Lactococcus, microbial inoculum and application of lactococcus and microbial inoculum in lowering cholesterol

Abstract

The invention relates to the field of microorganisms, in particular to lactococcus, a microbial agent and application of the lactococcus and the microbial agent in lowering cholesterol. The invention provides lactococcus. The lactococcus is selected from at least one of lactococcus with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 32756, lactococcus with the preservation number of CGMCC No. 32757 and lactococcus with the preservation number of CGMCC No. 32758. The lactococcus separated from yak ghee provided by the invention has good cholesterol lowering capacity and excellent acid resistance, cholate resistance and high temperature resistance, and provides a reference basis for application and development of the lactococcus in fermented food and development of probiotic functional milk products.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and specifically, to a Lactococcus, a bacterial agent, and their applications in reducing cholesterol. Background Art

[0002] Cholesterol, also known as cholesterin, is a lipid molecule containing a cyclopentanoperhydrophenanthrene skeleton and is an important lipid substance in living organisms. Cholesterol widely exists in animal bodies, especially in the brain and nerve tissues, and also has a relatively high content in the kidneys, spleen, skin, liver, and bile.

[0003] Cholesterol has very important physiological functions in the body. However, due to the changes in modern human lifestyles and dietary structures, excessive intake of cholesterol into the body is harmful to humans. High cholesterol content in the serum is an important factor inducing cardiovascular and cerebrovascular diseases. High concentrations of cholesterol increase the risk of atherosclerotic cardiovascular diseases and have become the main diseases threatening the health of humans globally. According to a study, for every 1% decrease in the cholesterol concentration in human serum, the incidence of cardiovascular and cerebrovascular diseases decreases by about 2% - 3%. According to the prediction of the World Health Organization in [year], up to 40% of deaths will be related to cardiovascular diseases caused by cholesterol by 2030, which is expected to affect the health of about 20 million people globally. Long-term intake of a high-fat diet will lead to the occurrence of metabolic diseases such as hypercholesterolemia. Currently, the clinical treatment methods for cholesterol include diet control and drug therapy. For diet control, it is difficult to adhere to in the long term, so the effect is not very good. However, the use of drugs also has side effects, such as rhabdomyolysis, liver function damage, and causing drowsiness, nausea, and vomiting.

[0004] With the enhancement of people's health awareness, how to effectively reduce the cholesterol level in the body has become one of the research hotspots of many scholars. Among many potential cholesterol-lowering sources, lactic acid bacteria have received extensive attention due to their probiotic characteristics. At present, the cholesterol degradation rate of commercial strains still needs to be further improved. Therefore, the development of lactic acid bacteria with high cholesterol-lowering functions and their related functional foods has become the first choice for the treatment of hypercholesterolemia. Summary of the Invention

[0005] In order to achieve the above object, the inventors of the present invention selected traditionally fermented yak butter, isolated lactic acid bacteria therefrom, and conducted morphological identification and physiological and biochemical identification. After determining their species, they measured their acid resistance, bile salt resistance, high-temperature resistance, and cholesterol-lowering ability. Finally, three strains of lactic acid bacteria with acid resistance, bile salt resistance, high-temperature resistance, and high-efficiency cholesterol-lowering ability were obtained, providing a reference basis for their application development in fermented foods and for the development of probiotic functional low-fat dairy products.

[0006] Therefore, in a first aspect, the present invention provides a Lactococcus bacterium, which is selected from at least one of the Lactococcus bacterium with the deposit number of CGMCC No. 32756, the Lactococcus bacterium with the deposit number of CGMCC No. 32757, and the Lactococcus bacterium with the deposit number of CGMCC No. 32758.

[0007] In a second aspect, the present invention provides a bacterial agent, which comprises the Lactococcus bacterium described in the first aspect.

[0008] In a third aspect, the present invention provides the use of the Lactococcus bacterium described in the first aspect or the bacterial agent described in the second aspect in the preparation of a drug or product for reducing cholesterol.

[0009] In a fourth aspect, the present invention provides the use of the Lactococcus bacterium described in the first aspect or the bacterial agent described in the second aspect in the preparation of a food with a low cholesterol content.

[0010] In a fifth aspect, the present invention provides a food, which contains the Lactococcus bacterium described in the first aspect or the bacterial agent described in the second aspect.

[0011] In a sixth aspect, the present invention provides a method for preparing a food with a low cholesterol content, which comprises: inoculating the Lactococcus bacterium described in the first aspect or the bacterial agent described in the second aspect into the food to be fermented to degrade the cholesterol therein.

[0012] In a seventh aspect, the present invention provides a food with a low cholesterol content prepared by the method described in the sixth aspect.

[0013] Through the technical solution of the present invention, the following beneficial effects can be obtained:

[0014] (1) The Lactococcus bacterium isolated from yak butter has good cholesterol-lowering ability, providing a reference basis for its application development in fermented foods and for the development of probiotic functional dairy products.

[0015] (2) The Lactococcus bacterium provided by the present invention has excellent acid resistance, bile salt resistance, and high temperature resistance, and has the characteristics of becoming a probiotic.

