Leuconostoc mesenteroides xd054 and application thereof in fermenting dendrobium candidum

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to Leuconostoc mesenteroides subsp. Mesenteroides XD054 and application thereof in fermentation of Dendrobium candidum. The Leuconostoc mesenteroides subsp. Mesenteroides XD054 is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M 20241728. The Dendrobium candidum fermentation liquor obtained by fermentation of the Dendrobium candidum after cellulase, pectinase and papain enzymolysis has obvious inhibitory effect on pancreatic lipase activity.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to Leuconostoc mesenteroides XD054 and its application in fermented Dendrobium officinale. Background Technology

[0002] Dendrobii caulis, considered the foremost of my country's "Nine Immortal Herbs," is a rare and precious traditional Chinese medicine among orchids. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is known for its tonifying and nourishing properties. It originates from the fresh or dried stems of cultivated varieties of perennial orchids such as Dendrobium nobile, Dendrobium officinale, Dendrobium chrysanthum, and Dendrobium fimbriatum, as well as closely related species. It is mainly distributed in Sichuan, Yunnan, and Guizhou provinces. Among these, Dendrobium officinale is widely used due to its high medicinal value and well-established large-scale cultivation. As a top-grade traditional Chinese medicine, Dendrobium officinale has no reported adverse reactions to date, demonstrating its reliable safety for consumption. On November 17, 2023, the "Announcement on the List of Substances that are Traditionally Both Food and Traditional Chinese Medicine" (No. 9 of 2023), officially included Dendrobium officinale, Astragalus membranaceus, Ganoderma lucidum, and 9 other substances in the list of substances that are traditionally both food and traditional Chinese medicine (referred to as food-medicine substances).

[0003] Dendrobium officinale contains glucose, inulin, fructose, and 17 kinds of amino acids, including the seven essential amino acids for the human body except tryptophan. It also contains various trace elements such as phosphorus, potassium, magnesium, sodium, zinc, manganese, and copper. It can be processed into functional foods, thus its application in health foods is becoming increasingly widespread, often used as a dietary supplement in teas, wines, soups, and porridges. Dendrobium officinale is sweet and slightly cold in nature, and enters the stomach and kidney meridians. It has the functions of nourishing the stomach and promoting the production of body fluids, nourishing yin and clearing heat. Clinically, it is mainly used for stomach yin deficiency, heat-related illnesses damaging body fluids, and kidney yin deficiency. Modern research shows that Dendrobium officinale includes various chemical components such as polysaccharides, flavonoids, coumarins, phenols, terpenes, alkaloids, amino acids, and tannins. It has various pharmacological effects such as protecting the gastric mucosa, enhancing immunity, anti-oxidation, lowering blood sugar, lowering blood lipids, and antibacterial properties.

[0004] Obesity is defined as a metabolic disorder characterized by elevated levels of total cholesterol (TC) and triglycerides (TG) in the blood plasma. Common clinical indicators include TC, TG, high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C), especially excessive TG accumulation leading to a thick layer of fat and excessive weight. According to estimates from the World Health Organization, by 2025, the global prevalence of obesity will reach 18% in men and over 21% in women. Obesity is also a root cause of many chronic diseases, including chronic inflammation, cardiovascular disease, and type 2 diabetes. It is another risk factor closely related to metabolic dysfunction and ranks fifth in the global mortality risk index.

[0005] Pancreatic lipase is a key lipolytic enzyme in the diet, which can hydrolyze fats into monoglycerides and free fatty acids, thereby being absorbed by the human body. Therefore, by inhibiting the activity of pancreatic lipase, the catalysis of fat decomposition can be reduced, thereby reducing fat absorption, achieving the effect of controlling and treating obesity. Orlistat, as an anti-obesity drug, can prevent the absorption of dietary fat by inhibiting the activity of pancreatic lipase in the small intestine, but it has many side effects, such as oily stool, oil spots and bloating.

[0006] And plant extracts have multiple active ingredients, usually have multiple target actions, and have a combined effect. Compared with synthetic drugs, natural products with minimal side effects can prevent or reduce the risk of chronic diseases, which is increasingly concerned in obesity treatment. Many animal and clinical trials have shown that plant extracts can improve lipid metabolism in obese patients as pancreatic lipase inhibitors, reduce the accumulation of fatty acids in the body, and reduce the levels of TC, TG and LDL-C in serum, and increase the level of HPL-C.

