Lactobacillus paracasei with bacteriostatic efficacy and application thereof

Abstract

This invention relates to the field of microbial technology, and discloses a strain of *Lactobacillus paracasei* with antibacterial effects and its applications, wherein the *Lactobacillus paracasei* (… Lactobacillus paracasei The strain has the accession number CGMCC No. 33154. This strain exhibits good resistance to gastric acid and bile salts, as well as strong self-aggregation and hydrophobicity. It shows significant inhibitory effects against Staphylococcus aureus and Salmonella. Furthermore, when preparing fermented butter, this strain can enhance the variety and content of flavor compounds in the fermented butter, resulting in a buttery aroma with a harmonious blend of lactic acid and fruit flavors. In particular, fermenting light cream at low temperatures (10-20℃) yields fermented butter with an even richer variety and content of flavor compounds.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Lactobacillus paracasei with antibacterial effects and its applications. Background Technology

[0002] Lactic acid bacteria are multifunctional microorganisms widely found in fermented dairy products, health supplements, and beverages. These microorganisms not only enhance the flavor of food but also provide the human body with a variety of beneficial nutrients. Probiotics, especially lactic acid bacteria, have become an important means of treating gastrointestinal diseases due to their natural, safe, and effective properties. They improve gut health by increasing the number of beneficial bacteria in the gut, competing with pathogenic bacteria for survival space. Furthermore, lactic acid bacteria have the ability to reduce the activity of inflammatory factors, which helps alleviate inflammatory responses. Therefore, probiotics, with their healthy, safe, and effective antibacterial properties, as well as their low-temperature fermentation characteristics and the unique flavor produced after fermentation, have significant research value and broad application prospects.

[0003] Lactobacillus paracasei ( Lactobacillus paracasei Lactobacillus paracasei is a common lactic acid bacteria with various biological characteristics. For example, it can inhibit the growth of harmful bacteria by producing lactic acid and hydrogen peroxide, and has certain antibacterial activity. It can effectively inhibit some pathogens and has probiotic functions such as regulating the intestines and enhancing immunity. In addition, Lactobacillus paracasei also has certain acid and heat resistance, making it play an important role in the food industry, health products, medicine and agriculture. However, there is relatively little research on the use of Lactobacillus paracasei in the production of fermented butter.

[0004] Fermented butter (also known as sour cream or fermented cream) is a dairy product made by fermenting milk or cream with specific lactic acid bacteria. It mainly consists of milk fat, milk protein, and water, and also contains phospholipids, sterols, small amounts of vitamins, carotene, minerals, and flavor components. Common strains used in fermented butter include Streptococcus thermophilus (Streptococcus thermophilus). Streptococcus thermophilus Lactobacillus bulgaricus ( Lactobacillus bulgaricus Lactobacillus ( ) Lactococcus lactis Bifidobacterium ( Bifidobacterium spp. These strains produce lactic acid during fermentation, which can increase the acidity of dairy products and enhance their taste. However, the variety and content of flavor substances in the prepared dairy products are relatively low, resulting in a single flavor and an unprominent creamy aroma in fermented butter, which cannot meet the needs of the general public. Summary of the Invention

[0005] The application aims to overcome the problems of low flavor substance type and content in the fermented butter in the prior art, and provide a Lactobacillus paracasei with bacteriostatic efficacy and application thereof.

[0006] To achieve the above-mentioned purpose, the application provides a Lactobacillus paracasei in a first aspect. Lactobacillus paracasei The preservation number of the Lactobacillus paracasei is CGMCC No.33154.

[0007] The application provides a fermentation agent in a second aspect.

[0008] The application provides a method for preparing the fermentation agent in a third aspect.

[0009] The application provides a method for preparing the fermentation agent in a third aspect.

[0010] The application provides a method for preparing the fermentation agent in a third aspect.

[0011] The application provides an application of the Lactobacillus paracasei or the fermentation agent in a fourth aspect.

[0012] The application provides a method for preparing the fermented butter in a fifth aspect.

[0013] The application provides a fermented butter prepared by the method in a sixth aspect.

[0014] By the technical scheme, the application can achieve the following beneficial effects:

[0015] (1) The Lactobacillus paracasei CGMCC No.33154 can improve the types and content of flavoring substances in the fermented butter, so that the fermented butter has the advantages of prominent butter aroma and coordinated lactic acid and fruit aroma; in addition, the Lactobacillus paracasei can ferment the whipped cream under low-temperature conditions (10-20℃), so that the fermented butter has more types and content of flavoring substances;

[0016] (2) The Lactobacillus paracasei CGMCC No.33154 has good resistance to gastric acid and bile salt, and strong self-aggregation and hydrophobicity, and the survival rate can reach 98.83% after 2h in simulated standard gastric juice (pH=3.0), the survival rate can reach 66.65% after 4h in simulated intestinal juice (bile salt concentration is 0.2wt%), the self-aggregation rate can reach 55.21%, and the hydrophobicity can reach 55.73%;

[0017] (3) The Lactobacillus paracasei CGMCC No.33154 has good bacteriostatic effect on common intestinal pathogenic bacteria such as Escherichia coli, Staphylococcus aureus and Salmonella, and has significant inhibitory effect on Staphylococcus aureus and Salmonella;

[0018] (4) The Lactobacillus paracasei CGMCC No.33154 is a natural strain isolated from traditional fermented dairy products, which is green and safe, and is suitable for application in health products, fermented dairy products and beverages, and can improve the self-independence of microbial preparations or leavening agents in China.

