CN116396868A - Microbial agent for preparing high-quality dietary fibers by fermenting peanut shells and method thereof - Google Patents
Microbial agent for preparing high-quality dietary fibers by fermenting peanut shells and method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
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Abstract
The invention provides a mould for high-yielding cellulase and hemicellulase and a bacterium for high-yielding novel xylanase, wherein the mould is aspergillus clavatus (Aspergillus clavatus) MZ211, the bacterium is Paenibacillus (Paenibacillus sp.) B1709, and the mould and the bacterium are respectively preserved in China general microbiological culture Collection center (CGMCC) at 22 months of 2021 and 11 months of 2017, and the preservation numbers are CGMCC NO.22410 and CGMCC NO.14870; the invention also discloses a method for preparing the microbial agent by utilizing the strain for preparing the high-quality dietary fiber by fermentation, and the dietary fiber obtained by the method has higher solubility, water holding capacity and oil holding capacity.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly provides a method for preparing high-quality dietary fibers by screening aspergillus clavus and paenibacillus for solid-state fermentation of peanut shells.
Background
In recent years, with rapid development of the food industry, the accuracy of food processing has been improved. The reduced dietary fiber content in the dietary structure of people leads to the general occurrence of sub-health diseases caused by various unhealthy diets, and prevention of such diseases has become a research hotspot in the fields of foods, nutrition and epidemiology. Many studies have shown that reasonable intake of dietary fiber can prevent the occurrence of sub-health diseases or reduce the risk of illness, such as kidney stones, inflammation, colon cancer and other cancers, obesity, cardiovascular diseases, and the like. This discovery has led to the rapid development of epidemiology of dietary fiber related diseases and the study of the underlying physiological mechanisms that reduce the risk of disease, while also allowing the public and food industry to rapidly accept dietary fiber as a beneficial special nutritional location in a healthy diet.
At present, the preparation methods of dietary fibers are mainly divided into three types: physical, chemical and biotechnological methods are also common to the above approaches. The biotechnology methods are classified into an enzymatic method and a microbial fermentation method. The enzyme method is to add biological enzymes such as protease, cellulase, saccharifying enzyme, hemicellulase and the like into the raw materials after adjusting the optimal pH value required by enzymolysis reaction, and degrade non-fibrous components and IDF components such as cellulose, hemicellulose and the like in the raw materials by controlling reaction conditions to generate SDF components such as micromolecular monosaccharides and the like. The enzymatic extraction of DF has high efficiency, and the obtained DF has small loss of physiological activity, light color and high purity, but the enzymatic preparation has high production cost and cannot be widely popularized in the aspect of practical operation. Microbial fermentation is a relatively safe, efficient and low cost process. The starch and protein in the raw materials are consumed by amylase, cellulase and other enzyme systems secreted by the amylase, the cellulase and other enzyme systems, and the starch and protein are loose in structure, so that insoluble macromolecules such as cellulose and hemicellulose are easier to degrade, IDF is converted into SDF, and the aim of improving the yield of the SDF is fulfilled.
Peanuts are an important edible vegetable oil raw material. At present, only a small part of peanut shells are used for manufacturing artificial boards and animal feeds, and most of peanut shells are used as fuel or are discarded, so that resource waste is caused. The peanut shell is rich in dietary fiber, generally more than 65%, has very low price and is easy to obtain. Therefore, the development of a novel preparation method of high-quality dietary fiber has great significance.
The invention relates to a mould for producing cellulase and hemicellulase and bacteria for producing novel xylanase and the like which are screened by self, and the proportion of high-quality dietary fibers in a treated object is effectively improved by using the solid-state composite fermentation of two strains. The product attribute related to the invention is characterized in that the high-performance strain screened by a specific area and a specific object is used as a basic characteristic for improving the specific quality of the specific object, and the characteristic is a main characteristic different from other similar products.
The invention comprises the following steps:
the invention aims to provide a high-quality dietary fiber fungus microbial agent prepared by enzyme catalytic hydrolysis and solid state fermentation of peanut shells and a method thereof.
