Weight-reducing Witzia ciliaris BCLD23 and application thereof

Abstract

The invention provides novel Weizwinia coagulans BCLD23 which is preserved in CGMCC (China General Microbiological Culture Collection Center) and has a preservation number of CGMCC No.28334 and is used for reducing body weight and application of the novel Weizwinia coagulans BCLD23. The Witzia ciliaris BCLD23 can degrade cholesterol, reduce cholesterol and triglyceride in body serum and reduce body weight, and can be applied to preparation of medicines, food and health care products.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a Weizmannella coagulans BCLD23 for reducing body weight and application thereof. Background Art

[0002] Weizmannella coagulans was first isolated by Hammer from spoiled condensed milk in 1915. It is a type of Gram-positive, facultative anaerobic to microaerobic bacteria. Weizmannella coagulans can be isolated from soil, canned food, tomato juice, gelatin, milk, pharmaceutical preparations and silage. Weizmannella coagulans is moderately thermophilic, with an optimal growth temperature of 35℃-50℃ and a maximum growth temperature of 57℃-61℃. So far, there are 44 Weizmannella coagulans genomes published on NCBI, with an average total genome length of 3.42Mb and a GC content of 46.5%. Weizmannella coagulans has been recognized as GRAS (generally recognized as safe) by the US Food and Drug Administration. In 2016, China also approved Weizmannella coagulans as a food-usable strain. Several strains of Weizmannella coagulans have been used in various probiotic foods worldwide.

[0003] In recent years, the global growth of obesity has approached epidemic status. With the advancement of economic development and globalization, my country's living standards have undergone earth-shaking changes, and residents' lifestyles and dietary structures have undergone tremendous changes. Traditional healthy diets have been replaced by high-calorie, high-fat and high-sugar processed foods, resulting in excessive energy intake and a decrease in labor intensity and physical activity intensity, which has led to a rapid increase in obesity rates among all types of people in urban and rural areas in my country. The World Health Organization (WHO) defines obesity as abnormal or excessive fat accumulation that may cause health damage. Obesity is related to the occurrence and development of a variety of diseases. Obesity is a risk factor for a variety of diseases such as type 2 diabetes, hypertension, dyslipidemia, and cardiovascular disease, and has become one of the most serious public health problems at home and abroad.

[0004] Currently, intervention and treatment for obesity include diet, exercise, drug control, and surgery. Among them, diet and exercise require a long cycle and are difficult to stick to; drug control may have side effects; surgery has a relatively high risk and may cause complications such as infection and bleeding.

[0005] At present, a large number of studies have shown that obesity is closely related to intestinal flora in addition to being affected by genetics and the environment. Intestinal microbial flora has become the main endogenous factor affecting obesity. Intestinal microorganisms can affect the host in different ways, and they play an important role in the body's fat metabolism. Intestinal flora ferments high-molecular polysaccharides that cannot be digested by the intestines and degrades them into oligosaccharides and short-chain fatty acids, thereby affecting the body's energy regulation. Summary of the invention

[0006] The present invention provides a new Weizmannella coagulans BCLD23 product and a preparation thereof. The strain has excellent acid production ability, can effectively reduce the content of triglyceride and cholesterol in serum, and can also achieve the purpose of reducing body weight.

[0007] In order to achieve the first invention object of the present invention, the present invention provides Weizmannia coagulans BCLD23, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee CGMCC, with a deposit number of CGMCC No. 28334 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 4, 2023, and the classification name is Weizmannia coagulans; Latin name: Weizmannia coagulans.

[0008] To achieve the second object of the present invention, the present invention provides the use of Weizmannella coagulans BCLD23 deposited in CGMCC with the accession number of CGMCC No. 28334 in degrading cholesterol.

[0009] To achieve the third object of the present invention, the present invention provides the use of Weizmannella coagulans BCLD23 deposited in CGMCC with the accession number of CGMCC No. 28334 in degrading cholesterol.

[0010] To achieve the fourth object of the present invention, the present invention provides the use of Weizmannella coagulans BCLD23, which is deposited in CGMCC with the deposit number of CGMCC No. 28334, in reducing cholesterol in body serum.

[0011] To achieve the fifth object of the present invention, the present invention provides the use of Weizmannella coagulans BCLD23, which is deposited in CGMCC with the deposit number of CGMCC No. 28334, in reducing body weight.

[0012] In order to achieve the sixth invention objective of the present invention, the present invention provides a probiotic preparation, wherein the probiotic preparation contains Weizmannella coagulans BCLD23 deposited in CGMCC with a deposit number of CGMCC No. 28334 as its effective ingredient.

