Search

CN-122012349-A - Bacillus and application thereof in extraction of enteromorpha dietary fibers

CN122012349ACN 122012349 ACN122012349 ACN 122012349ACN-122012349-A

Abstract

The invention discloses Bacillus KICET-3 strain which is separated from enteromorpha through natural fermentation and application thereof in extracting enteromorpha dietary fibers. The enteromorpha dietary fiber extraction process comprises the steps of firstly preprocessing enteromorpha and secondly extracting enteromorpha dietary fiber through fermentation. The method effectively improves the extraction rate of the dietary fibers from the enteromorpha, meets the requirement of industrial production on high-yield functional components, improves the functional characteristics of the enteromorpha dietary fibers, realizes the purposes of high efficiency, environmental protection, low cost and sustainability, and realizes the high-value utilization of the enteromorpha in green tide disasters.

Inventors

  • GUO LEI
  • FAN XIAOXUAN

Assignees

  • 青岛大学

Dates

Publication Date
20260512
Application Date
20260331

Claims (7)

  1. 1. A Bacillus is named as a Bacillus KICET-3 strain in a classification, and is characterized in that the strain is separated from enteromorpha through natural fermentation, and the specific separation process comprises the following steps: 1. bacterial strain source: (1) Fresh enteromorpha salvaged from the Qingdao sea area is placed in a sterilizing iron drum for natural fermentation for 30 days; (2) Collecting a sample of the middle part of the fermented enteromorpha, and preserving the enteromorpha in a refrigerating way at a temperature of 4 ℃ for later use; 2. strain screening: (3) Adding the sample into a test tube containing sterile water according to the ratio of 1:9 (w/v), and uniformly mixing for 30min by a shaker; (4) Diluting sterile water to 10 -2 -10 -8 in a gradient manner, coating the diluted solution on a cellulose Congo red culture medium, picking out colonies generating transparent circles, and repeatedly carrying out streak culture on a Potato Dextrose Agar (PDA) culture medium until a pure single strain is obtained; 3. identification of strains: (5) Performing DNA extraction, PCR amplification and sequencing on the pure single strain obtained in the step (4), searching homologous sequences through a BLAST tool of NCBI, constructing a phylogenetic tree by adopting a neighbor-joining algorithm of MEGA11.0 software, and identifying the phylogenetic tree as Bacillus sp KICET-3; 4. And (5) strain expansion culture and preservation: (6) Inoculating the pure single strain obtained in the step (4) to an LB liquid culture medium, performing shake culture at 37 ℃ and 150rpm, and controlling the strain concentration to 10 8 -10 9 CFU/mL to obtain seed liquid; (9) Uniformly mixing the bacterial liquid with glycerol with the mass fraction of 40% of that of the sterilization treatment according to the ratio of 1:1 (v/v), and preserving in a refrigerator with the temperature of-80 ℃.
  2. 2. The use of bacillus according to claim 1 for extracting enteromorpha dietary fiber.
  3. 3. The use according to claim 2, characterized in that the extraction process of enteromorpha dietary fiber comprises the following steps: 1. preprocessing enteromorpha: S1, cleaning fresh enteromorpha, drying at 65-80 ℃, crushing to 100 meshes, and performing high-pressure steam sterilization at 121 ℃ and 0.1MPa for 20min to obtain enteromorpha raw materials for later use; 2. And (3) fermenting and extracting enteromorpha dietary fibers: S2, preparing before fermentation, namely inoculating KICET-3 strain (Bacillus sp.) preserved in the step (9) in the step 1 to a seed culture medium from a glycerol joint, and shake-culturing the strain to the strain concentration of 10 8 -10 9 CFU/mL under the conditions of 35-38 ℃ and 130-155rpm to obtain seed liquid; s3, in the fermentation process, the enteromorpha raw material is mixed with distilled water according to a liquid-solid ratio of 30-33, seed liquid with the concentration of 2-5% (v/w) is added, the pH is regulated to 6-7.5, and shake culture is carried out for 28-36 hours under the conditions of 35-38 ℃ and 130-155rpm, so that an enteromorpha mixture after fermentation is obtained; S4, dietary fiber extraction, namely extracting SDF and IDF based on an AOAC enzyme weight method, adding thermostable alpha-amylase into the enteromorpha mixture after fermentation, adjusting pH to 5.5-6.5, stirring for 45min under the water bath condition of 65 ℃, and cooling to room temperature; S5, adding papain, regulating pH to 6-6.5, stirring for 120min under the water bath condition of 60 ℃, boiling for 10min to inactivate enzyme, and filtering to separate filtrate and filter residue; S6, adding a certain amount of 95% ethanol into the filtrate, mixing overnight, collecting precipitate, freeze-drying to obtain the fermentation soluble dietary fiber (XSDF), alternately washing the filter residue with 95% ethanol and acetone twice, and freeze-drying to obtain the fermentation insoluble dietary fiber (XIDF).
  4. 4. The method according to claim 3, wherein in S1, the drying temperature after washing of the fresh Enteromorpha prolifera is 70 ℃.
  5. 5. The method of claim 3, wherein the shaking culture is performed at 37℃and 150rpm in S2.
  6. 6. The application of the method according to claim 3, wherein in the step S3, the liquid-solid ratio of the enteromorpha raw material and distilled water is 32.061, the concentration of the added seed solution is 3.282% (v/w), the pH is regulated to 6.938, and the enteromorpha raw material and distilled water are subjected to shake culture at 37 ℃ and 150rpm for 30.294 hours.
  7. 7. The method according to claim 3, wherein in S6, the volume ratio of the added ethanol to the filtrate is 1:4.