[0016] Other features and advantages of the present invention will be described in detail in the subsequent specific implementation section.

[0017] Biological Deposit

[0018] The Lactococcus 1 provided by the present invention is Lactococcus formosensis, which was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 25, 2024. Its deposit number is CGMCC No. 32758, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC).

[0019] The Lactococcus 3 provided by the present invention is Lactococcus garvieae, which was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 25, 2024. Its deposit number is CGMCC No. 32756, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC).

[0020] The Lactococcus 5 provided by the present invention is Lactococcus garvieae, which was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 25, 2024. Its deposit number is CGMCC No. 32757, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings. Among them,

[0022] Figure 1 shows the colony morphology (left) of the Lactococcus provided by the present invention growing on MRS agar plate and the Gram staining result (right).

[0023] Figure 2 shows the survival rate of the Lactococcus provided by the present invention under different pH conditions.

[0024] Figure 3 shows the survival rate of the Lactococcus provided by the present invention under different bile salt concentrations.

[0025] Figure 4 shows the survival rate of the Lactococcus provided by the present invention under different temperature conditions.

[0026] Figure 5 shows the drawn cholesterol standard curve.

[0027] Figure 6 shows the cholesterol-lowering ability of the Lactococcus provided by the present invention in MRS-CHOL medium.

[0028] Figure 7 It shows the cholesterol-lowering ability of the Lactococcus provided by the present invention in fermented butter. Detailed implementation manners

[0029] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0030] In a first aspect, the present invention provides a strain of Lactococcus, which is selected from at least one of Lactococcus with deposit number CGMCC No. 32756, Lactococcus with deposit number CGMCC No. 32757, and Lactococcus with deposit number CGMCC No. 32758.

[0031] The present invention first used yak butter collected from Shangri-La City, Yunnan Province as a sample, and used MRS as a culture medium to isolate the lactic acid bacteria therein, obtaining three single colonies with good growth. Then, morphological observation and physiological and biochemical identification were carried out on them, and they were determined to be Lactococcus, named Lactococcus 1, Lactococcus 3, and Lactococcus 5 respectively, and were deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 25, 2024. Their deposit numbers are CGMCC No. 32758, CGMCC No. 32756, and CGMCC No. 32757 respectively, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC).

[0032] According to the present invention, the method for isolating lactic acid bacteria from yak butter can adopt conventional methods in the art. For example, isolation can be carried out in MRS culture medium. Specifically, yak butter is first enriched and cultured in LB culture medium, and then the culture is serially diluted and spread on MRS solid culture medium for culture. Single colonies with a calcium dissolution circle are selected and streaked on MRS solid culture medium, and finally single colonies with good growth are selected to obtain the Lactococcus of the present invention.

[0033] According to the present invention, the Lactococcus provided by the present invention can produce a large number of viable Lactococcus cells after liquid culture. There are no special requirements for the culture method, as long as it can make the Lactococcus proliferate. For example, viable cells of Lactococcus can be inoculated into MRS liquid culture medium and fermented according to the culture conditions of Lactococcus to obtain a fermentation broth containing a large number of viable cells.

[0034] In some embodiments, the preparation process of the live bacteria also includes activating and purifying the preserved Lactococcus on a solid plate, then inoculating it into a seed medium for cultivation to obtain a seed solution, and finally inoculating the seed solution into a fermentation medium. After fermentation culture, a fermentation broth is obtained.

[0035] The solid medium, seed medium, and fermentation medium can all be MRS medium. Those skilled in the art can understand that the solid medium can be obtained by adding agar to the liquid medium.

[0036] According to a preferred embodiment of the present invention, the Lactococcus is Lactococcus with the preservation number CGMCC No. 32756. Compared with commercial strains, Lactococcus with the preservation number CGMCC No. 32756 has more advantages in the efficacy of reducing cholesterol.

[0037] In a second aspect, the present invention provides a bacterial agent, which contains the Lactococcus as described above.

[0038] According to the present invention, the form of the bacterial agent may not be particularly limited. For example, it can be a liquid bacterial agent, semi-liquid bacterial agent, concentrated bacterial agent, compressed bacterial agent, or solid bacterial agent. Among them, the solid bacterial agent can be a dried bacterial agent. For example, it can be a freeze-dried bacterial agent, spray-dried bacterial agent, etc.

[0039] In some embodiments, the bacterial agent contains live bacteria of the Lactococcus. The number of live bacteria in the bacterial agent can be selected within a relatively wide range as long as the requirements of relevant standards are met. In some embodiments, the bacterial agent is liquid, and the effective content of Lactococcus can be 10 7 cfu / ml, 10 8 cfu / ml or 10 9 cfu / ml. In some embodiments, the bacterial agent is a solid bacterial agent, and the effective content of Lactococcus can be 10 9 cfu / g, 10 10 cfu / g or 10 11 cfu / g.

