Propionibacterium freudenreichii subsp. shermanii with high antibacterial activity and its use

Abstract

This invention belongs to the field of bio-fermentation technology, and relates to a highly antibacterial Propionibacterium fischeri subsp. Scheres and its uses, specifically involving a strain of Propionibacterium fischeri subsp. Scheres, which is classified and named as Propionibacterium fischeri subsp. Scheres strain ( Propionibacterium freudenreichii subsp. shermanii The strain CJPF0001, with accession number CCTCC No. M 20242477, produces fermentation products with high protein content and high antibacterial activity. These products can inhibit the growth of common spoilage microorganisms, thereby improving the nutritional value of food and extending its shelf life.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and particularly relates to a strain of Propionibacterium freudenreichii subspecies shermanii with high antibacterial activity and a use thereof. Background Art

[0002] Food is rich in nutrients and is easily contaminated by microorganisms and spoiled. In order to ensure the safety of food, people have adopted many methods to preserve food, such as salting, canning, refrigeration, adding chemical preservatives, etc. Under certain conditions, the use of preservatives as an auxiliary means of preservation has a significant effect on preventing food from spoiling. Therefore, preservatives are widely used in the food industry as one of the important food additives.

[0003] However, long-term use of chemical preservatives may affect the body's immune system, digestive system, endocrine system, and even pose a risk of cancer. It may also increase the drug resistance of spoilage bacteria, posing a potential risk to consumers' health. Biological preservatives or preservative functional ingredients refer to a class of highly effective preservative functional products that are obtained from organisms through biological culture, extraction and separation technology, and have the function of inhibiting and killing microorganisms. They mainly include organic acids, peptides or protein substances. Biological preservative functional products have the advantages of safety, non-toxicity, biodegradability, and a wide range of applications. They can reduce or completely replace the use of chemical preservatives.

[0004] Propionibacterium freudenreichii subsp. Propionibacterium freudenreichii subsp. shermanii ) is widely present in nature, especially in dairy products such as cheese, soil and other environments. It is also a food-grade safe strain on China's "List of Bacteria that Can Be Used in Food". During its growth, Propionibacterium freudenreichii subsp. shermanii can produce a variety of metabolites with antibacterial activity, such as organic acids and active peptides, and has varying degrees of inhibitory effects on foodborne bacteria, yeasts and molds. Its cells and / or fermentation products can be used in, including but not limited to, baked products, meat products, jams, condiments and cheese, and have the effect of extending the shelf life of food.

[0005] However, there are few resources of Propionibacterium freudenreichii subsp. schreberi with a broad antibacterial spectrum and strong antibacterial ability, which limits its application in the field of food and biological preservation. Therefore, it is necessary to screen Propionibacterium freudenreichii subsp. schreberi with strong antibacterial ability, simple fermentation, broad antibacterial spectrum and high nutrition, and develop it into a new type of food raw material to improve the nutritional value and antiseptic function of food, and also reduce the use of chemical preservatives. Summary of the invention

[0006] In order to overcome the shortcomings of the existing Propionibacterium freudenreichii subsp. shenkelii in antibacterial activity, the present disclosure provides a strain of Propionibacterium freudenreichii subsp. shenkelii with high antibacterial activity, whose fermentation product has a high protein content and antibacterial activity, can be used as a food ingredient, and has the function of improving the nutritional value of food and preservative function.

[0007] In one aspect, the present disclosure provides a strain of Propionibacterium freudenreichii subsp. schreiberi, which is classified as Propionibacterium freudenreichii subsp. schreiberi ( Propionibacterium freudenreichii subsp. shermanii )CJPF0001, its deposit number is CCTCC No. M 20242477.

[0008] The Propionibacterium freudenreichii subsp. siebelii strain described in the present invention is deposited in the China Center for Type Culture Collection; the address of the depository unit is Wuhan University, Wuhan, China; the deposit date is November 7, 2024.

[0009] On the other hand, the present disclosure provides a fermentation product of the aforementioned Propionibacterium freudenreichii subsp. shermanii strain, which contains 30% protein.