Description

Bacillus and application thereof in extraction of enteromorpha dietary fibers Technical Field The invention belongs to the technical field of microorganism screening and application, and particularly relates to bacillus and application thereof in extraction of enteromorpha dietary fibers. Background The problem of green tide caused by the outbreak of benthic algae is increasingly serious worldwide, and is disturbed in various places such as Asia, europe and the United states. The decay of Ulva on the beach of Bruyi, european, releases sulfur pollution and even causes death of horses, and the United states causes hypoxia death of fish along the sea green tide, which impacts fishery and travel industries. Enteromorpha prolifera is a dominant green tide algae species in the Qingdao sea area, has the characteristic of rapid proliferation, and the resource utilization is the core direction for relieving the pressure of green tide. Enteromorpha prolifera contains nutrients such as carbohydrate (43-51%), dietary Fiber (DF) is a key functional component, and is Soluble (SDF) and Insoluble (IDF), and SDF has the functions of preventing diabetes mellitus, relieving cardiovascular diseases and the like, but the current extraction rate is generally low. The existing dietary fiber extraction methods include physical methods (high energy consumption and easy structure damage), chemical methods (easy impurity introduction and environmental pollution) and biological methods (high enzyme cost, and the existing strain extraction efficiency and function enhancement effect are to be improved though the microbial fermentation method has advantages). For example, the yield of the enteromorpha SDF extracted by an enzymatic method is about 21.8%, and the obtained DF has improvement space in the performances of water retention, oil retention, cholesterol absorption, oxidation resistance and the like, and lacks a high-efficiency and sustainable enteromorpha dietary fiber extraction technology. In summary, how to effectively improve the extraction rate of dietary fibers from enteromorpha, meet the requirement of industrial production on high-yield functional components, improve the functional characteristics of the enteromorpha dietary fibers, realize high-efficiency, environment-friendly, low-cost and sustainable purposes, and realize the high-valued utilization of enteromorpha in green tide disasters, thus becoming a problem to be solved by those skilled in the microorganism field. Disclosure of Invention Aiming at the defects existing in the prior art, the technical problem to be solved by the invention is to provide a high-efficiency, environment-friendly, low-cost and sustainable extraction method, which effectively improves the extraction rate of dietary fibers, and simultaneously improves the bacillus with the functional characteristics of enteromorpha dietary fibers and the application of the bacillus in extracting enteromorpha dietary fibers. In order to solve the technical problems, the invention adopts the technical scheme that the Bacillus is classified and named as a Bacillus KICET-3 strain which is separated from enteromorpha naturally fermented, and the specific separation process comprises the following steps: 1. bacterial strain source: (1) Fresh enteromorpha salvaged from the Qingdao sea area is placed in a sterilizing iron drum for natural fermentation for 30 days; (2) Collecting a sample of the middle part of the fermented enteromorpha, and preserving the enteromorpha in a refrigerating way at a temperature of 4 ℃ for later use; 2. strain screening: (3) Adding the sample into a test tube containing sterile water according to the ratio of 1:9 (w/v), and uniformly mixing for 30min by a shaker; (4) Diluting sterile water to 10 ﹣2-10﹣8 in a gradient manner, coating the diluted solution on a cellulose Congo red culture medium, picking out colonies generating transparent circles, and repeatedly carrying out streak culture on a Potato Dextrose Agar (PDA) culture medium until a pure single strain is obtained; 3. identification of strains: (5) Performing DNA extraction, PCR amplification and sequencing on the pure single strain obtained in the step (4), searching homologous sequences through a BLAST tool of NCBI, constructing a phylogenetic tree by adopting a neighbor-joining algorithm of MEGA11.0 software, and identifying the phylogenetic tree as Bacillus sp KICET-3; 4. And (5) strain expansion culture and preservation: (6) Inoculating the pure single strain obtained in the step (4) to an LB liquid culture medium, performing shake culture at 37 ℃ and 150rpm, and controlling the strain concentration to 10 8-109 CFU/mL to obtain seed liquid; (9) Uniformly mixing the bacterial liquid with glycerol with the mass fraction of 40% of that of the sterilization treatment according to the ratio of 1:1 (v/v), and preserving in a refrigerator with the temperature of-80 ℃. The application of the bacillus in extracting enteromorpha die