Dendrobium officinale fermentation liquor with pancreatic lipase inhibition effect and preparation method thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine fermentation, and particularly relates to dendrobium officinale fermentation liquor with a pancreatic lipase inhibition effect and a preparation method of the dendrobium officinale fermentation liquor. The dendrobium officinale fermentation liquor is prepared by performing enzymolysis on dendrobium officinale and inoculating lactic acid bacteria for fermentation, wherein the dendrobium officinale fermentation liquor obtained by inoculating fermentation lactobacillus mucus DSM 13273 or leuconostoc mesenteroides subsp. Mesenteroides XD054 has a better pancreatic lipase activity inhibition effect.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine fermentation, and particularly relates to a Dendrobium officinale fermentation liquid with pancreatic lipase inhibition effect and a preparation method thereof. Background Art

[0002] 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.

[0003] 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.

[0004] 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 lipid metabolism in diabetic rat models [J]. Journal of Fujian University of Traditional Chinese Medicine. 2012, 22(03) confirmed that Dendrobium officinale mixture can reduce blood lipids, free fatty acids and cholesterol levels by regulating lipid metabolism, blocking the reduction of insulin secretion mediated by them, thereby achieving 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, and 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

[0005] To this end, the present invention provides a Dendrobium officinale fermented liquid with pancreatic lipase inhibitory effect, which is prepared by enzymatic hydrolysis of Dendrobium officinale and lactic acid bacteria fermentation. The obtained Dendrobium officinale fermented liquid can inhibit the activity of pancreatic lipase and can be used in the development of weight loss products.

[0006] 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.

[0007] In the present invention, "enzyme activity", "enzyme activity", "enzyme activity" and "enzyme specific activity" have the same meaning under some conditions.

[0008] In order to solve the above technical problems, the technical solution of the present invention is:

[0009] The invention discloses a Dendrobium officinale fermented liquid with the effect of inhibiting pancreatic lipase, which is prepared by enzymatic hydrolysis of the Dendrobium officinale and inoculation with lactic acid bacteria for fermentation.

[0010] Preferably, the lactic acid bacteria include one or more of 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 and Lactobacillus fermentans DSM 13273.

[0011] Preferably, the lactic acid bacteria include fermentative Lactobacillus mucilaginosus DSM 13273 and / or Leuconostoc mesenteroides subsp. mesenteroides XD054; wherein the deposit number of fermentative Lactobacillus mucilaginosus DSM 13273 is CCTCC AB 2013123; the deposit number of Leuconostoc mesenteroides subsp. mesenteroides XD054 is CCTCC NO: M 20241728.

[0012] Preferably, the lactic acid bacteria is Leuconostoc mesenteroides subspecies mesenteroides XD054, and its deposit number is CCTCC NO: M20241728.

[0013] Preferably, the enzyme preparations selected for enzymolysis include cellulase, pectinase and papain.

[0014] Preferably, the amount of cellulase added is 0.2-0.6%, the amount of pectinase added is 0.2-0.6%, and the amount of papain added is 0.05-0.2%;

[0015] More preferably, the added amount of cellulase is 0.5%, the added amount of pectinase is 0.5%, and the added amount of papain is 0.1%.

[0016] 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.

[0017] Preferably, the inoculation amount of the lactic acid bacteria is 1-3%, and more preferably, the inoculation amount is 2%.

[0018] Preferably, the lactic acid bacteria have a bacterial viability of 1.0×10 9 CFU / mL and above.

[0019] Further preferably, the activation method of the lactic acid bacteria is: 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.

[0020] A method for preparing a Dendrobium officinale fermentation liquid having pancreatic lipase inhibition effect comprises the following steps:

[0021] (1) mixing Dendrobium officinale and water, performing enzymolysis, inactivating the enzyme and sterilizing to obtain an enzymolysis solution; and (2) inoculating lactic acid bacteria into the enzymolysis solution for fermentation, performing solid-liquid separation, and obtaining a Dendrobium officinale fermentation solution.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Preferably, the above-mentioned Dendrobium officinale fermentation liquid and the Dendrobium officinale fermentation liquid obtained according to the above-mentioned preparation method are used in products for inhibiting pancreatic lipase activity.