[0010] In some embodiments, the fermentation product of the Propionibacterium freudenreichii subsp. shermanii strain has an antiseptic function.

[0011] On the other hand, the present disclosure provides a composition with an antiseptic function, comprising an effective amount of Propionibacterium freudenreichii subsp. schreberi strain, and / or a fermentation product of Propionibacterium freudenreichii subsp. schreberi strain in a fermentation medium, and acceptable excipients;

[0012] The deposit number of the Propionibacterium freudenreichii subsp. schreiberi strain is CCTCC No. M 20242477.

[0013] In another aspect, the present disclosure provides a method for preparing the aforementioned fermentation product, which comprises at least the following steps:

[0014] 1) Culturing the aforementioned Propionibacterium freudenreichii subsp. schreiberi strain CJPF0001 on a solid culture medium plate;

[0015] 2) Pick the single colony obtained in step 1) and activate the culture;

[0016] 3) expanding the bacterial solution obtained in step 2);

[0017] 4) inoculating the bacterial solution obtained in step 3) into the fermentation medium for batch fed-batch fermentation with an inoculation amount of 0.5%-10%;

[0018] 5) The inactivated fermentation product is directly freeze-dried or spray-dried, or the bacterial liquid is separated by centrifugation, and the supernatant and the bacterial cells are dried separately to obtain the fermentation product.

[0019] On the other hand, the present disclosure provides use of the aforementioned Propionibacterium freudenreichii subsp. shermanii strain, the aforementioned fermentation product or the aforementioned composition in the preparation of food, medicine or cosmetics.

[0020] In another aspect, the present disclosure provides use of the aforementioned Propionibacterium freudenreichii subsp. shermanii strain, the aforementioned fermentation product, and the aforementioned composition for inhibiting the growth of Staphylococcus aureus, Escherichia coli, Aspergillus flavus, or Aspergillus niger.

[0021] The beneficial effects of this application are at least as follows:

[0022] The present application discloses a new strain of Propionibacterium freudenreichii subspecies schreberi, which is classified and named as Propionibacterium freudenreichii subspecies schreberi ( Propionibacterium freudenreichii subsp. shermanii ) CJPF0001, and its deposit number is CCTCC No. M 20242477. The fermentation product of this strain has high protein and high antibacterial activity, which can inhibit the growth of common spoilage microorganisms, improve the nutritional value of food and extend the shelf life of food. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the protein content of the fermentation product of Propionibacterium freudenreichii subsp. cheleriana CJPF0001.

[0024] Figure 2 The results show the inhibition of the fermentation product of Propionibacterium freudenreichii subsp. cheleriana CJPF0001 against Staphylococcus aureus (A) and Escherichia coli (B).

[0025] Figure 3 The results show the inhibition of the fermentation products of Propionibacterium freudenreichii subsp. schreberi CJPF0001 on Aspergillus flavus (A), Aspergillus niger (B) and baker's yeast (C).

[0026] Figure 4 The preservation / antiseptic effect of the fermentation product of Propionibacterium freudenreichii subsp. chelery CJPF0001 in the toast bread challenge experiment. DETAILED DESCRIPTION

[0027] I. Terminology

[0028] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0029] The term "and / or" should be understood to mean either or both of the alternatives.

[0030] The terms “comprising” or “including” generally imply the inclusion of explicitly stated features, but not the exclusion of other elements.

[0031] The term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0032] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not occur.

[0033] The term "fermentation" refers to the process of preparing the cell itself, or direct metabolites or secondary metabolites by the life activities of Propionibacterium freudenreichii subsp. schreberi under facultative anabolic conditions. The term "fermentation product" described in this application refers to a product produced by a method including a fermentation step using a fermenting organism, and may include the Propionibacterium freudenreichii subsp. schreberi strain CJPF0001 cell and / or its fermentation broth in this application.

