Kluyveromyces lactis with weight loss and lipid-lowering functions and application thereof

Abstract

The application discloses a kind of Kluyveromyces lactis with weight loss and lipid-lowering function and application thereof, and belongs to the field of microbial technology.The Kluyveromyces lactis JSA18 has been preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.26714, and the preservation date is February 27, 2023.The application utilizes Kluyveromyces lactis JSA18, and uses it for maintaining high-fat diet mice, then obtains liver and serum samples, and detects body weight change and total cholesterol TC, triglyceride TG level, to evaluate the influence of yeast on obesity and blood lipids.The application shows by experiment that Kluyveromyces lactis JSA18 tolerates gastrointestinal environment stress in vivo well.The results show that Kluyveromyces lactis JSA18 can effectively reduce hyperlipidemia and fat accumulation caused by obesity, and provide a new treatment approach for weight loss and lipid-lowering.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a type of Kluyveromyces lactis with weight loss and lipid-lowering functions and its applications. Background Technology

[0002] Modern high-sugar, high-fat diets lead to energy intake exceeding the body's energy expenditure, resulting in fat accumulation and obesity. Obesity has become a global epidemic, not only affecting personal appearance but also seriously harming health, such as chronic diseases caused by obesity, including type 2 diabetes, dyslipidemia, hypertension, coronary heart disease, and obstructive sleep apnea syndrome. Hyperlipidemia refers to excessively high blood lipid levels, which can directly cause diseases such as atherosclerosis, coronary heart disease, and pancreatitis. Furthermore, excess lipids that the body cannot break down accumulate in the liver, leading to fatty liver and liver fibrosis. Obesity is often accompanied by hyperlipidemia; therefore, many of their complications are similar and mutually influential. To suppress obesity, currently available weight-loss drugs are lipid absorption inhibitors or diuretics, but their side effects can cause endocrine disorders. Therefore, non-pharmacological treatments for obesity, including probiotics, are being explored to alleviate obesity.

[0003] Probiotics generally refer to microorganisms that benefit their hosts by improving the gut microecological environment. A potential probiotic strain should produce beneficial effects on the human body. Based on recent research on the functions of probiotics, an ideal probiotic should possess characteristics such as tolerance and adhesion. Research on the probiotic functions of yeast began with the discovery of *Saccharomyces boulardii*. With in-depth research on yeast, it has been discovered that other yeasts also possess probiotic functions, such as cholesterol-lowering and antibacterial properties. Compared to well-known probiotic species such as *Bifidobacterium* and *Lactobacillus*, the probiotic functions of yeast require further exploration.

[0004] Kluyveromyces lactis is a species of yeast in the genus Kluyveromyces. Its cell morphology is generally spherical or olive-shaped, and most strains are isolated from dairy products where lactose is the primary carbon source. When cultured, Kluyveromyces lactis emits a distinctive fruity aroma. It can utilize a wide range of carbon sources, such as cellobiose, sorbitol, and 2,3-butanediol, which are difficult for other yeasts to utilize. As a food-grade yeast, it has strong protein secretion capabilities, a safe expression system, and a safe fermentation process, making it widely used in the food and pharmaceutical industries. Recent studies have shown that Kluyveromyces lactis possesses potential probiotic characteristics. However, to date, there are no reported studies on the application of Kluyveromyces lactis in the treatment of weight loss and lipid-lowering diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a Kluyveromyces lactis with weight loss and lipid-lowering functions and its application, so as to solve the problems existing in the prior art. The Kluyveromyces lactis JSA18 provided by this invention can effectively reduce hyperlipidemia and fat accumulation caused by obesity, and provide a new treatment approach for weight loss and lipid-lowering.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a Kluyveromyces lactis JSA18, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26714 and deposit date of February 27, 2023.

[0008] The present invention also provides the application of the aforementioned Kluyveromyces lactis JSA18 in the preparation of weight-loss and lipid-lowering drugs.