[0007] Currently, there are few studies on the regulation of lipid metabolism by Dendrobium candidum. Lin Ya et al. Influence of Dendrobium Mixture on Blood Glucose and Lipid Metabolism in Diabetic Rat Models [J] Journal of Fujian University of Traditional Chinese Medicine. 2012, 22(03) confirmed that Dendrobium mixture can block the reduction of insulin secretion mediated by reducing blood lipids, free fatty acids and cholesterol levels, and achieve the purpose of reducing blood glucose. The latest research results show that the mechanism of Dendrobium mixture in reducing blood lipids is related to the improvement of the expression of multiple gene pathways, which can regulate WNT, PPARβ / δ and other signal pathways, and play a role in regulating lipid metabolism and protecting the liver. There is no report on the pancreatic lipase inhibitory activity of Dendrobium candidum. SUMMARY

[0008] Therefore, in one aspect, the present application provides a Leuconostoc mesenteroides XD054, and in another aspect, the application provides the use of the strain in the fermentation of Dendrobium candidum. The Dendrobium candidum fermentation supernatant obtained can inhibit the activity of pancreatic lipase and can be applied in the development of weight loss products. In another aspect, the present application provides a bacterial agent that can be used for the fermentation of Dendrobium candidum.

[0009] In the present application, "bacterial activity", "bacterial activity", "viable bacterial count", "viable bacterial content", "viable bacterial amount" have the same meaning under some conditions.

[0010] In the present application, "enzyme activity", "enzyme activity", "enzyme activity", "enzyme specific activity" have the same meaning under some conditions.

[0011] To solve the above technical problems, the technical scheme of the present application is:

[0012] A Leuconostoc mesenteroides XD054, which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20241728.

[0013] The application of the Leuconostoc mesenteroides XD054 in preparing a Dendrobium candidum fermentation liquor or fermenting Dendrobium candidum.

[0014] The application of the Leuconostoc mesenteroides XD054 in fermenting Dendrobium candidum for improving the pancreatic lipase inhibitory activity of the Dendrobium candidum fermentation liquor.

[0015] A Dendrobium candidum fermentation liquor, which is prepared by fermenting Dendrobium candidum through enzymolysis and inoculation of Leuconostoc mesenteroides XD054.

[0016] Preferably, the enzyme preparation for the enzymolysis of Dendrobium candidum includes cellulase, pectinase and papain.

[0017] Preferably, the addition amount of cellulase is 0.2-0.6%, the addition amount of pectinase is 0.2-0.6%, and the addition amount of papain is 0.05-0.2%; further preferably, the addition amount of cellulase is 0.5%, the addition amount of pectinase is 0.5%, and the addition amount of papain is 0.1%.

[0018] Further preferably, the enzyme activity of cellulase is 10,000 U / g, the enzyme activity of pectinase is 60,000 U / g, and the enzyme activity of papain is 100,000 U / g.

[0019] Preferably, the inoculation amount of the Leuconostoc mesenteroides XD054 strain is 1-3%, and further preferably, the inoculation amount is 2%.

[0020] Preferably, the viable bacterial count of the Leuconostoc mesenteroides XD054 is more than 1.0×10 9 CFU / mL.

[0021] Further preferably, the activation method of the Leuconostoc mesenteroides XD054 is as follows: inoculating the strain in MRS medium, incubating at 37°C for 24 h, and continuously passing for 3 times to fully activate the strain to a viable bacterial count of more than 1.0×10 9 CFU / mL.

[0022] A preparation method of a Dendrobium candidum fermentation liquor, which comprises the following steps: (1) mixing Dendrobium candidum and water, and then enzymolysis, enzyme inactivation and sterilization to obtain an enzymolysis liquor; (2) inoculating Leuconostoc mesenteroides XD054 strain in the enzymolysis liquor for fermentation, and then solid-liquid separation to obtain the Dendrobium candidum fermentation liquor.

[0023] Preferably, the solid-liquid ratio of Dendrobium candidum and water is 1:(10-20); further preferably, the solid-liquid ratio of Dendrobium candidum and water is 1:15.

[0024] Preferably, the enzymatic hydrolysis temperature is 40-60℃, and the enzymatic hydrolysis time is 1-3h; further preferably, the enzymatic hydrolysis temperature is 50℃, and the enzymatic hydrolysis time is 2h.