[0034] The term "Oxford cup method" is a commonly used in vitro drug sensitivity test method, which is mainly used to detect the sensitivity of microorganisms (such as bacteria, fungi, etc.) to different drugs. Its detection is based on the principle of drug diffusion in agar medium. Filter paper containing a certain concentration of the drug to be tested, Oxford cup (a small cup-shaped device made of stainless steel, usually with an inner diameter of 6mm, an outer diameter of 8mm, and a height of about 10mm) and the like are placed on the surface of the agar plate medium inoculated with microorganisms as drug carriers. The drug will diffuse from the carrier to the surrounding agar medium, and a drug concentration gradient will be formed in the area where the drug diffuses. The closer to the carrier, the higher the drug concentration, and the farther away, the lower the drug concentration. If the microorganism is sensitive to the drug, then the growth of the microorganism will be inhibited in the area covered by the effective antibacterial concentration of the drug, thereby forming an obvious inhibition zone on the agar plate. By measuring the size of the inhibition zone, the sensitivity of the microorganism to the drug can be preliminarily judged. The larger the inhibition zone, the more sensitive the microorganism is to the drug.

[0035] II. Detailed description of embodiments

[0036] In one aspect, the present disclosure provides a strain of Propionibacterium freudenreichii subsp. schreiberi, which is classified as Propionibacterium freudenreichii subsp. schreiberi ( Propionibacterium freudenreichii subsp. shermanii) CJPF0001, whose deposit number is CCTCC No. M 20242477. In the present disclosure, the Propionibacterium freudenreichii subsp. schreiberi may also be referred to as CJPF0001.

[0037] The Propionibacterium freudenreichii subsp. siebelii strain described in the present invention is deposited in the China Center for Type Culture Collection; the address of the depository unit is Wuhan University, Wuhan, China; the deposit date is November 7, 2024.

[0038] When the inventors were separating and purifying microbial strains of Yunnan Shangri-La cheese, they unexpectedly discovered a strain of Propionibacterium freudenreichii subsp. schreberi, whose fermentation product had high protein content and high antibacterial activity. The inventors believe that the Propionibacterium freudenreichii subsp. schreberi strain of the present application will produce a variety of organic acids (including but not limited to propionic acid, acetic acid, citric acid, etc.) and antimicrobial peptides and other substances during the fermentation process. The types, contents, proportions, etc. of these substances are different from those of the existing Propionibacterium freudenreichii subsp. schreberi strains, so it exhibits higher protein content and better antibacterial activity.

[0039] On the other hand, the present disclosure provides a fermentation product of the aforementioned Propionibacterium freudenreichii subsp. shermanii strain, which contains 30% protein.

[0040] On the other hand, the present disclosure provides a composition with an antiseptic function, comprising an effective amount of Propionibacterium freudenreichii subsp. schreberi strain, and / or a fermentation product of Propionibacterium freudenreichii subsp. schreberi strain in a fermentation medium, and acceptable excipients;

[0041] The deposit number of the Propionibacterium freudenreichii subsp. schreiberi strain is CCTCC No. M 20242477.

[0042] In some embodiments, the acceptable excipient is an excipient acceptable in the field of food science.

[0043] In some embodiments, the composition is a liquid or a solid.

[0044] In some embodiments, the composition is a tablet, granules, powder, solution, suspension, emulsion or premix.

[0045] In some embodiments, the fermentation medium comprises at least a carbon source and a nitrogen source.

[0046] In some embodiments, the carbon source includes at least sodium lactate. In some embodiments, the carbon source also includes at least one of glucose, sucrose, glycerol, xylose, molasses, and lactose.

[0047] In some embodiments, the nitrogen source includes at least peptone or yeast powder. In some embodiments, the nitrogen source also includes at least one of whey powder, corn steep liquor, corn flour, ammonium sulfate, and yeast extract.

[0048] In some embodiments, the mass ratio of the carbon source to the nitrogen source is (1-10): 1. The inventors found that the ratio is conducive to rapid proliferation of bacteria and obtains a high-protein and high-antibacterial activity fermentation product.