[0019] Biological preservation

[0020] The strain provided by the application is named Lactobacillus paracasei Lactobacillus paracasei , which is isolated from Xinjiang koumiss and has been preserved in the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Beichen West Road, Haidian District, Beijing, China on December 23, 2024, and the preservation number is CGMCC No.33154. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the colony morphology diagram of the Lactobacillus paracasei of the application.

[0022] Figure 2 is the gram staining result and cell morphology diagram of the Lactobacillus paracasei of the application. DETAILED DESCRIPTION

[0023] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not to be understood as being crucial to the application. Any numeric range recited is intended to include all values from the lower value to the upper value. For a numeric range recited as between two specific values, the intent is to include all numeric values (integers, fractions, or decimals) that fall within the range. Numeric ranges can be expressed herein with "approximately" or "about" for convenience in connection with a standard, approximate, well-understood measure of the value with a small tolerance before or after the stated quantity that still performs the desired function or achieves the desired end result. For example, "approximately 10" includes 10.0, 10.1, 10.2, etc.

[0024] In the present application, "CCNH577" is the number of Lactobacillus paracasei provided by the inventor during the research process, and "CGMCC No." is the preservation number of the strain, both of which represent the same strain, and the numbers can be used interchangeably in the following.

[0025] The inventors of the present application isolated a strain of Lactobacillus paracasei CCNH577 from Xinjiang sour mare milk during the research process. It was found that the strain has both probiotic properties and fermentation properties, has good gastric acid and bile salt tolerance and in vitro non-specific adhesion performance, and has significant antibacterial activity against common intestinal pathogenic bacteria. Moreover, it can ferment dairy products (especially fermented butter) at a lower temperature (10-20℃) to produce rich fruit flavor, cream flavor and lactic acid flavor, with coordinated aroma.

[0026] The first aspect of the present application provides a Lactobacillus paracasei (Lactobacillus paracasei) strain. Lactobacillus paracasei The preservation number of the Lactobacillus paracasei is CGMCC No. 33154.

[0027] The second aspect of the present application provides a fermentation inoculant, wherein the fermentation inoculant comprises the Lactobacillus paracasei according to the first aspect of the present application.

[0028] According to some embodiments of the present application, the content of the Lactobacillus paracasei is 10 7 -10 12 CFU / g (liquid starter can be converted according to 1g=1mL).

[0029] In the present application, "10 7 -10 12 CFU / g" means that the content of Lactobacillus paracasei CCNH577 in the starter reaches the order of magnitude of 10 7 -10 12 CFU / g, for example, the order of magnitude of 10 7 CFU / g represents a range of greater than or equal to 1x10 7 CFU / g to less than 1x10 8 CFU / g, that is, for example, 1x10 7 CFU / g, 5x10 7CFU / g, 9.9 x 10 7 CFU / g, 9.9 x 10 7 CFU / g, 9.9 x 10 7 CFU / g, 9.9 x 10 13 CFU / g, 9.9 x 10 7 CFU / g, 9.9 x 10 7 CFU / g, 9.9 x 10 7 CFU / g, 9.9 x 10 8 CFU / g, 9.9 x 10 8 CFU / g, 9.9 x 10 8 CFU / g, 9.9 x 10 9 CFU / g, 9.9 x 10 9 CFU / g, 9.9 x 10 9 CFU / g, 9.9 x 10 10 CFU / g, 9.9 x 10 10 CFU / g, 9.9 x 10 10 CFU / g, 9.9 x 10 11 CFU / g, 9.9 x 10 11 CFU / g, 9.9 x 10 11 CFU / g, 9.9 x 10 12 CFU / g, 9.9 x 10 12 CFU / g, 9.9 x 10 12 CFU / g, 9.9 x 10 13 CFU / g, 9.9 x 10

[0030] The third aspect of the present application provides a method for preparing the fermentation starter according to the present application.

[0031] In the present application, the specific type of the fermentation starter is not particularly limited, and any type of fermentation starter commonly used in the art, especially the type of fermentation starter used for the preparation of fermented food, can be applied to the present application. For example, it can be a liquid fermentation starter, a solid fermentation starter, or a semi-solid fermentation starter, etc.

[0032] According to some embodiments of the present application, the method comprises inoculating the Lactobacillus paracasei according to the present application into a liquid culture medium for seed culture to obtain a liquid fermentation starter.

[0033] According to some embodiments of the present application, the method comprises: inoculating the Lactobacillus paracasei of the present application into a liquid culture medium for seed culture to obtain a seed culture solution; culturing the seed culture solution in a fermentation culture medium for fermentation culture to obtain a fermentation solution; and performing solid-liquid separation on the fermentation solution to obtain a semi-solid fermentation inoculum.

[0034] In the present application, after the fermentation solution is subjected to solid-liquid separation (centrifugation), most of the liquid (preferably more than 90% by weight) in the fermentation solution is removed, and the obtained bacterial slurry (fermentation solution content in the bacterial slurry is less than 10% by weight) can be used as a semi-solid fermentation inoculum.

[0035] According to some embodiments of the present application, the method comprises: inoculating the Lactobacillus paracasei of the present application into a liquid culture medium for seed culture to obtain a seed culture solution; culturing the seed culture solution in a fermentation culture medium for fermentation culture to obtain a fermentation solution; and performing solid-liquid separation on the fermentation solution to obtain a semi-solid fermentation inoculum.