[0025] Preferably, the enzyme inactivation condition is boiling water bath treatment for 5-20min, and further preferably, the boiling water bath treatment is 10min.

[0026] Preferably, the sterilization condition is 100-120℃, and the sterilization time is 10-30min; further preferably, the sterilization condition is 115℃, and the sterilization time is 20min.

[0027] Preferably, the fermentation temperature is 30-45℃, and the fermentation time is 32-54h; further preferably, the fermentation temperature is 37℃, and the fermentation time is 48h.

[0028] A microbial agent, the active ingredient of which comprises the above-mentioned Leuconostoc mesenteroides XD054; preferably, further comprising an auxiliary agent for maintaining the long-term preservation of the microorganism.

[0029] The technical scheme of the present application has the following advantages:

[0030] 1. The present application provides a Leuconostoc mesenteroides XD054 and its application in the fermentation of Dendrobium candidum. Fermentation of Dendrobium candidum with the strain can significantly improve the pancreatic lipase inhibition rate of the Dendrobium candidum fermentation broth.

[0031] 2. The present application provides a Dendrobium candidum fermentation broth, which is prepared by inoculating Leuconostoc mesenteroides XD054 after cellulase, pectinase and papain enzymolysis. The obtained Dendrobium candidum fermentation broth has a very good pancreatic lipase inhibition rate.

[0032] 3. The present application provides a preparation method of a Dendrobium candidum fermentation broth. The method has few process steps, is convenient for industrial production, and the obtained Dendrobium candidum fermentation product has a significant pancreatic lipase inhibition activity effect.

[0033] 4. The present application uses Dendrobium candidum as raw material and adopts natural plant preparation without chemical addition, which is safe and reliable.

[0034] Preservation information

[0035] Leuconostoc mesenteroides XD054 was preserved in China Center for Type Culture Collection on August 2, 2024, with the preservation number CCTCC NO: M 20241728, Latin name Leuconostoc mesenteroides, and address China. Wuhan. Wuhan University.

[0036] Lactiplantibacillus plantarum XD117 was deposited in China Center for Type Culture Collection on August 2, 2024, with the accession number CCTCC NO: M 20241730, Latin name Lactiplantibacillus plantarum, and address China. Wuhan. Wuhan University. On August 25, 2022, the updated "List of strains that can be used for food" was officially released, and Lactobacillus plantarum was renamed Lactiplantibacillus plantarum. Lactobacillus plantarum XD117 and Lactiplantibacillus plantarum XD117 are the same strain.

[0037] Lactiplantibacillus plantarum GXL94 was deposited in China Center for Type Culture Collection, with the accession number CCTCC NO: M20242281, Latin name Lactiplantibacillus plantarum, and address China. Wuhan. Wuhan University. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the present application clearer, the embodiments of the present application will be further described in detail below. All other embodiments obtained by those of ordinary skill in the art based on the embodiments disclosed in the present application without creative labor shall fall within the scope of the present disclosure.

[0039] The specific experimental steps or conditions not specified in the embodiments can be performed according to the conventional steps described in the literature or the operation or conditions. All reagents not specified by the manufacturer are conventional reagent products that can be obtained by purchase.

[0040] In the following examples, the source information of some products is as follows:

[0041] The cellulase was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., with an enzyme activity of 10,000 U / g; the pectinase was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., with an enzyme activity of 60,000 U / g; and the papain was purchased from Guangzhou Solabio Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g.

[0042] Fermented Limosilactobacillus fermentu DSM 13273 was obtained from China Center for Type Culture Collection, with the accession number CCTCC AB 2013123, Latin name Limosilactobacillus fermentu, original number S1L18, and DSM 13273 was a self-named number by the applicant.

[0043] Leuconostoc mesenteroides Leul, from China Center for Type Culture Collection, preservation number CCTCC AB2010207; Leuconostoc mesenteroides LM2, from China Center for Type Culture Collection, preservation number CCTCC HB20082735; Leuconostoc mesenteroides subsp. mesenteroides XZ9302, from China Center for Type Culture Collection, preservation number CCTCC LB2008044; Leuconostoc mesenteroides subsp. mesenteroides XZ39303, from China Center for Type Culture Collection, preservation number CCTCC LB2008141.

[0044] Lactiplantibacillus plantarum LPA, from China Center for Type Culture Collection, preservation number CCTCC AB2010210; Lactiplantibacillus plantarum BX6-6, from China Center for Type Culture Collection, preservation number CCTCC LB2008011.

