Lactobacillus paracasei with antibacterial effect and application of lactobacillus paracasei

Abstract

The invention relates to the technical field of microorganisms, and discloses lactobacillus paracasei with an antibacterial effect and application of the lactobacillus paracasei, and the preservation number of the lactobacillus paracasei is CGMCC (China General Microbiological Culture Collection Center) No.33154. The strain has good gastric acid resistance, cholate resistance and strong self-aggregation and hydrophobicity, has a remarkable inhibition effect on staphylococcus aureus and salmonella, and in addition, when the strain is used for preparing the fermented butter, the variety and content of flavor substances in the fermented butter can be increased, so that the fermented butter has the characteristics of prominent cream fragrance, strong flavor and the like. Meanwhile, the lactic acid flavor and the fruit flavor are coordinated, and particularly, the single cream is fermented under the low-temperature condition (10-20 DEG C), so that the prepared fermented butter is richer in variety and content of flavor substances.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus paracasei with antibacterial effect and application thereof. Background Art

[0002] Lactic acid bacteria are multifunctional microorganisms that are widely found in fermented dairy products, health products and beverages. These microorganisms can not only enhance the flavor of food, but also provide a variety of beneficial nutrients to the human body. Probiotics, especially lactic acid bacteria, have become an important means of treating gastrointestinal diseases due to their natural, safe and effective characteristics. They increase the number of beneficial bacteria in the intestines and compete with pathogens for living space, thereby reducing the number of harmful bacteria and improving intestinal health. In addition, lactic acid bacteria also have the ability to reduce the activity of inflammatory factors, which helps to reduce inflammatory responses. Therefore, probiotics have important research value and broad application prospects due to their healthy, safe and effective antibacterial properties, low-temperature fermentation properties and the unique flavor produced after fermenting foods.

[0003] Lactobacillus paracasei ( 副干酪乳杆菌 ) is a common lactic acid bacteria with multiple biological characteristics. For example, Lactobacillus paracasei can inhibit the growth of harmful bacteria by producing lactic acid, hydrogen peroxide and other substances. It has certain antibacterial activity, can effectively inhibit some pathogenic bacteria, and can regulate the intestines and enhance immunity and other probiotic functions. In addition, Lactobacillus paracasei also has certain acid and heat resistance, making it an important player in the food industry, health products, medicine and agriculture. However, there is little research on the use of Lactobacillus paracasei in the production of fermented butter.

[0004] Fermented butter (also known as sour cream or fermented butter) is a dairy product made by fermenting milk or cream with specific lactic acid bacteria. It is mainly composed of milk fat, milk protein and water, and also contains phospholipids, sterols, a small amount of vitamins, carotene, minerals and flavor components. Common strains used in fermented butter are mainly thermophilic Streptococcus ( 链球菌属 嗜热的 bulgaricus ( 保加利亚乳杆菌 )、Lactobacillus lactis( 乳球菌属 乳酸的 )、Bifidobacterium( 双歧杆菌属 ) etc. These strains produce lactic acid during the fermentation process, which can increase the acidity of dairy products and enhance the taste of dairy products. However, the types and contents of flavor substances in the prepared dairy products are relatively low, which makes the flavor of fermented butter single and the cream aroma not prominent, which cannot meet the needs of the public. Summary of the invention

[0005] The invention aims to overcome the problem that the types and contents of flavor substances in fermented butter in the prior art are relatively low, so that the flavor of the fermented butter is single and the cream aroma is not prominent, and to provide a strain of Lactobacillus paracasei with antibacterial effect and application thereof. The Lactobacillus paracasei provided by the invention has good gastric acid and bile salt resistance, strong self-aggregation and hydrophobicity, and good antibacterial effect (especially significant inhibition effect on Staphylococcus aureus and Salmonella). When the strain (Lactobacillus paracasei) is used for the production of fermented butter, the types and contents of flavor substances in the fermented butter can be increased, so that the fermented butter has a prominent cream aroma, and has the advantages of lactic acid taste, fruity aroma, and the like.

[0006] In order to achieve the above object, the present invention provides a first aspect of a Lactobacillus paracasei strain ( 乳杆菌属 副干酪 ), wherein the preservation number of the Lactobacillus paracasei is CGMCC No.33154.

[0007] The second aspect of the present invention provides a fermentation bacterial agent, wherein the fermentation bacterial agent includes the Lactobacillus paracasei described in the first aspect of the present invention.

[0008] The third aspect of the present invention provides a method for preparing the fermentation agent of the present invention, wherein the method comprises: inoculating the Lactobacillus paracasei of the present invention into a liquid culture medium for seed culture to obtain a liquid fermentation agent; Alternatively, the Lactobacillus paracasei of the present invention is inoculated into a liquid culture medium for seed culture to obtain a seed culture solution; the seed culture solution is fermented in a fermentation medium to obtain a fermentation solution; the fermentation solution is solid-liquid separated to obtain a semi-solid fermentation agent; Alternatively, the Lactobacillus paracasei described in the present invention is inoculated into a liquid culture medium for seed culture to obtain a seed culture solution; the seed culture solution is fermented and cultured in a fermentation medium to obtain a fermentation solution; the fermentation solution is solid-liquid separated, and the obtained bacterial mud is mixed with an optional auxiliary agent and dried to obtain a solid fermentation bacterial agent.