[0036] In the present application, the solid separation method is not particularly limited, such as filtration or centrifugation, preferably centrifugation.

[0037] Preferably, the centrifugation conditions include a rotation speed of 5000-6000 rpm and a time of 25-35 min.

[0038] In the present application, the drying method is not particularly limited, and preferably, the drying method is freeze-drying.

[0039] In the present application, the liquid culture medium and the fermentation culture medium are not particularly limited, such as conventional MRS liquid culture medium (MRS liquid culture medium is purchased from Qingdao Haibo Biology, and the product number is HB0384-1).

[0040] In the present application, preferably, the solid fermentation inoculum of the present application can further contain an auxiliary material. The auxiliary material can be an auxiliary material commonly used in the preparation of bacterial inoculum or fermentation inoculum in the art, such as a buffer (such as a buffer solution, a culture medium, etc.), a protective agent (such as a freeze-drying protective agent, such as skimmed milk powder, trehalose, sodium glutamate, glycerol, etc.), etc.

[0041] In the present application, the addition method of the auxiliary material comprises mixing the bacterial slurry obtained by solid-liquid separation with the auxiliary material and then drying to obtain a solid fermentation inoculum.

[0042] Preferably, the auxiliary material is a water solution of skimmed milk powder; the content of skimmed milk powder in the water solution of skimmed milk powder is 5%-15% (w / v). "% (w / v)" means weight percentage, for example, "the content of skimmed milk powder in the water solution of skimmed milk powder is 12% (w / v)" means that the weight of skimmed milk powder in unit volume of the water solution of skimmed milk powder is 12% (for example, 12g of skimmed milk powder in 100mL of the water solution of skimmed milk powder).

[0043] Preferably, the volume ratio of the bacterial slurry to the auxiliary material is 1: (3-5).

[0044] Preferably, the inoculation amount of the Lactobacillus paracasei is 10 1 -10 11 CFU / mL.

[0045] Preferably, the inoculation amount of the seed culture solution is 3v%-10v% based on the volume of the fermentation medium. "% (v / v)" means volume percentage, for example, "adding the seed solution into the medium at a ratio of 3% (v / v)" means that the volume of the seed solution added into the medium is 3% of the volume of the medium.

[0046] According to some embodiments of the present application, the conditions of the seed culture include: temperature of 33-37℃, time of 24-48h, and pH of 5-7.

[0047] According to some embodiments of the present application, the conditions of the fermentation culture include: temperature of 33-37℃, time of 24-48h, and pH of 5-7.

[0048] The fourth aspect of the present application provides the use of the Lactobacillus paracasei or the fermentation inoculant of the present application in dairy products or improving flavor substances of fermented butter.

[0049] According to some embodiments of the present application, the dairy product is selected from at least one of fermented butter, cheese, condensed milk and yogurt.

[0050] According to some embodiments of the present application, the flavor substances include at least one of ethyl hexanoate, nonanoic acid, δ-octanolide, butyric acid, acetic acid, hexanoic acid, δ-decanolide and ethyl octanoate.

[0051] The fifth aspect of the present application provides a method for preparing the fermented butter of the present application, wherein the method comprises inoculating the Lactobacillus paracasei or the fermentation inoculant of the present application into whipped cream for fermentation.

[0052] According to some embodiments of the present application, the fermentation temperature is 10-40℃, preferably 10-20℃. For example, the fermentation temperature can be any value within a range defined by any two of 10℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃ and 20℃.

[0053] The inventors of the present application have found in their research that the Lactobacillus paracasei provided by the present application can ferment the light cream under low or medium temperature conditions to prepare the fermented butter. However, after fermentation at a higher temperature, the obtained fermentation product needs to be refrigerated for post-ripening, and if the fermentation is carried out under low temperature conditions, the post-ripening is not needed, which can greatly shorten the fermentation and post-ripening time, improve the production efficiency, and the fermented butter prepared by the Lactobacillus paracasei provided by the present application under low temperature conditions (10-20℃) has more types and contents of flavor substances.

[0054] The fermentation time in the method provided by the present application is not particularly limited, as long as the fermented butter with good flavor can be obtained. For example, the specific fermentation time can be determined according to the changes in the texture properties, pH value, odor and other properties of the fermentation product.

[0055] Preferably, the fermentation time is 10-30h, preferably 18-24h.

[0056] According to some preferred embodiments of the present application, the pH value at the end of the fermentation is 5.2-6. That is, when the pH value of the fermentation product reaches the above range, the fermentation can be stopped. If the fermentation time does not reach the above requirement when the pH value reaches the above range, the fermentation time can be appropriately extended or shortened to make the fermented butter have better quality.

[0057] Preferably, the pH value at the end of the fermentation is 5.4-5.8, for example, the pH value at the end of the fermentation can be any value within a range defined by any two of 5.4, 5.45, 5.5, 5.55, 5.6, 5.65, 5.7, 5.75 and 5.8.

[0058] According to some embodiments of the present application, the amount of the Lactobacillus paracasei or the fermentation agent is such that the content of the Lactobacillus paracasei in the inoculated light cream is ≥10 6 CFU / mL, preferably 10 7 -10 8 CFU / mL.