[0009] Furthermore, the Kluyveromyces lactis JSA18 exerts its weight loss and lipid-lowering effects by slowing down the rate of weight gain and reducing the levels of triglycerides and cholesterol in the serum.

[0010] The present invention also provides a weight loss and lipid-lowering drug comprising an effective dose of the aforementioned Kluyveromyces lactis JSA18.

[0011] Furthermore, the effective dose of the Kluyveromyces lactis JSA18 is 5 × 10⁻⁶. 9 CFU / kg.

[0012] The present invention discloses the following technical effects:

[0013] This invention utilizes *Kluyveromyces lactis* JSA18 and administers it to mice on a high-fat diet. Liver and serum samples were then obtained, and changes in body weight, total cholesterol (TC), and triglyceride (TG) levels were measured to assess the effects of the yeast on obesity and blood lipids. Experiments showed that *Kluyveromyces lactis* JSA18 tolerates gastrointestinal stress well in vivo. The results indicate that *Kluyveromyces lactis* JSA18 can effectively reduce hyperlipidemia and fat accumulation caused by obesity, providing a new therapeutic approach for weight loss and lipid reduction. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is the phylogenetic tree of Kluyveromyces lactis JSA18 in this invention;

[0016] Figure 2 Results of body weight change and gain for mice in each group over 8 weeks; A: Body weight change results; B: Body weight gain results:

[0017] Figure 3 The values ​​of TC and TG in the serum of mice in each group are: A: TC; B: TG. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1

[0024] Isolation, screening and identification of Kluyveromyces lactis

[0025] All the following procedures were performed under aseptic conditions.

[0026] Enrichment of strain 1

[0027] Take 1g of traditional yak milk sample and dilute it in 9mL of physiological saline. Perform a 10-fold serial dilution. Take 10 drops of the diluted sample. -3 -10 -5 100 μL of the sample was spread on YPD medium and incubated upside down at 30°C for 48 h to observe colony growth.

[0028] The YPD culture medium is prepared as follows: 20g peptone, 20g glucose, 10g yeast extract, 10g agarose, 1L distilled water, pH 6.4±0.2, sterilized at 121℃ for 20min.

[0029] Isolation of 2 strains

[0030] Single colonies with regular morphology, large and thick, oval shape, moist to slightly dry, uniform texture, and easy to pick up were isolated; the isolated single colonies were subcultured on YPD solid medium;

[0031] Purification of 3 strains

[0032] Pick the colonies obtained in step 2 and streak them on a YPD plate. Repeat the purification process until no other bacteria are present.

[0033] Identification of 4 strains

[0034] Select strain JSA18 obtained in step 3 for microscopic examination.

[0035] The colonies were observed to be regular in shape, large and thick, oval, moist or moist and slightly dry, with uniform texture, easy to pick up, and have a fermented and fruity aroma. Microscopic examination revealed that the cell morphology was spherical or olive-shaped, similar to that of brewer's yeast.

[0036] Identification of 5 strains

[0037] The genome of yeast strain JSA18 was extracted and used as a template for PCR amplification. The successfully amplified PCR products were sequenced and assembled to obtain the DNA sequence. Sequence homology analysis was performed using the NCBI BLAST search system. The phylogenetic evolution of yeast strain JSA18 constructed from this sequence is as follows: Figure 1 As shown, yeast strain JSA18 was identified as Kluyveromyces lactis.

[0038] The method for extracting the genome is as follows: (1) Yeast JSA18 is inoculated into an Erlenmeyer flask containing 5 mL of LYPD medium and cultured overnight at 30°C and 220 r / min on a shaker; the next day, 1 mL of bacterial culture is taken into an EP tube, centrifuged at 8000 r / min at room temperature for 2 min to collect the bacteria, and the supernatant is discarded.

[0039] (2) Add 200 μL of sorbitol solution (1M) and 25 μL of Zymolyase solution (1 mg / mL), place in a constant temperature incubator, and incubate at 37°C for 1 h;

[0040] (3) Centrifuge at maximum speed for 15 seconds, discard the supernatant; add 200 μL of SDS solution (2%), vortex shaker to mix thoroughly, place in a shaking incubator, shake at 1500 r / min at room temperature for 15 min;

[0041] (4) Transfer to a 65℃ constant temperature water bath for 30 min; add 200 μL potassium acetate solution (5M), invert to mix thoroughly, and place on ice for 20 min.