[0009] The fourth aspect of the present invention provides the use of the Lactobacillus paracasei of the present invention or the fermentation agent of the present invention in dairy products or in improving the flavor of fermented butter.

[0010] A fifth aspect of the present invention provides a method for preparing the fermented butter of the present invention, wherein the method comprises inoculating the Lactobacillus paracasei or the fermentation agent of the present invention into cream for fermentation.

[0011] A sixth aspect of the present invention provides a fermented butter prepared by the method of the present invention.

[0012] Through the above technical solution, the present invention can at least achieve the following beneficial effects: (1) When preparing fermented butter, the Lactobacillus paracasei CGMCC No. 33154 provided by the present invention can increase the types and contents of flavor substances in the fermented butter, so that the fermented butter has the advantages of prominent cream aroma, harmonious lactic acid flavor and fruity aroma, etc.; in addition, the Lactobacillus paracasei provided by the present invention ferments the cream under low temperature conditions (10-20° C.), and the types and contents of flavor substances in the prepared fermented butter are richer; (2) The Lactobacillus paracasei CGMCC No. 33154 provided by the present invention has good gastric acid resistance, bile salt resistance, strong self-aggregation and hydrophobicity, and its survival rate reaches 98.83% after 2 hours in simulated standard gastric fluid (pH = 3.0), and its survival rate can reach 66.65% after 4 hours in simulated intestinal fluid conditions (bile salt concentration of 0.2wt%, 4 hours), the self-aggregation rate can reach 55.21%, and the hydrophobicity can reach 55.73%; (3) The Lactobacillus paracasei CGMCC No. 33154 provided by the present invention exhibits good antibacterial effects on common intestinal pathogens such as Escherichia coli, Staphylococcus aureus, and Salmonella, especially on Staphylococcus aureus and Salmonella; (4) The Lactobacillus paracasei CGMCC No. 33154 provided by the present invention is a natural strain isolated from traditional fermented dairy products. It is green and safe, suitable for use in health products, fermented dairy products and beverages, and can improve the autonomy of my country's microbial preparations or starter cultures.

[0013] Biological Deposit The strain provided by the present invention is classified and named as Lactobacillus paracasei 副干酪乳杆菌 , isolated from Xinjiang sour mare's milk, was deposited in the General Microbiology Center of China Microorganism Culture Collection (abbreviated as CGMCC) on December 23, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No.33154. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a colony morphology diagram of Lactobacillus paracasei of the present invention.

[0015] Figure 2 It is the Gram staining result and cell morphology diagram of Lactobacillus paracasei of the present invention. DETAILED DESCRIPTION

[0016] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0017] In the present invention, "CCNH577" is the number of the Lactobacillus paracasei provided by the present invention during the research process by the inventor, and "CGMCC No." is the preservation number of the strain. The two represent the same strain, and their numbers can be used interchangeably in the following text.

[0018] The inventors of the present invention isolated a strain of Lactobacillus paracasei CCNH577 from Xinjiang sour mare's milk during the research process. The research found that the strain has both probiotic properties and fermentation properties, has good gastric acid and bile salt resistance and in vitro nonspecific adhesion performance, and has significant antibacterial activity against common intestinal pathogens. It can also ferment dairy products (especially fermented butter) at a relatively low temperature (10-20°C) to produce rich fruit flavor, cream flavor and lactic acid flavor with a harmonious aroma.

[0019] The first aspect of the present invention provides a strain of Lactobacillus paracasei ( 副干酪乳杆菌 ), wherein the preservation number of the Lactobacillus paracasei is CGMCC No.33154.

[0020] The second aspect of the present invention provides a fermentation bacterial agent, wherein the fermentation bacterial agent includes the Lactobacillus paracasei described in the first aspect of the present invention.

[0021] According to some embodiments of the present invention, the content of Lactobacillus paracasei is 10 7 -10 12 CFU / g (liquid starter culture can be converted according to 1g=1mL).

[0022] In the present invention, "10 7 -10 12 CFU / g means that the content of Lactobacillus paracasei CCNH577 in the starter reaches 10 7 -10 12 CFU / g level, for example 10 7 The CFU / g level represents a value greater than or equal to 1×10 7 CFU / g to less than 1×10 8 CFU / g range, that is, 1×10 7 CFU / g, 5×10 7CFU / g, 9.9×10 7 CFU / g and so on are all within 10 7 CFU / g. Therefore, in the method provided by the present invention, the content of Lactobacillus paracasei CCNH577 is greater than or equal to 1×10 7 CFU / g to less than 1×10 13 CFU / g. For example, in the starter provided by the present invention, the content of Lactobacillus paracasei CCNH577 can be 1×10 7 CFU / g, 5×10 7 CFU / g, 8×10 7 CFU / g, 1×10 8 CFU / g, 5×10 8 CFU / g, 8×10 8 CFU / g, 1×10 9 CFU / g, 5×10 9 CFU / g, 8×10 9 CFU / g, 1×10 10 CFU / g, 5×10 10 CFU / g, 8×10 10 CFU / g, 1×10 11 CFU / g, 5×10 11 CFU / g, 8×10 11 CFU / g, 1×10 12 CFU / g, 5×10 12 CFU / g, 8×10 12 CFU / g and 1×10 13 Any value within the numerical range formed by any two values ​​of CFU / g.