[0013] To achieve the seventh object of the present invention, the present invention provides the use of Weizmannella coagulans BCLD23 deposited in CGMCC with a deposit number of CGMCC No. 28334 in the preparation of medicines, foods and health products.

[0014] Different from the prior art, the above technical solution includes at least the following beneficial effects: providing a new coagulant Weizmannella BCLD23 and preparations which are deposited in CGMCC with the deposit number of CGMCC No. 28334. The coagulant Weizmannella BCLD23 can lower cholesterol, reduce cholesterol and triglycerides in serum, and reduce body weight, and can be used in the preparation of medicines, foods, and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a comparison chart of the lactic acid production performance of Weizmannella coagulans BCLD23 in Example 2.

[0016] Figure 2 This is a graph showing the degradation rate of cholesterol by Weizmannella coagulans BCLD23 at different times in Example 3.

[0017] Figure 3 This is a graph showing the number of weeks the mice were raised and their average daily feed intake per week in Example 4.

[0018] Figure 4 This is a graph showing the number of weeks of feeding and body weight of mice in Example 5. DETAILED DESCRIPTION

[0019] In order to explain in detail the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved, the following is a detailed description of the specific embodiments listed in conjunction with the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0020] Reference to "embodiment" herein means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or association with other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the various technical features mentioned in the embodiments can be combined in any way to form a corresponding implementable technical solution.

[0021] Unless otherwise defined, the technical terms used in this document have the same meanings as those generally understood by those skilled in the art to which this application belongs; the use of relevant terms in this document is only for describing specific embodiments and is not intended to limit this application.

[0022] In the description of this application, the term "and / or" is an expression used to describe the logical relationship between objects, indicating that three relationships may exist, for example, A and / or B, which means: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this article generally indicates that the objects before and after are in an "or" logical relationship.

[0023] In the present application, terms such as “first” and “second” are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship of quantity, priority or sequence between these entities or operations.

[0024] Without further limitations, in this application, the words "include", "comprises", "has" or other similar expressions used in the sentences are intended to cover non-exclusive inclusion. These expressions do not exclude the presence of additional elements in the process, method or product including the elements, so that the process, method or product including a series of elements may include not only those limited elements, but also other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0025] Similar to the understanding in the Examination Guidelines, in this application, expressions such as "greater than", "less than", "exceed" and the like are understood to exclude the number itself; expressions such as "above", "below", "within" and the like are understood to include the number itself. In addition, in the description of the embodiments of this application, "multiple" means more than two (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups", "multiple times", etc., unless otherwise clearly and specifically limited.

[0026] In this embodiment, the sources of materials are as follows:

[0027] 1. Experimental animals: 30 SPF-grade adult male healthy nude mice, weighing 19±1g, provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0028] 2. Reagents:

[0029] MRS agar medium and MRS broth were purchased from Guangdong Huankai Microbiology Technology Co., Ltd.;

[0030] Triglyceride (TG) kit and total cholesterol (TC) kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0031] Example 1: Isolation, purification and identification of Weizmannella coagulans BCLD23

[0032] The coagulant Weizmannella BCLD23 strain was isolated from kimchi that was naturally fermented by farmers using traditional methods for 20 days.

[0033] 1. Strain Isolation and Screening

[0034] Separation and purification: Take 10g of naturally fermented kimchi and place it in a triangular flask filled with small glass beads, add 90ml of sterile water and shake well, oscillate for 30min, let it stand for 30s, take 10ml of the supernatant and heat it in an 80℃ water bath for 10min, then spread it on MRS solid culture medium, and place it in a 37℃ constant temperature incubator for anaerobically cultured 48h, pick a single colony, and perform Gram staining and microscopic examination to obtain rod-shaped, Gram-positive spore-forming bacteria, further purify and separate, and store them in glycerol tubes.

[0035] Strain screening: The purified spore-forming bacteria were inoculated on MRS medium containing triglycerides, cultured at 37°C for 48 hours, the colonies were observed, and the strain BCLD23 with a larger transparent circle was screened out.

[0036] 2. Identification of strain species

[0037] Bacteriological morphological identification:

[0038] The screened BCLD23 strain was inoculated into MRS agar medium and cultured anaerobically at 37°C for 48 h. The colony morphology was round, with neat edges and no wrinkles. The surface of the colony was milky white, smooth and moist.

