Marine-derived talaromyces brachypus fermentation extract and application thereof

Abstract

The invention discloses a marine talaromyces brachypodius extract and application thereof, the strain is talaromyces brachypodius QDU-WH004 preserved in China Center for Type Culture Collection on November 24, 2023, the preservation number is CCTCC NO: M 20232329, and the preservation address is Wuhan University Life Science College, Luojia Hill, Wuchang District, Wuchang District, Hubei Province, China. The fermented extract of talaromyces brachypus can effectively inhibit the activity of pancreatic lipase and further inhibit the hydrolysis of dietary triglyceride, so that the effects of reducing fat and losing weight are achieved.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to a marine-derived short-rod Bacillaceae fermentation extract and application thereof. Background Art

[0002] Obesity is a highly prevalent, chronic, relapsing, and progressive disease. The World Health Organization (WHO) has declared obesity a major public health and medical issue of the 21st century, listing it as one of the top ten threats to human health. Its primary characteristic is excessive fat accumulation, which is closely linked to the development of numerous chronic diseases, including immune dysfunction, respiratory illnesses, cardiovascular disease, and diabetes. The World Obesity Federation (https: / / www.worldobesity.org) predicts that by 2035, the proportion of the global population who are overweight or obese will reach 51%, making it a significant global health issue.

[0003] Lipases present in the digestive system include lingual lipase, gastric lipase, and pancreatic lipase. The most important of these is pancreatic lipase, which directly influences intestinal fatty acid absorption. Pancreatic lipase, the primary lipase secreted by the pancreas, hydrolyzes dietary lipids in the digestive system, converting triacylglycerol substrates in ingested fats and oils into monoglycerides and free fatty acids. Within the intestine, monoglycerides and free fatty acids are then transferred to the intestinal epithelial cells for absorption. Pancreatic lipase plays a crucial role in triglyceride hydrolysis, processing 50-70% of total dietary fat. Therefore, inhibiting pancreatic lipase activity is an effective approach to combating obesity (Liu TT, Liu XT, Chen QX, Shi Y. Biomed Pharmacother, 2020, 128:1103-14). Currently, weight loss drugs on the market fall into three main categories: those that suppress appetite, those that increase consumption, and those that inhibit gastrointestinal absorption. Pancreatic lipase inhibitors do not stimulate the nervous system or enter the bloodstream; they achieve their weight loss effects simply by inhibiting food absorption in the gastrointestinal tract, making them relatively safe. Currently, orlistat is a commonly used weight loss drug for inhibiting pancreatic lipase activity, but it can cause side effects such as gastrointestinal adverse reactions, liver damage, kidney damage, and malnutrition. Therefore, the search for safe and effective pancreatic lipase inhibitors remains a hot topic in natural product research (Kumar A, Chauhan S. Life Sci. 2021, 271:119-115).

[0004] The unique characteristics of the marine environment (such as high pressure, high salinity, lack of oxygen, low temperature, darkness, and oligotrophic factors) and the complex and extensive ecological interactions between marine species have endowed marine microorganisms with unique metabolic pathways and adaptive mechanisms, enabling the production of structurally distinct and bioactive secondary metabolites, which are an important source of new drugs. Marine microorganisms have a short growth and reproduction period and can be produced on a large scale through fermentation. Therefore, the research and development of pancreatic lipase inhibitors from microorganisms and their metabolites is not only promising but also highly feasible. Summary of the Invention

[0005] The present invention provides a strain of Talaromyces brevis (QDU-WH004), originating from the marine intertidal zone and collected from soil near Swan Lake in Rongcheng, Weihai City, Shandong Province. The strain of Talaromyces brevis QDU-WH004 is deposited with the following information: Depository Name: China Center for Type Culture Collection (CCTCC); Address: School of Life Sciences, Wuhan University, Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China; Deposit Date: November 24, 2023; Deposit Number: CCTCC NO: M 20232329.