[0023] The third aspect of the present invention provides a method for preparing the fermentation agent of the present invention.

[0024] In the present invention, there is no particular limitation on the specific type of the leavening agent, and any leavening agent type commonly used in the art, especially leavening agent types used for fermented food preparation, can be applicable to the present invention, for example, liquid leavening agent, solid leavening agent, or semi-solid leavening agent.

[0025] According to some embodiments of the present invention, the method comprises: inoculating the Lactobacillus paracasei described in the present invention into a liquid culture medium for seed culture to obtain a liquid fermentation bacterial agent.

[0026] According to some embodiments of the present invention, the method includes: inoculating the Lactobacillus paracasei described in the present invention into a liquid culture medium for seed culture to obtain a seed culture solution; fermenting the seed culture solution in a fermentation medium to obtain a fermentation solution; and performing solid-liquid separation on the fermentation solution to obtain a semi-solid fermentation agent.

[0027] In the present invention, after the fermentation broth is subjected to solid-liquid separation (centrifugation), most of the liquid components (preferably more than 90% by weight) in the fermentation broth are removed, and the obtained bacterial sludge (the fermentation broth content in the bacterial sludge is less than 10% by weight) can be used as a semi-solid starter.

[0028] According to some embodiments of the present invention, the method includes: inoculating the Lactobacillus paracasei described in the present invention into a liquid culture medium for seed culture to obtain a seed culture solution; fermenting the seed culture solution in a fermentation medium to obtain a fermentation solution; performing solid-liquid separation on the fermentation solution, and drying the obtained bacterial mud to obtain a solid fermentation bacterial agent.

[0029] In the present invention, there is no particular limitation on the method of solid separation, and for example, filtration or centrifugation may be used, preferably centrifugation.

[0030] Preferably, the centrifugal conditions include: a rotation speed of 5000-6000 rpm and a time of 25-35 min.

[0031] In the present invention, there is no particular limitation on the drying method. Preferably, the drying method is freeze drying.

[0032] In the present invention, there is no particular limitation on the culture medium for liquid culture and fermentation culture. For example, the culture medium used for liquid culture and fermentation culture can be conventional MRS liquid culture medium (MRS liquid culture medium is purchased from Qingdao Haibo Biological, with the product number HB0384-1).

[0033] In the present invention, preferably, the solid starter of the present invention may further contain auxiliary materials. The auxiliary materials may be auxiliary materials commonly used in the preparation of bacterial agents or starter in the art, such as buffers (such as buffers, culture media, etc.), protective agents (such as freeze-drying protective agents, such as skim milk powder, trehalose, sodium glutamate, glycerol, etc.), etc.

[0034] In the present invention, the method of adding the auxiliary material includes: mixing the bacterial mud obtained by solid-liquid separation with the auxiliary material and then drying to obtain a solid fermentation bacterial agent.

[0035] Preferably, the auxiliary material is a skim milk powder aqueous solution; the skim milk powder content in the skim milk powder aqueous solution is 5%-15% (w / v). "% (w / v)" refers to the weight volume percentage, for example, "the skim milk powder content in the skim milk powder aqueous solution is 12% (w / v)" means that the weight of skim milk powder in the skim milk powder aqueous solution per unit volume is 12% (e.g., the weight of skim milk powder in 100 mL of milk powder aqueous solution is 12g).

[0036] Preferably, the volume ratio of the bacterial mud to the auxiliary material is 1:(3-5).

[0037] Preferably, based on the total volume of the liquid culture medium, the inoculum amount of the Lactobacillus paracasei is 10 1 -10 11 CFU / mL.

[0038] Preferably, based on the volume of the fermentation medium, the inoculation amount of the seed culture solution is 3v%-10v%. "% (v / v)" refers to volume percentage, for example, "adding the seed solution to the culture medium at a ratio of 3% (v / v)" means that the volume of the seed solution added to the culture medium is 3% of the volume of the culture medium.

[0039] According to some embodiments of the present invention, the conditions for seed culture include: temperature of 33-37° C., time of 24-48 h, and pH of 5-7.

[0040] According to some embodiments of the present invention, the fermentation culture conditions include: temperature of 33-37°C, time of 24-48h, and pH of 5-7.

[0041] The fourth aspect of the present invention provides the use of the Lactobacillus paracasei of the present invention or the fermentation agent of the present invention in dairy products or in improving the flavor of fermented butter.

[0042] According to some embodiments of the present invention, the dairy product is selected from at least one of fermented butter, cheese, condensed milk and yogurt.

[0043] According to some embodiments of the present invention, the flavor substance includes at least one of ethyl caproate, nonanoic acid, δ-octanolactone, butyric acid, acetic acid, caproic acid, δ-decanoic acid and ethyl caprylate.

[0044] A fifth aspect of the present invention provides a method for preparing the fermented butter of the present invention, wherein the method comprises inoculating the Lactobacillus paracasei or the fermentation agent of the present invention into cream for fermentation.