[0040] According to the present invention, the bacterial agent can be prepared according to the conventional methods in the art. According to a specific embodiment of the present invention, the preparation method of the bacterial agent includes: fermenting and culturing the Lactococcus as described above in a fermentation medium to make the viable bacteria count reach 10 8 cfu / mL or more to obtain a liquid bacterial agent; concentrating the liquid bacterial agent, for example, by solid-liquid separation, to obtain a semi-liquid bacterial agent; then adding a freeze-drying protectant to the semi-liquid bacterial agent and adjusting the viable bacteria concentration to 10 10 cfu / mL or more, and drying the obtained mixed material to obtain a dried bacterial agent.

[0041] According to the present invention, the method of solid-liquid separation can refer to conventional technical means in the art. For example, it can be centrifugation, filtration, etc., as long as the activity of Lactococcus is not significantly affected. According to a preferred embodiment of the present invention, for the centrifugation, for example, it can be centrifuged at a speed of 5000 - 12000 rpm for 5 - 20 min in a refrigerated centrifuge to obtain cell precipitation.

[0042] According to the present invention, the lyoprotectant can be various conventional cryoprotectants in the art. For example, it can be at least one of skim milk powder, maltodextrin, trehalose, dextran, glycerol, etc.

[0043] According to a preferred embodiment of the present invention, before adding the lyoprotectant to the semi-liquid bacterial agent, the method further includes washing the semi-liquid bacterial agent with a buffer solution. Among them, the buffer solution can be a conventional buffer solution for rinsing bacteria in the art. For example, it can be physiological saline or PBS buffer solution.

[0044] Among them, the drying method can not be particularly limited. For example, it can be freeze-drying, drying, air-drying, spray-drying, etc.

[0045] In some embodiments, the bacterial agent is a composition containing the Lactococcus provided by the present invention as the first active ingredient. In addition to Lactococcus, the composition further contains at least one additional probiotic as the second active ingredient for enhancing or supplementing the probiotic effect of Lactococcus. The additional probiotic can be any known probiotic, usually having a complementary effect to Lactococcus, and capable of promoting intestinal health or improving immune function. In some embodiments, the additional probiotic includes but is not limited to Lactobacillus, Bifidobacterium, Lactobacillus acidophilus, Streptococcus thermophilus, or other Lactococcus strains different from the Lactococcus described in the present invention.

[0046] In some preferred embodiments, the active ingredients of the bacterial agent composition can be co-cultured, mixed, or used in combination at a specific ratio to obtain the best biological effect. According to different application requirements, the ratio of the composition can be adjusted according to the actual situation to enhance the probiotic activity of a specific strain or improve the intestinal microecological balance.

[0047] In some preferred embodiments, the composition can further include other auxiliary components, such as prebiotics, carrier substances, protectants, or other substances that can optimize the probiotic effect, to further improve its stability and effectiveness in application.

[0048] In the third aspect, the present invention provides the application of the above-mentioned Lactococcus or the above-mentioned bacterial agent in the preparation of a drug or product for reducing cholesterol.

[0049] The Lactococcus of the present invention not only has remarkable cholesterol-lowering ability, has potential health care and therapeutic value, and is suitable for the development of drugs or related products. At the same time, this Lactococcus has excellent acid tolerance, bile salt tolerance and high temperature tolerance, and can survive stably in the gastrointestinal tract, ensuring its biological activity and efficacy in oral use. Therefore, the Lactococcus of the present invention provides a solid foundation for the development of new health products and treatment programs and has broad application prospects.

[0050] In the fourth aspect, the present invention provides the use of the Lactococcus as described above, or the bacterial agent as described above, in the preparation of a food with a low cholesterol content.

[0051] The Lactococcus provided by the present invention has a remarkable cholesterol-lowering effect and can be widely used in the preparation of foods with a low cholesterol content. By adding this Lactococcus or its bacterial agent to daily foods, it can effectively reduce cholesterol absorption, help maintain a healthy blood lipid level, and thus has an important health promotion effect. These foods not only meet the requirements of a low-cholesterol diet, but also play an active role in promoting cardiovascular health and preventing related diseases caused by high cholesterol. Therefore, the application of the Lactococcus and its bacterial agent of the present invention in the food industry provides an innovative healthy food solution, with broad market prospects and social value.

[0052] In the fifth aspect, the present invention provides a food that contains the Lactococcus as described above, or the bacterial agent as described above.

[0053] The Lactococcus and its bacterial agent provided by the present invention can be widely used in a variety of foods with a low cholesterol content, including but not limited to dairy products, beverages, functional foods, health foods, ready-to-eat foods, pastries, cereal products, snacks, and seasonings, etc. By adding the Lactococcus of the present invention to these foods, it can not only reduce cholesterol absorption, help maintain a healthy blood lipid level, but also provide other potential health benefits, such as improving the intestinal microecological balance and enhancing immune function. Especially in populations with high cholesterol and high fat intake, the elderly, and populations susceptible to cardiovascular diseases, it has a significant health promotion effect. Therefore, the Lactococcus and its bacterial agent of the present invention not only provide innovative technical support for the research and development of low-cholesterol foods, but also open up new market opportunities for the food industry, meeting the growing demand of consumers for healthy diets and functional foods.