[0049] In some embodiments, the fermentation time is 24-120 hours. The inventors have found that appropriately extending the fermentation time can increase the yield of the fermentation product, but too long a fermentation time will cause bacterial aging, affecting the protein content and antibacterial activity of the fermentation product.

[0050] In some embodiments, the fermentation temperature is 25-45° C. The inventors have found that when the temperature is too low during the fermentation process, the fermentation speed slows down, and when the fermentation temperature is too high, the bacterial protein content and antibacterial activity are affected. When the fermentation temperature is controlled within the temperature range, it is beneficial to increase the fermentation speed and the protein content and antibacterial activity of the fermentation product.

[0051] In some embodiments, the inoculation amount of the Propionibacterium freudenreichii subsp. shermanii strain is 0.5%-10%. If the inoculation amount is too low, it is not conducive to the rapid proliferation of the bacteria. If the inoculation amount is too high, the proliferation rate is too fast, the bacteria are prone to aging, and the protein content and antibacterial activity of the fermentation product are affected.

[0052] In some embodiments, the growth and activation medium is a de Man-Rogosa-Sharp medium (MRS medium). The inventors found that the Propionibacterium marinum subsp. schreiberi strain grows fast and has a high biomass in the above medium. In the present application, the formulas of the culture medium used are all conventional formulas in the art. Taking MRS medium as an example, it can contain peptone: 10.0 g / L; beef extract: 10.0 g / L; yeast extract: 5.0 g / L; glucose: 20.0 g / L; sodium acetate: 5.0 g / L; diammonium hydrogen citrate: 2.0 g / L; Tween 80: 1.0 ml / L; dipotassium hydrogen phosphate: 2.0 g / L; magnesium sulfate: 0.2 g / L; manganese sulfate: 0.05 g / L. If it is a solid culture medium, it can also contain 20 g / L agar; the culture medium can be obtained by sterilizing at 121°C for 15-20 min.

[0053] In some embodiments, the fermentation medium comprises the following components: 10 g / L tryptone, 16.7 mL / L sodium lactate, 10 g / L yeast powder, 0.005 g / L manganese sulfate, and 0.25 g / L potassium dihydrogen phosphate.

[0054] On the other hand, the present disclosure provides a method for preparing the aforementioned biological preservative, which at least comprises the following steps:

[0055] 1) Culturing the aforementioned Propionibacterium freudenreichii subsp. schreiberi strain CJPF0001 on a solid culture medium plate;

[0056] 2) Pick the single colony obtained in step 1) and activate the culture;

[0057] 3) expanding the bacterial solution obtained in step 2);

[0058] 4) inoculating the bacterial solution obtained in step 3) into the fermentation medium for batch fed fermentation with an inoculation amount of 0.5%-10%;

[0059] 5) The inactivated fermentation product is directly freeze-dried or spray-dried, or the bacterial liquid is separated by centrifugation, and the supernatant and the bacterial cells are dried separately to obtain the fermentation product.

[0060] In some embodiments, the preparation method is specifically:

[0061] 1) CJPF0001 strain was cultured on MRS solid medium plates and incubated at 30°C for 48 h;

[0062] 2) Pick a single colony obtained in step 1), activate it with 5 mL of MRS liquid medium, and culture it at 30°C for 48-72 hours;

[0063] 3) The bacterial solution obtained in step 2) was expanded and cultured in 50 mL MRS liquid medium at 30°C for 48-72 hours, and the culture was terminated when OD = 5;

[0064] 4) The bacterial solution obtained in step 3) was inoculated into a fermentation medium (tryptone 10 g / L, sodium lactate 16.7 mL / L, yeast powder 10 g / L, manganese sulfate 0.005 g / L, potassium dihydrogen phosphate 0.25 g / L), and a 1.5 L tank was used for batch feeding fermentation, with an inoculation amount of 2-10% and a liquid volume of 1000 mL; fermentation conditions: a rotation speed of 100 rpm, NaOH to control pH=5.5-6.5, anaerobic fermentation culture at 30°C for 96h-120h, and inactivation at 90°C for 30min after the fermentation was completed;

[0065] 5) The inactivated fermentation product is directly freeze-dried or spray-dried, or the bacterial liquid is separated by centrifugation, and the supernatant and the bacterial cells are dried separately.