[0045] According to some embodiments of the present invention, the fermentation temperature is 10-40° C., preferably 10-20° C. For example, the fermentation temperature can be any value within a numerical range consisting of any two values ​​of 10° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C. and 20° C.

[0046] The inventors of the present invention have found in their research that the Lactobacillus paracasei provided by the present invention can ferment cream under medium and low temperature conditions to prepare fermented butter. However, after fermentation at a higher temperature, the obtained fermentation product needs to be refrigerated and ripened, while if fermentation is carried out under low temperature conditions, refrigeration and ripening are not required, the fermentation and ripening time can be greatly shortened, and the production efficiency can be improved. Moreover, the Lactobacillus paracasei provided by the present invention ferments cream under low temperature conditions (10-20°C), and the prepared fermented butter has richer flavor substances and content.

[0047] There is no particular limitation on the fermentation time in the method provided by the present invention, as long as fermented butter with excellent flavor can be obtained. For example, the specific fermentation time can be determined based on changes in the texture characteristics, pH value, odor and other properties of the fermentation product.

[0048] Preferably, the fermentation time is 10-30 hours, preferably 18-24 hours.

[0049] According to some preferred embodiments of the present invention, the pH value at the fermentation end point 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 meet the above requirements when the above pH is reached, the fermentation time can be appropriately extended or shortened to make the fermented butter have better quality.

[0050] Preferably, the pH value at the fermentation endpoint is 5.4-5.8. For example, the pH value at the fermentation endpoint can be any value within a numerical range consisting of any two values ​​of 5.4, 5.45, 5.5, 5.55, 5.6, 5.65, 5.7, 5.75 and 5.8.

[0051] According to some embodiments of the present invention, the amount of the Lactobacillus paracasei or the fermentation agent is such that the content of the Lactobacillus paracasei in the inoculated cream is ≥10 6 CFU / mL, preferably 10 7 -10 8 CFU / mL.

[0052] Preferably, based on the total volume of the cream, the amount of the Lactobacillus paracasei or the fermentation agent can be 0.5-5% (w / v), that is, the amount of the Lactobacillus paracasei or the fermentation agent added in every 100 mL of cream can be 0.5-5 g. For example, the amount of the Lactobacillus paracasei or the fermentation agent can be any value within the numerical range consisting of any two 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).

[0053] Preferably, in the fermented product (i.e., fermented cream), the effective viable count of Lactobacillus paracasei is ≥ 2×10 7 CFU / mL, preferably 1×10 8 -1×10 10 CFU / mL; More preferably, in the product obtained by fermentation, the content of Lactobacillus paracasei is 2×10 8 -5×10 9 CFU / mL.

[0054] Preferably, the method further comprises stirring, filtering, washing and squeezing the prepared fermentation product, so as to remove buttermilk and water in the fermentation product.

[0055] Preferably, the stirring is performed in a stirrer; the stirring conditions include: a stirring temperature of 10-15° C. and a stirring speed of 750-1000 rpm.

[0056] In the present invention, there is no particular limitation on the filtering method. Preferably, the filtering method includes filtering using 3-4 layers of medical gauze.

[0057] Preferably, the washing method comprises washing the filtered fermentation product with cold water at 0-4° C. for 1-3 times to remove buttermilk in the fermentation product.

[0058] Preferably, the extrusion method includes using a mill to extrude the washed product to remove moisture and shape it to obtain the fermented butter.

[0059] A sixth aspect of the present invention provides a fermented butter prepared by the method of the present invention.

[0060] According to a particularly preferred embodiment of the present invention, a method for preparing fermented butter is provided, the method comprising the following steps: (1) Lactobacillus paracasei CGMCC No. 33154 was inoculated into MRS liquid culture medium and cultured at 33-37°C for 24-48 hours to obtain seed culture medium; Based on the total volume of the liquid culture medium, the inoculum size of the Lactobacillus paracasei is 10 1 -10 9 CFU / mL; (2) Add MRS liquid culture medium to the fermentation tank, sterilize at 115-121°C for 15-20 min, cool to room temperature after sterilization, add the above seed culture solution at a ratio of 3-10% (v / v), and continue fermenting at 33-37°C and 110-200 rpm for 24-48 h. During the fermentation process, maintain the pH of the fermentation solution at 6±1 to obtain a strain fermentation solution; (3) Centrifuging the fermentation liquid at 5000-6000 rpm for 25-35 min, collecting the bacterial sludge; then mixing the bacterial sludge with a skim milk powder protective agent (i.e., a skim milk powder aqueous solution containing 5-12% (w / v) skim milk powder) at a volume ratio of 1:(3-5), pre-freezing at -80°C overnight after thorough mixing, and then freeze-drying the mixture to obtain a freeze-dried powder, thereby obtaining a solid starter culture; (4) pasteurizing fresh cream, then inoculating the solid starter at a ratio of 1-5% (w / v), fermenting at 10-20° C. until the pH value of the fermentation system reaches 5.4-5.8, and fermenting for 18-24 hours; (5) After fermentation, the fermentation product is stirred at a high speed of 750-1000 rpm at 10-15°C to aggregate the fat particles therein, and then the stirred fermentation product is filtered and washed with cold water (0-4°C) 1-3 times to remove the buttermilk in the fermentation product; the washed product is squeezed with a mill to remove moisture and set to obtain fermented butter, which is then stored in a refrigerator at 0-4°C.

