Multifunctional fermentation agent strain and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a multifunctional starter strain and its application. The Lactobacillus delbrueckii subspecies bulgaricus Ldelb-1 strain and the thermophilic Streptococcus salivarius subspecies Ssali-1 strain of the present invention can be used as starters to produce fermented dairy products with excellent texture and taste, and the two strains have the effects of anti-oxidation, anti-inflammatory, antibacterial, cholesterol-lowering, inhibiting body fat absorption, inhibiting skin fat secretion, and improving acne, and are potential strains integrating fermentation and multiple prebiotic functions.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a multifunctional fermentation agent strain and application thereof. Background Art

[0002] As consumers pay more and more attention to healthy eating, the demand for yogurt, a dairy product rich in probiotics and easy to digest and absorb, has shown a steady growth trend in the Chinese market. The production of yogurt depends on specific starter cultures, which are mainly composed of lactic acid bacteria that can convert lactose into lactic acid. Fermentation gives yogurt a unique flavor and texture. However, existing yogurt starter cultures still have some limitations, such as insufficient bacterial diversity, low fermentation efficiency, short product shelf life, and failure to fully meet consumers' special nutritional and functional needs.

[0003] Therefore, the market demand for high-performance fermentation agents is becoming increasingly urgent. Globally, Europe is the largest dairy fermentation agent market, accounting for more than 40% of the market share, while Asia Pacific and North America account for more than 45% of the market share. In this field, international giants such as Chr. Hansen, Danisco, and DSM have a market share of more than 85% in China, which means that the market share of domestic brands is less than 15%, indicating that my country has huge potential and development space in the development of fermentation agents with independent intellectual property rights.

[0004] Traditional yogurt starters mainly use Lactobacillus bulgaricus (full name Lactobacillus delbrueckii subsp. bulgaricus) and Streptococcus thermophilus (Streptococcus salivarius subsp. thermophilus). Although these two strains can realize the basic yogurt fermentation process, they lack functionality and diversity. In order to cater to the market demand for specific health benefits, such as antioxidants, enhancing immunity and improving intestinal health, Bifidobacterium longum ( Bifidobacterium longum 、Bifidobacterium animalis( Bifidobacterium animalis )、Lactobacillus plantarum( Lactobacillus plantarum However, this approach also brings new challenges, such as whether the added functional probiotics will affect the activity of the basic fermentation flora, whether it will change the taste of yogurt or affect its fermentation characteristics.

[0005] In view of the above situation, the present invention proposes a new type of yogurt starter, which aims to overcome the shortcomings of existing starters, provide better quality yogurt products, optimize the taste and bring additional health benefits to consumers. Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus salivarius subspecies thermophilic in the starter have multiple intestinal probiotic functions and are also beneficial to the skin, and have the potential for multiple application scenarios. Summary of the invention

[0006] The present invention first provides a yogurt starter, wherein the starter contains Lactobacillus delbrueckii subspecies bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) Ldelb-1 strain and Streptococcus salivarius subsp. thermophilus ( Streptococcus salivarius subsp. thermophilus ) Ssali-1 strain, the microbial preservation number of Ldelb-1 is CCTCC NO: M 20241013, and the preservation number of Ssali-1 is CCTCC NO: M 20241014.

[0007] In some specific embodiments, the ratio of the Lactobacillus delbrueckii subsp. bulgaricus Ldelb-1 strain and the Streptococcus salivarius subsp. thermophilus Ssali-1 strain in the aforementioned yogurt starter is 1:100 to 1:1.

[0008] In some specific embodiments, the ratio of the Lactobacillus delbrueckii subsp. bulgaricus Ldelb-1 strain and the Streptococcus salivarius subsp. thermophilus Ssali-1 strain in the aforementioned yogurt starter is 1:100 to 1:10.

[0009] In some specific embodiments, the ratio of the Lactobacillus delbrueckii subsp. bulgaricus Ldelb-1 strain and the Streptococcus salivarius subsp. thermophilus Ssali-1 strain in the aforementioned yogurt starter is 1:10 to 1:1.

[0010] The present invention also provides a fermented dairy product, which contains the aforementioned Lactobacillus delbrueckii subspecies bulgaricus Ldelb-1 strain or Streptococcus salivarius thermophilic subspecies Ssali-1 strain.

[0011] Furthermore, the present invention provides a fermented dairy product, which is obtained by fermenting the aforementioned Lactobacillus delbrueckii subsp. bulgaricus Ldelb-1 strain and Streptococcus salivarius thermophilic subsp. Ssali-1 strain.

[0012] In some embodiments, the fermented dairy product is yogurt.

[0013] The present invention also provides a method for preparing a fermented dairy product, the method comprising: inoculating the aforementioned starter into a milk base, and fermenting at 35-50°C.

[0014] Secondly, the present invention provides a multifunctional strain, which is: Lactobacillus delbrueckii subspecies bulgaricus Ldelb-1 strain with a preservation number of CCTCC NO: M20241013, or Streptococcus salivarius subspecies thermophilic Ssali-1 strain with a preservation number of CCTCC NO: M 20241014.

[0015] Furthermore, the present invention provides a method for culturing the aforementioned multifunctional strain, comprising inoculating the Ldelb-1 strain or the Ssali-1 strain into a culture medium, performing proliferation culture, and obtaining a proliferated strain.

[0016] The present invention also provides the use of a multifunctional strain in non-disease treatment purposes, wherein the non-disease treatment purposes are selected from one or more of the following: anti-oxidation, anti-inflammatory, antibacterial, cholesterol-lowering, inhibition of body fat absorption, inhibition of skin fat secretion, and improvement of acne, and the multifunctional strain is selected from the Ldelb-1 strain and the Ssali-1 strain.

[0017] In some specific embodiments, the antibacterial activity includes inhibiting pathogenic Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Shigella dysenteriae, Yersinia enterocolitica, Staphylococcus aureus, and Epidermidis herpes simplex.

[0018] The invention also provides application of the multifunctional strain in preparing medicine for treating acne or obesity.

[0019] Finally, the present invention provides a composition, which is food, medicine or cosmetics, and contains the Ldelb-1 strain or the Ssali-1 strain.

[0020] The results of the present invention are based on two excellent strains: Lactobacillus delbrueckii subsp. bulgaricus Ldelb-1 and Streptococcus salivarius thermophilic subsp. Ssali-1. The two strains are highly efficient in yogurt fermentation, and the texture and taste of yogurt fermented by them are better than those produced by commercial starter cultures. What is even more surprising is that the two strains themselves have multiple probiotic functions, which can avoid the introduction of functional probiotics that makes it impossible to take into account the quality of the fermented products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The frontal photos of the colony morphology of the strains. A. Lactobacillus delbrueckii subsp. bulgaricus Ldelb-1 B. Streptococcus salivarius subsp. thermophilus Ssali-1.

