Leuconostoc mesenteroides subsp. Mesenteroides XD054 and application thereof in fermentation of dendrobium officinale

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to leuconostoc mesenteroides subsp. Mesenteroides XD054 and application thereof in fermentation of dendrobium officinale. The leuconostoc mesenteroides subsp. Mesenteroides XD054 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20241728. Dendrobium officinale is subjected to enzymolysis by cellulase, pectinase and papain and then fermented by the strain to obtain dendrobium officinale fermentation liquor, and the dendrobium officinale fermentation liquor has an obvious pancreatic lipase activity inhibition effect.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to Leuconostoc mesenteroides subspecies mesenteroides XD054 and application thereof in fermenting Dendrobium officinale. Background Art

[0002] Dendrobii caulis, as the first of the "Nine Immortal Grasses" in my country, is a rare and precious Chinese medicinal material in the orchid family. It was first seen in the "Shennong Bencao Jing" and is known for strengthening yin and replenishing deficiency. It comes from the cultivated products of the perennial herbs of the orchid family, such as Dendrobium candidum, Dendrobium officinale, Dendrobium chrysotoxum, Dendrobium tasselii, and the fresh or dried stems of similar species of plants of the same genus. It is mainly distributed in Sichuan, Yunnan, Guizhou and other places. Among them, Dendrobium officinale is widely used due to its high medicinal value and relatively complete large-scale planting. At the same time, as a top-grade Chinese medicine, Dendrobium has no adverse reactions reported so far, which shows that its edible safety is reliable. On November 17, 2023, the "Notice on the Implementation of the Traditional Both Food and Chinese Medicinal Materials for Codonopsis and Other 9 Substances" (No. 9 of 2023) officially included 9 substances such as Dendrobium officinale, Astragalus, and Ganoderma lucidum into the catalog of substances that are both food and Chinese medicinal materials according to tradition (referred to as food and drug substances).

[0003] Dendrobium candidum contains glucose, inulin, fructose and 17 kinds of amino acids, including 7 essential amino acids for human body except tryptophan. In addition, it also contains P, K, Mg, Na, Zn, Mn, Cu and other trace elements. It can be processed into functional food. Therefore, Dendrobium candidum is more and more widely used in health food. It is often used as tea, wine, soup, porridge and other dietary supplements. Dendrobium candidum is sweet, slightly cold, and belongs to stomach and kidney meridians. It has the functions of benefiting stomach and producing body fluid, nourishing yin and clearing heat. It is mainly used clinically for stomach yin deficiency, heat disease damage to body fluid syndrome, and kidney yin deficiency syndrome. Modern research shows that Dendrobium candidum includes polysaccharides, flavonoids, coumarins, phenols, terpenes, alkaloids, amino acids, tannins and other chemical components. It has multiple pharmacological effects such as protecting gastric mucosa, enhancing immunity, anti-oxidation, lowering blood sugar, lowering blood lipids, and antibacterial.

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

[0005] Pancreatic lipase is a key fat hydrolyzing enzyme in the diet, which can hydrolyze fat into monoglycerides and free fatty acids, which are then absorbed by the human body. Therefore, by inhibiting the activity of pancreatic lipase, the catalytic effect of fat decomposition can be reduced, thereby reducing fat absorption, to achieve 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 stools, oily spots and flatulence.

[0006] Plant extracts have multiple active ingredients and usually have multiple targets, which can treat both the symptoms and the root cause. Compared with synthetic drugs, natural products with minimal side effects can prevent or reduce the risk of chronic diseases, which is gaining more and more attention in the treatment of obesity. Currently, 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 serum TC, TG and LDL-C levels, while increasing HPL-C levels.

[0007] At present, there are few studies on the regulation of lipid metabolism by Dendrobium officinale. Lin Ya et al. Effects of Dendrobium officinale mixture on blood glucose and blood lipid metabolism in diabetic rat models [J]. Journal of Fujian University of Traditional Chinese Medicine. 2012, 22 (03) confirmed that Dendrobium officinale mixture can regulate lipid metabolism, reduce blood lipids, free fatty acids and cholesterol levels, and block the reduction of insulin secretion mediated by them to achieve the purpose of lowering blood sugar. The latest research results show that the mechanism of action of Dendrobium officinale mixture in lowering blood lipids is related to improving the expression of multiple gene pathways. It can regulate lipid metabolism and protect the liver by regulating signaling pathways such as WNT and PPARβ / δ. There is no literature reporting the inhibitory activity of Dendrobium officinale on pancreatic lipase. Summary of the invention

[0008] To this end, the present invention provides, on one hand, Leuconostoc mesenteroides subsp. mesenteroides XD054; on the other hand, provides the use of the strain in fermenting Dendrobium officinale. The Dendrobium officinale fermentation clear liquid obtained thereby can inhibit the activity of pancreatic lipase and can be used in the development of weight loss products. In another aspect, the present invention provides a bacterial agent that can be used for fermenting Dendrobium officinale.