[0061] The present invention will be described in detail below through examples.

[0062] The cream in the following examples was purchased from Arla Cheese Co., Ltd.

[0063] In the following embodiments, "% (v / v)" refers to volume percentage, for example, "adding the seed liquid 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, "adding the starter to the cream at a ratio of 1% (w / v)" means that the weight of the starter added to the unit volume of the cream is 1% (for example, the weight of the starter added to 100 mL of cream is 1 g).

[0064] Lactobacillus rhamnosus LGG was purchased from the American Type Culture Collection with the strain number ATCC 53103.

[0065] Lactobacillus paracasei CICC 6108 was purchased from China Industrial Microbiological Culture Collection Center.

[0066] Unless otherwise specified in the following examples and comparative examples, the reagents and materials used are all commercial products purchased from regular chemical or biological reagent and material suppliers, and the reagents are all analytically pure.

[0067] Preparation Example 1 Isolation, purification, identification, performance testing and preservation of Lactobacillus paracasei CGMCC No.33154.

[0068] 1. Isolation and purification of strains A lactobacillus strain CCNH577 was isolated from sour mare's milk collected from Xinjiang by gradient dilution and spreading method, and a pure culture of the strain was obtained by streaking method.

[0069] 2. Identification of strains (1) Morphological identification: The colony morphology of strain CCNH577 was observed. It was found that its colonies on the MRS plate were milky white regular circles. The colonies were moist, smooth, and opaque. Figure 1 ); then the strain CCNH577 was subjected to Gram staining, and the staining results and cell morphology of the strain were observed under a microscope, and it was found that it was a Gram-positive bacterium, with rod-shaped cells, arranged singly, in short chains, or in a palisade shape ( Figure 2 ).

[0070] (2) Molecular identification The strain CCNH577 was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the NCBI database and it was found that the 16S rDNA of strain CCNH577 was similar to that of Lactobacillus paracasei ( 乳杆菌属 副干酪 ) has a homology of 99.58%.

[0071] Combining the results of physiological ecology, morphology and molecular identification, strain CCNH577 was identified as Lactobacillus paracasei ( 副干酪乳杆菌 ).

[0072] (III) Basic performance evaluation (1) Growth performance evaluation: After Lactobacillus paracasei CCNH577 was activated for 2 generations, it was inoculated into MRS broth at a rate of 3% (v / v). After culturing for 18 h, the OD value of the bacterial solution at 600 nm was measured and the OD value was expressed as 18h The blank culture medium was used as a control; the results showed that the OD of Lactobacillus paracasei CCNH577 18h It is about 4.63, which indicates that the strain has good growth ability and can reproduce at a faster rate.

[0073] (2) Evaluation of acid production capacity: After Lactobacillus paracasei CCNH577 was activated for two generations, it was inoculated into MRS broth at an inoculum ratio of 3% (v / v). A control group was set up (Lactobacillus rhamnosus LGG and Lactobacillus paracasei CICC 6108 were used as control group strains). The lactic acid content in the culture medium was detected after culturing for 18 h. The lactic acid content (g / L) in the culture medium was determined by HPLC. The test results are shown in Table 1.

[0074] The determination results in Table 1 show that the lactic acid content in the culture solution of Lactobacillus paracasei CCNH577 is 23.99 g / L, indicating that the strain has a high lactic acid production capacity and can produce lactic acid efficiently.

[0075] Table 1

[0076] (3) Enzyme production capacity test: After strain CCNH577 was activated for two generations, it was inoculated into MRS broth at an inoculum ratio of 3% (v / v). A control group was set up (Lactobacillus rhamnosus LGG and Lactobacillus paracasei CICC 6108 were used as control group strains). After culturing for 18 h, the enzyme activities of protease and lipase in the culture medium were determined by the Folin-phenol method and the p-nitrophenol method, respectively. The test results are shown in Table 2.

[0077] The measurement results in Table 2 show that the enzyme activity of protease in the culture medium of strain CCNH577 is 34.43U / mL, and the enzyme activity of lipase is 1.72U / mL, indicating that the strain has a strong enzyme production ability and can produce a high degradation efficiency for fat and protein during fermentation.

[0078] Table 2

[0079] (4) Evaluation of probiotic performance Evaluation of gastric acid and bile salt resistance: Refer to T / CNHFA435-2024 "Gastric Juice Tolerance Test Method for Probiotics" to evaluate the gastric acid resistance of candidate strains; refer to this method to evaluate the bile salt resistance of candidate strains.

[0080] The results of the evaluation of the candidate strains' resistance to gastric acid and bile salts are shown in Table 3.

[0081] The test results in Table 3 show that the survival rate of strain CCNH577 reached 98.83% after 2 hours in simulated standard gastric fluid (pH=3.0), and the survival rate reached 66.65% after 4 hours in simulated intestinal fluid conditions (bile salt concentration of 0.2wt%, 4 hours), indicating that the strain has good resistance to gastric acid and bile salts.