[0054] The application of the Lactococcus of the present invention and its bacterial agent in clarified butter is particularly important. As a traditional food with high fat and high cholesterol content, the widespread use of clarified butter in daily diet may have an adverse impact on blood lipid levels. However, by adding the Lactococcus of the present invention, the cholesterol content in clarified butter can be effectively reduced, helping to reduce the adverse effects on the human body, thus making it a healthier choice. In addition, the excellent acid tolerance, bile salt tolerance and high temperature tolerance of Lactococcus ensure its stability during the processing of clarified butter, and can maintain its biological activity, so as to play a cholesterol-lowering role after consumption. Through this innovative application, the present invention not only provides technical support for the health of clarified butter products, but also provides food choices that better meet the modern health needs for consumers.

[0055] In a sixth aspect, the present invention provides a method for preparing a food with low cholesterol content, the method comprising: inoculating the Lactococcus as described above, or the bacterial agent as described above, into the food to be fermented to degrade the cholesterol therein.

[0056] In some embodiments, the preparation method of the present invention includes steps such as Lactococcus inoculation, fermentation and subsequent treatment, ensuring the effective activity of Lactococcus in the food, and being able to degrade the cholesterol in the food to the greatest extent. This method can be widely applied to various food types such as dairy products, clarified butter, beverages, functional foods, ready-to-eat foods, etc.

[0057] The Lactococcus or bacterial agent of the present invention has a significant cholesterol-lowering effect and can maintain a high activity in an acidic environment and at high temperatures. In some embodiments, the Lactococcus provided by the present invention can be added to the food to be fermented by direct inoculation, or added in the form of a bacterial agent. The inoculation concentration of Lactococcus can be 10^6 to 10^9 cfu / g of food, and is adjusted according to the type of specific food and fermentation conditions.

[0058] In some embodiments, the Lactococcus or bacterial agent can be added to the food to be fermented by liquid inoculation. For example, the Lactococcus or bacterial agent is dissolved in a suitable solution (such as nutrient solution, diluted food raw materials, etc.), and then added to the food to be fermented for uniform mixing.

[0059] In some embodiments, the Lactococcus or bacterial agent can be added to the food to be fermented by dry powder inoculation. For example, the Lactococcus or bacterial agent is directly sprinkled into the food raw material to be fermented in the form of dry powder, and then mixed.

[0060] In some embodiments, Lactococcus has good fermentation activity in the temperature range of 25°C to 40°C. Those skilled in the art can also adjust according to the nature of the food and the fermentation target. For example, during the fermentation of dairy products, the temperature is generally controlled at 30-37°C.

[0061] In some embodiments, the length of the fermentation time affects the effect of Lactococcus in degrading cholesterol. Generally, the fermentation time can be between 6 and 48 hours, and the specific time depends on the type of food and the activity of Lactococcus. Too short a time may result in an insignificant degradation effect, while too long a time may affect the taste and other nutritional components of the food.

[0062] During the fermentation process, Lactococcus can degrade cholesterol in food and convert it into other beneficial substances through metabolic activities, reducing the cholesterol content. The cholesterol-lowering effect of Lactococcus gradually increases during the fermentation process. Therefore, appropriate fermentation time and environmental conditions are crucial for maximizing the degradation effect.

[0063] In some embodiments, after fermentation is completed, post-treatment steps may be performed on the fermented food to ensure the quality and stability of the final product. In some embodiments, the post-treatment steps include:

[0064] Cooling: After fermentation is completed, the food needs to be quickly cooled to inhibit the further growth of Lactococcus and ensure its appropriate activity in the food.

[0065] Packaging: The fermented food should be packaged as soon as possible to prevent the external environment from affecting the food quality. Especially for foods such as dairy products and clarified butter, aseptic operation needs to be ensured during packaging.

[0066] Quality control: During the preparation process, quality control of the food is required to ensure that the addition amount of Lactococcus, cholesterol degradation rate, fermentation conditions, etc. meet the predetermined requirements.

[0067] In some embodiments, the food is clarified butter. In some embodiments, the method for preparing the clarified butter includes: inoculating Lactococcus with cholesterol-lowering ability into the clarified butter and using its biological activity to decompose and convert the cholesterol in the clarified butter, thereby obtaining clarified butter with a low cholesterol content.

[0068] In some embodiments, the method for preparing the clarified butter includes: preparation of Lactococcus seed liquid, preparation of clarified butter, inoculation, and fermentation.

[0069] In some embodiments, the Lactococcus provided by the present invention is cultured in a suitable medium (such as MRS medium or a customized lactic acid bacteria medium) and cultured at 30 - 40 °C until the logarithmic growth phase to ensure that the bacteria are in a highly active state. In some embodiments, the cultured Lactococcus is obtained as a bacterial liquid by centrifugation or other solid-liquid separation methods, and the concentration of the bacterial liquid can be controlled at 10^8 to 10^9 cfu / ml to ensure the inoculation effect.

[0070] In some embodiments, the clarified butter is clarified butter prepared by traditional handcraft or industrially produced clarified butter. In some embodiments, the clarified butter is heated to remove the moisture therein, obtaining a pure fat component.