[0022] Figure 2 The growth curves of the two bacterial strains are shown in Figure 2.

[0023] Figure 3 This is the result of the interaction between strains Ldelb-1 and Ssali-1.

[0024] Figure 4 These are the sensory evaluation results of different compound ratios of Lactobacillus bulgaricus and Streptococcus thermophilus.

[0025] Figure 5 This is the total score of skin lesions in each group at the end of the acne model mouse experiment.

[0026] Figure 6 These are the skin thickness results of each group at the end of the acne model mouse experiment.

[0027] Figure 7 These are the results of TNF-α detection in skin tissues of each group of acne model mice.

[0028] Figure 8 Representative pictures of the back skin of mice in each group at the end point of the experiment. DETAILED DESCRIPTION

[0029] The meaning of the "strain" with the specific deposit number claimed in the present invention includes but is not limited to:

[0030] (1) A strain with a specific accession number deposited in the above-mentioned depository;

[0031] (2) Strains with the same genome as the strain described in (1), including clonal strains;

[0032] (3) A non-mutated strain based on the above (1) or (2), including progeny strains;

[0033] (4) A bacterial strain based on (1), (2) or (3) above that accumulates minor mutations during subculture but has no substantial changes in toxicity, immunogenicity or biological activity.

[0034] As is known in the art, minor mutations are inevitably introduced into strains through passage and application. When mutations occur in non-coding sequence regions or synonymous mutations in coding regions or mutations that do not affect the toxicity, immunogenicity and biological activity of the strain (for example, it may be a connecting amino acid residue between two domains, or a minor mutation residue located inside the higher-level structure of a protein that does not contact immune cells and does not affect toxicity, immunogenicity and biological activity), it can be reasonably expected that when these minor changes do not significantly affect the toxicity, immunogenicity and biological activity of the offspring strains, the purpose of the present invention can still be achieved, and they are derived from the strains contributed by the present invention, and therefore are still within the scope of the substantial technical contribution of the present invention. These minor mutations are still non-substantial mutations and should be regarded as mutant strains with no changes in toxicity, immunogenicity and biological activity.

[0035] There is no substantial change in toxicity, immunogenicity and biological activity, including but not limited to, within the limits of detection technology such as detection sensitivity and detection limit and acceptable or unavoidable errors, the toxicity, immunogenicity and biological activity are considered to be the same. When the toxicity, immunogenicity and biological activity of the offspring of the strain are measured using cells and animals, there is no substantial change due to differences in cell strains, animal species, age, sex, health status, culture conditions, etc., as well as foreseeable or unavoidable systematic errors.

[0036] "Progeny strain" refers to a descendant strain produced by an original strain or parent strain, including those strains that have undergone multiple generations but still retain the key characteristics and functional properties described in this patent application.

[0037] "Clone strain" means a genetically identical offspring strain that is directly derived from a single parent strain or its progeny through asexual reproduction (such as cell division or gene duplication).

[0038] "Fermented dairy products" are a class of products made through the action of microorganisms (mainly lactic acid bacteria, but can also include yeast and other bacteria) to make controlled biochemical changes in milk or milk components. These microorganisms use sugars in milk (such as lactose) as an energy source to produce metabolites such as lactic acid, which leads to a decrease in the pH value of the milk and coagulation of proteins, forming a unique texture, flavor and extending the shelf life. Common fermented dairy products include yogurt, cheese, curd, fermented milk drinks, etc.

[0039] "Milk-based" usually refers to products or formulas that use milk (cow's milk, sheep's milk, goat's milk and other animal milk) as the main raw material.

[0040] “Ratio” refers to the quantitative relationship between different components. Unless otherwise specified in the present invention, the ratio of bacteria refers to the ratio of bacterial counts (CFU).

[0041] The reagents / culture media used in the examples are prepared as follows. Those not specifically described can be prepared according to conventional methods or obtained commercially.

[0042] Preparation of anaerobic MRS & M17 liquid culture medium: weigh 27 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312), 21.15 g of M17 broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB0391), 1.5 g of oligofructose (Shanghai Yuanye Biotechnology Co., Ltd., S11133), and 1.5 g of oligogalactose (Shanghai Yuanye Biotechnology Co., Ltd., S11138), and dissolve them in 1 L of distilled water. 2 Replace and deoxygenate, repackage, sterilize at 121℃ for 30 min, and store in a cool, dry place.

[0043] Preparation of anaerobic MRS&M17 solid culture medium: Add 12g of agar powder to the anaerobic MRS&M17 liquid culture medium. The other ingredients and steps are the same.

[0044] Preparation of anaerobic BHI & MRS & GAM liquid culture medium: weigh 19.25 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-5), 13.5 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312), and 15 g of modified GAM broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8518-3), and dissolve them in 1 L of distilled water. 2 Replace and deoxygenate, repackage, sterilize at 121℃ for 30 min, and store in a cool, dry place.

[0045] Preparation of aerobic BHI liquid culture medium: weigh 38.5 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-5), dissolve it in 1 L of distilled water, sterilize it at 121°C for 30 min, and store it in a cool and dry place.

[0046] Preparation of anaerobic BHI & TSB liquid medium: weigh 19.8 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-5) and 15 g of TSB broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB4114-19), dissolve in 1 L of distilled water, N 2 Replace and deoxygenate, repackage, sterilize at 121℃ for 30 min, and store in a cool, dry place.

[0047] Preparation of anaerobic BHI&TSB&cholesterol liquid culture medium: Weigh 0.5 g of cholesterol (Sigma-Aldrich Trading Co., Ltd., C8667), add it to 25 mL of Tween 80 (Shanghai MacLean Biochemical Technology Co., Ltd., T6336), heat it in a 90℃ water bath until the cholesterol is dissolved (about 40-60 min), then add the mixture to the packaged anaerobic BHI&TSB liquid culture medium (add 10 mL of cholesterol & Tween 80 mixture to every 400 mL of anaerobic BHI&TSB liquid culture medium), sterilize it at 121℃ for 15 min, and store it in a cool and dry place.

[0048] Example 1 Isolation and identification of strains

[0049] Lactobacillus bulgaricus Ldelb-1 was isolated from traditional fermented yogurt in Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, and was obtained by facultative anaerobic culture at 37°C for 4 days using MRS medium (Guangdong Huankai Biotechnology Co., Ltd., 027312). Streptococcus thermophilus Ssali-1 was isolated from traditional fermented yogurt in Yushu City, Qinghai Province, and was obtained by facultative anaerobic culture at 37°C for 2 days using MRS medium (Guangdong Huankai Biotechnology Co., Ltd., 027312).