[0059] Preferably, the amount of Lactobacillus paracasei or starter culture used can be 0.5-5% (w / v) based on the total volume of the whipping cream, i.e. the amount of Lactobacillus paracasei or starter culture added can be 0.5-5 g per 100 mL of whipping cream. For example, the amount of Lactobacillus paracasei or starter culture used can be any value within the range of any two values selected from the group consisting of 0.5% (w / v), 1% (w / v), 1.5% (w / v), 2% (w / v), 2.5% (w / v), 3% (w / v), 3.5% (w / v), 4% (w / v), 4.5% (w / v) and 5% (w / v).

[0060] Preferably, the number of viable Lactobacillus paracasei in the product obtained by fermentation (i.e. the whipping cream after fermentation) is ≥ 2 x 10 7 CFU / mL, preferably 1 x 10 8 CFU / mL; more preferably, the number of Lactobacillus paracasei in the product obtained by fermentation is 2 x 10 10 CFU / mL. 8 CFU / mL. 9 CFU / mL.

[0061] Preferably, the method further comprises stirring, filtering, washing and pressing the fermentation product prepared, so as to remove buttermilk and water from the fermentation product.

[0062] Preferably, the stirring is performed in a blender, and the stirring conditions include a stirring temperature of 10-15°C and a stirring speed of 750-1000 rpm.

[0063] In the present application, the filtering method is not particularly limited. Preferably, the filtering method comprises filtering with 3-4 layers of medical gauze.

[0064] Preferably, the washing method comprises washing the filtered fermentation product with cold water at 0-4°C for 1-3 times, so as to remove buttermilk from the fermentation product.

[0065] Preferably, the pressing method comprises pressing the washed product with a churning machine, so as to remove water and obtain the fermented butter.

[0066] The present application provides a fermented butter prepared by the method of the present application.

[0067] According to a particularly preferred embodiment of the present application, there is provided a method for preparing a fermented butter, which comprises the following steps:

[0068] (1) inoculating Lactobacillus paracasei CGMCC No. 33154 into MRS liquid medium, and culturing at 33-37°C for 24-48 h to obtain a seed culture;

[0069] The inoculation amount of the Lactobacillus paracasei is 10 1 -10 9 CFU / mL;

[0070] (2) MRS liquid culture medium is added into a fermentation tank, sterilized at 115-121℃ for 15-20 min, and then cooled to room temperature. The seed culture solution is added at a ratio of 3-10% (v / v), and continuous fermentation is carried out at 33-37℃ and 110-200 rpm for 24-48 h. The pH of the fermentation liquid is maintained at 6±1 during the fermentation process, and a strain fermentation liquid is obtained.

[0071] (3) The strain fermentation liquid is centrifuged at 5000-6000 rpm for 25-35 min, and the bacterial slurry is collected. Then the bacterial slurry is mixed with a skimmed milk powder protective agent (an aqueous solution of skimmed milk powder containing 5-12% (w / v) of skimmed milk powder) at a volume ratio of 1: (3-5), and then placed in a freezer at -80℃ for pre-freezing overnight. Then, the freeze-dried powder is prepared by vacuum freeze-drying, and a solid starter culture is obtained.

[0072] (4) Fresh whipping cream is pasteurized, and then the solid starter culture is inoculated into the whipping cream at a ratio of 1-5% (w / v), and fermented at 10-20℃ until the pH of the fermentation system is 5.4-5.8, and the fermentation time is 18-24 h.

[0073] (5) After the fermentation is completed, the fermentation product is stirred at high speed at 10-15℃ and 750-1000 rpm to make the fat particles in the fermentation product gather into groups. Then, the stirred fermentation product is filtered, and washed with cold water (0-4℃) for 1-3 times to remove buttermilk from the fermentation product. The washed product is extruded by a press machine to remove water and shape, and then fermented butter is obtained and stored in a refrigerator at 0-4℃.

[0074] The application will be described in detail below through examples.

[0075] The whipping cream in the following examples is purchased from Inner Mongolia Mengniu Dairy Products Co., Ltd.

[0076] In the following examples, "% (v / v)" refers to volume percentage, for example, "the seed liquid is added to the culture medium at a ratio of 3% (v / v)" means that the volume of the seed liquid added to the culture medium is 3% of the volume of the culture medium; "% (w / v)" refers to weight volume percentage, for example, "the starter culture is added to the whipping cream at a ratio of 1% (w / v)" means that the weight of the starter culture added to the whipping cream per unit volume is 1% (for example, 1g of starter culture is added to 100mL of whipping cream).

[0077] Lactobacillus rhamnosus LGG was purchased from American Type Culture Collection, and the strain number was ATCC 53103.

[0078] Lactobacillus paracasei CICC 6108 was purchased from China General Microbiological Culture Collection Center.

[0079] The reagents and materials used in the following examples and comparative examples were commercially available products purchased from regular chemical or biological reagent and material suppliers, and the reagents were all of analytical purity, unless otherwise specified.

[0080] Preparation Example 1

[0081] Isolation, purification, identification, performance testing and preservation of Lactobacillus paracasei CGMCC No.33154.

[0082] (I) Strain isolation and purification

[0083] A lactobacillus strain was isolated from acid mare milk collected from Xinjiang by gradient dilution coating method, and the number was CCNH577, and the pure culture of the strain was obtained by streaking method.