[0042] (5) Centrifuge at maximum speed at room temperature for 5 minutes, aspirate the supernatant into a new centrifuge tube, being careful not to aspirate the precipitate, centrifuge again at maximum speed at room temperature for 3 minutes, carefully aspirate the supernatant, and transfer it to a new centrifuge tube containing 250 μL of isopropanol.

[0043] (6) Invert the bowl to mix thoroughly, centrifuge at maximum speed for 15 seconds, and discard the supernatant;

[0044] (7) Add 500 μL of 70% ethanol, vortex thoroughly, centrifuge for 15 seconds, and discard the supernatant;

[0045] (8) Place the centrifuge tubes with the opening facing down and air dry them on filter paper at room temperature for about 10 minutes until the residual ethanol is removed; add 40 μL of LTE buffer, mix well, and centrifuge at low speed using a handheld centrifuge.

[0046] Preservation of 6-Lactobacillus lactis JSA18

[0047] Kluyveromyces lactis JSA18, accession number: CGMCCNo.26714, accession date: February 27, 2023, accession address: No.3, No.1 Beichen West Road, Chaoyang District, Beijing, depositary institution: China General Microbiological Culture Collection Center.

[0048] Example 2

[0049] 1. Experiment on artificial synthesis of gastrointestinal fluid

[0050] Artificial gastric fluid was prepared (6.2 g / L sodium chloride, 2.2 g / L potassium chloride, 0.22 g / L calcium chloride, 1.2 g / L sodium bicarbonate, 0.3% pepsin, pH 3.0), and sterilized by filtration (0.22 μm).

[0051] Artificial duodenal fluid was prepared (6.4 g / L sodium bicarbonate, 0.239 g / L potassium chloride, 1.28 g / L sodium chloride, 0.1% pancreatic enzyme, 10% bovine bile, pH 7.4), and then sterilized by filtration (0.22 μm).

[0052] To activate Kluyveromyces lactis cells, samples were cultured twice in 5 mL YPD, and the cells were collected by centrifugation at 3000 rpm for 10 min and washed twice with PBS. The cells were then added to 10 mL of simulated gastric fluid and incubated at 37°C and 150 rpm for 90 min. After incubation, 17.5 mL of simulated duodenal fluid was added, and the cells were incubated at 37°C and 150 rpm for 180 min. Yeast viability was then assessed by growth on YPD plates.

[0053] 2. Cell adhesion experiment

[0054] The density of the recovered HT-29 cells was adjusted to 1×10⁻⁶. 5 Cell suspensions were seeded into 96-well plates, and 100 μL of DMEM-HG medium was added to each well. The cells were cultured overnight until they were fully adhered, approximately 12 hours later.

[0055] The activated Kluyveromyces lactis cells were diluted to a concentration of 5 × 10⁻⁶ using PBS. 8 CFU / mL was added to 100 μL of each well in a 96-well cell culture plate. After incubation at 37°C for 1 h, the cells were washed three times with PBS to remove any unadhered cells. The absorbance of the yeast cells after PBS washing, the unwashed yeast cells, and the untreated blank wells was measured using the MTT assay and denoted as A (total bacteria), A (adhered bacteria), and A (control), respectively. The adhesion rate was calculated using the following formula:

[0056]

[0057] The obtained Kluyveromyces lactis JSA18 was studied in a simulated gastrointestinal environment and its cell adhesion ability. It was found that the survival rate of the yeast in the artificially synthesized in vivo gastrointestinal environment was 66.93%, and its cell adhesion rate was 41.3%, which showed good in vivo survival ability.

[0058] Example 3

[0059] animal experiments

[0060] This invention uses a high-fat diet to establish an obesity animal model using C57BL / 6 mice.