[0066] In some embodiments, the fermentation medium comprises the following components: 10 g / L tryptone, 16.7 mL / L sodium lactate, 10 g / L yeast powder, 0.005 g / L manganese sulfate, and 0.25 g / L potassium dihydrogen phosphate.

[0067] On the other hand, the present disclosure provides use of the aforementioned Propionibacterium freudenreichii subsp. shermanii strain, the aforementioned fermentation product or the aforementioned composition in the preparation of food, medicine or cosmetics.

[0068] In some embodiments, the Propionibacterium freudenreichii subsp. shermanii strain can be used to ferment the corresponding substrate to prepare a food product as a food raw material. In other embodiments of the present application, the Propionibacterium freudenreichii subsp. shermanii strain can be directly added to the food product.

[0069] In another aspect, the present disclosure provides use of the aforementioned Propionibacterium freudenreichii subsp. shermanii strain, the aforementioned fermentation product or the aforementioned composition for inhibiting the growth of Staphylococcus aureus, Escherichia coli, Aspergillus flavus or Aspergillus niger.

[0070] In the following detailed description of the embodiments of the present disclosure, reference is made to the accompanying drawings. Similar reference numerals in the accompanying drawings indicate similar elements, and specific embodiments in which the present disclosure can be practiced are shown in the accompanying drawings by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to implement the present disclosure. In other cases, well-known processes, structures, and techniques are not shown in detail to avoid confusing the understanding of this specification. Therefore, the following detailed description should not be understood as limiting, and the technical solutions of the present disclosure are limited only by the attached claims.

[0071] Example

[0072] A further understanding of the present disclosure can be obtained by referring to some specific embodiments given herein, which are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. Obviously, various modifications and changes can be made to the present disclosure without departing from the essence of the present disclosure, and therefore, these modifications and changes are also within the scope of the protection claimed in this application.

[0073] Example 1: Screening and identification of Propionibacterium freudenreichii subsp. schreiberi strains

[0074] 1. Strain Isolation

[0075] (1) Add 10 g of the sample (cheese) collected from Shangri-La, Yunnan in October 2023 to 90 mL of sterile saline, mix thoroughly, and perform ultrasound for 30 min. -3 Up to 10 -8The diluted solution was spread on solid MRS, placed in an anaerobic bag, and cultured at 30°C for 48 h under anaerobic conditions.

[0076] (2) Streak a single colony similar to Propionibacterium freudenreichii subsp. shermanii onto solid medium MRS for culture. Select a single colony and repeat the streaking 2-3 times to obtain a pure strain of microorganism.

[0077] (3) The purified single colony was inoculated into MRS liquid culture medium and cultured at 30°C for 24-72 h. The strain with the highest protein content and antibacterial activity was obtained through protein content detection and antibacterial effect evaluation. It was named CJPF0001 and the obtained bacterial solution was stored in a glycerol tube.

[0078] In the above separation step, the components of the solid separation medium MRS are: peptone: 10.0 g / L; beef extract powder: 10.0 g / L; yeast extract powder: 5.0 g / L; glucose: 20.0 g / L; sodium acetate: 5.0 g / L; diammonium hydrogen citrate: 2.0 g / L; Tween 80: 1.0 ml / L; dipotassium hydrogen phosphate: 2.0 g / L; magnesium sulfate: 0.2 g / L; manganese sulfate: 0.05 g / L, and agar 20 g / L.

[0079] The components of MRS liquid culture medium are: peptone: 10.0 g / L; beef extract powder: 10.0 g / L; yeast extract powder: 5.0 g / L; glucose: 20.0 g / L; sodium acetate: 5.0 g / L; diammonium hydrogen citrate: 2.0 g / L; Tween 80: 1.0 mL / L; dipotassium hydrogen phosphate: 2.0 g / L; magnesium sulfate: 0.2 g / L; manganese sulfate: 0.05 g / L; sterilize at 121°C for 15 min.