[0084] (II) Strain identification

[0085] (1) Morphological identification: the colony morphology of strain CCNH577 was observed, and it was found that the colony on MRS plate was milky white regular round, and the colony was wet, smooth and opaque (Fig. 1). Figure 1 );Then the strain CCNH577 was subjected to Gram staining, and the staining results and cell morphology were observed under a microscope, and it was found that the strain was Gram-positive, and the cells were rod-shaped, single, short chain or arranged in a fence shape (Fig. 2). Figure 2

[0086] (2) Molecular identification

[0087] Strain CCNH577 was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing; the sequencing results were compared in NCBI database, and it was found that the homology of 16S rDNA of strain CCNH577 and Lactobacillus paracasei (Lactobacillus paracasei) reached 99.58%. Lactobacillus paracasei

[0088] Combined with the results of physiological ecology and morphological identification and molecular identification, it was determined that strain CCNH577 was Lactobacillus paracasei (Lactobacillus paracasei). Lactobacillus paracasei

[0089] (III) Basic performance evaluation

[0090] ​​​(1) Growth performance evaluation: after the L. paracasei CCNH577 was activated for 2 generations, it was inoculated into MRS broth culture medium at an inoculation amount of 3% (v / v), and the OD value of the bacterial liquid at 600 nm was measured after 18 hours of culture, and the OD value was represented by OD 18h The determination results showed that the OD 18h of the L. paracasei CCNH577 was about 4.63, indicating that the growth ability of the strain was good and it could reproduce at a faster speed.

[0091] (2) Acid production capacity evaluation: after the L. paracasei CCNH577 was activated for 2 generations, it was inoculated into MRS broth culture medium at an inoculation amount of 3% (v / v), and a control group (Lactobacillus rhamnosus LGG and L. paracasei CICC 6108 were used as control group strains) was set up, and the lactic acid content in the culture liquid was detected after 18 hours of culture; the lactic acid content (g / L) in the bacterial liquid was determined by the HPLC method, and the test results are shown in Table 1.

[0092] Table 1 shows that the lactic acid content in the culture liquid of the L. paracasei CCNH577 is 23.99 g / L, indicating that the strain has high lactic acid production capacity and can produce lactic acid efficiently.

[0093] Table 1

[0094]

[0095] (3) Enzyme production capacity detection: after the strain CCNH577 was activated for 2 generations, it was inoculated into MRS broth culture medium at an inoculation amount of 3% (v / v), and a control group (Lactobacillus rhamnosus LGG and L. paracasei CICC 6108 were used as control group strains) was set up, and the enzyme activity of protease and the enzyme activity of lipase in the culture liquid were determined by Folin-phenol method and p-nitrophenol method after 18 hours of culture, and the test results are shown in Table 2.

[0096] Table 2 shows that the enzyme activity of protease in the culture liquid of the strain CCNH577 is 34.43 U / mL, and the enzyme activity of lipase is 1.72 U / mL, indicating that the strain has strong enzyme production capacity, and it can produce high degradation efficiency for fat and protein during fermentation.

[0097] Table 2

[0098]

[0099] (4) Evaluation of probiotic performance

[0100] Gastric acid and bile salt tolerance evaluation: the gastric acid tolerance of the candidate strain was evaluated according to T / CNHFA435-2024 "Probiotic agent gastric juice tolerance detection method"; the bile salt tolerance of the candidate strain was evaluated according to the method.

[0101] The results of the evaluation of the gastric acid and bile salt tolerance of the candidate strain are shown in Table 3.

[0102] Table 3 shows that the survival rate of strain CCNH577 reached 98.83% after 2h in simulated standard gastric juice (pH=3.0), and the survival rate could reach 66.65% after 4h in simulated intestinal juice conditions (bile salt concentration of 0.2wt%), indicating that the strain has good gastric acid and bile salt tolerance.

[0103] Table 3

[0104]

[0105] (5) Evaluation of adhesion performance

[0106] Self-aggregation performance evaluation: inoculate the glycerol tubes of strains CCNH577, LGG and CICC6108 into fresh MRS liquid medium at 2‰ (v / v), and incubate at 37°C overnight for 18h. Inoculate at 2% (v / v) into fresh MRS liquid medium, and incubate at 37°C, 200 rpm for 18h. Detect the OD 600 , wash twice with physiological saline, resuspend to OD 600 =1, take 100μL of the bacterial solution to the enzyme-labeled plate, detect the initial OD 600 value, and record it as A0; then let the bacterial solution stand for 4h, take 100μL of the upper layer bacterial solution to the enzyme-labeled plate, detect the OD 600 value after standing, and record it as A1. Calculate the self-aggregation using the following formula: self-aggregation rate (%)=(1- )×100%, and the results are shown in Table 4.

[0107] Hydrophobicity evaluation: take 100μL of the bacterial solution with OD 600 =1 to the enzyme-labeled plate, detect the initial OD 600 value, and record it as A0; then take 900μL of the bacterial solution, add 180μL of dodecane, vortex, stand for 20min, take the lower layer aqueous phase to detect the OD 600 value, and record it as A. Calculate the hydrophobicity using the following formula: hydrophobicity rate (%)= ×100%, and the results are shown in Table 4.