[0071] In some embodiments, the clarified butter is first heated to 50 - 60 °C to keep it in a liquid state, ensuring that Lactococcus can be smoothly inoculated and fermented. In some embodiments, the prepared Lactococcus solution is added to the clarified butter at a predetermined concentration (10^8 to 10^9 cfu / g of clarified butter). Then the bacterial solution and the clarified butter are fully mixed to ensure that Lactococcus is evenly distributed in the clarified butter. In some embodiments, the clarified butter inoculated with Lactococcus is placed at a suitable fermentation temperature (usually 30 - 40 °C) and fermented at a constant temperature. At this time, Lactococcus will gradually degrade cholesterol in the clarified butter. In some embodiments, the fermentation time is 24 - 72 hours. For example, it can be 24 hours, 48 hours, and 72 hours. The fermentation process can be monitored as needed to ensure that the activity of Lactococcus in the clarified butter is not affected.

[0072] In the present invention, the term "clarified butter" refers to a milk fat refined through a special process, usually made from cow's milk or goat's milk. The production process of clarified butter generally includes extracting the fat in milk and washing it to remove the moisture and solid impurities therein, finally obtaining a pure and storable fat substance. Usually, the moisture content of clarified butter is extremely low, which enables it to be stored for a long time without spoilage easily. Since the moisture and milk solids are removed from the clarified butter, its smoke point is relatively high, making it suitable for high-temperature cooking and frying. In addition, clarified butter has a unique nutty aroma and a rich milk fragrance, which makes it commonly used to add flavor in cooking. Additionally, in traditional medicine (such as Ayurveda in India), clarified butter is considered to have certain nutritional value and therapeutic effects, such as improving digestion and relieving constipation.

[0073] In a seventh aspect, the present invention provides a food with a low cholesterol content prepared by the method as described above.

[0074] Examples

[0075] The methods and materials in the following examples are conventional methods unless otherwise specified.

[0076] MRS agar medium, with the following components: peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween-80 1.0 g / L. Weigh 66.2 g of MRS agar medium, heat and dissolve it in 1000 ml of distilled water, sterilize it at 121 °C under high pressure for 15 min, cool it to 50 - 60 °C, pour it into plates and use it after solidification.

[0077] MRS liquid medium, with the following components: peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween-80 1.0 g / L. Dispense 100 ml into each conical flask, sterilize it at 121 °C under high pressure for 15 min, and reserve it for use.

[0078] The yak butter samples were collected from Shangri-La City, Yunnan Province, and transported back to the laboratory in a refrigerator and stored at -20 °C for bacterial isolation.

[0079] Cholesterol was provided by the China-Malaysia National Joint Laboratory of Northwest Minzu University.

[0080] Example 1

[0081] This example is used to illustrate the isolation and identification of Lactococcus

[0082] (1) Isolation of strains

[0083] Add 2.5 g of yak butter to 100 mL of LB liquid medium to produce a suspension, and enrich and culture it at 37 °C and a rotation speed of 160 r / min for 24 - 48 h to obtain a bacterial suspension. Use the multiple proportion dilution method to dilute the suspension into 10-1, 10-2, 10-3, 10-4, 10-5 suspensions respectively, and coat the suspensions on 0.7% calcium carbonate MRS solid medium, and culture it at 37 °C for 24 - 48 h. Pick the single colonies with a calcium dissolution circle and streak them on MRS solid medium, and culture them at 37 °C for 24 - 48 h. Select the single colonies with better growth in the medium, transfer them to LB liquid medium, continue to enrich and culture them at 37 °C and 120 r / min for 24 - 48 h, then mix the enriched bacterial liquid with 40% sterile glycerol in equal volume and store it at -80 °C for standby. Each time the strain used is activated from the bacterial suspension stored in glycerol at -80 °C.

[0084] (2) Observation of colony morphology

[0085] The selected target strains were streaked and purified on MRS agar medium. After culturing at 37 °C for 24 h, the state of the purified single colonies was observed. The specific operation is as follows:

[0086] The Gram staining method was used. Place a drop of 0.9% normal saline on a glass slide. Use an inoculation needle to pick a single colony and disperse it evenly in the normal saline. Fix it at the flame of an alcohol lamp. It is sufficient to pass through the flame 1 - 2 times, with the glass slide not being too hot to touch. After fixation, perform Gram staining: generally including four steps: primary staining, mordanting, decolorization, and counterstaining.

[0087] 1) Primary staining: Stain the smear with several drops of ammonium oxalate crystal violet staining solution for 1 min, then wash with water.

[0088] 2) Mordanting: Stain the smear with several drops of iodine solution for 1 min, then wash with water.

[0089] 3) Decolorization: Decolorize the smear with several drops of 95% ethanol for 20 - 40 s, then wash with water.

[0090] 4) Counterstaining: Counterstain the smear with several drops of safranin staining solution for 1 min, then wash with water.

[0091] 5) Air dry and observe under an oil immersion microscope.