[0080] 2. Molecular identification of CJPF0001

[0081] 2.1. CJPF0001 genome extraction

[0082] After CJPF0001 was cultured in MRS medium at 30°C for 24-72 h, the strain was collected and the genomic DNA of Propionibacterium freudenreichii subsp. schreiberi strain CJPF0001 was extracted using a genome extraction kit (Tiangen DP302-02). The specific operation steps are shown in the kit instructions. In order to study the taxonomy of unknown bacterial strains, the 16SrRNA gene fragment was amplified and sequenced, and its classification information was determined after comparison with the NCBI database. The above method is usually used for molecular identification of common microorganisms.

[0083] 2.2. 16S sequence amplification, sequencing and alignment

[0084] The genomic DNA of strain CJPF0001 was used as a template to amplify the 16S rRNA gene fragment by PCR. The primers used were 27F and 1492R. The sequences are shown in Table 1. The PCR reaction system (20 μL) included 10 × buffer 2.5 μL, metal ion Mg 2+ (25 mmol / L) 1.5 μL, polymerase Taq enzyme 0.25 μL, base dNTP (25 mmol / L) 0.3 μL, 27 F primer (10 mmol / L) 0.5 μL, 1492 R primer (10 mmol / L) 0.5 μL, PCFR extension template DNA 0.1 μL, deionized water 14.35 μL.

[0085] The PCR amplification conditions were as follows: denaturation temperature 94°C, annealing temperature 55°C, extension temperature 72°C, extension time 1.5 min, cycle number 30-35 times, then extension at 72°C for 10 min, and PCR reaction products were stored at 4°C for later use. DNA markers containing fragments of various lengths were used as DNA Maker, and 1% agarose was added to the nucleic acid dye GelRed (0.1%) to prepare gel blocks. An appropriate amount of PCR product was added to the gel blocks, and placed in an electrophoresis instrument filled with TBE (Tris boric acid) buffer. After working at 120-140 V for 20 min, the gel was taken out and observed using a gel imaging system to verify whether the PCR amplification reaction was successful. The PCR product was then sent to BGI Sequencing Company for sequencing, and the primer required for sequencing was 27 F.

[0086] The results of sequencing the core region 16S rRNA gene fragment are shown in SEQ ID NO.1, as follows:

[0087] GGGGGTACACGAGTGGCGAACGGGTGAGTAACACGTGAGGAACGTGCCCTTGACTTCGGTATAGCTCCAGGAAACTGGTGGTAATCCCGAATATGAGCCTGGCCTGCATGGGTTGGGTTGGAAAGCTTTATGCGGTCAGGGATCGTCTCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCAGCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCAACGCCGCGTGCGGGATGACGGCCTTCGGGTTGTAAACCGCTTTCATCCATGACGAAGCGCAAGTGACGGTAGTGGGAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTGATACGTAGGGTGCGAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTTGTAGGCGGTTGATCACGTCGGAAGTGAAATTCCAGGGCTTAACTCTGGGCTTGCTTTCGATACGGGTTGACTTGAGGAATGTAGGGGAGAATGGAACTCTCGGTGGAGCGGTGGAATGCGCAGATATCGGGAAGAACACCAGTGGCGAAGGCGGTTCTCTGGACATTTCCTGACGCTGAGAAGCGAAAGCGTGGGGAGCAAACAGGCTTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTCCCTTCCACGGGGTCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATT。

[0088] The result of NCBI sequence alignment is *Propionibacterium shermanii* subsp. *fermentans*, and the inventor named it CJPF0001.

[0089] Table 1

[0090]

[0091] The CJPF0001 strain of the present application has been deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China) on November 7, 2024, with the deposit number of CCTCC No. M 20242477, and the classification name is Propionibacterium freudenreichii subsp. Propionibacterium freudenreichii subsp. shermanii )CJPF0001.