[0108] Table 4

[0109]

[0110] High hydrophobicity of bacteria facilitates their adhesion to the intestinal mucosa; the higher the hydrophobicity, the stronger their adhesion ability. Table 4 shows that strain CCNH577 has a hydrophobicity of 55.73%, higher than strains LGG and CICC 6108. The self-aggregation of strains is positively correlated with their adhesion ability; strain CCNH577 has the highest self-aggregation rate at 55.21%, higher than the two control strains.

[0111] (6) Evaluation of antibacterial properties

[0112] Indicator strains of Escherichia coli, Staphylococcus aureus, and Salmonella were inoculated into LB broth, while Candida albicans was inoculated into YPD broth. After incubating the indicator cultures overnight, they were transferred to their respective fresh media and cultured until OD200. 600 =1 or viable count greater than 10 8 CFU / mL, dilute the viable bacteria count in the culture medium to 10. 5 CFU / mL was used as the indicator bacterial solution.

[0113] Glycerol tubes of strains CCNH577, LGG, and CICC 6108 were inoculated into fresh, sterile MRS liquid medium and cultured overnight at 37°C for 18 hours. Then, 2% (v / v) of the inoculum was added to fresh MRS liquid medium, and the mixture was incubated statically at 37°C for 18 hours. The supernatant was collected by centrifugation, sterilized by membrane filtration, and used as the test sample. MRS liquid medium served as a negative control. Reaction system S1: 100 μL fermentation broth supernatant, 100 μL indicator bacteria dilution; Reaction system S2: 50 μL fermentation broth supernatant, 150 μL indicator bacteria dilution; Reaction system S3: 25 μL fermentation broth supernatant, 175 μL indicator bacteria dilution. Inhibition rate (%) = ×100%, where: A0 is the OD of the negative control sample. 600 Value; A is the sample OD. 600 Values ​​(where A0 and A are the OD values ​​after culture) 600 and OD before culture 600 The results are shown in Table 5.

[0114] Table 5

[0115]

[0116] As can be seen from Table 5, the inhibitory ability of the strain CCNH577 to Escherichia coli CICC 10421 (purchased from China Industrial Microbial Culture Collection Center), Staphylococcus aureus CMCC 26001 (purchased from China Medical Bacterial Preservation Center), Staphylococcus aureus CMCC 26003 (purchased from China Medical Bacterial Preservation Center), Salmonella ATCC 14028 (purchased from American Type Culture Collection) and Salmonella CVCC 3378 (purchased from National Veterinary Microbial Culture Collection) are all superior to LGG and CICC 6108. It is shown that the strain CCNH577 has strong bacteriostatic ability, especially significant bacteriostatic ability to Staphylococcus aureus and Salmonella.

[0117] (Four) Strain preservation

[0118] The Lactobacillus paracasei CCNH577 obtained by the above screening was preserved in the China General Microbiological Culture Collection Center (abbreviated as CGMCC) on December 23, 2024, and the address of the preservation center is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 33154.

[0119] Preparation Example 2

[0120] The Lactobacillus paracasei CGMCC No. 33154 screened in Preparation Example 1 was inoculated into MRS liquid medium, and cultured at 37°C for 24h to obtain a seed culture liquid (the number of viable bacteria was about 8×10 9 CFU / mL);

[0121] MRS liquid medium was added to the fermenter, sterilized at 121°C for 20min, and after sterilization, cooled to room temperature, and the above seed culture liquid was added at a ratio of 3% (v / v), and continuously fermented at 37°C, 110rpm for 24h, and the pH of the fermentation liquid was maintained at 6±1 during the fermentation process. A strain fermentation liquid (the number of viable bacteria was about 5×10 8 CFU / mL) was obtained;

[0122] The strain fermentation liquid was centrifuged at 5000rpm for 25min, and the bacterial slurry was collected; then the bacterial slurry was mixed with skim milk powder protectant (i.e. an aqueous solution of skim milk powder, containing 12% (w / v) skim milk powder) at a volume ratio of 1:3, and after mixing, it was placed in -80°C for pre-freezing overnight, and then vacuum freeze-dried to obtain a freeze-dried powder, i.e. a solid starter culture (the number of viable bacteria was about 3.4×10 10 CFU / g).

[0123] Example 1

[0124] Fermentation: Fresh cream was pasteurized (95°C, 30s) and then inoculated with solid starter culture (Preparation Example 2) at 1% (w / v) and fermented at 10°C until the pH of the fermentation system reached 5.72, for 24h;

[0125] Stirring and removal of buttermilk: After the fermentation, the fermented product was stirred at 10°C at 750 rpm for 30 min to make the fat particles in the fermented product aggregate, and then the stirred fermented product was filtered using 4 layers of medical gauze and washed twice with cold water (4°C) to remove buttermilk.

[0126] The washed product was pressed using a churn to remove water and to shape, and then fermented butter A1 was obtained and stored at 4°C.

[0127] Example 2

[0128] Fermentation: Fresh cream was pasteurized (85°C, 2min) and then inoculated with solid starter culture (Preparation Example 2) at 2% (w / v) and fermented at 20°C until the pH of the fermentation system reached 5.69, for 26h;

[0129] Stirring and removal of buttermilk were performed under the same conditions as in Example 1.

[0130] The washed product was pressed using a churn to remove water and to shape, and then fermented butter A2 was obtained and stored at 4°C.