[0028] The technical solution of the present invention has the following advantages:

[0029] The present invention provides a fermented Dendrobium officinale liquid with pancreatic lipase inhibitory effect. The fermented Dendrobium officinale liquid is prepared by inoculating lactic acid bacteria after enzymatic hydrolysis. The obtained fermented Dendrobium officinale liquid has excellent pancreatic lipase inhibitory activity.

[0030] 2. The present invention provides a fermented Dendrobium officinale liquid with pancreatic lipase inhibitory effect. When Lactobacillus mucilaginosus DSM 13273 or Leuconostoc mesenteroides subsp. mesenteroides XD054 is used to ferment Dendrobium officinale, the fermented liquid has better pancreatic lipase inhibitory activity.

[0031] The present invention provides a method for preparing a fermented Dendrobium officinale liquid, which has few process steps and is convenient for industrial production. The obtained fermented Dendrobium officinale liquid has an obvious effect of inhibiting the activity of pancreatic lipase.

[0032] 4 The present invention uses Dendrobium officinale as raw material, is prepared from natural plants, has no chemical additives, and is safe and reliable.

[0033] Storage Information

[0034] 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.

[0035] Lactobacillus plantarum XD117 is deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20241730 on August 2, 2024. The address is Wuhan University, Wuhan, China. The updated "List of Bacteria that Can Be Used in Food" has renamed Lactobacillus plantarum as Lactiplantibacillus plantarum. Lactobacillus plantarum XD117 and Lactiplantibacillus plantarum XD117 are the same strain.

[0036] 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

[0037] 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.

[0038] 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.

[0039] In the following examples, the source information of some products is as follows:

[0040] 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.

[0041] Fermented mucus lactobacillus DSM 13273 is from the China Center for Type Culture Collection, with the preservation number of CCTCCAB 2013123, Latin Limosilactobacillus fermentu), the original number of which is S1L18, and DSM 13273 is the number named by the applicant.

[0042] 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.

[0043] 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.

[0044] Embodiment Dendrobium officinale fermentation liquid

[0045] This embodiment provides a fermented Dendrobium officinale liquid, which specifically comprises the following steps:

[0046] (1) After mixing the powder of Dendrobium officinale with water in a ratio of 1:15 (w / v), 0.5% cellulase, 0.5% pectinase, and 0.1% papain were added, homogenized for 2 minutes, and enzymolyzed in a 50°C water bath for 2 hours. After enzymolysis, the enzyme was inactivated by boiling water bath for 10 minutes. 10% fructose syrup was added, and the mixture was sterilized at 115°C for 20 minutes to obtain the enzymolysis solution of Dendrobium officinale;

[0047] (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.

[0048] (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;

[0049] 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.

[0050] (4) Inoculate 2% lactic acid bacteria (the 10 strains in step (3)) into the Dendrobium officinale hydrolysate, keep the number of live bacteria consistent during inoculation, ferment at 37° C. for 48 h, centrifuge at 8000 rpm for 10 min, collect the supernatant, and obtain the Dendrobium officinale fermentation liquid.

[0051] The addition amount of cellulase, pectinase, papain and fructose syrup refers to w / v, such as 0.5% means that the volume ratio of cellulase to the fermentation system is 0.5%; 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 ratio of the fermentation system.

[0052] Comparative Example

[0053] This comparative example provides a Dendrobium officinale enzymatic hydrolysate, wherein the Dendrobium officinale powder and water are mixed in 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.

[0054] Experimental Example 1 Method for determining pancreatic lipase inhibition rate

[0055] 1. Reagents and materials are shown in Table 1.

[0056] Table 1 Reagents and materials

[0057] 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.

[0058] 2. Reagent preparation:

[0059] 25mM pH=7.4 PBS: weigh 4.477g disodium hydrogen phosphate dodecahydrate and 1.95g sodium dihydrogen phosphate dihydrate, dissolve in 500ml distilled water, mix well and adjust the pH to 7.4;

[0060] 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;

[0061] 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.

[0062] 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.

[0063] 4. Experimental Procedure

[0064] Mix 500 μL of the sample to be tested, 500 μL of pancreatic lipase, and 500 μL of PBS, and incubate in a 37°C water bath for 10 min. Then add 500 μL 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 405 nm and record the value.