[0045] Embodiment

[0046] The present embodiment provides a Dendrobium candidum fermentation liquor, which specifically comprises the following steps:

[0047] (1) Dendrobium candidum powder is mixed with water at a ratio of 1:15 (w / v), 0.5% cellulase, 0.5% pectinase, and 0.1% papain are added, and homogenized for 2 minutes, and then enzymolysis is performed in a 50°C water bath for 2 hours. After enzymolysis, boiling water bath treatment is performed for 10 minutes to inactivate the enzyme. 10% fructose-glucose syrup is added, and sterilization is performed at 115°C for 20 minutes to obtain a Dendrobium candidum enzymolysis liquor;

[0048] (2) Selection of fermentation strains: Leuconostoc mesenteroides XD054, Leuconostoc mesenteroides Leul, Leuconostoc mesenteroides LM2, Leuconostoc mesenteroides subsp. mesenteroides XZ9302, Leuconostoc mesenteroides subsp. mesenteroides XZ39303; Lactiplantibacillus plantarum XD117, Lactiplantibacillus plantarum LPA, Lactiplantibacillus plantarum GLX94, Lactiplantibacillus plantarum BX6-6; and Lactobacillus muci laplantarum DSM 13273.

[0049] (3) Activation of lactic acid bacteria: the strains are inoculated in MRS medium and incubated at 37°C for 24 hours, and then subcultured for 3 times to fully activate the strains to a viable bacterial count of more than 1.0×10 9 CFU / mL;

[0050] The composition and preparation method of the MRS medium are as follows: 10 g of proteose peptone, 5 g of beef extract powder, 4 g of yeast extract powder, 20 g of glucose, 1 g of Tween-80, 2 g of potassium phosphate dibasic, 5 g of sodium acetate, 2 g of triammonium citrate, 0.2 g of magnesium sulfate (MgSO4.7H2O), 0.5 g of manganese sulfate (MnSO4.4H2O), 1 L of distilled water, pH 6.2±0.2, 121 ℃ sterilization treatment for 20 min.

[0051] (4) 2% of lactic acid bacteria (10 strains in step (3)) were inoculated in the enzyme hydrolysate of D. candidum, and fermentation was carried out at 37°C for 48 h. The supernatant was collected by centrifugation at 8000 rpm for 10 min to obtain the fermentation liquor of D. candidum.

[0052] The addition amount of cellulase, pectinase, papain and fructose syrup refers to w / v, for example, 0.5% refers to the volume ratio of the amount of cellulase to the fermentation system being 0.5%; the inoculation amount of the strain refers to v / v, for example, the addition amount is 2%, which refers to the volume ratio of the inoculation volume to the fermentation system being 2%.

[0053] Comparative Example

[0054] The present comparative example provides an enzyme hydrolysate of D. candidum. D. candidum powder is mixed with water at a ratio of 1:15 (w / v), 0.5% cellulase, 0.5% pectinase and 0.1% papain are added, and homogenized for 2 min. Enzymolysis is carried out in a water bath at 50°C for 2 h. After enzyme hydrolysis, boiling water bath treatment is carried out for 10 min to inactivate the enzyme. 10% fructose syrup is added, and sterilization is carried out at 115°C for 20 min to obtain the enzyme hydrolysate of D. candidum. The enzyme hydrolysate of D. candidum is centrifuged at 8000 rpm for 10 min, and the supernatant is collected to obtain the enzyme hydrolysate supernatant of D. candidum.

[0055] Experimental Example 1: Determination method of pancreatic lipase inhibition rate

[0056] 1. Reagents and materials are shown in Table 1

[0057] Table 1: Reagents and materials

[0058] Reagents / materials Company 4-nitrophenyl butyrate (p-NPB) Macclin pancrelipase Solabia disodium hydrogen phosphate dodecahydrate Hunan Huihong Reagent Co., Ltd. sodium dihydrogen phosphate dihydrate Hunan Huihong Reagent Co., Ltd.

[0059] 2. Preparation of reagents:

[0060] 25 mM pH=7.4 PBS: 4.477 g of diphosphoric acid disodium dodecahydrate and 1.95 g of phosphoric acid disodium diphosphate were weighed into 500 mL of distilled water, respectively, and mixed uniformly. The pH was adjusted to 7.4;

[0061] 5 mg / mL pancreatic lipase: 250 mg of pancreatic lipase was weighed into 50 mL of sterile water, then centrifuged at 8000 rpm for 5 min, the precipitate was discarded, and the supernatant was taken. The enzyme was stored in an ice box for use.