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

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

[0011] In order to solve the above technical problems, the technical solution of the present invention is:

[0012] A strain of Leuconostoc mesenteroides subspecies XD054 was deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241728.

[0013] The application of the above-mentioned Leuconostoc mesenteroides subspecies mesenteroides XD054 in preparing Dendrobium officinale fermentation liquid or fermenting Dendrobium officinale.

[0014] The above-mentioned Leuconostoc mesenteroides subspecies mesenteroides XD054 is used in the fermentation of Dendrobium officinale to improve the pancreatic lipase inhibitory activity of the Dendrobium officinale fermentation liquid.

[0015] The invention discloses a Dendrobium officinale fermentation liquid, which is prepared by enzymatic hydrolysis of the Dendrobium officinale and inoculation with Leuconostoc mesenteroides subspecies mesenteroides XD054 bacteria for fermentation.

[0016] Preferably, the enzyme preparations selected for enzymatic hydrolysis of Dendrobium officinale include cellulase, pectinase and papain.

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

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

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

[0020] Preferably, the viable count of the Leuconostoc mesenteroides subspecies XD054 is 1.0×10 9 CFU / mL and above.

[0021] Further preferably, the activation method of Leuconostoc mesenteroides subspecies XD054 is as follows: inoculating the strain into MRS medium, culturing at 37°C for 24 hours, and continuously subculturing for 3 times to fully activate the strain until the number of viable bacteria is 1.0×10 9 CFU / mL and above.

[0022] A method for preparing a fermented Dendrobium officinale liquid comprises the following steps: (1) mixing the Dendrobium officinale with water and performing enzymolysis, inactivating the enzyme and sterilizing the mixture to obtain an enzymolysis liquid; and (2) inoculating the enzymolysis liquid with Leuconostoc mesenteroides subsp. mesenteroides XD054, fermenting the mixture, and performing solid-liquid separation to obtain a fermented Dendrobium officinale liquid.

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

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

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

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

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

[0028] A microbial agent, the active ingredients of which include the above-mentioned Leuconostoc mesenteroides subspecies mesenteroides XD054; preferably, it also includes an auxiliary agent for maintaining the long-term preservation of the microorganisms.

[0029] The technical solution of the present invention has the following advantages:

[0030] 1 The present invention provides a strain of Leuconostoc mesenteroides subsp. mesenteroides XD054 and its application in fermenting Dendrobium officinale. Fermenting Dendrobium officinale with the strain can significantly improve the pancreatic lipase inhibition rate of the Dendrobium officinale fermentation liquid.

[0031] 2 The present invention provides a Dendrobium officinale fermentation liquid, which is prepared by enzymatic hydrolysis with cellulase, pectinase and papain followed by inoculation with Leuconostoc mesenteroides subsp. mesenteroides XD054 for fermentation. The obtained Dendrobium officinale fermentation liquid has a very good pancreatic lipase inhibition rate.

[0032] 3 The present invention provides a method for preparing a fermented Dendrobium officinale liquid. The method has few process steps and is convenient for industrial production. The obtained fermented Dendrobium officinale liquid has a significant effect of inhibiting the activity of pancreatic lipase.

[0033] 4 The present invention uses Dendrobium officinale as raw material, is prepared from natural plants, has no chemical additives, and is safe and reliable.

[0034] Storage Information

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

[0036] Lactobacillus plantarum XD117 was deposited in the China Center for Type Culture Collection on August 2, 2024, with the deposit number CCTCC NO: M 20241730, Latin name Lactobacillus plantarum, and the address is Wuhan University, Wuhan, China. On August 25, 2022, the updated "List of Bacteria that Can Be Used in Food" was officially released, and Lactobacillus plantarum was renamed Lactiplantibacillus plantarum. Lactobacillus plantarum XD117 and Lactiplantibacillus plantarum XD117 are the same strain.