[0050] After picking the single clones, they were sent to the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA gene sequencing results obtained by sequencing were analyzed, and the sequences were compared with the NCBI Nucleotide database to preliminarily identify the species of the isolated strains. They were Lactobacillus delbrueckii subspecies bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ), named Ldelb-1, and Streptococcus salivarius subsp. thermophilus ( Streptococcus salivarius subsp. thermophilus), named Ssali-1. After culturing in anaerobic MRS&M17 solid medium for 48 hours, the colony morphology was white opaque round colonies with a convex middle and a smooth and moist surface. Figure 1 , A is Lactobacillus delbrueckii subspecies bulgaricus Ldelb-1, and B is Streptococcus salivarius thermophilic subspecies Ssali-1.

[0051] The deposit information of Ldelb-1 and Ssali-1 strains are as follows:

[0052] Strain name: Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus )Ldelb-1

[0053] Date of deposit: May 20, 2024

[0054] Depository: China Center for Type Culture Collection (CCTCC), Address: Wuhan University, Wuhan, Hubei, Postal Code: 430072, Tel: 027-68754052

[0055] Deposit number: CCTCC NO: M 20241013.

[0056] Strain name: Streptococcus salivarius subsp. thermophilus ( Streptococcus salivarius subsp. thermophilus )Ssali-1

[0057] Date of deposit: May 20, 2024

[0058] Depository: China Center for Type Culture Collection (CCTCC), Address: Wuhan University, Wuhan, Hubei, Postal Code: 430072, Tel: 027-68754052

[0059] Deposit number: CCTCC NO: M 20241014.

[0060] Example 2 Whole genome analysis and identification

[0061] The strains Ldelb-1 and Ssali-1 were inoculated into 5 mL of anaerobic MRS&M17 liquid culture medium at a 2% (v / v) inoculation rate, cultured to the late logarithmic growth stage, and the whole genome DNA of the strains was extracted. The whole genome was sequenced using the Illumina high-throughput sequencing platform NovaSeq 6000. After assembly and annotation by conventional methods, the protein sequences were compared with the VFDB (Virulence Factor Databases) and CARD (The Comprehensive Antibiotic Resistance Database) databases for virulence factor and drug resistance gene analysis. The results showed that strains Ldelb-1 and Ssali-1 did not have virulence factors and drug resistance genes, and were safe at the genetic level.

[0062] The average nucleotide identity (ANI) was used to analyze the novelty of strains Ldelb-1 and Ssali-1. A total of 304 published Lactobacillus delbrueckiisubsp.bulgaricus ( Lactobacillus bulgaricus ) The whole genome of the species was compared with that of Ldelb-1 by fastANI (v1.33). The two strains most similar to the whole genome of Ldelb-1 were ASM1871933v1 (ANI= 99.65%) and ASM1871956v1 (ANI= 99.63%). There are 538 published Streptococcus salivarius subsp. thermophilus ( Streptococcus thermophilus ) species, and compared with the whole genome of the genus Ssali-1 using fastANI (v1.33), it was found that the two strains most similar to the whole genome of Ssali-1 were GCA_021293505.1 (ANI=99.96%) and GCA_022024355.1 (ANI=99.95%), so Ldelb-1 and Ssali-1 are both new strains with novelty.

[0063] In addition, according to literature reports, there is a type of cell wall protease PrtS in Streptococcus thermophilus, which is crucial for quickly reducing the pH value and improving fermentation efficiency during fermentation. It can decompose milk protein, provide peptides and amino acids, and enable Streptococcus thermophilus to grow rapidly in milk, but it only exists in a few strains. The PrtS sequence information of Streptococcus thermophilus (AAG09771.1) was downloaded from NCBI, and the key enzyme sequence index was constructed using blast 2.16.0. The protein sequence of the predicted gene (prokka 1.14.5) of the whole genome of strain Ssali-1 was compared, and the PrtS protein sequence of strain Ssali-1 was obtained as follows SEQ ID NO: 1:

[0064]

[0065] The cell wall-bound protease PrtB exists in Lactobacillus bulgaricus, which can degrade casein into large molecular polypeptides. It is very important for the rapid fermentation and flavor formation of yogurt, and affects the acidity and aroma of yogurt. In mixed culture with Streptococcus thermophilus, the presence of PrtB is also necessary for the growth of Streptococcus thermophilus. Its absence will lead to an increase in the final pH value, affecting the acidification of yogurt. After downloading the sequence information of Lactobacillus bulgaricus PrtB from NCBI (AAC41529.1) and using blast 2.16.0 to construct a key enzyme sequence index, the protein sequence of the predicted gene (prokka1.14.5) of the whole genome of strain Ldelb-1 was compared. The predicted PrtB protein sequence of strain Ldelb-1 is as follows SEQ ID NO:2:

[0066]

[0067] Example 3 Strain Growth Characteristics

[0068] The preserved Ldelb-1 and Ssali-1 were inoculated into 5 mL of anaerobic MRS&M17 liquid medium, respectively. The activated strains were transferred into 5 mL of anaerobic MRS&M17 liquid medium, and 3 parallels were set for each strain. The culture was carried out anaerobically at 42°C, and the OD was measured at 0 h, 2 h, 4 h, 6 h, 9 h, 12 h, 24 h, 36 h, 48 h, and 72 h. 600 The growth curve is drawn as Figure 2 The lag phase of strain Ldelb-1 was about 6 h, and it reached the initial stable state at about 24 h. 600 The value can reach about 1.0, and the curve is still stable when the stable period reaches 72 hours; the lag period of strain Ssali-1 is about 2 hours, and it reaches the initial stable stage at about 12 hours, with a faster growth rate, and the OD 600 The value can reach about 0.8, and the curve is still stable when the stable period reaches 72 hours. The strain grows faster in the early stage and has strong stability in the later stage, which is in line with the characteristics of fermentation strain selection.

[0069] Example 4 Low temperature stability

[0070] The preserved Ldelb-1 and Ssali-1 were inoculated into 5 mL of anaerobic MRS&M17 liquid culture medium, and the activated strains were transferred into 5 mL of anaerobic MRS&M17 liquid culture medium. Three parallels were set for each strain, and anaerobically cultured at 42°C until the late logarithmic growth period. The viable bacteria in the cultured solution were counted and recorded as D0. The remaining culture solution was then stored in a 4°C refrigerator. The viable bacteria were sampled and tested on the 7th, 14th, and 21st days (D7, D14, and D21) to explore the stability of the strains under low-temperature storage. The viable bacteria counting method was carried out in accordance with GB 4789.35-2023 National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test, and the results were expressed as Mean±SD. As shown in Tables 1 and 2, after 21 days of storage at 4°C, the viable bacteria counts of strains Ldelb-1 and Ssali-1 decreased by less than one order of magnitude, and the low-temperature stability was good, which was suitable for long-term storage.

