 v / v% respectively. Under anaerobic conditions, ferment and culture at 37 °C for 50 h, filter through a 0.22 μm microporous membrane, collect the bacterial sludge, ultrafilter the filtrate to collect the solution with a molecular weight of 2-10 KDa, freeze-dry to obtain the antibacterial peptide, and freeze-dry the remaining liquid to obtain the fermentation product.

[0075] Comparative Example 2

[0076] Compared with Example 3, the difference is that Lactobacillus rhamnosus LR05 was not inoculated in step S3.

[0077] Specifically as follows:

[0078] S3. Fermentation: Inoculate the seed solutions of Lactobacillus plantarum JLA-9 and Propionibacterium acidipropionici FPHC0659 prepared in step S2 into the culture medium prepared in step S1, with inoculation amounts of 2 v / v% and 6.5 v / v% respectively. Under anaerobic conditions, ferment and culture at 37 °C for 50 h, filter through a 0.22 μm microporous membrane, collect the bacterial sludge, ultrafilter the filtrate to collect the solution with a molecular weight of 2-10 KDa, freeze-dry to obtain the antibacterial peptide, and freeze-dry the remaining liquid to obtain the fermentation product.

[0079] Comparative Example 3

[0080] Compared with Example 3, the difference lies in that Propionibacterium acidipropionici FPHC0659 was not inoculated in step S3.

[0081] Specifically as follows:

[0082] S3. Fermentation: The seed solutions of Lactobacillus plantarum JLA-9 and Lactobacillus rhamnosus LR05 prepared in step S2 were inoculated into the culture medium prepared in step S1, and the inoculation amounts were 2 v / v% and 6.5 v / v% respectively. Under anaerobic conditions, at 37 °C, fermentation culture was carried out for 50 h, filtered through a 0.22 μm microporous filter membrane, the bacterial sludge was collected, the filtrate was ultrafiltered to collect the solution with a molecular weight of 2-10 KDa, freeze-dried to obtain antibacterial peptides, and the remaining liquid was freeze-dried to obtain fermentation products.

[0083] Comparative Example 4

[0084] Compared with Example 3, the difference lies in that the antibacterial peptide-chitosan condensate in step S5 was replaced by an equal mass of antibacterial peptide.

[0085] S5. Preparation of sustained-release microspheres: 12 parts by weight of the antibacterial peptides prepared in step S3, 10 parts by weight of sodium alginate, and 1.5 parts by weight of lecithin were added to 250 parts by weight of water, 28 parts by weight of the bacterial sludge prepared in step S3 was added, stirred and mixed for 20 min, dropped into 500 parts by weight of fish oil, emulsified at 7000 r / mmin for 15 min, 25 parts by weight of 2 wt% calcium chloride solution was dropped, cured at room temperature for 25 min, centrifuged, washed, and dried to obtain sustained-release microspheres.

[0086] Comparative Example 5

[0087] Compared with Example 3, the difference lies in that the antibacterial peptide-chitosan condensate in step S5 was replaced by an equal mass of carboxymethyl chitosan.

[0088] Specifically as follows:

[0089] S5. Preparation of sustained-release microspheres: 12 parts by weight of carboxymethyl chitosan, 10 parts by weight of sodium alginate, and 1.5 parts by weight of lecithin were added to 250 parts by weight of water, 28 parts by weight of the bacterial sludge prepared in step S3 was added, stirred and mixed for 20 min, dropped into 500 parts by weight of fish oil, emulsified at 7000 r / mmin for 15 min, 25 parts by weight of 2 wt% calcium chloride solution was dropped, cured at room temperature for 25 min, centrifuged, washed, and dried to obtain sustained-release microspheres.

[0090] Comparative Example 6

[0091] Compared with Example 3, the difference lies in that no embedding was carried out in step S5.

[0092] The details are as follows:

[0093] S1. Preparation of the culture medium: Add 13 parts by weight of desalted whey powder, 12 parts by weight of glucose, 4 parts by weight of yeast extract powder, and 3 parts by weight of anhydrous sodium acetate to 300 parts by weight of water, sterilize to obtain the culture medium;

[0094] S2. Activation of probiotics: Inoculate Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium propionicum FPHC0659 into MRS broth medium respectively. Under anaerobic conditions at 37 °C, activate and culture for 21 h to obtain a bacterial strain seed solution with a bacterial content of 10 8 -10 9 cfu / mL;

[0095] S3. Fermentation: Inoculate the bacterial strain seed solutions of Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium propionicum FPHC0659 prepared in step S2 into the culture medium prepared in step S1, with inoculation amounts of 2 v / v%, 2.5 v / v%, and 4 v / v% respectively. Under anaerobic conditions at 37 °C, ferment and culture for 50 h, filter with a 0.22 μm microporous filter membrane, collect the bacterial sludge, ultrafilter the filtrate to collect the solution with a molecular weight of 2-10 KDa, freeze-dry to obtain the antibacterial peptide, and freeze-dry the remaining liquid to obtain the fermentation product;