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

[0038] To make the purpose, technical solutions and advantages of the present invention clearer, the embodiments of the present invention are described in further detail below. Based on the embodiments disclosed in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0039] If no specific experimental steps or conditions are specified in the examples, the experiments can be carried out according to the conventional steps or conditions described in the literature in the field. All reagents without manufacturer specified are conventional reagent products that can be obtained through commercial purchase.

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

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

[0042] Fermented Lactobacillus fermentu DSM 13273 is from the China Center for Type Culture Collection, with the preservation number of CCTCCAB 2013123, Latin Limosilactobacillus fermentu), the original number is S1L18, and DSM 13273 is the number named by the applicant.

[0043] Leuconostoc mesenteroides subsp. mesenteroides Leu1 is from the China Center for Type Culture Collection, with the accession number CCTCCAB 2010207; Leuconostoc mesenteroides subsp. mesenteroides LM2 is from the China Center for Type Culture Collection, with the accession number CCTCC HB 20082735; Leuconostoc mesenteroides subsp. mesenteroides XZ9302 is from the China Center for Type Culture Collection, with the accession number CCTCC LB 2008044; Leuconostoc mesenteroides subsp. mesenteroides XZ39303 is from the China Center for Type Culture Collection, with the accession number CCTCC LB 2008141.

[0044] Lactobacillus plantarum LPA is from China Center for Type Culture Collection, with the accession number CCTCC AB 2010210; Lactobacillus plantarum BX6-6 is from China Center for Type Culture Collection, with the accession number CCTCC LB 2008011.

[0045] Example

[0046] This embodiment provides a fermented Dendrobium officinale liquid, which specifically comprises the following steps:

[0047] (1) After mixing the powder of Dendrobium officinale with water at a ratio of 1:15 (w / v), add 0.5% cellulase, 0.5% pectinase, and 0.1% papain, homogenize for 2 minutes, and perform enzymolysis in a 50℃ water bath for 2 hours. After enzymolysis, boil the water bath for 10 minutes to inactivate the enzyme. Add 10% fructose syrup and sterilize at 115℃ for 20 minutes to obtain the enzymolysis solution of Dendrobium officinale.

[0048] (2) Selection of fermentation bacteria: Leuconostoc mesenteroides subsp. mesenteroides XD054, Leuconostoc mesenteroides subsp. mesenteroides Leu1, Leuconostoc mesenteroides subsp. mesenteroides LM2, Leuconostoc mesenteroides subsp. mesenteroides XZ9302, Leuconostoc mesenteroides subsp. mesenteroides XZ39303; Lactobacillus plantarum XD117, Lactobacillus plantarum LPA, Lactobacillus plantarum GLX94, Lactobacillus plantarum BX6-6; fermentation Lactobacillus mucosa DSM 13273.

[0049] (3) Activation of lactic acid bacteria: The strains were inoculated into MRS medium and cultured at 37°C for 24 h. The strains were subcultured three times to fully activate the strains until the viable count was 1.0×10 9 CFU / mL or above;

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

[0051] (4) Inoculate 2% lactic acid bacteria (the 10 strains in step (3) to ensure that the number of viable bacteria of the strains is consistent) into the enzymatic hydrolysate of Dendrobium officinale, ferment at 37°C for 48 h, centrifuge at 8000 rpm for 10 min, collect the supernatant, and obtain the fermented Dendrobium officinale liquid.

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

[0053] Comparative Example

[0054] This comparative example provides a Dendrobium officinale enzymatic hydrolysate, wherein the Dendrobium officinale powder and water are mixed at a ratio of 1:15 (w / v), 0.5% cellulase, 0.5% pectinase, and 0.1% papain are added, homogenized for 2 minutes, and enzymolyzed in a 50°C water bath for 2 hours, and then the enzyme is inactivated by boiling water bath for 10 minutes after enzymolysis. 10% fructose syrup is added, and the Dendrobium officinale enzymatic hydrolysate is obtained by sterilization at 115°C for 20 minutes, and the Dendrobium officinale enzymatic hydrolysate is centrifuged at 8000rpm for 10 minutes, and the supernatant is collected to obtain the Dendrobium officinale enzymatic hydrolysate supernatant.

[0055] Experimental Example 1 Method for determining pancreatic lipase inhibition rate

[0056] 1 Reagents and materials See Table 11

[0057] Table 1 Reagents and materials

[0058] Reagents / Materials company 4-Nitrophenylbutyrate (p-NPB) Maclean Pancreatic lipase Solebao Disodium hydrogen phosphate dodecahydrate Hunan Huihong Reagent Co., Ltd. Sodium dihydrogen phosphate dihydrate Hunan Huihong Reagent Co., Ltd.