[0082] Table 3

[0083] (5) Adhesion performance evaluation Evaluation of self-aggregation performance: The glycerol tubes of strains CCNH577, LGG and CICC6108 were inoculated into fresh MRS liquid medium at 2‰ (v / v) and cultured at 37°C overnight for 18 h. The OD 600 After washing twice with saline, resuspend the 600 =1, pipette 100 μL of bacterial solution into the ELISA plate and detect the initial OD 600 The bacterial solution was then allowed to stand for 4 h, and 100 μL of the upper bacterial solution was taken to an ELISA plate to detect the OD value after standing. 600 The value is recorded as A1, and the self-aggregation is calculated using the following formula: Self-aggregation rate (%) = (1- )×100%, the results are shown in Table 4.

[0084] Hydrophobic performance evaluation: absorb the above OD 600 =1, add 100 μL of bacterial solution to the ELISA plate and detect the initial OD 600 Then, 900 μL of bacterial solution was taken, 180 μL of dodecane was added, and the mixture was vortexed and allowed to stand for 20 min to separate layers. The lower aqueous phase was taken to measure its OD 600 Value, recorded as A, use the following formula to calculate hydrophobicity, hydrophobicity (%) = ×100%, the results are shown in Table 4.

[0085] Table 4

[0086] High bacterial hydrophobicity can help bacteria adhere to the intestinal mucosa. The higher the bacterial hydrophobicity, the stronger its adhesion ability. As shown in Table 4, the hydrophobicity of strain CCNH577 is 55.73%, which is higher than that of strain LGG and strain CICC 6108. The self-aggregation of the strain is positively correlated with the adhesion ability of the strain. The self-aggregation rate of strain CCNH577 is the highest, reaching 55.21%, which is higher than the two control strains.

[0087] (6) Evaluation of antibacterial performance The indicator strains Escherichia coli, Staphylococcus aureus, and Salmonella were inoculated into LB liquid medium, and Candida albicans was inoculated into YPD liquid medium. After the indicator bacteria were cultured overnight, they were transferred to the corresponding fresh medium and cultured until OD 600 =1 or the viable count is greater than 10 8 CFU / mL, dilute the viable bacteria in the culture medium to 105 CFU / mL was used as the indicator bacterial solution.

[0088] Glycerol tubes of strains CCNH577, LGG, and CICC 6108 were inoculated into fresh sterile MRS liquid culture medium, cultured overnight at 37°C for 18 h, and then inoculated into fresh MRS liquid culture medium at a 2% (v / v) inoculation amount. After static culture at 37°C for 18 h, the supernatant was centrifuged and sterilized by membrane filtration as test samples; MRS liquid culture medium was used 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 sample OD 600 Values ​​(where A0 and A are the OD 600 and OD before culture 600 ), the results are shown in Table 5.

[0089] Table 5

[0090] As can be seen from Table 5, the inhibitory ability of strain CCNH577 on Escherichia coli CICC10421 (purchased from China Industrial Microbiological Culture Collection Center), Staphylococcus aureus CMCC 26001 (purchased from China Medical Bacteria Collection Center), Staphylococcus aureus CMCC 26003 (purchased from China Medical Bacteria Collection Center), Salmonella ATCC 14028 (purchased from American Type Culture Collection Center) and Salmonella CVCC 3378 (purchased from National Veterinary Microbiological Culture Collection Center) is better than LGG and CICC 6108. This shows that strain CCNH577 has a strong antibacterial ability, especially for Staphylococcus aureus and Salmonella.

[0091] 4. Strain preservation The Lactobacillus paracasei CCNH577 obtained by the above screening was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (abbreviated as CGMCC) on December 23, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No.33154.

[0092] Preparation Example 2 The Lactobacillus paracasei CGMCC No.33154 obtained in Preparation Example 1 was inoculated into MRS liquid culture medium and cultured at 37°C for 24 h to obtain a seed culture solution (the number of viable bacteria was about 8×10 9 CFU / mL); MRS liquid culture medium was added to the fermenter and sterilized at 121°C for 20 min. After sterilization, it was cooled to room temperature and the above seed culture medium was added at a ratio of 3% (v / v). The fermentation was continued at 37°C and 110 rpm for 24 h. During the fermentation process, the pH of the fermentation liquid was maintained at 6±1 to obtain the bacterial fermentation liquid (the number of viable cells was about 5×10 8 CFU / mL); The fermentation liquid was centrifuged at 5000 rpm for 25 min to collect the bacterial sludge. The bacterial sludge was then mixed with skim milk powder protective agent (i.e., skim milk powder aqueous solution containing 12% (w / v) skim milk powder) at a volume ratio of 1:3. After being fully mixed, it was pre-frozen at -80°C overnight and then freeze-dried by vacuum freeze drying to obtain freeze-dried powder, thus obtaining a solid starter (the number of viable cells was about 3.4×10 10 CFU / g).

[0093] Example 1 Fermentation: pasteurize fresh cream (95°C, 30s), then inoculate the solid starter (Preparation Example 2) at a ratio of 1% (w / v), and ferment at 10°C until the pH value of the fermentation system reaches 5.72, and the fermentation time is 24h; Stirring and removing buttermilk: After fermentation, stir the fermented product at 10°C and 750 rpm to aggregate the fat particles (stirring for about 30 minutes), then filter the stirred fermented product (using 4 layers of medical gauze for filtration), and then wash it twice with cold water (4°C) to remove the buttermilk; The washed product was squeezed by a mill to remove moisture and was shaped to obtain fermented butter A1, which was then stored in a refrigerator at 4°C.