[0071] Table 1 Number of viable bacteria after low temperature storage

[0072]

[0073] Table 2 Results of the order of magnitude decrease in the number of viable bacteria after low-temperature storage

[0074]

[0075] Example 5 Mutual influence of strain growth

[0076] Recent studies have shown that not all Lactobacillus bulgaricus and Streptococcus thermophilus The strains are all symbiotic, and their interactions may also be antagonistic.

[0077] The preserved Ldelb-1 and Ssali-1 were inoculated into 5 mL of anaerobic MRS&M17 liquid medium, respectively. The culture solution was streaked crosswise on anaerobic MRS&M17 solid medium (first streaking horizontally for Ssali-1 and then vertically for Ldelb-1), and cultured anaerobically at 42°C for 48 h. The results were as follows. Figure 3 As shown, there is no mutual inhibition between strains Ldelb-1 and Ssali-1. Therefore, the two strains can be used in combination with each other.

[0078] Example 6 Strain Functionality Test

[0079] This example investigates the probiotic functions of strains Ldelb-1 and Ssali-1 to evaluate whether the two strains meet the requirements for diverse efficacy.

[0080] (1) Self-agglutination ability

[0081] The preserved Ldelb-1 and Ssali-1 were inoculated into 5 mL of anaerobic MRS&M17 liquid culture medium respectively, and the activated strains were transferred into 5 mL of anaerobic MRS&M17 liquid culture medium respectively. Three parallels were set for each strain and cultured anaerobically at 42°C until the late logarithmic growth stage.

[0082] The culture solution was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the solution was resuspended with PBS. This process was repeated twice. PBS was used as a blank control. The OD value of each resuspended solution sample was calculated. 600 Adjust to 0.5±0.1, recorded as A 0 , incubate at 42°C for 7 h, 18 h, and 24 h, and measure OD 600 , denoted as A x . Calculation method of self-agglutination: Self-agglutination rate (%) = (A 0 –A x ) / A 0 ×100%, and the results are expressed as Mean±SD. As shown in Table 4, after 24 h of static standing, the self-agglutination rate of strain Ldelb-1 reached 95.41%±0.85%, and the self-agglutination rate of strain Ssali-1 reached 89.69%±3.39%.

[0083] The above results showed that both strains had strong self-aggregation ability, potential adhesion, and were easy to colonize in the gastrointestinal tract.

[0084] (2) Antioxidant capacity

[0085] The preserved Ldelb-1 and Ssali-1 were inoculated into 5 mL of anaerobic MRS&M17 liquid culture medium respectively, and the activated strains were transferred into 5 mL of anaerobic MRS&M17 liquid culture medium respectively. Three parallels were set for each strain and cultured anaerobically at 42°C until the late logarithmic growth stage.

[0086] Sample processing: 1 mL of culture solution was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the sample was resuspended with 1 mL of the extract solution (pre-cooled) in the total antioxidant capacity detection kit; it was transferred to a sterilized screw-cap tube containing beads (Sigma-Aldrich, G4649-1KG), and the sample was broken by oscillation once with a rapid sample preparation instrument (parameter setting: 4.5 m / s, 30 s), and then centrifuged at 12000 rpm at 4℃ for 10 min, and the supernatant was placed on ice for testing. The BCA protein concentration determination kit (the kit was purchased from Beijing Solebold Technology Co., Ltd., PC0020) was used to draw a standard curve and detect BCA samples according to the instructions of the kit. The total antioxidant capacity detection kit of the strain (the kit was purchased from Beijing Solebold Technology Co., Ltd., BC1315) was used to determine the antioxidant capacity of the sample in combination with the standard curve according to the instructions of the kit. The total antioxidant capacity unit is μmol / mg prot, and the results are expressed as Mean±SD.

[0087] The results are shown in Table 3. The total antioxidant capacity of strain Ldelb-1 reached 0.556 ± 0.009 μmol / mg prot, and the total antioxidant capacity of strain Ssali-1 reached 0.414 ± 0.016 μmol / mg prot, indicating that both strains have certain antioxidant capacity.

[0088] (3) Cholesterol-lowering ability

[0089] The preserved Ldelb-1 and Ssali-1 were inoculated into 5 mL of anaerobic MRS&M17 liquid culture medium, respectively. The activated strains were transferred into 1 mL of anaerobic BHI&TSB&cholesterol liquid culture medium. Three parallels were set for each strain. The culture was anaerobically cultured at 42°C until the late logarithmic growth period, and the cholesterol content was detected.

[0090] Take 1 mL of culture solution and centrifuge at 12000 rpm for 10 min, and take the supernatant to obtain the test sample. The total cholesterol (TC) content detection kit (Beijing Solebold Technology Co., Ltd., BC1985) was used to draw a standard curve and detect the total cholesterol content according to the instructions of the kit. The cholesterol degradation rate was calculated as follows: cholesterol degradation rate % = (cholesterol content in blank culture medium - cholesterol content in experimental group) / cholesterol content in blank culture medium × 100%, and the results were expressed as Mean ± SD.

[0091] The results are shown in Table 3. The cholesterol clearance rate of strain Ldelb-1 was 83.19±6.30%, and the cholesterol clearance rate of strain Ssali-1 was 71.19±9.10%, indicating that both strains have strong cholesterol-lowering ability.

[0092] Table 3 Results of strain functional test

[0093]

[0094] (4) Antibacterial ability against pathogenic bacteria

[0095] Seven common pathogens that cause diarrhea and / or skin problems were selected for antibacterial ability testing. The source information of the pathogenic strains is as follows:

[0096] Table 4 Information on the source of pathogenic bacteria beads

[0097]

[0098] The preserved Ldelb-1 and Ssali-1 were inoculated into 5 mL of anaerobic MRS&M17 liquid medium, and the activated strains to be tested were transferred into 5 mL of anaerobic MRS&M17 liquid medium. Three parallels were set for each strain and anaerobically cultured at 42°C for 48 h. The preserved pathogens shown in Table 4 were inoculated into 5 mL of aerobic BHI liquid medium (ATCC 6919 was inoculated into anaerobic BHI&MRS&GAM liquid medium), and the activated pathogens were transferred into the same medium and cultured at 37°C until the late logarithmic growth period.