[0065] Inhibition rate (%) = [1-(A4-A3) / (A2-A1)] × 100%

[0066] Control background group A1: substrate

[0067] Control group A2: enzyme and substrate

[0068] Experimental background group A3: test sample + substrate

[0069] Experimental group A4: Experimental group

[0070] A1: PBS 1.5 mL + p-NPB 0.5 mL

[0071] A2: PBS 1mL + pancreatic lipase 0.5mL + p-NPB 0.5mL

[0072] A3: PBS 1mL + test sample 0.5mL + p-NPB 0.5mL

[0073] A4: PBS 0.5mL + test sample 0.5mL + pancreatic lipase 0.5mL + p-NPB 0.5mL

[0074] 5. Test results are shown in Table 2

[0075] Table 2 Pancreatic lipase inhibition rate

[0076]

[0077] Note: Different letters in the same column represent significant differences, p≤0.05.

[0078] Analysis of results: 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, the activity of pancreatic lipase can be inhibited to reduce the catalytic effect of fat decomposition, thereby reducing fat absorption, to achieve the effect of controlling and treating obesity. The higher the inhibition rate, the better the lipid-lowering effect. As shown in Table 2, the pancreatic lipase inhibition rates of the 10 lactic acid bacteria fermented by the present invention are all higher than those of the supernatant of the enzymatic hydrolyzate of Dendrobium officinale, while the pancreatic lipase inhibition rates of the fermented Dendrobium officinale by Leuconostoc mesenteroides subsp. XD054 and the fermented Dendrobium officinale by Lactobacillus mucilaginosus DSM 13273 are the highest.

[0079] 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 fermented liquid of Dendrobium officinale having the effect of inhibiting pancreatic lipase, characterized in that: The Dendrobium officinale fermented liquid is prepared by enzymatic hydrolysis of Dendrobium officinale and inoculation with lactic acid bacteria for fermentation; The lactic acid bacteria include one or more of 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 and Lactobacillus fermentans DSM 13273.

2. The Dendrobium officinale fermented liquid according to claim 1, characterized in that The lactic acid bacteria include fermentative Lactobacillus mucilaginosus DSM 13273 and / or Leuconostoc mesenteroides subspecies mesenteroides XD054; wherein the preservation number of fermentative Lactobacillus mucilaginosus DSM 13273 is CCTCC AB 2013123; the preservation number of Leuconostoc mesenteroides subspecies mesenteroides XD054 is CCTCC NO: M20241728.

3. The Dendrobium officinale fermented liquid according to claim 1, characterized in that The lactic acid bacteria is Leuconostoc mesenteroides subspecies mesenteroides XD054, and the preservation number of Leuconostoc mesenteroides subspecies mesenteroides XD054 is CCTCC NO: M 20241728.

4. The Dendrobium officinale fermented liquid according to claim 1, characterized in that The enzyme preparations selected for enzymolysis include cellulase, pectinase and papain.

5. The Dendrobium officinale fermented liquid according to claim 1, characterized in that The inoculation amount of the lactic acid bacteria is 1-3%, and the viable bacteria of the lactic acid bacteria are 1.0×10 9 CFU / mL and above.

6. The Dendrobium officinale fermented liquid according to claim 4, 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. A method for preparing the Dendrobium officinale fermentation liquid according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: (1) mixing Dendrobium officinale and water, performing enzymolysis, inactivating the enzyme and sterilizing to obtain an enzymolysis solution; and (2) inoculating lactic acid bacteria into the enzymolysis solution for fermentation, and performing solid-liquid separation to obtain Dendrobium officinale fermentation solution.

8. The preparation method according to claim 7, characterized in that: The enzymatic hydrolysis temperature is 40-60° C., and the enzymatic hydrolysis time is 1-3 hours; the enzyme inactivation condition is a boiling water bath treatment for 5-20 minutes; the sterilization condition is 100-120° C., and the sterilization time is 10-30 minutes.

9. The preparation method according to claim 7, characterized in that: The fermentation temperature is 30-45°C and the fermentation time is 32-54h.

10. Use of the Dendrobium officinale fermented liquid according to any one of claims 1 to 6 and the Dendrobium officinale fermented liquid prepared by the method according to any one of claims 7 to 9 in a product for inhibiting pancreatic lipase activity.

Images