[0006] Another embodiment of the present invention provides a fermentation extract of marine-derived Talaromyces brevis QDU-WH004 and a method for preparing the same. The method comprises the following steps: (1) Strain activation: Under sterile conditions, the preserved short-rod Basilicum fungus QDU-WH004 was inoculated onto a PDA solid culture medium plate for activation, and cultured at a constant temperature of 25-35°C for 5-10 days; (2) Fermentation culture: inoculate the activated culture block in (1) into GPY liquid fermentation medium, culture at a constant temperature of 25-35°C for 30-50 days to obtain a fermentation product; (3) Extraction: The fermentation product obtained in step (2) was sterilized with ethyl acetate, the cells were broken, and ultrasonic treatment was performed for 30 minutes. The filtrate was filtered through 8 layers of sterile gauze to obtain the filtrate, which was extracted three times by shaking with an equal volume of ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain the fermentation extract of the short-rod Basilicum fungus QDU-WH004.

[0007] Another embodiment of the present invention provides the use of marine-derived Talaromyces brachyrhizium QDU-WH004 in the preparation of a pancreatic lipase inhibitor.

[0008] Another embodiment of the present invention provides a fermentation extract of marine-derived short-rod Bacillaceae QDU-WH004 that inhibits pancreatic lipase activity and can be used in the preparation of pancreatic lipase inhibitors. By inhibiting pancreatic lipase activity, the hydrolysis of dietary triglycerides is inhibited, and the product can be used in the preparation of products for treating obesity by inhibiting pancreatic lipase activity.

[0009] Another embodiment of the present invention provides a pharmaceutical composition characterized in that the pharmaceutical composition contains the aforementioned extract as an active ingredient. The pharmaceutical composition may further include an appropriate amount of a pharmaceutically acceptable excipient (e.g., a pharmaceutically acceptable carrier, diluent, or excipient). The pharmaceutical composition may be in the form of a solid preparation, a liquid preparation, or a semisolid preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Colony characteristics of marine-derived short-rod Bacillaceae QDU-WH004.

[0011] Figure 2 Phylogenetic tree of marine-derived short-rod Bacillaceae QDU-WH004. DETAILED DESCRIPTION

[0012] To better understand the content of the present invention, the following specific embodiments are described in detail, but the following examples do not limit the scope and implementation of the present invention.

[0013] Example 1 Isolation and identification of marine-derived Talaromyces brachyrhizium QDU-WH004

[0014] 1. Isolation and identification of marine fungi

[0015] The present invention collected soil samples from the intertidal zone of Swan Lake, Rongcheng, Weihai, Shandong Province, China. Part of the soil was suspended in 10 ml of sterile double-distilled water, and then the suspension was continuously diluted to 10 -2 , inoculate the diluted liquid onto PDA solid medium using the three-zone streak method, and incubate the medium at a constant temperature of 28°C. Use an inoculation loop to pick up yellow colonies and transfer them to a new PDA plate using the streak method until a single colony is formed.

[0016] 2. Identification methods

[0017] (1) Morphological identification: The purified fungus was inoculated onto a PDA solid flat culture medium using the dot-planting method and the culture medium was placed in a constant temperature culture at 28°C. After the fungus grew into a single colony, the colony was observed for its physical characteristics. The colony was initially yellow in the center with a white flocculent edge, a regular circle, and expanded in all directions with neat edges and slightly lighter color. After 15 days, the colony turned orange-red. A sterile coverslip was inserted obliquely at the edge of the colony. When the hyphae grew and extended onto the coverslip, the coverslip with hyphae was removed with tweezers and attached to a slide. The morphological characteristics of hyphae, conidiophores, and conidia were observed under an optical microscope. Morphological identification was performed with reference to the "Handbook of Fungal Identification" compiled by Wei Jingchao.

[0018] (2) A strain belonging to the genus Talaromyces was identified and further molecular biological identification was performed: the strain was inoculated on a PDA plate and the culture medium was kept at a constant temperature of 28°C for 7 days. After the fungus was fully grown, genomic DNA extraction, 18S amplification, PCR product detection and purification were carried out in sequence. The purified product was directly sequenced using a gene sequencer, and the sequencing results were compared with the sequences in the Genbank database by BLAST. Based on the sequence similarity, the strain was identified as Talaromyces brevis, and named Talaromyces brevis QDU-WH004.

[0019] Example 2 Preparation of Fermentation Extract of Marine-derived Talaromyces QDU-WH004

[0020] 1. Preparation of Culture Medium

[0021] (1) Potato solid culture medium (PDA): Peel and cut 200 g of potatoes into pieces, add 1000 mL of distilled water, and boil for 10-20 min. Filter through gauze and add distilled water to 1000 mL. Add 20 g of glucose, 25 g of agar, and 33 g of NaCl, heat to dissolve, and divide into Erlenmeyer flasks. Sterilize in a high-pressure steam autoclave at 121°C for 25 min. After sterilization, move the Erlenmeyer flask to a clean bench. After cooling slightly, quickly pour the PDA liquid into a culture dish and let it cool and solidify before use.