[0039] Biological identification:

[0040] The 16S rRNA fragment of the strain was amplified using bacterial 16s rDNA universal primers (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, 1492: 5′-GGTTACCTTGTTACGACTT-3′), and the amplified product was sequenced. Finally, the sequence obtained after sequencing was compared and analyzed in the NCBI database, and it was confirmed to be Weizmannella coagulans and named Weizmannella coagulans BCLD23.

[0041] Example 2: BCLD23 acid production performance test

[0042] Activation of strains: The BCLD23 strain seed liquid was taken out from the glycerol, inoculated into MRS liquid culture medium under sterile operation, cultured in a constant temperature shaker at 37°C for 48 hours, and then technically prepared for use.

[0043] The bacterial liquid was inoculated into MRS liquid medium under sterile conditions, and the bacterial count was 10 9CFU / ml, 37℃ constant temperature static culture for 24h; take the fermentation broth after 8, 12, and 24h, centrifuge for 10min, set the speed to 5000, and analyze the supernatant by liquid chromatography to detect the content of lactic acid, acetic acid, propionic acid and other organic acids in the fermentation broth. The content is shown in Table 1. The lactic acid production performance of BCLD23 is shown in Figure 1 As shown, BCLD23 has excellent acid-generating properties.

[0044] Table 1. Organic acid content of LDBC23 culture medium at different times (mg / ml)

[0045]

[0046] As can be seen from Table 1, BCLD23 has excellent acid production performance. In vitro culture shows that the acid produced by BCLD23 after 24 hours of culture is mainly lactic acid and acetic acid.

[0047] Example 3 BCLD23 in vitro cholesterol degradation test

[0048] Preparation of cholesterol-MRS medium: Weigh 1 g of cholesterol, dissolve it in anhydrous ethanol, and make up to 100 mL. Filter and sterilize it with a 0.22 μm microporous filter membrane under sterile conditions, and add it to sterilized MRS liquid medium containing 0.2% bile salts to prepare a liquid medium with a final cholesterol concentration of 0.1 mg / mL.

[0049] Activation of strains: The seed liquid was taken out from the glycerol and inoculated into MRS liquid medium under sterile operation. The culture was incubated in a constant temperature shaker at 37°C for 48 hours and the number of live bacteria in the bacterial liquid was counted. The number of live bacteria was 2.0×10 9 CFU / mL.

[0050] The activated Weizmann Bacillus coagulans BCLD23 bacterial suspension was inoculated into 10 ml of cholesterol-MRS liquid medium prepared in advance at a 1% inoculation rate, and the cholesterol content was measured after static culture at 37°C for 12, 24, 48, and 72 hours. Figure 2 As shown, the cholesterol content was measured by o-phthalaldehyde colorimetric method, the wavelength was set at 550nm, and its absorbance was measured.

[0051] Table 2. Cholesterol concentrations of BCLD23 culture medium at different times

[0052]

[0053] Draw a standard curve: Use cholesterol concentration as the horizontal axis and absorbance at 550nm as the vertical axis to draw a standard curve. The standard curve equation is

[0054] Y = 0.0233X - 0.0219, R2 =0.9935

[0055] Sample processing: The cultured cholesterol-MRS was centrifuged at a speed of 8000 r / min for 10 minutes, the supernatant was taken, and the colorimetric analysis was performed using o-phthalaldehyde as a colorimetric agent, and the degradation rate of cholesterol was calculated.

[0056]

[0057] Where C0 is the cholesterol concentration before treatment; C n is the cholesterol concentration in the culture medium of the experimental group after culture for n hours.

[0058] The degradation rate of cholesterol by Weizmannella coagulans BCLD23 was calculated according to formula ①, as shown in Table 3 and Figure 2 shown.

[0059] Table 3. Cholesterol degradation rate of BCLD23 at different time

[0060]

[0061] According to the results in Table 3, Weizmannella coagulans BCLD23 can degrade cholesterol in the presence of 0.1 mg / ml cholesterol-MRS. The degradation rate of cholesterol can reach 8% in the first 12 hours, and the degradation rate of cholesterol is significantly enhanced with the increase of time. The degradation rate of cholesterol reaches 26% within 24 hours, 40% within 48 hours, and 52% within 72 hours. The rate of cholesterol degradation decreases with the increase of time, which may be caused by the insufficient concentration of the substrate in the culture medium. Figure 2 It can be seen that the coagulated Weizmann's BCLD23 has excellent ability to degrade cholesterol in vitro.