[0131] Example 3

[0132] Fermentation: Fresh cream was pasteurized (85°C, 2min) and then inoculated with solid starter culture (Preparation Example 2) at 3% (w / v) and fermented at 37°C until the pH of the fermentation system reached 5.42, for 16h;

[0133] Post-ripening: The fermented product was left to stand at 8°C for 24h.

[0134] Stirring and removal of buttermilk were performed under the same conditions as in Example 1.

[0135] The washed product was pressed using a churn to remove water and to shape, and then fermented butter A3 was obtained and stored at 4°C.

[0136] Comparative Example 1

[0137] The method of Example 1 was followed, except that the fermenting agent was replaced with a fermenting agent containing Lactobacillus paracasei CICC6108 (the preparation method of the fermenting agent was the same as that of Preparation Example 2), and the other operations and conditions were the same as those of Example 1 to obtain fermented butter D1, which was stored at 4°C.

[0138] Comparative Example 2

[0139] The method of Example 1 was followed, except that the fermenting agent was replaced with a commercial agent Advance 2.0 (the agent was purchased from Kewenhan (Beijing) Trade Co., Ltd.), and the other operations and conditions were the same as those of Example 1 to obtain fermented butter D2, which was stored at 4°C.

[0140] Test Example 1

[0141] The viable cell count in the whipped cream inoculated with the fermenting agent (before fermentation) and the fermented whipped cream (after fermentation) in the above examples and comparative examples was detected; the fermentation and post-ripening time of the fermented butter and the pH value of the fermentation product (fermentation broth) in the above examples and comparative examples were counted; the water content and fat content of the fermented butter obtained in the above examples and comparative examples were detected, and the results are shown in Table 7.

[0142] Test Example 2

[0143] Twenty people with experience in sensory evaluation in the field were employed as evaluators, and the fermented butter obtained in the above examples and comparative examples was subjected to sensory evaluation according to the evaluation criteria in Table 6, and the results are shown in Table 8.

[0144] Table 6

[0145]

[0146] Test Example 3

[0147] The aroma component and content of the fermented butter of Example 1 and commercially available domestic fermented butter were detected, and the specific detection results are shown in Tables 9-10 (Table 9 corresponds to the fermented butter obtained in Example 1, and Table 10 corresponds to the commercially available domestic fermented butter, which was purchased from Mengniu Dairy Company).

[0148] The test method of the aroma component and content in the fermented butter: the sample (fermented butter) was pretreated by solid phase microextraction technology (SPME), and the volatile flavor components of the above fermented butter were detected by gas chromatography-mass spectrometry (GC-MS), and the key aroma components were analyzed and identified.

[0149] SPME conditions: Accurately weigh about 5 g of the sample to be tested into a 20 mL headspace bottle, add an appropriate amount of internal standard solvent 4-octanol, use a 50 / 30 μm DVB / CAR / PDMS extraction head, extraction temperature 65℃, extraction time 60 min.

[0150] GC conditions: HP-INNOWax (60 m x 0.25 mm x 0.25 μm) chromatographic column; carrier gas He, flow rate 2 mL / min; injection port temperature 250℃, no split; detector temperature 280℃; temperature program: initial 40℃, hold for 2 min; increase by 3℃ / min to 180℃, hold for 3 min; increase by 5℃ / min to 230℃, hold for 5 min.

[0151] MS conditions: EI ionization source, electron energy 70 eV, ion source temperature 250℃, mass scan range 29-350 amu, interface temperature 250℃.

[0152] The collected mass spectrum was searched using NIST11.L spectrum library, combined with retention time, CAS number and English name for manual spectrum analysis to determine the volatile flavor components of fermented butter; the characteristic aroma component evaluation was evaluated by odor activity value method (odor activity value, OAV, odor activity value method can be referred to: Zhao Y, Zhang YH, Li JK, et al. GC-O combined with OAV to identify key aroma components of Longnan virgin olive oil [J]. Food Science, 2022, 43(08): 184-189) to evaluate the contribution of each volatile component to the overall aroma of the sample, i.e. the odor activity value (OAV) is the ratio of the concentration (C) of each aroma component to the sensory threshold (T): OVA = C / T, when OAV < 1, it means that the substance has no effect on the overall aroma; when OAV > 1, it means that the substance may have a direct impact on the overall aroma; within a certain range, the larger the OAV value, the greater the contribution of the substance to the overall flavor.

[0153] Table 7

[0154]

[0155] Table 8

[0156]

[0157] Table 9

[0158]

[0159] Table 10

[0160]

[0161] Note: The aroma substances not listed in the table are not detected.

[0162] From the data in Table 7, compared with Example 3, the L. paracasei CCNH577 provided by the application can ferment the whipping cream under low and medium temperature conditions (10-37℃) to prepare fermented butter, but after fermentation at a higher temperature (37℃), the obtained fermented product needs to be refrigerated for post-ripening, while fermentation under low temperature conditions (10-20℃) does not need to be refrigerated for post-ripening, which can greatly shorten the fermentation and post-ripening time and improve the production efficiency; compared with Comparative Example 1-2, the L. paracasei CCNH577 of the application can rapidly proliferate in the whipping cream, and the fermentation intensity (i.e. the number of L. paracasei increased per unit time, 10 7 CFU / (mL . h) is high.

[0163] From the data in Table 8, the fermented butter prepared by the L. paracasei CCNH577 provided by the application has rich flavor substances, and has the advantages of coordinated buttery aroma, lactic acid flavor and fruit aroma.