[0022] (2) GPY medium: Place 20 g / L glucose, 5 g / L peptone, 2 g / L yeast extract powder, and 33 g / L NaCl in a conical flask, seal with double-distilled water, and sterilize in a high-pressure steam autoclave at 121°C for 25 min. After sterilization, move the conical flask to a clean bench and let it cool before use.

[0023] (3) Fungal Culture Medium No. 2: Place 20 g / L mannitol, 3 g / L yeast extract powder, 0.3 g / L MgSO4·7H2O, 10 g / L MSG, 10 g / L glucose, 0.5 g / L KH2PO4, 20 g / L maltose, 1 g / L corn steep liquor, 20 g / L CaCO3, and 33 g / L NaCl in a conical flask. Add double-distilled water, seal the flask, and sterilize it in a high-pressure steam sterilizer at 121°C for 25 min. After sterilization, move the flask to a clean bench and let it cool before use.

[0024] (4) Peter's medium: Add 3 g / L yeast extract, 3 g / L malt extract, 5 g / L peptone, 20 g / L glucose, and 33 g / L NaCl to a conical flask, seal it with double-distilled water, and sterilize it in a high-pressure steam sterilizer at 121°C for 25 min. After sterilization, move the conical flask to a clean bench and let it cool before use.

[0025] (5) Glycerol culture medium: Place 20 g / L glycerol, 2 g / L peptone, 2 g / L yeast extract powder, and 33 g / L NaCl in a conical flask, seal it with double-distilled water, and sterilize it in a high-pressure steam sterilizer at 121°C for 25 min. After sterilization, move the conical flask to a clean bench and let it cool before use.

[0026] (6) Potato liquid medium (PDB): Peel and dice 200 g of potatoes, add 1000 mL of distilled water, and boil for 10–20 min. Filter through gauze and add distilled water to 1000 mL. Add 20 g of glucose and 33 g of NaCl, heat to dissolve, and divide into Erlenmeyer flasks. Autoclave at 121°C for 20 min. After sterilization, move the Erlenmeyer flasks to a clean bench and allow to cool before use.

[0027] 2. Fungal fermentation and extraction

[0028] (1) Strain activation: Under sterile conditions, the preserved short-rod Basilicum fungus QDU-WH004 was inoculated onto a PDA solid culture medium plate for activation and cultured at a constant temperature of 28°C for 7 days; (2) Fermentation culture: inoculate the activated culture block in (1) into the liquid fermentation medium, culture it at a constant temperature of 28°C for 40 days, and obtain a fermentation product; (3) Extraction: The fermentation product obtained in step (2) was sterilized with ethyl acetate, the cells were broken, and ultrasonic treatment was performed for 30 minutes. The filtrate was filtered through 8 layers of sterile gauze to obtain the filtrate, which was extracted three times by shaking with an equal volume of ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain the fermentation extract of the short-rod Basilicum fungus QDU-WH004.

[0029] Example 3 Determination of pancreatic lipase inhibitory activity of marine fungi

[0030] 1. Measurement Principle

[0031] The inhibitory ability of the sample on pancreatic lipase was evaluated by measuring the change in the amount of the reaction product (4-methylumbelliferone) generated by pancreatic lipase-catalyzed substrate (4-methylumbelliferyl oleate) after the addition of the sample by HPLC, that is, the change in the product peak area.

[0032] Pancreatic lipase inhibition rate (%) = (A C -A S ) / A C × 100%

[0033] Among them, A C is the peak area of ​​the reaction product under blank conditions, A S is the peak area of ​​the reaction product after adding the sample.

[0034] 2. Measurement system

[0035] (1) Prepare 0.05 M phosphate buffer solution: Solution A: Accurately weigh 1.9504 g of NaH2PO4·2H2O in a 250 ml volumetric flask and dilute to volume with deionized water to obtain a 0.05 M NaH2PO4 stock solution; Solution B: Accurately weigh 4.4768 g of Na2HPO4 in a 250 ml volumetric flask and dilute to volume with deionized water to obtain a 0.05 M Na2HPO4 stock solution; Mix 19 ml of Solution A and 81 ml of Solution B and adjust the pH to 7.4 to obtain a 0.05 M phosphate buffer solution.