[0099] The fermentation liquid (including the bacteria) of the strain to be tested after culturing for 48 hours was sterilized by high pressure steam at 121°C for 20 minutes, and the supernatant was taken and filtered with a 0.22 μm filter to obtain the inactivated sample to be tested (i.e., the fermentation product of the strain to be tested and the bacterial lysate) and stored at 4°C for later use. 600 , and add an appropriate amount of the corresponding culture medium of the pathogenic bacteria to adjust the OD 600 To 0.1±0.02 (ATCC 6919 was adjusted to 0.2±0.04). Take 0.1 mL and adjust the OD600 The pathogenic bacteria suspension was plated in a 96-well plate, and 0.1 mL of the inactivated sample was added. An equal volume of pathogenic bacteria culture medium was used as a positive control. Each group had 3 parallels and was cultured according to the culture conditions of the pathogenic bacteria. The OD at 0 h and 24 h of culture was detected. 600 , calculate △OD 600 The results were expressed as Mean ± SD, and the calculation formula of the inhibition rate was as follows:

[0100]

[0101] Experimental results: As shown in Table 5, strain Ldelb-1 has inhibitory ability against pathogenic Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Shigella dysenteriae, Yersinia enterocolitica, Staphylococcus aureus, and Epidermidis herpes; strain Ssali-1 has inhibitory ability against pathogenic Escherichia coli, Salmonella enterica, Shigella dysenteriae, Yersinia enterocolitica, Staphylococcus aureus, and Epidermidis herpes.

[0102] Table 5 Results of strain functional test

[0103] Pathogens Ldelb-1 antibacterial ability (%) Ssali-1 antibacterial ability (%) Pathogenic Escherichia coli 94.91 ± 2.90 64.53 ±2.94 Pseudomonas aeruginosa 95.25 ±0.37 0.00 ±0.00 Salmonella enterica (Salmonella paratyphi B) 85.93 ±1.79 50.57 ±2.69 Shigella dysenteriae 98.42 ±2.23 99.30 ±0.57 Yersinia enterocolitica 96.42 ±1.36 98.18 ±0.50 Staphylococcus aureus 86.36 ±4.34 49.93 ±1.83 Epidermidis herpes simplex (Propionibacterium acnes) 95.84 ±0.84 77.49 ±4.21

[0104] (5) In vitro cell inflammation inhibition test

[0105] Cell culture and plating: Human colon cancer cells HT29 were cultured in DMEM medium (Gibco, C11995500BT) containing 10% (v / v) FBS (Gibco, 16000-044). When the cell density reached 70%-80%, the cells were rinsed twice with PBS buffer (Wuhan Boster Biotechnology Co., Ltd.), and then 2 mL of trypsin cell digestion solution (Gibco, C25200-056) was added and placed in 37°C 5% CO 2 After digestion in the incubator for 3-5 min, add DMEM medium containing 10% (v / v) FBS to terminate digestion. Centrifuge the cell suspension at 800 rpm for 5 min. Discard the supernatant and resuspend in DMEM medium containing 5% (v / v) FBS. 4 HT29 cells were seeded in a 96-well plate in a 5% CO 2 Incubator, 37℃ for 24 h.

[0106] Strain culture: 200 μL of Ldelb-1 and Ssali-1 bacterial suspension were inoculated into 5 mL of anaerobic MRS&M17 liquid medium from the bacterial storage, and cultured anaerobically in a 42°C electric constant temperature incubator for 24 h. After one transfer, cultured anaerobically for 8 h. Take 1 mL of bacterial suspension and centrifuge at 5000 rpm / min for 15 min. Dilute to 1×10 with DMEM medium containing 1 μg / mL LPS and 1% (v / v) FBS. 7 CFU / mL is reserved.

[0107] Grouping and administration and testing: After HT29 cells were plated and cultured for 24 h, the old culture medium was removed and the cells were divided into normal control group, model group, dexamethasone group and sample group, with 3 parallels in each group. 200 μL of DMEM culture medium containing 1% (v / v) FBS was added to the normal control group; 200 μL of DMEM culture medium containing 1 μg / mL LPS and 1% (v / v) FBS was added to the model group; dexamethasone group was supplemented with dexamethasone (Sigma-Aldrich Company, D4902-25) with a final concentration of 25 μg / mL on the basis of the model group; 200 μL of Ldelb-1 and Ssali-1 bacterial solution samples prepared in the previous step were added to the sample group on the basis of the model group. Cells in each group were placed in 5% CO 2 After culturing in an incubator at 37°C for 24 h, 100 μL of cell culture medium was taken from each group and centrifuged for 10 min (4°C, 1000 rpm / min). The supernatant was collected and the IL-8 content was detected using a Human IL-8 ELISA kit (Wuhan Elaruite Biotechnology Co., Ltd., E-EL-H6008).

[0108] The test results are shown in Table 6. The expression of IL-8 in the model control group was significantly higher than that in the normal control group (P<0.01); the expression of IL-8 in the dexamethasone group was significantly lower than that in the model group, showing anti-inflammatory effects; the Ldelb-1 sample group and the Ssali-1 sample group were able to significantly reduce the expression of the pro-inflammatory factor IL-8 compared with the model group (P<0.01), indicating that the two strains of bacteria have good anti-inflammatory effects in vitro. The results are expressed as Mean±SD, and One-way ANOVA was used for multiple comparison analysis. ** indicates that compared with the model group, p<0.01; * indicates that compared with the model group, p<0.05.

[0109] Table 6 Expression of inflammatory factor IL-8 in each group

[0110]

[0111] (6) Lipid removal ability

[0112] Many skin problems usually involve abnormal lipid synthesis. Linoleic acid is used to induce human sebaceous gland cells SZ95 to synthesize lipids, forming a cellular lipid accumulation model. The ability of bacteria to clear lipid accumulation is evaluated by detecting the amount of cellular lipid accumulation.

[0113] SZ95 cells were collected at 3×10 4 The number of cells / well was inoculated in a black light-proof 96-well plate and placed in a 5% CO 2 Incubator, culture at 37℃ for 24 h, then remove the supernatant. The cells were divided into blank control group, model group, isotretinoin group, Ldelb-1 sample group and Ssali-1 sample group, and the required working solution for each group was prepared.

[0114] 1 mM linoleic acid working solution: Linoleic acid was dissolved in a mixture of DMSO and ethanol (1:1, v / v) to prepare a 1 M linoleic acid stock solution, which was then diluted 1000-fold using serum-free DMEM medium.

[0115] 100 μM isotretinoin working solution: Isotretinoin was dissolved in DMSO to prepare a 100 mM isotretinoin stock solution, which was then diluted 1000-fold with linoleic acid working solution.

[0116] The Ldelb-1 and Ssali-1 linoleic acid working solutions were prepared by adding the corresponding Ldelb-1 and Ssali-1 bacteria to the linoleic acid working solution, 200 μL was added to each well, and the final concentration was 3×10 6 CFU / well.

[0117] According to the experimental plan, 200 μL of serum-free DMEM medium was added to the blank control group, 200 μL of linoleic acid working solution was added to the model group, 200 μL of isotretinoin working solution was added to the isotretinoin group, and 200 μL of Ldelb-1 linoleic acid working solution and Ssali-1 linoleic acid working solution were added to the sample groups, respectively.