[0062] 11.2 mol / L p-NPB solution preparation: 0.11715 g p-NPB solution was dissolved in 50 mL PBS with a pipette and placed in an ice box for standby.

[0063] 3. Sample to be tested: the fermented Dendrobium officinale K. Koch fermentation broth obtained in the examples and the Dendrobium officinale K. Koch enzymatic hydrolysis supernatant obtained in the comparative examples.

[0064] 4. Experimental process

[0065] 500 μL of the sample to be tested, 500 μL of pancrelipase, 500 μL of PBS were mixed and placed in a 37°C water bath for incubation for 10 min, then 500 μL of p-NPB solution was added, mixed gently, placed in a 37°C water bath for incubation for 20 min, placed in an ice bath for 2 min, and the absorbance was measured at 405 nm wavelength and the value was recorded.

[0066] Inhibition rate (%) = [1- (A4-A3) / (A2-A1)] x 100%

[0067] Control background group A1: substrate

[0068] Control group A2: enzyme and substrate

[0069] Experimental background group A3: sample to be tested + substrate

[0070] Experimental group A4: experimental group

[0071] A1: PBS 1.5 mL + p-NPB 0.5 mL

[0072] A2: PBS 1 mL + pancrelipase 0.5 mL + p-NPB 0.5 mL

[0073] A3: PBS 1 mL + sample to be tested 0.5 mL + p-NPB 0.5 mL

[0074] A4: PBS 0.5 mL + sample to be tested 0.5 mL + pancrelipase 0.5 mL + p-NPB 0.5 mL

[0075] 5. The detection results are shown in Table 2

[0076] Table 2: Inhibition rate of pancrelipase

[0077]

[0078] Note: The same column with different letters represents significant difference, p≤0.05.

[0079] Result analysis: pancreatic lipase is the key fat hydrolysis enzyme in diet, which can hydrolyze fat into monoglyceride and free fatty acid, so as to be absorbed by human body. Therefore, the catalysis of fat decomposition can be reduced by inhibiting the activity of pancreatic lipase, so as to reduce the absorption of fat, so as to achieve the effect of controlling and treating obesity. The higher the inhibition rate is, the better the lipid-lowering effect is. As shown in Table 2, the pancreatic lipase inhibition rate of the fermentation liquor of D. hansenii XD054 fermented by D. hansenii XD054 is the highest.

[0080] Finally, it should be pointed out that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A type of Leuconostoc mesenteroides ( Leuconostoc mesenteroides XD054, characterized in that, The preservation number of the Leuconostoc mesenteroides XD054 is CCTCC NO: M 20241728, which is preserved in the China Center for Type Culture Collection on August 2, 2024.

2. The Leuconostoc mesenteroides XD054 for use in the preparation of a Dendrobium candidum fermentation broth or fermented Dendrobium candidum according to claim 1.

3. Use according to claim 2, characterized in that, The Leuconostoc mesenteroides XD054 is used in the fermentation of Dendrobium candidum to improve the pancreatic lipase inhibitory activity of the Dendrobium candidum fermentation broth.

4. Use according to claim 2, characterized in that, The Dendrobium candidum fermentation broth is prepared by inoculating Leuconostoc mesenteroides XD054 after enzymatic hydrolysis of Dendrobium candidum.

5. Use according to claim 4, characterized in that, The enzyme preparations selected for the enzymatic hydrolysis include cellulase, pectinase, and papain.

6. Use according to claim 5, characterized in that, The addition amount of cellulase is 0.2-0.6% (w / v), the addition amount of pectinase is 0.2-0.6% (w / v), and the addition amount of papain is 0.05-0.2% (w / v).

7. Use according to claim 4, characterized in that, The temperature of the enzymatic hydrolysis is 40-60℃, and the enzymatic hydrolysis time is 1-3h; the temperature of the fermentation is 30-45℃, and the fermentation time is 32-54h.

8. A microbial inoculant, characterized in that, The active ingredients include the Leuconostoc mesenteroides XD054 according to claim 1.

9. The microbial inoculant of claim 8, wherein, It also includes a microbial preservation aid. The active ingredients include the Leuconostoc mesenteroides XD054 according to claim 1. It also includes a microbial preservation aid.

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