[0092] The colony morphology of the strain growing on the MRS agar plate and the Gram staining results are as Figure 1 shown. As Figure 1 can be seen, the colonies are round and slightly convex, with a diameter of about 0.5 - 1.0 mm, white and opaque, with a smooth surface and regular edges, and a slightly buttery texture. The strain was Gram stained and examined under a microscope. The Gram staining result of the strain was purple, indicating that the strain is a Gram - positive bacterium. The microscopic morphology of the bacterium is spherical, distributed singly, in pairs, or in chains, and no spore and capsule structures were observed.

[0093] (3) Physiological and biochemical identification of the strain

[0094] The physiological and biochemical test items of the strain include: aesculin, lactose, maltose, cellobiose, sucrose, raffinose, sorbitol, mannitol, salicylic acid, inulin, and sodium hippurate. Take a test tube containing 2 ml of normal saline. Use an inoculation loop to pick a single colony from the purified culture plate and grind it carefully in the normal saline to prepare a uniform bacterial suspension with a turbidity of 0.5 McFarland, for standby. Take out the physiological and biochemical identification tubes from the physiological and biochemical identification kit. Break the upper end of the identification tube with a grinding wheel scratch. Use a pipette to transfer the bacterial suspension and inoculate it into the identification tube to be tested. Wipe the mouth of the identification tube with 75% alcohol and seal it with a sealing film. Incubate it in a 37 °C incubator at a constant temperature. The judgment results of the physiological and biochemical identification tubes are shown in Table 1.

[0095] Table 1 Comparison table for judging the results of physiological and biochemical identification tubes

[0096]

[0097] The results of physiological and biochemical identification are shown in Table 2. The esculin test of R3 was positive, and the tests for cellobiose, maltose, mannitol, salicin, sucrose, raffinose, lactose, sodium hippurate, sorbitol, and inulin were all negative, indicating that this strain can ferment esculin but not cellobiose, maltose, mannitol, salicin, sucrose, raffinose, lactose, sodium hippurate, sorbitol, and inulin. This result is basically consistent with the determination results of Lactococcus garvieae by Shi Jinlian et al. Considering the colony growth morphology and Gram staining results of the strain, it can be preliminarily considered that R1, R3, and R5 are Lactococcus. After 16S rRNA sequencing and sequence alignment, it was determined that R1 is Lactococcus formosensis, and R3 and R5 are Lactococcus garvieae.

[0098] Table 2 Results of Physiological and Biochemical Identification

[0099] Name R1 R3 R5 Esculin + + + Lactose - - - Maltose - - - Cellobiose - - - Sucrose + - + Raffinose - - - Sorbitol - - - Mannitol - - - Salicylic acid - - - Inulin - - - Hippuric acid + - +

[0100] Note: "-" indicates negative, and "+" indicates positive. R1 is Lactococcus 1, R3 is Lactococcus 3, and R5 is Lactococcus 5.

[0101] Example 2

[0102] This example is used to illustrate the in vitro acid tolerance experiment of the strain.

[0103] Adjust the pH values of MRS liquid medium to 2.5, 3.5, 4.5, 5.5, 6.0, and 7.0 respectively; sterilize at 121 °C for 20 min. After cooling, inoculate the strain into the MRS liquid medium at 1% (v / v) under sterile conditions, and incubate at 37 °C for 4 h. Perform serial dilution, inoculate 100 μl of the strain diluted to 10-4 into the MRS agar medium, and after activation for 24 h, sample and count the viable cell number.

[0104] Survival rate of the strain (%) = (A1 / A0) x 100%

[0105] Where: A1 is the number of viable cells after treatment; A0 is the number of viable cells in the control group.

[0106] The reason for testing the acid tolerance of Lactococcus is mainly that this index is crucial for its survival ability and efficacy as a probiotic in the human digestive tract. The human digestive system, especially the stomach, has a very acidic environment with a pH value as low as between 1.5 and 3. Such an environment is extremely unfavorable for most microorganisms, easily leading to the inactivation or even death of microorganisms. Therefore, for probiotics intended to be supplemented orally, having a certain degree of acid tolerance is a prerequisite for them to successfully reach the intestine, colonize and play a role in the intestine.

[0107] The survival rates of Lactococcus under different pH conditions are as Figure 2 shown. As Figure 2 can be seen, for Lactococcus 1, Lactococcus garvieae, Lactococcus 5 and commercial strains, the survival rates of Lactococcus under different pH values are different, but the differences are not obvious and are almost similar. Taking the survival rate of Lactococcus garvieae under different pH conditions as an example, as the pH value gradually increases, the survival rate of Lactococcus garvieae gradually increases. Under the condition of pH 1.0, the survival rate of Lactococcus garvieae is 31.23%, while under the condition of pH 5.5, the survival rate of Lactococcus garvieae can reach 94.27%. The above results indicate that this strain can grow in an acidic environment and has a sufficiently high tolerance to the acidic environment.

[0108] Example 3

[0109] This example is used to illustrate the in vitro bile salt tolerance experiment of bacterial strains

[0110] Use bovine bile salt to adjust the bile salt mass fraction of MRS liquid medium to 0.1%, 0.2%, 0.3%, 0.5%, 1.0%, 2.0% respectively, and sterilize at 121 °C for 20 min. Cool down, inoculate the bacterial strain into the MRS liquid medium under aseptic conditions at 2% (v / v), and incubate at 37 °C for 4 h. Perform gradient dilution, inoculate 100 μl of the bacterial strain diluted to 10-4 into the MRS agar medium, and culture for 24 h to sample and count the viable bacteria.