[0094] Example 2 Fermentation: pasteurize fresh cream (85°C, 2 min), then inoculate the solid starter (Preparation Example 2) at a ratio of 2% (w / v), and ferment at 20°C until the pH value of the fermentation system reaches 5.69, and the fermentation time is 26 h; The operation and conditions of stirring and removing buttermilk are the same as those in Example 1; The washed product was squeezed by a mill to remove moisture and shape to obtain fermented butter A2, which was then stored in a refrigerator at 4°C.

[0095] Example 3 Fermentation: pasteurize fresh cream (85°C, 2 min), then inoculate solid starter (Preparation Example 2) at a ratio of 3% (w / v), and ferment at 37°C until the pH value of the fermentation system reaches 5.42, and the fermentation time is 16 h; Post-ripening: place the fermentation product at 8°C for 24 hours; The operation and conditions of stirring and removing buttermilk are the same as those in Example 1; The washed product was squeezed by a mill to remove moisture and shape to obtain fermented butter A3, which was then stored in a refrigerator at 4°C.

[0096] Comparative Example 1 The method of Example 1 is followed, except that the starter is replaced with a starter containing Lactobacillus paracasei CICC6108 (the preparation method of the starter is the same as that of Preparation Example 2), and the remaining operations and conditions are the same as those of Example 1 to obtain fermented butter D1, which is refrigerated and stored at 4°C.

[0097] Comparative Example 2 The method of Example 1 was followed, except that the starter was replaced with the commercial bacterial agent Advance 2.0 (the bacterial agent was purchased from Chr. Hansen (Beijing) Trading Co., Ltd.), and the remaining operations and conditions were the same as those of Example 1 to obtain fermented butter D2, which was refrigerated and stored at 4°C.

[0098] Test Example 1 The number of live bacteria in the cream inoculated with the starter (before fermentation) and the cream after fermentation (after fermentation) in the above embodiments and comparative examples was detected; the fermentation and ripening time of the fermented butter in the above embodiments and comparative examples and the pH value of the fermentation product (fermentation liquid) were counted; the water content and fat content of the fermented butter obtained in the above embodiments and comparative examples were detected, and the results are shown in Table 7.

[0099] Test Example 2 Twenty persons with experience in sensory evaluation in this field served as evaluators, and sensory evaluation was performed on the fermented butters obtained in the above examples and comparative examples with reference to the evaluation criteria in Table 6. The results are shown in Table 8.

[0100] Table 6

[0101] Test Example 3 The aroma substance components and contents of Example 1 and commercially available domestic fermented butter were tested respectively. The specific test results are shown in Tables 9-10 (Table 9 corresponds to the fermented butter obtained in Example 1, and Table 10 corresponds to commercially available domestic fermented butter, and the domestic fermented butter was purchased from Mengniu Dairy Company).

[0102] Testing method for aroma components and contents in fermented butter: Use solid phase microextraction (SPME) to pre-treat the sample (fermented butter), and combine it with gas chromatography-mass spectrometry (GC-MS) to detect the volatile flavor components of the fermented butter, analyze and identify its key aroma components.

[0103] SPME conditions: Accurately weigh about 5 g of the sample to be tested and add it to 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 ° C, extraction time 60 min.

[0104] GC conditions: HP-INNOWax (60m×0.25mm×0.25μm) chromatographic column; carrier gas: He, flow rate: 2mL / min; inlet temperature: 250℃, no split; detector temperature: 280℃; temperature program: start at 40℃, hold for 2min; increase to 180℃ at 3℃ / min and hold for 3min; increase to 230℃ at 5℃ / min and hold for 5min.

[0105] MS conditions: EI ionization source, electron energy 70 eV, ion source temperature 250 °C, mass scanning range 29-350 amu, interface temperature 250 °C.

[0106] The collected mass spectra were searched using the NIST11.L spectral library, and manual spectral analysis was performed in combination with retention time, CAS number and English name to determine the volatile flavor components of fermented butter; the characteristic aroma components were evaluated using the odor activity value method (OAV, for details, please refer to: Zhao Yu, Zhang Yuhuan, Li Jianke, et al. GC-O combined with OAV identification of key aroma components of extra virgin olive oil in Longnan [J]. Food Science, 2022, 43(08): 184-189) to evaluate the contribution of each volatile component to the overall aroma of the sample, that is, 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.

[0107] Table 7

[0108] Table 8

[0109] Table 9

[0110] Table 10

[0111] Note: Aroma substances not listed in the table were not detected.

[0112] It can be seen from the data in Table 7 that, when comparing Example 1-2 with Example 3, the Lactobacillus paracasei CCNH577 provided by the present invention can ferment cream under medium and low temperature conditions (10-37°C) to prepare fermented butter. However, after fermentation at a higher temperature (37°C), the obtained fermentation product needs to be refrigerated and ripened, while fermentation under low temperature conditions (10-20°C) does not require refrigeration and ripening, which can greatly shorten the fermentation and ripening time and improve production efficiency. When comparing Example 1-3 with Comparative Example 1-2, the Lactobacillus paracasei CCNH577 of the present invention can be rapidly proliferated when inoculated in cream, and the fermentation intensity (i.e., the number of Lactobacillus paracasei increased per unit time, 10 7 CFU / (mL . h)) High.