[0096] S4. Preparation of the antibacterial peptide-chitosan condensate: Add 12 parts by weight of the antibacterial peptide prepared in step S3 to water, add 11.5 parts by weight of N-hydroxysuccinimide and 15 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, activate at 2 °C for 35 min, add 20 parts by weight of chitosan, and stir and react at 37 °C for 21 h to obtain the antibacterial peptide-chitosan condensate;

[0097] S5. Preparation of the mixture: Mix 12 parts by weight of the antibacterial peptide prepared in step S3 and 28 parts by weight of the bacterial sludge prepared in step S3 evenly to obtain the mixture;

[0098] S6. Preparation of the natural preservative composition: Stir and mix 8.5 parts by weight of lysozyme and 3 parts by weight of vitamin C for 10 min to obtain the natural preservative composition;

[0099] S7. Preparation of the probiotic preservative composition: Stir and mix 13.5 parts by weight of the fermentation product prepared in step S3, 8.5 parts by weight of the mixture prepared in step S5, and 2.5 parts by weight of the natural preservative composition prepared in step S6 for 15 min to obtain the probiotic preservative composition.

[0100] Comparative Example 7

[0101] Compared with Example 3, the difference is that lysozyme is not added in step S6.

[0102] The details are as follows:

[0103] S6. Natural preservative composition: Using lysozyme as the natural preservative composition.

[0104] Comparative Example 8

[0105] Compared with Example 3, the difference lies in that vitamin C was not added in step S6.

[0106] The details are as follows:

[0107] S6. Natural preservative composition: Using vitamin C as the natural preservative composition.

[0108] Comparative Example 9

[0109] Compared with Example 3, the difference lies in that the natural preservative composition was not added in step S7.

[0110] The details are as follows:

[0111] S7. Preparation of probiotic preservative composition: Mixing 13.5 parts by weight of the fermentation product obtained in step S3 and 8.5 parts by weight of the sustained-release microspheres obtained in step S5 and stirring for 15 min to obtain the probiotic preservative composition.

[0112] Comparative Example 10

[0113] Compared with Example 3, the difference lies in that the sustained-release microspheres were not added in step S7.

[0114] The details are as follows:

[0115] S7. Preparation of probiotic preservative composition: Mixing 13.5 parts by weight of the fermentation product obtained in step S3 and 2.5 parts by weight of the natural preservative composition obtained in step S6 and stirring for 15 min to obtain the probiotic preservative composition.

[0116] Test Example 1

[0117] Adding the probiotic preservative compositions prepared in Examples 1 - 3 and Comparative Examples 1 - 10 of the present invention into water, stirring and mixing evenly, spraying on the surface of the cooled bread, drying, sealing and packaging, and storing in a constant temperature environment at 37°C, wherein the addition amount of the probiotic preservative composition is 0.02 wt%.

[0118] 1. Sensory quality evaluation

[0119] The sensory evaluation method of bread can be carried out with reference to the sensory indicators of "National Food Safety Standard Pastries and Bread" (GB 7099-2015). The sensory evaluation test method is to take more than 50g of samples, observe whether their color, smell, taste and texture are normal. The evaluation criteria are that they should have the normal color, smell, taste and texture of pastries and bread respectively, and there should be no off-flavors such as rancidity and mold, and there should be no mildew, insect infestation and other foreign contaminants inside and outside the food. The sensory scoring panel consists of 20 professional personnel, and the average value of the sensory scores is calculated as the final result.

[0120] 2. Detection of microorganisms in bread

[0121] With reference to "National Food Safety Standard Microbiological Examination of Foods - Determination of Total Number of Colonies" (GB 4789.2-2016) implemented in China

[0122] (GB 4789.2-2016) for detection to understand the changes of bread microorganisms and determine the storage date of bread.

[0123] The results are shown in Table 1.

[0124] Table 1

[0125]

[0126]

[0127] As can be seen from the above table, the probiotic preservation compositions prepared in Examples 1-3 of the present invention have good preservation and anti-corrosion effects on bakery products such as bread.

[0128] Test Example 2 Antibacterial experiment

[0129] The probiotic preservation compositions prepared in Examples 1-3 and Comparative Examples 1-10 of the present invention were added to water to prepare a 0.1wt% probiotic preservation composition suspension. Take 0.1 mL of the spore-forming Clostridium bacterium (ATCC 11437) suspension and spread it. Use a punch to punch holes equidistantly on the culture medium, and add 50 μL of the probiotic preservation composition suspension to each hole for antibacterial ability determination. Use sterile water as a control and culture at 37 °C for 24 h, and measure the diameter of the antibacterial zone. The results are shown in Table 2.