[0036] (2) Take 5µL of the sample of different concentrations, 300µL of pancreatic lipase solution (75U / mL, prepared with phosphate buffer solution), and 195µL of phosphate buffer solution in an EP tube, place in a vortex mixer to mix well, and then incubate in a 37℃ water bath for 15min. Add 5µL of 4-methylumbelliferyl oleate (0.1mM, prepared with DMSO), mix well, and then incubate in a 37℃ water bath for 5min. Add 505µL of glacial acetonitrile to terminate the reaction. DMSO is the blank control group, and orlistat is the positive drug. The experiment was repeated three times. The amount of product (4-methylumbelliferyl) generated was determined by HPLC and the inhibition rate of the sample on pancreatic lipase was calculated.

[0037] (3) Chromatographic conditions: Chromatographic column: Agilent ZORBAX SB-C18 (4.6 mm × 150 mm × 5 µm); mobile phase: 0.1% trifluoroacetic acid solution-acetonitrile = 80:20; flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 20 µL.

[0038] 3. Measurement results

[0039] Table 1: Results of pancreatic lipase inhibitory activity screening of different fermentation extracts of Talaromyces brevis QDU-WH004 (400 μg / mL) culture medium Inhibition rate Fungus No. 2 46.4% GPY 82.3% Peter 38.5% glycerin 26.8% PDB 11.3% Orlistat (0.5 μg / mL) 81.4%

[0040] The results showed that the ethyl acetate extract of the fermentation broth of Talaromyces brevis QDU-WH004 GPY exhibited strong pancreatic lipase inhibitory activity, with an inhibition rate of 82.3% at a concentration of 400 μg / mL.

Claims

1. A short-rod basket fungus of marine origin ( Talaromyces brevis ) QDU-WH004 fermentation extract, characterized in that The extract is obtained by extracting the fermentation broth of Talaromyces brachyrhizome QDU-WH004, which was deposited in the China Center for Type Culture Collection on November 24, 2023, with a deposit number of CCTCC NO: M 20232329. The deposit address is School of Life Sciences, Wuhan University, Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The extract has the ability to inhibit pancreatic lipase activity. The preparation method of the extract comprises the following steps: (1) Strain activation: Under sterile conditions, the preserved short-rod Basilicum fungus QDU-WH004 was inoculated onto a PDA solid culture medium plate for activation, and cultured at a constant temperature of 25-35°C for 5-10 days; (2) Fermentation culture: inoculate the activated culture block in (1) into GPY liquid fermentation medium, culture at a constant temperature of 25-35°C for 30-50 days to obtain a fermentation product; (3) Extraction: The fermentation product obtained in step (2) was sterilized with ethyl acetate, the cells were broken, and ultrasonic treatment was performed for 30 minutes. The filtrate was filtered through 8 layers of sterile gauze to obtain the filtrate, which was extracted three times by shaking with an equal volume of ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain the fermentation extract of the short-rod Basilicum fungus QDU-WH004.

2. The method for preparing the fermentation extract of Talaromyces brevis QDU-WH004 according to claim 1, characterized in that: The steps include: (1) Strain activation: Under sterile conditions, the preserved short-rod Basilicum fungus QDU-WH004 was inoculated onto a PDA solid culture medium plate for activation, and cultured at a constant temperature of 25-35°C for 5-10 days; (2) Fermentation culture: inoculate the activated culture block in (1) into the liquid fermentation medium, culture it at a constant temperature of 25-35°C for 30-50 days to obtain a fermentation product; (3) Extraction: The fermentation product obtained in step (2) was sterilized with ethyl acetate, the cells were broken, and ultrasonic treatment was performed for 30 minutes. The filtrate was filtered through 8 layers of sterile gauze to obtain the filtrate, which was extracted three times by shaking with an equal volume of ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain the fermentation extract of the short-rod Basilicum fungus QDU-WH004.

3. Use of the fermentation extract of the short-rod Talaromyces QDU-WH004 according to claim 1 in the preparation of weight loss products.

4. Use of the fermentation extract of Talaromyces brachyrhizium QDU-WH004 according to claim 1 in the preparation of a pancreatic lipase inhibitor product.

Images