[0111] The calculation method is the same as that in Example 2.

[0112] Testing the bile salt tolerance of Lactococcus is closely related to its adaptability and survival ability as a probiotic in the human digestive tract. Bile salts, secreted by the liver and entering the small intestine through the gallbladder, are important cofactors for digesting fats and fat-soluble vitamins. However, bile salts also constitute a highly challenging environment because they can not only disrupt the integrity of the bacterial cell membrane but also interfere with intracellular physiological activities. For probiotics such as Lactococcus intended for intestinal health, it is particularly important to be able to survive in a bile salt environment and maintain its biological activity. Through the above tests, not only can Lactococcus strains with good bile salt tolerance be screened out, but also a foundation can be laid for further studying its mechanism of action in intestinal health, thereby promoting the development and optimization of probiotic products to better meet the special needs of the human digestive system.

[0113] The survival rates of the Lactococcus strains screened in this invention at different bile salt concentrations are as Figure 3 shown. As Figure 3 can be seen, for Lactococcus 1, Lactococcus garvieae, Lactococcus 5, and the commercial strain, the survival rates of Lactococcus are different under different bile salt concentration conditions, but the differences are not obvious and are almost similar. Taking Lactococcus garvieae as an example for analysis: as the bile salt concentration increases, the survival rate of Lactococcus garvieae begins to decline. In addition, at bile salt concentrations of 0.1% - 0.3%, the survival rates of Lactococcus garvieae are relatively high, being 97.93%, 92.34%, and 84.29% respectively, and there are significant differences in the survival rates between different bile salt concentrations (P < 0.05). When the bile salt concentration is 0.5%, the survival rate of Lactococcus garvieae is still 49.97%.

[0114] Example 4

[0115] This example is used to illustrate the in vitro high-temperature experiment of the bacterial strain

[0116] The bacterial strain liquid is inoculated into MRS liquid medium at 1% (v / v). The water bath temperature gradients are 20°C, 30°C, 40°C, 50°C, and 60°C, and static culture is carried out for 20 min. Serial dilution is performed, and 100 μL of the bacterial suspension at the 10-4 dilution gradient is spread on an MRS agar plate, and viable cell counting is carried out after 24 h. In the experiment, culture at 37°C is used as a control to calculate the survival rate of the bacterial strain.

[0117] The calculation method is the same as that in Example 2.

[0118] Many foods containing Lactococcus, such as cheese, yogurt, and other fermented dairy products, undergo a heat treatment stage during production, such as pasteurization, to kill harmful microorganisms and extend the shelf life. The relatively high heat resistance of Lactococcus allows it to remain active within a wide temperature range, maintaining its functionality and nutritional value even during transportation and storage at non-refrigerated temperatures. Therefore, to ensure that Lactococcus remains active under such heat treatment and can exert its probiotic effects in the finished product, it is crucial to test its high-temperature tolerance.

[0119] The survival rates of Lactococcus under different temperature conditions are as Figure 4 shown. As Figure 4 can be seen, for Lactococcus 1, Lactococcus garvieae, Lactococcus 5, and the commercial strain, the survival rates of Lactococcus under different temperature conditions are different, but the differences are not obvious and are almost similar. Taking Lactococcus garvieae as an example for analysis: as the temperature increases, the survival rate of Lactococcus garvieae decreases, and the temperature has a significant effect on the survival rate (P < 0.05). In addition, after treatment at 50°C and 60°C for 15 min, the survival rates of Lactococcus garvieae are still at relatively high levels, with survival rates of 90.18% and 85.63% respectively, indicating that it grows well under the conditions of 37 - 60°C. This result can provide a reference for the development and preservation of probiotic preparations in the later stage.

[0120] Example 5

[0121] This example is used to illustrate the cholesterol-lowering ability of the strain in vitro

[0122] 1) Preparation of culture media and reagents

[0123] Cholesterol standard stock solution (1.0 mg / ml): Accurately weigh 0.1 g of cholesterol, dissolve it in absolute ethanol, and make up the volume to 100 ml with absolute ethanol.

[0124] Cholesterol standard solution (0.1 mg / ml): Take 10.0 ml of the cholesterol standard stock solution and dilute it to 100 ml with absolute ethanol.

[0125] Ammonium ferric sulfate stock solution: Accurately weigh 4.46 g of solid ammonium ferric sulfate and dissolve it in 100 ml of 85% phosphoric acid

[0126] Ammonium ferric sulfate solution: Take 10.0 ml of the ammonium ferric sulfate stock solution and dilute it to 100 ml with concentrated sulfuric acid. Place this solution in a silica gel desiccator for later use.

[0127] MRS-CHOL medium (0.01 mg / ml): Add 1 ml of the cholesterol stock solution to 100 ml of MRS liquid medium to obtain a high-cholesterol medium of 0.01 mg / ml.