[0092] Example 2: Fermentation of strain CJPF0001

[0093] 1) The CJPF0001 strain obtained in Example 1 was cultured on an MRS solid medium plate and statically cultured at 30° C. for 48 h;

[0094] 2) Pick a single colony obtained in step 1), activate it with 5 mL of MRS liquid medium, and culture it at 30°C for 48-72 hours;

[0095] 3) The bacterial solution obtained in step 2) was expanded and cultured in 50 mL MRS liquid medium at 30°C for 48-72 hours, and the culture was terminated when OD = 5;

[0096] 4) The bacterial solution obtained in step 3) was inoculated into a fermentation medium (tryptone 10 g / L, sodium lactate 16.7 mL / L, yeast powder 10 g / L, manganese sulfate 0.005 g / L, potassium dihydrogen phosphate 0.25 g / L), and a 1.5 L tank was used for batch feeding fermentation, with an inoculation amount of 0.5-10% and a liquid volume of 1000 mL; fermentation conditions: a rotation speed of 100 rpm, NaOH to control pH=5.5-6.5, anaerobic fermentation culture at 30°C for 96h-120h, and inactivation at 90°C for 30min after the fermentation was completed;

[0097] 5) The inactivated fermentation product is directly freeze-dried or spray-dried, or the bacterial solution is separated by centrifugation, and the supernatant and the bacterial cells are dried separately;

[0098] 6) The protein content of CJPF0001 fermentation product was determined by Kjeldahl method.

[0099] The results are as follows Figure 1 As shown, the protein content by mass is about 30%. Compared with the common biological preservatives of the same type on the market (Yinong Bio SPX / D whey fermentation product) (protein content is 4.9%), the protein content in the CJPF0001 fermentation product is significantly increased.

[0100] Example 3: Evaluation of antibacterial properties of CJPF0001 fermentation products

[0101] 3.1. Inhibition of common spoilage bacteria by CJPF0001 fermentation products

[0102] The fermentation product obtained in Example 2 of the present application was tested for its antibacterial activity against Staphylococcus aureus, Escherichia coli, Aspergillus niger, Aspergillus flavus and baker's yeast.

[0103] The Oxford cup method was used to detect the effects of CJPF0001 fermentation products on Staphylococcus aureus ( Figure 2 A) and Escherichia coli ( Figure 2 B) of the inhibitory effect. With the increase of the addition amount (concentration) of CJPF0001 fermentation product, the transparent zone of Staphylococcus aureus and Escherichia coli gradually increased. The results of colony changes after 24 hours are shown in Figure 2 Middle A and Figure 2 B in the middle indicates that it has a good inhibitory effect on Staphylococcus aureus and Escherichia coli.

[0104] A certain amount of CJPF0001 fermentation product (0.5%-1.5%) was added to the solid culture medium for culturing Aspergillus flavus and Aspergillus niger. After 7 days of cultivation, the results were shown in Figure 3 Middle A and Figure 3 In medium B, the growth of Aspergillus flavus and Aspergillus niger on the plate was significantly inhibited. Even at the lowest added concentration of 0.5% in this experiment, the growth of Aspergillus flavus and Aspergillus niger was significantly inhibited.

[0105] In addition, the effect of CJPF0001 fermentation products on the growth of baker's yeast in the plate was tested. The results are shown in Figure 3 Figure C: Compared with the control, after 48 h of culture, the addition of CJPF0001 fermentation products did not affect the growth of baker's yeast, while common products of the same type on the market had a strong inhibitory effect on baker's yeast.

[0106] The above results show that the fermentation product of CJPF0001 has a good antibacterial effect on Escherichia coli, Staphylococcus aureus, Aspergillus niger and Aspergillus flavus, but will not affect the growth of baker's yeast, and can be added to bread as a raw material.

[0107] 3.2. Application of CJPF0001 fermentation products in bread

[0108] The fermentation product of CJPF0001 was added into toast bread dough in different amounts as bread raw material. After the toast bread was prepared, a challenge experiment was carried out on the bread using Aspergillus niger and Aspergillus flavus.