[0118] After adding the sample, place in 5% CO 2 Incubator, 37°C for 24 h. According to the instructions of lipid droplet red fluorescence detection kit (Biyuntian Biotechnology Co., Ltd., C2051S), 100 μL of dye was added to each well for staining, incubated at 37°C in the dark for 10 min, and then detected. The excitation light was 485 nm and the emission light was 535 nm.

[0119] The test results are shown in Table 7. After linoleic acid induction, cell lipid secretion increased and obvious lipid accumulation occurred. The lipid accumulation in the model group was significantly higher than that in the normal control group (p<0.01); the lipid accumulation in the isotretinoin group was significantly reduced compared with the model group (p<0.01); the lipid accumulation in the Ldelb-1 sample group and the Ssali-1 sample group was significantly reduced compared with the model group (p<0.05, p<0.01); indicating that both have skin oil control effects. The results are expressed as Mean±SD, and One-way ANOVA was used for multiple comparison analysis. ** indicates that compared with the model group, p<0.01; * indicates that compared with the model group, p<0.05.

[0120] Table 7 Lipid accumulation removal ability test results

[0121]

[0122] It can be seen from the above that strains Ldelb-1 and Ssali-1 have multiple excellent probiotic functions.

[0123] Example 7 Ldelb-1 and Ssali-1 inhibit fat synthesis in zebrafish

[0124] Sample preparation:

[0125] The preserved Ldelb-1 and Ssali-1 were inoculated into 5 mL of anaerobic MRS&M17 liquid medium, and the activated strains were transferred into 50 mL of anaerobic MRS&M17 liquid medium, and cultured anaerobically at 42°C until the late logarithmic growth period. The bacterial solution was centrifuged at 5000 rpm / min for 15 min, and the supernatant was discarded. The bacterial cells were diluted and mixed with physiological saline to make concentrations of 1×10 8 CFU / mL of bacterial suspension.

[0126] Experimental grouping and administration: Wild-type AB zebrafish (purchased from Hangzhou Huante Biotechnology Co., Ltd.) were selected and randomly divided into 5 groups, namely normal control group, model control group, orlistat group, Ldelb-1 group, and Ssali-1 group, with 30 fish in each group. The normal control group and the model control group were given PBS solution, the positive control orlistat group was given orlistat at a concentration of 15.0 μg / mL (purchased from Shandong New Era Pharmaceutical Co., Ltd.), and the Ldelb-1 group and Ssali-1 group were given 1×10 8CFU / mL of Ldelb-1 and Ssali-1 bacterial suspension, the final volume of zebrafish solution in each group was 20 mL, and after incubation at 28 ℃ for 1 hour, except for the normal control group, the other groups were fed with high-fat food (egg yolk powder, 38% glyceride, 19% lecithin, 30% protein, a small amount of cholesterol, sugars, minerals, etc.), and then continued to incubate at 28 ℃ for 24 hours. After the experiment, oil red O fat staining was performed (Oil Red O is a strong lipid solvent and lipid dye that can combine with neutral fat droplets such as triglycerides to form an orange-red color, which can quantitatively reflect the fat content in the intestine and blood vessels of zebrafish). Ten zebrafish were randomly selected from each group and photographed under a dissecting microscope to analyze the fat staining intensity of the intestine and tail blood vessels. The results are expressed as Mean±SEM, and one-way ANOVA was used for multiple comparison analysis.

[0127] The test results are shown in Table 8. Orlistat, Ldelb-1 group and Ssali-1 group can significantly inhibit fat absorption in zebrafish.

[0128] Table 8 Experimental results on inhibiting fat absorption in zebrafish

[0129]

[0130] Note: ** indicates that compared with the model control group, p < 0.01; *** indicates that compared with the model control group, p < 0.001.

[0131] Example 8 Exploration of the ratio of fermentation agent strains

[0132] The two strains of bacteria, Ldelb-1 and Ssali-1, were combined to explore the effects of different inoculation ratios on the sensory evaluation of fermented milk and screen out the best ratio. The starter product was named YY-J254.

[0133] The commercially available Huaxi Dangdang milk was used as the milk base (protein content 3.6%). The milk was heated to 42°C ± 1°C in a water bath. Sucrose was added to 700 mL of the milk base at a rate of 6% (wt%), and the mixture was shaken evenly and dissolved. The milk base was kept in a water bath at 42°C ± 1°C. The fermentation strains were inoculated, and the inoculation ratio was shown in Table 9 (the ratio in the table is Ldelb-1: Ssali-1). After inoculation, the mixture was inverted and mixed. After the strains were evenly dispersed, they were placed in a 43°C fermentation box for fermentation. When the fermentation reached the end point (acidity was 70°T), the sample was taken out for demulsification and stored at 4°C for post-ripening. After 12 hours of post-ripening, the best ratio was selected through sensory evaluation, and commercial fermentation agents YF-L904 and Yo-Mix800 were used as controls.

[0134] Table 9 Inoculation amount of different compound ratios

[0135]

[0136] Sensory evaluation is the most direct way to evaluate fermentation indicators of fermented milk, such as smell, taste, texture, sweet-sour ratio, etc. It can also reflect the degree of consumer preference for the fermented milk. In this evaluation, 15 people with experience in tasting were invited to conduct a blind evaluation of fermented milk. The sensory evaluation table is shown in Table 10. The final score is recorded. When calculating the score, the highest and lowest scores are removed, and the optimal ratio is selected by averaging the scores.

[0137] Table 10 Sensory evaluation details

[0138]

[0139] The total score of the evaluation results is shown in Table 11, and the detailed scores are as follows: Figure 4 . Among them, YY-J254 (1:100) had a total score of 88.2, YY-J254 (1:1) had a total score of 85.7, and YY-J254 (1:10) had a total score of 84.8, ranking in the top three. The total sensory evaluation scores of the three groups were all higher than those of the commercial starter control group. It can be seen that fermented milk that satisfies consumers can be obtained by combining starters at a ratio of 1:100 to 1:1 with Ldelb-1:Ssali-1. Among them, YY-J254 (1:100) is the optimal ratio. The fermented milk in this group has a uniform milky white color, a rich fermented milk flavor, no bitterness, a refreshing taste, a moderate sweet-sour ratio, a wire-drawing state, a fine texture, and no whey precipitation, and is most popular with consumers. Subsequent experiments were carried out with this optimal ratio.

[0140] Table 11 Sensory evaluation scores

[0141]

[0142] Example 9 Fermentation performance of starter YY-J254

[0143] The commercial starter was used as a control to further study the fermentation performance of YY-J254 (1:100). The grouping and inoculation conditions are shown in Table 12. After fermentation according to the process of Example 8, the fermentation indexes such as the acid production capacity of the starter, water holding capacity after 12 h of post-ripening, viscosity, diacetyl, and acid after D1, D7, D14, and D21 were determined. Each index was replicated 3 times, and the results were expressed as Mean±SD. One-way ANOVA was used for multiple comparison analysis. "**" indicates that compared with the control group, p < 0.01; "*" indicates that compared with the control group, p < 0.05.