[0061] 1. Animal experimental grouping

[0062] Forty male C57BL / 6 mice (4 weeks old, 17±2g) were randomly divided into four groups of 10 mice each after a 5-day acclimatization period, as shown in Table 1. The control group was fed a basal diet, while the other three groups were fed a high-fat diet (purchased from Nanjing Xietong Biotechnology Co., Ltd.) for 8 weeks. Mice were housed at room temperature with free access to food. After one week of acclimatization, they were randomly divided into a normal control group (ND), a model group (HFD), a K. Lactis experimental group, and an Orlistat positive control group, with 6 mice in each group. Except for the normal control group, which was fed a basal diet, the other groups were fed a high-fat diet. The K. Lactis experimental group underwent a gavage experiment, administered once daily at a dose of 5 × 10⁻⁶ mg / L. 9 CFU / kg Kluyveromyces lactis JSA18 culture was administered via gavage to the Orlistat positive control group once daily at a dose of 10 mg / kg. The control and model groups were administered PBS solution via gavage.

[0063] Table 1 Grouping of Animal Experiments

[0064]

[0065] Note: High-fat diet D12492, basal diet D12450J; PBS (phosphate buffer: 135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4, and 8mM K2HPO4, pH 7.2); Oselita: a commercially available and relatively good weight loss drug.

[0066] 2. Measurement of mouse body weight

[0067] Each mouse was weighed before the experiment began, and weighed every 7 days during the experiment. The mice were fasted for 12 hours before each weighing. Results are as follows: Figure 2 As shown in AB.

[0068] After 8 weeks of dietary and gavage intervention, the weight gain in each group was assessed. Compared with the ND group, the HFD group showed a 21% increase in weight, meeting the criteria for an obesity model (≥20%). By observing the weight gain in each group from week 1 to week 8, compared with the HFD group, the weight gain in the Kluyveromyces lactis JSA18 intervention group was significantly lower, indicating that Kluyveromyces lactis JSA18 delayed obesity induced by a high-fat diet.

[0069] 3. Measurement of mouse serum

[0070] Mice were fed according to the method described in Table 1, administered via gavage at 9:00 AM daily. After 56 days of experimentation, mice were fasted for 12 hours. Blood was collected from each group of mice using a retrobulbar vein capillary method. After standing for 15 minutes, the blood was centrifuged at 3000g for 15 minutes to collect serum. The serum cholesterol (TC) and triglyceride (TG) levels were measured using a lipid assay kit from Nanjing Jiancheng Bioengineering Institute. The results are as follows: Figure 2 As shown in AB (Note: Figure 2 and Figure 3 middle * P<0.05, ** P<0.01, and *** P < 0.005 vs. ND; # P<0.05, ## Pand ### P<0.005 vs. HFD).

[0071] Depend on Figure 3 It can be seen that TC and TG levels in the HFD group were significantly higher than those in the ND group, indicating that the hyperlipidemia mouse model was successfully established. Meanwhile, the TC and TG levels in the Kluyveromyces lactis group were significantly lower than those in the HFD group, suggesting that Kluyveromyces lactis JSA18 delayed the onset of hyperlipidemia induced by a high-fat diet.

[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Kluyveromyces lactis ( Kluyveromyces lactis The application of JSA18 in the preparation of weight-loss and lipid-lowering drugs is characterized by, The Kluyveromyces lactis JSA18 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26714 and deposit date of February 27, 2023. The lactic acid kluyveromycin JSA18 exerts its weight loss effect by slowing down the rate of weight gain in the body.

2. The application according to claim 1, characterized in that, The Kluyveromyces lactis JSA18 exerts its lipid-lowering effect by reducing the levels of triglycerides and cholesterol in serum.

3. A weight-loss and lipid-lowering drug, characterized in that, Contains an effective dose of Kluyveromyces lactis JSA18 as described in claim 1.

4. The weight-loss and lipid-lowering drug according to claim 3, characterized in that, The effective dose of Kluyveromyces lactis JSA18 is 5 × 10⁻⁶. 9 CFU / kg.

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