[0109] The bacterial solution containing 100-300 spores / mL of Aspergillus flavus or Aspergillus niger was inoculated in S-type on the surface of different toasts. Among them, the blank control did not add any preservatives and was stored at room temperature for 7 days.

[0110] The results of microbial growth on the surface of toast after 7 days are shown in Figure 4 It can be seen that after 7 days of storage, the blank control had obvious growth of Aspergillus niger and Aspergillus flavus S type, while no obvious colonies were observed on the toast bread with 0.5% addition of CJPF0001 fermentation product, indicating that the growth of Aspergillus flavus and Aspergillus niger was significantly inhibited.

[0111] In summary, the fermentation product of CJPF0001 can inhibit the growth of Aspergillus flavus and Aspergillus niger in bread in practical applications, prevent bread from spoiling, and has the effect of extending the shelf life of bread.

[0112] Without being limited to any theory, the inventors believe that the Propionibacterium freudenreichii subsp. shermanii strain disclosed herein will produce a variety of organic acids (including but not limited to propionic acid, acetic acid, citric acid, etc.) and antimicrobial peptides and other substances during the fermentation process. The types, contents, proportions, etc. of these substances are different from those of existing Propionibacterium freudenreichii subsp. shermanii strains, and therefore it exhibits higher protein content and better antibacterial activity.

[0113] The above-mentioned embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be subject to the attached claims.

Claims

1. A strain of Propionibacterium freudenreichii subsp. schreberi, which is classified and named Propionibacterium freudenreichii subsp. schreberi ( Propionibacterium freudenreichii subsp. shermanii )CJPF0001, its deposit number is CCTCCNo. M 20242477.

2. A composition with an antiseptic function, comprising an effective amount of Propionibacterium freudenreichii subsp. schreberi strain and a fermentation product of Propionibacterium freudenreichii subsp. schreberi strain in a fermentation medium, and acceptable excipients; The deposit number of the Propionibacterium freudenreichii subsp. schreiberi strain is CCTCC No. M 20242477.

3. The composition according to claim 2, wherein The acceptable excipients are excipients acceptable in the field of food science.

4. The composition according to claim 2, wherein The composition is liquid or solid.

5. The composition according to claim 4, wherein The composition is in the form of tablets, granules, powders, solutions, suspensions, emulsions or premixes.

6. The composition according to claim 2, wherein The fermentation medium contains at least a carbon source and a nitrogen source.

7. A method for preparing the fermentation product in the composition as claimed in claim 2, comprising at least the following steps: 1) using a solid culture medium plate to culture the Propionibacterium freudenreichii subsp. schreberi strain CJPF0001 described in claim 1; 2) Pick the single colony obtained in step 1) and activate the culture; 3) expanding the bacterial solution obtained in step 2); 4) inoculating the bacterial solution obtained in step 3) into the fermentation medium for batch fed-batch fermentation with an inoculation amount of 0.5%-10%; 5) The inactivated fermentation product is directly freeze-dried or spray-dried, or the bacterial liquid is separated by centrifugation, and the supernatant and the bacterial cells are dried separately to obtain the fermentation product.

8. Use of the Propionibacterium freudenreichii subsp. shermanii strain according to claim 1 and the composition according to any one of claims 2 to 6 in the preparation of food, medicine or cosmetics.

9. Use of the composition according to any one of claims 2 to 6 in the preparation of food, medicine or cosmetics.

10. Use of the Propionibacterium freudenreichii subsp. shermanii strain of claim 1 and the composition of any one of claims 2 to 6 for inhibiting the growth of Staphylococcus aureus, Escherichia coli, Aspergillus flavus or Aspergillus niger, wherein the use is for non-disease treatment or diagnosis purposes.

11. Use of the composition according to any one of claims 2 to 6 for inhibiting the growth of Staphylococcus aureus, Escherichia coli, Aspergillus flavus or Aspergillus niger, wherein the use is for non-disease treatment or diagnosis purposes.

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