[0130] Table 2

[0131]

[0132]

[0133] As can be seen from the above table, the probiotic preservation compositions prepared in Examples 1-3 of the present invention have good antibacterial effects.

[0134] Test Example 3 Antioxidant experiment

[0135] The probiotic preservation compositions prepared in Examples 1-3 and Comparative Examples 1-10 of the present invention were added to water to prepare a 0.1 wt% suspension of the probiotic preservation composition.

[0136] 1. DPPH· scavenging activity test:

[0137] Take 50 μL of the suspension of the probiotic preservation composition and mix it evenly with 150 μL of 0.1 mmol / L DPPH solution. After standing in the dark at room temperature for 30 min, measure its absorbance at 517 nm, using absolute ethanol as the blank control.

[0138] DPPH· scavenging rate (%) = [1 - (A i - A j ) / A0] × 100%

[0139] In the formula: A i is the absorbance of the test group; A j is the absorbance of the control group; A0 is the absorbance of the blank group.

[0140] 2. ABTS+ radical scavenging activity test:

[0141] Mix 20 μL of the suspension of the probiotic preservation composition with 180 μL of ABTS + working solution and react in the dark at room temperature for 60 min. Using distilled water as the blank control, measure the absorbance at 734 nm.

[0142] ABTS + radical scavenging rate (%) = (A0 - A1) / A0 × 100%

[0143] In the formula: A0 is the absorbance of the test group; A1 is the absorbance of the blank group.

[0144] The results are shown in Table 3.

[0145] Table 3

[0146]

[0147]

[0148] As can be seen from the above table, the probiotic preservation compositions prepared in Examples 1-3 of the present invention have good antioxidant activity.

[0149] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a probiotic preservation composition, characterized in that, It includes the following steps: S1. Preparation of the culture medium: Add desalted whey powder, glucose, yeast extract powder, and sodium acetate anhydrous into water, sterilize it to obtain the culture medium; S2. Activation of probiotics: Inoculate Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Propionibacterium acidipropionici ( Acidipropionibacterium acidipropionici ), FPHC0659 into MRS broth medium respectively, and perform activation culture to obtain bacterial strain seed liquid; S3. Fermentation: Inoculate the seed solutions of Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Acidipropionibacterium acidipropionici FPHC0659 prepared in step S2 into the culture medium prepared in step S1, conduct fermentation culture, filter, collect the bacterial sludge, ultrafilter the filtrate to collect the solution with a molecular weight of 2 - 10KDa, conduct freeze-drying to obtain the antibacterial peptide, and conduct freeze-drying on the remaining liquid to obtain the fermentation product; S4. Preparation of the antibacterial peptide-chitosan condensate: Activate the antibacterial peptide prepared in step S3 with N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and react it with chitosan to obtain the antibacterial peptide-chitosan condensate; S5. Preparation of the sustained-release microspheres: Add the antibacterial peptide-chitosan condensate, sodium alginate, and lecithin prepared in step S4 into water, add the bacterial sludge prepared in step S3, disperse evenly, drop it into fish oil, emulsify, dropwise add calcium chloride solution, solidify at room temperature, centrifuge, wash, and dry to obtain the sustained-release microspheres; S6. Natural preservative composition: Mix lysozyme and vitamin C evenly to obtain the natural preservative composition; S7. Preparation of the probiotic preservative composition: Mix the fermentation product prepared in step S3, the sustained-release microspheres prepared in step S5, and the natural preservative composition prepared in step S6 evenly to obtain the probiotic preservative composition; The Lactobacillus plantarum ( Lactobacillus plantarum ), JLA-9, with the preservation number of CGMCC NO.10686, the preservation date of April 2, 2015, the preservation unit of China General Microbiological Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode: 100101; The Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), namely LR05, with the preservation number of CGMCC NO.22773, the preservation date of June 24, 2021, and the preservation unit of the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101; The Propionibacterium acidipropionici ( Acidipropionibacterium acidipropionici ) FPHC0659, with the preservation number of CGMCC NO.29345, the preservation date of December 18, 2023, and the preservation unit of the General Microbiology Center of the China Microbial Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode: 100101.

2. The preparation method according to claim 1, wherein The mass ratio of the desalted whey powder, glucose, yeast extract powder, sodium acetate anhydrous, and water described in step S1 is 12 - 15:10 - 15:3 - 5:2 - 4:200 - 400.