[0144] Table 12 Experimental groups and inoculum size of each group

[0145]

[0146] (1) Fermentation time and acid production capacity of YY-J254

[0147] The speed of acid production of starter is one of the important indicators that enterprises pay attention to. At the same time, acidity is also an important parameter that affects the coagulation time, texture, taste, flavor and even the limit of bacteria in fermented milk. Appropriate acidity can not only increase the flavor and taste of fermented milk, but also improve shelf stability and extend the storage period. According to the national standard GB 19302-2010, the titrated acidity of fermented milk shall not be less than 70°T. The fermented milk acidity detection method refers to the "phenolphthalein indicator method" specified in GB 5009.239-2016.

[0148] The test results are shown in Table 13. According to the fermentation process described in Example 8, the time taken for the acidity of YY-J254 to reach 70°T was 3.4 h, and the time taken for the acidity of YF-L904 and Yo-Mix800 to reach 70°T was 4.8 h and 4.9 h respectively.

[0149] In addition, excellent starter strains require not only strong acid production ability, but also weak post-acid ability. If the acidification is severe after the shelf life, the product will have a sharp sour taste, which seriously affects the taste and quality of the product and shortens the shelf life of the product. As shown in Table 13, when stored at 4°C for 21 days, the acidity of all groups showed an upward trend. Among them, the acidity value of the YY-J254 group on the 21st day was 92.5±0.2°T, an increase of 10.7°T from the first day; the control group YF-L904 was 89.4±0.4°T, an increase of 9°T from the first day; Yo-Mix800 was 87.9±0.2°T, an increase of 9.6°T from the first day. The three have similar post-acidification degrees, and the acidity range is small, with weak post-acid properties. It has been reported in the literature that fermented milk tastes best when the acidity is less than 110°T, and is most easily accepted by consumers. Therefore, YY-J254 can meet moderate acidity during the fermentation process and shelf life.

[0150] Table 13 Acid production capacity of fermented milk and post-acidification test

[0151]

[0152] (2) Determination of water holding capacity, apparent viscosity and diacetyl content of YY-J254

[0153] (a) The water holding capacity of fermented milk is determined by the three-dimensional network structure of proteins in fermented milk. It is an important parameter for evaluating the gelation, whey precipitation and quality of fermented milk, and is also one of the important factors affecting the taste of fermented milk. The higher the water holding capacity of fermented milk, the better the structure of the product. The lower the whey precipitation, the more stable the product. On the contrary, if the water holding capacity is too low, the looser the structure, the more serious the whey precipitation, the worse the taste, and the worse the stability. It is not conducive to the transportation and storage of the product, and it also affects the consumer experience.

[0154] Water holding capacity determination method: weigh a certain mass (W 2 ) was placed in a 50 mL centrifuge tube and centrifuged at 4°C and 4500 rpm / min for 10 min. The supernatant was poured out and the mass W of the remaining sample in the centrifuge tube was determined. 1 , the pipe weight is recorded as W 0 , the water holding capacity (X) of fermented milk is calculated as follows:

[0155] The results are shown in Table 14. The water holding capacity of the experimental group YY-J254 was 70.01±0.67%, the water holding capacity of the control group YF-L904 was 70.15±0.34%, and the water holding capacity of the control group Yo-Mix800 was 69.12±0.71%. The experimental group had a water holding capacity similar to that of the control group and was around 70%, indicating that the leavening agent of the present invention has a good water holding capacity.

[0156] (b) Viscosity is an important fermentation index of fermented milk. The increase of viscosity can not only improve the texture of fermented milk, enhance the apparent body feel, reduce whey precipitation, but also increase the dense taste of fermented milk. Therefore, screening strains with better viscosity production characteristics is also an important part of starter development.

[0157] Determination of viscosity: Take 30 mL of fermented milk sample, use NDJ-8S digital viscometer, No. 3 rotor, speed of 12 rpm, test at room temperature, and select the data with torque close to 50%.

[0158] The results showed that the viscosity value of the experimental group YY-J254 was 5343±92 mpa·s, which was significantly higher than that of the control groups YF-L904 and Yo-Mix800 (Table 14). The apparent characteristics of the fermented milk in the YY-J254 group were good in the drawing state, indicating that the starter YY-J254 had the excellent characteristics of high viscosity and drawing ability.

[0159] (c) Diacetyl is a product of citric acid metabolism by lactic acid bacteria. An appropriate amount of diacetyl will give fermented milk a rich milky flavor.

[0160] The determination method of diacetyl content is based on the research of Hu Kaili et al. (Hu Kaili, et al . 2018), by making a diacetyl standard curve, the absorbance of the sample at 335 nm was measured and the diacetyl content was counted.

[0161] The results are shown in Table 14. The diacetyl content of the fermented milk of the YY-J254 group of the present invention is 10.65±1.20 μg / mL, and the diacetyl contents of YF-L904 and Yo-Mix800 are 8.39±0.47 and 8.05±0.42, respectively. The ability of the YY-J254 group to produce diacetyl is significantly higher than that of the two control groups, indicating that it has a higher aroma production ability and the fermented milk has a stronger aroma, which is consistent with the results of the sensory evaluation in Example 8.

[0162] Table 14 Water holding capacity, viscosity and diacetyl detection of fermented milk

[0163] Group Water holding capacity (%) Viscosity (mpa·s) Diacetyl content (μg / mL) YY-J254 70.01±0.67 5343±92 10.65±1.20 YF-L904 70.15±0.34 4552±164* 8.39±0.38* Yo-Mix800 69.12±0.71 4709±231** 8.05±0.42**

[0164] Example 10 Application of starter YY-J254 in the field of skin acne

[0165] In the antibacterial test, it was found that two strains of bacteria in the fermentation agent YY-J254 could inhibit the growth of skin pathogens, so the application of YY-J254 in the field of skin acne was explored.

[0166] Preparation of test samples: 1×10 of each of YF-L904, Yo-mix 800 and YY-J254 starter cultures were taken. 11 The CFU (total bacterial count) was dissolved in 20 mL of normal saline to make the final concentration of the sample 5×10 9 CFU / mL, and then add 0.4 g of hydroxyethyl cellulose (thickener, purchased from Shandong Heda Co., Ltd.), mix well, and then divide them into portions with 1 ml sterile syringes for storage at -20°C, and take one piece per day for application.