[0062] Example 4 BCLD23 mouse test

[0063] Experimental animals: 30 SPF mice, each weighing 19±1g

[0064] The main ingredients of standard daily feed are: corn, soybean meal, flour, wheat bran, salt, sucrose, multiple minerals, multiple vitamins, amino acids, etc.

[0065] High-fat diet: Add 20% sucrose, 15% lard, and 1.2% cholesterol to the standard diet. Probiotics: Cultivate Weizmannella coagulans BCLD23 to the logarithmic phase and dilute the bacterial count to 1.0×10 10 CFU / ml.

[0066] Mouse feeding and management: The mice were fed with a normal diet for one week, with free access to water and food, 12 hours per day and night, the temperature was controlled at 25°C, and the humidity was controlled at around 60%.

[0067] Three groups of experiments were set up, each group had 10 SPF adult mice, including NG (normal group), CG (control group), and EG (experimental group). The daily diet and drinking conditions of the three groups of mice were as follows: NG group: fed with standard diet every day, free diet and drinking water, and gavage of 0.2 ml of normal saline.

[0068] Group CG: The mice were fed a high-fat diet daily, had free access to food and water, and were gavaged with 0.2 ml of normal saline.

[0069] EG group: The mice were fed with high-fat diet daily, and had free access to food and water. They were intragastrically administered with 0.2 ml of Weizmannella coagulans BCLD23 bacterial solution.

[0070] Trial period: 8 weeks

[0071] The food intake of each group of mice was recorded every week. Figure 3 The weight of mice was measured as shown in Figure 4 shown.

[0072] At 28 weeks of the experiment, blood was collected from mice that had fasted for 12 h, centrifuged, and triglyceride (TG) and total cholesterol (TC) levels were measured using a triglyceride (TG) kit and a total cholesterol (TC) kit, respectively. The results were recorded and shown in Table 4.

[0073] Table 4. Changes of serum TG and TC levels in mice

[0074]

[0075] Note: Compared with the control group, # P<0.05, compared with the normal group, * P<0.05

[0076] Daily food intake of mice Figure 3 As shown in the figure, by tracking the food intake of mice fed weekly, it can be found that the food intake of the EG group and the CG group was significantly different from the fourth week (P < 0.05), and from the fifth week onwards, the food intake of the EG group was significantly lower than that of the CG group (P < 0.01). This may be because gavage of Bacillus coagulans BCLD23 suppressed the appetite of mice.

[0077] After 8 weeks of feeding, the weight of mice was Figure 4As shown in the figure, the potential effect of Weizmannella coagulans BCLD23 bacterial solution on the body weight of mice fed a high-fat diet. After 8 consecutive weeks of feeding intervention, the body weights of the two groups of mice were accurately measured and statistically analyzed. The results showed that compared with the CG group, the body weight of mice in the EG group showed a significant downward trend (P < 0.01), and their body weight decreased by an average of 8.99% compared with the CG group. This data shows that the Weizmannella coagulans BCLD23 bacterial solution has a significant effect in regulating the body weight of mice, which may be because the Weizmannella coagulans BCLD suppressed the appetite of mice, thereby causing the weight loss of mice.

[0078] After 8 weeks of feeding, the TC and TG contents in the serum of mice are shown in Table 4. The TC and TG in the serum of the experimental group mice were significantly lower than those in the control group, with significant differences (P < 0.05). Compared with the blank group, TC and TG increased, among which the TC content increased, but the difference was not significant (P > 0.05). This shows that Weizmannella coagulans BCLD23 can effectively reduce the serum TG and TC of mice fed a high-fat diet.

Claims

1. Weizmannella coagulans BCLD23, deposited in CGMCC with the deposit number CGMCC No.28334.

2. Application of Weizmannella coagulans BCLD23, which is deposited in CGMCC with the deposit number CGMCC No.28334, in the degradation of cholesterol.

3. Application of Weizmannella coagulans BCLD23, which is deposited in CGMCC with the deposit number CGMCC No.28334, in lowering cholesterol in body serum.

4. Use of Weizmannella coagulans BCLD23, which is deposited in CGMCC with the deposit number CGMCC No. 28334, in reducing triglycerides in body serum.

5. Use of Weizmannella coagulans BCLD23, which is deposited in CGMCC with the deposit number of CGMCC No. 28334, in reducing body weight.

6. A probiotic preparation, characterized in that: The probiotic preparation contains the Weizmannella coagulans BCLD23 according to claim 1 as its effective ingredient.

7. Application of Weizmannella coagulans BCLD23, which is deposited in CGMCC with the deposit number CGMCC No. 28334, in the preparation of medicines, foods and health products.

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