[0128] 2) Standard curve

[0129] Respectively pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 mL of cholesterol standard solution, add absolute ethanol to 4 mL, add 2 mL of ammonium ferric sulfate color-developing solution, shake well, let stand for 20 min, and measure the absorbance value (A) at 560 nm using a spectrophotometer. Plot a standard curve with cholesterol content (mg) as the abscissa and absorbance (A560nm) as the ordinate. As Figure 5 shown, the fitted curve is: y = 3.6x + 0.01 (R 2 = 0.9988).

[0130] 3) Determination of the cholesterol-lowering ability of strains

[0131] Inoculate the strain suspension into MRS-CHOL medium at an inoculation amount of 1%, and incubate it in a constant-temperature shaking incubator. The incubation conditions are 37 °C and the shaking speed is 60 - 80 r·min. After culturing for 72 h, determine the cholesterol-degrading ability of the strain respectively.

[0132] Determination of the cholesterol-degrading ability of the strain: After shaking the strain liquid culture and MRS-CHOL medium well, pipette 0.5 mL and transfer it to a centrifuge tube containing 9.5 mL of absolute ethanol, and mix well; centrifuge at 4000 r / min for 5 min after 5 min; take 2 mL of the supernatant and put it into a test tube, add 2 mL of ammonium ferric sulfate, shake well, use 2 mL of absolute ethanol added with 2 mL of ammonium ferric sulfate as the blank, measure the absorbance of the sample at 560 nm after 20 min, and calculate the cholesterol degradation rate.

[0133] Calculation method: Cholesterol degradation rate (%) = (D1 - D2) / D1 × 100%.

[0134] In the formula: D1 is the total amount of cholesterol; D2 is the cholesterol content in the supernatant.

[0135] The results of the cholesterol-lowering experiment are shown in Figure 6 , and there are significant differences in the cholesterol degradation rates of the 3 isolated lactic acid bacteria strains compared with the commercial strain. Among them, the strain Lactococcus garvieae has the best cholesterol-lowering effect, with a cholesterol degradation rate of 58.6%; followed by strains Lactococcus sp. 1 and Lactococcus sp. 5, with cholesterol degradation rates of 26.7% and 36.4% respectively; while the commercial strain has a relatively poor cholesterol-lowering effect, with a cholesterol degradation rate of only 17.0%. The results show that Lactobacillus garvieae has a relatively high cholesterol-lowering efficiency and is a potential lactic acid bacterium with high cholesterol-lowering function.

[0136] 4) Cholesterol-lowering ability of the strain in fermented butter

[0137] The strains with the best degradation ability and commercial bacteria were inoculated into butter and fermented at 37°C for 24 h, 48 h, and 72 h respectively, and the cholesterol removal rate of the strains was measured.

[0138] The determination of cholesterol content was carried out with reference to GB / T 5009.128—2006 Determination of Cholesterol in Foods.

[0139] The cholesterol removal rate was calculated according to the following formula for each weighed oil sample after determining the cholesterol content.

[0140] Cholesterol removal rate = (W1 - W2) / W1 × 100%

[0141] Where: W1 is the cholesterol content of the original butter, mg / g; W2 is the measured cholesterol content, mg / g.

[0142] As Figure 7 shown, the cholesterol content of Lactococcus garvieae decreased to 32.92 mg / 100 g after 48 h of cultivation, and the cholesterol removal rate was as high as 63.25%, which was 11.71% higher than the cholesterol removal rate of 51.54% after 24 h of fermentation culture. The cholesterol removal rate after 72 h of fermentation culture did not increase significantly and was similar to that after 48 h of culture; the cholesterol removal rate of commercial bacteria was also relatively high at 35.04% after 48 h of fermentation culture. Therefore, the results showed that the cholesterol removal effect of the strain in fermented butter was the best after 48 h of fermentation culture.

[0143] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A lactococcus, characterized in that The lactococcus is selected from at least one of the lactococcus with a preservation number of CGMCC No.32756, the lactococcus with a preservation number of CGMCC No.32757 and the lactococcus with a preservation number of CGMCC No.32758.

2. The lactococcus according to claim 1, wherein The lactococcus is the lactococcus with the preservation number of CGMCC No.32756.

3. A bacterial agent, characterized in that: The bacterial agent comprises the lactococcus according to claim 1 or 2.

4. Use of the lactococcus according to claim 1 or 2, or the bacterial agent according to claim 3, in the preparation of a medicine or product for lowering cholesterol.

5. Use of the lactococcus according to claim 1 or 2, or the bacterial agent according to claim 3, in the preparation of foods with low cholesterol content.

6. A food, characterized in that The food contains the lactococcus according to claim 1 or 2, or the bacterial agent according to claim 3.

7. The food according to claim 6, wherein The food is a dairy product, preferably ghee.

8. A method for preparing a food with low cholesterol content, characterized in that: The method comprises: inoculating the lactococcus described in claim 1 or 2, or the bacterial agent described in claim 3, into the food to be fermented, so as to degrade the cholesterol therein.

9. The method according to claim 8, wherein: The food is ghee.

10. The food with low cholesterol content prepared by the method according to claim 8 or 9.

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