3. The preparation method according to claim 1, characterized in that, The conditions for the activation culture described in step S2 are anaerobic conditions, 36 - 38 °C, and culturing for 18 - 24 h. The bacterial content of the bacterial strain seed liquid is 10 8 -10 9 cfu / mL.

4. The preparation method according to claim 1, characterized in that, The inoculation amounts of the seed solutions of Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Acidipropionibacterium acidipropionici FPHC0659 described in step S3 are 1 - 3 v / v%, 2 - 3 v / v%, and 3 - 5 v / v% respectively. The conditions for the fermentation culture are under anaerobic conditions, at 36 - 38°C, and ferment for 48 - 52h. The filtration is carried out using a 0.22μm microporous membrane.

5. The preparation method according to claim 1, wherein The mass ratio of the antibacterial peptide, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and chitosan described in step S4 is 10 - 15:11 - 12:14 - 16:17 - 22. The temperature of the activation reaction is 0 - 4°C, and the time is 30 - 40min. The temperature of the reaction is 35 - 40°C, and the time is 18 - 24h.

6. The preparation method according to claim 1, wherein In step S5, the mass ratio of the antibacterial peptide-chitosan condensate, sodium alginate, lecithin, water, bacterial sludge, and fish oil is 10 - 15:7 - 12:1 - 2:200 - 300:25 - 30:

500.

7. The preparation method according to claim 1, characterized in that In step S6, the mass ratio of lysozyme and vitamin C is 7 - 10:2 - 4; in step S7, the mass ratio of the fermentation product, sustained-release microspheres, and natural preservation composition is 12 - 15:7 - 10:2 - 3.

8. The preparation method according to claim 1, wherein Specifically, it includes the following steps: S1. Preparation of the culture medium: Add 12 - 15 parts by weight of desalted whey powder, 10 - 15 parts by weight of glucose, 3 - 5 parts by weight of yeast extract powder, and 2 - 4 parts by weight of anhydrous sodium acetate to 200 - 400 parts by weight of water, sterilize to obtain the culture medium; S2. Activation of probiotics: Lactobacillus plantarum JLA-9, Lactobacillus rhamnosus LR05, and Propionibacterium acidipropionici FPHC0659 were respectively inoculated into MRS broth medium, and cultured under anaerobic conditions at 36-38 °C for 18-24 h to obtain a strain seed solution with a bacterial content of 10 8 -10 9 cfu / mL; S3. Fermentation: Inoculate the seed solutions of Lactobacillus plantarum JLA - 9, Lactobacillus rhamnosus LR05, and Propionibacterium acidipropionici FPHC0659 prepared in step S2 into the culture medium prepared in step S1, with the inoculation amounts being 1 - 3 v / v%, 2 - 3 v / v%, and 3 - 5 v / v% respectively. Under anaerobic conditions, ferment and culture at 36 - 38 °C for 48 - 52 h. Filter using a 0.22 μm microporous membrane, collect the bacterial sludge, ultrafilter the filtrate to collect the solution with a molecular weight of 2 - 10 KDa, freeze-dry to obtain the antibacterial peptide, and freeze-dry the remaining liquid to obtain the fermentation product; S4. Preparation of the antibacterial peptide-chitosan condensate: Add 10 - 15 parts by weight of the antibacterial peptide prepared in step S3 to water, add 11 - 12 parts by weight of N-hydroxysuccinimide and 14 - 16 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, activate at 0 - 4 °C for 30 - 40 min, add 17 - 22 parts by weight of chitosan, and stir and react at 35 - 40 °C for 18 - 24 h to obtain the antibacterial peptide-chitosan condensate; S5. Preparation of the sustained-release microspheres: Add 10 - 15 parts by weight of the antibacterial peptide-chitosan condensate prepared in step S4, 7 - 12 parts by weight of sodium alginate, and 1 - 2 parts by weight of lecithin to 200 - 300 parts by weight of water, add 25 - 30 parts by weight of the bacterial sludge prepared in step S3, disperse evenly, dropwise add to 500 parts by weight of fish oil, emulsify, dropwise add 20 - 30 parts by weight of a 1 - 3 wt% calcium chloride solution, cure at room temperature for 20 - 30 min, centrifuge, wash, and dry to obtain the sustained-release microspheres; S6. Natural preservative composition: Mix 7 - 10 parts by weight of lysozyme and 2 - 4 parts by weight of vitamin C evenly to obtain the natural preservative composition; S7. Preparation of the probiotic preservation composition: Mix 12 - 15 parts by weight of the fermentation product prepared in step S3, 7 - 10 parts by weight of the sustained-release microspheres prepared in step S5, and 2 - 3 parts by weight of the natural preservation composition prepared in step S6 evenly to obtain the probiotic preservation composition.

9. A probiotic preservation composition prepared by the preparation method according to any one of claims 1 - 8.

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