[0113] It can be seen from the data in Table 8 that the fermented butter prepared by using the Lactobacillus paracasei CCNH577 provided by the present invention is rich in flavor substances and has the advantages of coordinated cream flavor, lactic acid flavor and fruit flavor.

[0114] As shown in Table 9, a total of 26 volatile substances were detected in the fermented butter prepared by Lactobacillus paracasei CCNH577, including 8 acids, 5 alcohols, 5 esters, 4 ketones, 1 phenol, 1 aldehyde, and 2 others. Among them, the OAV values ​​of ethyl caproate, nonanoic acid, δ-octanolactone and butyric acid are all greater than 1, which are the key flavor compounds in the above fermented butter. Ethyl caproate has fruity, sweet and cellar aromas, nonanoic acid has soapy and grassy flavors, δ-octanolactone has fruity and coconut aromas, and butyric acid has cheese and sour cream aromas. These key flavor substances have made a major contribution to the unique flavor of fermented butter. Aroma compounds with OAVs between 0.1-1 include acetic acid, caproic acid, δ-decanoic acid and ethyl caprylate, and these aroma substances have a modifying effect on the flavor of fermented butter products.

[0115] By comparing the data in Table 9 and Table 10, it can be seen that the types and contents of flavor substances in commercially available fermented butter are significantly lower than those in the fermented butter obtained in Example 1. Twelve flavor substances were detected in commercially available fermented butter, and only acetoin had an OAV value greater than 1, which is the key flavor compound of fermented butter and mainly has the flavor of butter and cream; butyric acid has an OAV value between 0.1 and 1, which has a cheese aroma and modifies the flavor of fermented butter.

[0116] In summary, the Lactobacillus paracasei CCNH577 provided by the present invention has both probiotic properties and fermentation properties, has good gastric acid and bile salt resistance and in vitro nonspecific adhesion performance, and has significant antibacterial activity against common intestinal pathogens. Moreover, it can ferment dairy products (especially fermented butter) at a relatively low temperature (10-20°C) to produce rich fruit flavor, cream flavor and lactic acid flavor with a harmonious aroma.

[0117] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A strain of Lactobacillus paracasei with antibacterial effect ( Lactobacillus paracasei ), characterized in that, The preservation number of the Lactobacillus paracasei is CGMCC No.33154.

2. A fermentation agent, characterized in that: The fermentation bacteria agent comprises the Lactobacillus paracasei described in claim 1.

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

4. A method for preparing a fermentation agent, characterized in that: The method comprises: inoculating the Lactobacillus paracasei described in claim 1 into a liquid culture medium for seed culture to obtain a liquid fermentation bacterial agent; Alternatively, the Lactobacillus paracasei of claim 1 is inoculated into a liquid culture medium for seed culture to obtain a seed culture solution; the seed culture solution is fermented in a fermentation medium to obtain a fermentation solution; the fermentation solution is solid-liquid separated to obtain a semi-solid fermentation agent; Alternatively, the Lactobacillus paracasei described in claim 1 is inoculated into a liquid culture medium for seed culture to obtain a seed culture solution; the seed culture solution is fermented and cultured in a fermentation medium to obtain a fermentation solution; the fermentation solution is solid-liquid separated, and the obtained bacterial mud is dried to obtain a solid fermentation bacterial agent.

5. The method according to claim 4, wherein: The liquid culture medium and the fermentation medium are each independently an MRS liquid culture medium; And / or, the solid-liquid separation method is centrifugation; And / or, based on the total volume of the liquid culture medium, the inoculum amount of the Lactobacillus paracasei is 10 1 -10 11 CFU / mL; and / or, the inoculation amount of the seed culture solution is 3v%-10v% of the volume of the fermentation medium; And / or, the seed culture conditions include: temperature of 33-37°C, time of 24-48h, pH of 5-7; And / or, the fermentation culture conditions include: temperature of 33-37°C, time of 24-48h, and pH of 5-7.

6. Application of the Lactobacillus paracasei described in claim 1 or the fermentation bacteria agent described in claim 2 or 3 in dairy products or improving fermented butter flavor substances.

7. The use according to claim 6, wherein: The dairy product is selected from at least one of fermented butter, cheese, condensed milk and yogurt; And / or, the flavor substance includes at least one of ethyl caproate, nonanoic acid, δ-octanolactone, butyric acid, acetic acid, caproic acid, δ-decanoic acid and ethyl caprylate.

8. A method for preparing fermented butter, characterized in that: The method comprises inoculating the Lactobacillus paracasei described in claim 1 or the fermentation agent described in claim 2 or 3 into cream for fermentation.

9. The method according to claim 8, wherein: The fermentation conditions include: temperature of 10-40°C, time of 10-30h, pH of 5.2-6; And / or, the amount of the Lactobacillus paracasei or the fermentation agent is such that the number of viable Lactobacillus paracasei in the cream after inoculation is ≥10 6 CFU / mL.

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

Images