[0059] 2. Reagent preparation:

[0060] 25 mM pH=7.4 PBS: weigh 4.477 g of disodium hydrogen phosphate dodecahydrate and 1.95 g of sodium dihydrogen phosphate dihydrate, respectively, and dissolve them in 500 mL of distilled water. After mixing, adjust the pH to 7.4.

[0061] 5mg / mL pancreatic lipase: weigh 250mg of pancreatic lipase and dissolve it in 50mL of sterile water, then centrifuge at 8000rpm for 5min, discard the precipitate and take the supernatant, and place the enzyme in an ice box before use;

[0062] Preparation of 11.2 mol / L p-NPB solution: Use a pipette to dissolve 0.11715 g of p-NPB solution in 50 mL of PBS and place in an ice box for later use.

[0063] 3. Samples to be tested: the fermented Dendrobium officinale broth obtained in the example and the supernatant of the enzymatic hydrolyzate of Dendrobium officinale obtained in the comparative example.

[0064] 4. Experimental Procedure

[0065] Mix 500µL of the sample to be tested, 500µL of pancreatic lipase, and 500µL of PBS, and incubate in a 37°C water bath for 10 min. Then add 500µL of p-NPB solution, gently shake to mix, incubate in a 37°C water bath for 20 min, place in an ice bath for 2 min, measure the absorbance at a wavelength of 405nm and record the value.

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

[0067] Control background group A1: substrate

[0068] Control group A2: enzyme and substrate

[0069] Experimental background group A3: test sample + substrate

[0070] Experimental group A4: Experimental group

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

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

[0073] A3: PBS 1 mL + test sample 0.5 mL + p-NPB 0.5 mL

[0074] A4: PBS 0.5 mL + test sample 0.5 mL + pancreatic lipase 0.5 mL + p-NPB 0.5 mL

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

[0076] Table 2 Pancreatic lipase inhibition rate

[0077]

[0078] Note: Different letters in the same column represent significant differences, p≤0.05.

[0079] Results analysis: Pancreatic lipase is a key fat hydrolyzing enzyme in the diet, which can hydrolyze fat into monoglycerides and free fatty acids, which are then absorbed by the human body. Therefore, by inhibiting the activity of pancreatic lipase, reducing the catalytic effect of fat decomposition, and thus reducing fat absorption, the effect of controlling and treating obesity can be achieved. The higher the inhibition rate, the better the lipid-lowering effect. As shown in Table 2, the pancreatic lipase inhibition rate of the fermented liquid of Dendrobium officinale fermented by Leuconostoc mesenteroides subsp. mesenteroides XD054 was the highest.

[0080] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A Leuconostoc mesenteroides subspecies mesenteroides XD054, characterized in that The deposit number of the Leuconostoc mesenteroides subspecies mesenteroides XD054 is CCTCC NO: M 20241728.

2. Application of Leuconostoc mesenteroides subspecies mesenteroides XD054 in preparing Dendrobium officinale fermentation liquid or fermenting Dendrobium officinale.

3. The use according to claim 2, characterized in that: The Leuconostoc mesenteroides subspecies mesenteroides XD054 is used in the fermentation of Dendrobium officinale to improve the pancreatic lipase inhibitory activity of the Dendrobium officinale fermentation liquid.

4. The use according to claim 2, characterized in that: The Dendrobium officinale fermented liquid is prepared by inoculating the Dendrobium officinale with Leuconostoc mesenteroides subspecies XD054 and fermenting the enzymatic hydrolysis of the Dendrobium officinale.

5. The use according to claim 4, characterized in that: The enzyme preparations selected for enzymolysis include cellulase, pectinase and papain.

6. The use according to claim 5, characterized in that: The added amount of the cellulase is 0.2-0.6%, the added amount of the pectinase is 0.2-0.6%, and the added amount of the papain is 0.05-0.2%.

7. The use according to claim 4, characterized in that: The temperature of the enzymolysis is 40-60°C, and the enzymolysis time is 1-3h; the temperature of the fermentation is 30-45°C, and the time is 32-54h.

8. A microbial agent, characterized in that: The active ingredient includes the Leuconostoc mesenteroides subspecies XD054.

9. The microbial agent according to claim 8, characterized in that: It also includes additives for maintaining the long-term preservation of microorganisms.

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