[0164] From Table 9, it can be seen that 26 volatile substances are detected in the fermented butter prepared by the L. paracasei CCNH577, including 8 acids, 5 alcohols, 5 esters, 4 ketones, 1 phenol, 1 aldehyde and 2 others. The OAV values of ethyl hexanoate, nonanoic acid, δ-octanolide and butyric acid are all greater than 1, which are the key flavor compounds in the above fermented butter. Ethyl hexanoate has fruit aroma, sweet aroma and cellar aroma, nonanoic acid has soap aroma and grassy flavor, δ-octanolide has fruit aroma and coconut aroma, and butyric acid has cheese aroma and sour cream aroma. These key flavor substances make a major contribution to the unique flavor of the fermented butter. The aroma compounds with OAV between 0.1 and 1 include acetic acid, hexanoic acid, δ-decanolide and octanoic acid ethyl ester. These aroma substances have a modifying effect on the flavor of the fermented butter product.

[0165] From the comparison of the data in Table 9 and Table 10, it can be seen that the types and contents of aroma substances in the commercially available fermented butter are significantly lower than those in the fermented butter obtained in Example 1. There are mainly 12 aroma substances detected in the commercially available fermented butter, and only the OAV value of acetoin is greater than 1, which is the key flavor compound of the fermented butter, and it mainly has buttery aroma and creamy aroma; the OAV value of butyric acid is between 0.1 and 1, and it has cheese aroma, which has a modifying effect on the flavor of the fermented butter.

[0166] In summary, the Lactobacillus paracasei CCNH577 provided by the application has probiotic properties and fermentation properties, has good gastric acid tolerance and bile salt tolerance, and has non-specific adhesion performance in vitro, and has significant bacteriostatic activity on common intestinal pathogenic bacteria, and can ferment dairy products (especially fermented butter) at a lower temperature (10-20 DEG C) to produce rich fruit flavor, milk fat flavor and lactic acid flavor, and the aroma is harmonious.

[0167] The preferred embodiments of the application are described in detail above, but the application is not limited thereto. Within the technical concept of the application, various simple modifications can be made to the technical solutions of the application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the application and fall within the protection scope of the application.

Claims

1. A Lactobacillus paracasei strain (ATCC® 33423®) Lactobacillus paracasei ) or a fermentation inoculum prepared from Lactobacillus paracasei for use in improving the flavour of fermented butter. wherein The preservation number of the Lactobacillus paracasei is CGMCC No.33154. The flavoring substances in the fermented butter include at least one of ethyl hexanoate, nonanoic acid, δ-octanolactone, butyric acid, acetic acid, hexanoic acid, δ-decanolactone and ethyl octanoate.

2. The use according to claim 1, wherein, The content of the Lactobacillus paracasei is 10 7 -10 12 CFU / g, based on the total weight of the fermentation agent.

3. Lactobacillus paracasei (ATCC 43506) having the function of inhibiting bacteria and the function of improving the flavor of fermented butter, characterized in that, Lactobacillus paracasei The preservation number of the Lactobacillus paracasei is CGMCC No.33154. ​ 4. A fermentation inoculant characterized by, The fermentation agent includes the Lactobacillus paracasei according to claim 3.

5. The fermentation inoculant of claim 4, wherein, The content of the Lactobacillus paracasei is 10 7 -10 12 CFU / g.

6. A method for preparing a fermentation inoculum, characterized by, The method includes inoculating the Lactobacillus paracasei according to claim 3 into a liquid culture medium for seed culture to obtain a liquid fermentation agent; or inoculating the Lactobacillus paracasei according to claim 3 into a liquid culture medium for seed culture to obtain a seed culture liquid; fermenting the seed culture liquid in a fermentation culture medium to obtain a fermentation liquid; and performing solid-liquid separation on the fermentation liquid to obtain a semi-solid fermentation agent; or inoculating the Lactobacillus paracasei according to claim 3 into a liquid culture medium for seed culture to obtain a seed culture liquid; fermenting the seed culture liquid in a fermentation culture medium to obtain a fermentation liquid; and performing solid-liquid separation on the fermentation liquid to obtain a semi-solid fermentation agent.

7. The method of claim 6, wherein, The liquid culture medium and the fermentation culture medium are each independently MRS liquid culture medium; and / or, the solid-liquid separation is performed by centrifugation; and / or the inoculum of Lactobacillus paracasei is 10 1 - 10 11 CFU / mL; and / or, the inoculation amount of the seed culture liquid is 3v%-10v% of the volume of the fermentation culture medium; and / or, the seed culture is performed under conditions of a temperature of 33-37℃, a time of 24-48h and a pH of 5-7; and / or, the fermentation culture is performed under conditions of a temperature of 33-37℃, a time of 24-48h and a pH of 5-7.

8. A method of preparing a fermented butter, characterized in that, The method includes inoculating the Lactobacillus paracasei according to claim 3 or the fermentation agent according to claim 4 or 5 into whipped cream for fermentation.

9. The method of claim 8, wherein, The fermentation is performed under conditions of a temperature of 10-40℃, a time of 10-30h and a pH of 5.2-6; and / or the Lactobacillus paracasei or the fermentative inoculant is used in an amount such that the number of viable Lactobacillus paracasei cells in the whipped cream after inoculation is > 10 6 CFU / mL.

10. The fermented butter prepared by the method according to claim 8 or 9.

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