[0167] Experimental grouping and administration: 30 SPF female ICR mice aged 8-9 weeks and weighing 23-26 g were purchased from Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd. and kept in an SPF animal room. After 7 days of adaptive feeding, the mice were randomly divided into 6 groups according to their body weight, namely normal control group, model control group, adapalene group, YF-L904 group, Yo-mix 800 group and YY-J254 group, with 5 mice in each group. Before the start of the experiment, all mice were depilated on their backs. Except for the normal control group, the other groups were injected with Propionibacterium acnes modeling for two consecutive days between 9:00 and 10:00 am on the first and second days of the experiment. The injection sites were the left and right sides of the back of the mice, and 50 μL was injected on each side. The injection volume was 1×10 8CFU, after the injection on the first day, 0.1g of artificial sebum (purchased from Beijing Solebow Technology Co., Ltd.) was immediately applied to the injection site for 7 consecutive days, and the sebum application was performed in the morning. The normal control group was injected with the same amount of saline and no artificial sebum was applied. The positive drug adapalene group was applied with adapalene (purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.) at 0.1 g / mouse, and the YF-L904 group, Yo-mix 800 group, and YY-J254 group were all applied with the above-prepared test product at 0.2 mL / mouse, once a day, for 7 consecutive days, and the administration was performed in the afternoon. On the 8th day, the backs of all mice were photographed and all mice were dissected, and the skin tissues at the modeling site were collected and stored for later use.

[0168] Skin lesion score: The diameter of the papule / pustule / erythema formed at the modeling site was measured with a vernier caliper, and the skin lesion status of all mice was scored according to the scoring criteria in Table 15. The total score of skin lesions was obtained by adding the scores of each index in the table below. The test results were expressed as Mean±SEM, and the differences between the groups were tested by non-parametric tests, Kruskal-Wallis test. Compared with the model control group, * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

[0169] Table 15 Skin lesion scoring details

[0170] Lesion type Rating content Diameter of papule / pustule / erythema 0~1.9mm 0 points 2~3.9mm 1 point 4~5.9mm 2 points 6~8mm 3 points Abscess (degree of swelling) None 0 points Small 1 point Medium 2 points Large 3 points Black scab None 0 points Mild 1 point Moderate 2 points Severe 3 points Dry skin None 0 points Mild 1 point Moderate 2 points Severe 3 points Desquamation None 0 points Mild 1 point Moderate 2 points Severe 3 points

[0171] Skin thickness: After the experiment, all mice were dissected, the back skin was cut open, and the skin thickness of the modeling sites on both sides was measured with a vernier caliper.

[0172] Inflammatory factor detection: According to the instructions, the mouse ELISA detection kit was used to detect the TNF-α content in mouse skin tissue. The results of skin thickness and inflammatory factor tests were expressed as Mean±SEM, and one-way ANOVA was used for multiple comparison analysis. Compared with the model control group, * p <0.05;** p <0.01;*** p <0.001; **** p <0.0001.

[0173] Test results: Figure 5 The total score of skin lesions at the end of the experiment was as follows. Compared with the normal control group, the total score of skin lesions in the model control group was significantly increased. Compared with the model control group, adapalene and YY-J254 could significantly reduce the skin lesion score. There was no significant difference in the skin lesion scores of the other groups compared with the model control group.

[0174] Figure 6As for the skin thickness results, compared with the normal control group, the skin thickness of the model control group at the modeling site was significantly increased. Compared with the model control group, adapalene and YY-J254 could significantly reduce the skin thickness of the modeling site. There was no significant difference in skin thickness between the other groups and the model control group.

[0175] Figure 7 The results of TNF-α detection showed that compared with the normal control group, the TNF-α content in the skin tissue of the model control group was significantly increased. Compared with the model control group, adapalene and YY-J254 could significantly reduce the TNF-α content in the skin tissue. There was no significant difference in the TNF-α content in the skin tissue of the other groups compared with the model control group.

[0176] Figure 8 The following are representative pictures of the back skin of mice in each group at the end point of the experiment. The back skin of mice in the normal control group was normal, while the back skin of mice in the model control group was rough, hardened, and had increased dandruff. The modeling site was raised, with black scabs formed by the transformation of pustules. The other groups all had different degrees of improvement. Adapalene had a significant improvement effect, but increased dandruff was observed, with certain side effects. Among the other groups, YY-J254 had a more obvious improvement effect. At the end of the administration, the modeling site was completely healed, and the dandruff and skin roughness were improved.

Claims

1. A yogurt starter, the starter containing Lactobacillus delbrueckii subspecies bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) Ldelb-1 strain and Streptococcus salivarius subsp. thermophilus ( Streptococcus salivarius subsp. thermophilus ) Ssali-1 strain, the microbial preservation number of the Ldelb-1 strain is CCTCC NO: M 20241013, and the preservation number of the Ssali-1 strain is CCTCC NO: M 20241014.

2. The yogurt starter according to claim 1, characterized in that The ratio of Ldelb-1 strain and Ssali-1 strain in the starter is 1:100~1:

1.

3. The yogurt starter according to claim 1, characterized in that The ratio of Ldelb-1 strain to Ssali-1 strain is 1:100~1:10 or 1:10~1:

1.

4. A fermented dairy product, characterized in that The invention contains Lactobacillus delbrueckii subspecies bulgaricus Ldelb-1 strain or Streptococcus salivarius thermophilic subspecies Ssali-1 strain, wherein the microbial preservation number of the Ldelb-1 strain is CCTCC NO: M 20241013, and the preservation number of the Ssali-1 strain is CCTCC NO: M 20241014.

5. A fermented dairy product, characterized in that The yogurt starter is fermented by the yogurt starter according to claim 1.

6. A method for preparing a fermented dairy product, the method comprising: The starter is inoculated into the milk base and fermented at 35-50° C., wherein the starter is the yogurt starter according to claim 1.

7. A multifunctional strain, characterized in that The strain is selected from Lactobacillus delbrueckii subspecies bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) Ldelb-1 strain, or Streptococcus salivarius subsp. thermophilus with a deposit number of CCTCC NO: M 20241014 ( Streptococcus salivarius subsp. thermophilus ) Ssali-1 strain.

8. The method for culturing a multifunctional strain according to claim 7, comprising inoculating the Ldelb-1 strain or the Ssali-1 strain into a culture medium, performing proliferation culture, and obtaining a proliferated strain.

9. The use of the multifunctional strain as claimed in claim 7 for non-disease treatment purposes, wherein the non-disease treatment purposes are selected from one or more of the following: anti-oxidation, anti-inflammatory, antibacterial, cholesterol-lowering, inhibition of body fat absorption, inhibition of skin fat secretion, and the antibacterial refers to inhibition of pathogenic Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Shigella dysenteriae, Yersinia enterocolitica, Staphylococcus aureus or Epidermidis herpes.

10. Use of the multifunctional strain according to claim 7 in preparing a drug for treating acne or obesity.

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