In order to achieve the purpose, the technical scheme of the invention screens and provides mould and bacteria which can realize that peanut shells are well utilized for fermentation to produce high-quality dietary fibers. The strain is respectively identified as aspergillus clavatus MZ211 (Aspergillus clavatusMZ) by the institute of microbiology of China (the strain is preserved in China general microbiological culture Collection center (China center for type culture Collection) on 4 months and 22 days in 2021, and has the address of CGMCC No.22410, the institute of microbiology of China, national institute of advanced technology, north Chen West Lu No.1, beijing, and the preservation number of CGMCC No.14870, the strain is preserved in Paenibacillus sp.B1709 (the strain is preserved in China center for general microbiological culture collection at 11 months and 8 days in 2017).
The first aspect of the invention provides an aspergillus clavatus (Aspergillus clavatus) MZ211 for producing high-quality dietary fibers by fermenting peanut shells, wherein the strain is preserved in China general microbiological culture collection center (China center for type culture collection) at the year 22 of 2021, and the addresses are: the collection number of the national institute of microbiology, national academy of sciences, no. 3, north Chen West Lu 1, chao yang, beijing, is: CGMCC No.22410.
The second aspect of the present invention provides a microbial agent for producing high quality dietary fiber by fermenting peanut shells, comprising aspergillus clavatus and Paenibacillus B1709 (Paenibacillus sp.) according to the first aspect, wherein the Paenibacillus is deposited in the chinese common microorganism strain deposit management center at 11 months 8 in 2017, at the following addresses: the collection number of the national institute of microbiology, national academy of sciences, no. 3, north Chen West Lu 1, chao yang, beijing, is: CGMCC No.14870.
The third aspect of the invention provides a method for preparing high-quality peanut shell dietary fiber, which mainly comprises the following steps:
(1) Screening of high-yield and high-yield multiple macromolecular degrading enzymes and fermentation adaptive microorganisms
And (5) respectively carrying out plate primary screening and fermentation secondary screening on the screened strains. Wherein the plate primary screening culture mediums are respectively as follows: (1) xylanase screening medium: 1.0% of beech xylan, 0.3% of beef peptone, 0.2% of yeast extract and KH 2 PO 4 0.6％，K 2 HPO 4 0.15％，MgSO 4 ·7H 2 O0.05％，FeSO 4 ·7H 2 O0.001%, agar 2.0% and pH 6.0. Sterilizing at 121deg.C for 20min. (2) StarchPowder enzyme screening medium: beef extract 0.3%, sodium chloride 0.5%, tryptone 1.0%, agar powder 2.0%, soluble starch 2.0%, pH natural, and sterilizing at 121deg.C for 20min. (3) Protease screening medium: beef extract 0.3%, sodium chloride 0.5%, tryptone 1.0%, agar powder 2.0%, skimmed milk powder 1.5%, pH naturally, and sterilizing at 121deg.C for 20min. Shake flask rescreening medium: 4.0% of peanut shell powder (dried and screened by a 65-mesh sieve), 1.0% of tryptone, 0.6% of yeast extract powder and NaNO 3 0.4％，KH 2 PO 4 0.2％，K 2 HPO 4 0.1％，MgSO 4 ·7H 2 O0.05％，FeSO 4 ·7H 2 O0.001%, pH 6.0. Sterilizing at 121deg.C for 20min. And (5) respectively measuring amylase, xylanase and protease from the fermentation liquid.
(2) Preparation of fermenting microorganism
Preparation of aspergillus clavatus MZ211 mould spore bacterial suspension: collecting fungus spores with platinum ring, suspending in sterile water, and adjusting concentration to about 10 6 ～10 7 Individual spores/mL. 10mL (about 10%) of the suspension (10 6 ～10 7 spores/mL) was inoculated into 100mL of a sterile seed medium, then the medium inoculated with the suspension was cultured at 150r/min at 28℃for 2d, and the liquid medium after fermentation was subjected to filtration to remove mycelia, thereby obtaining a seed solution for use.
Paenibacillus B1709 seed fermentation broth preparation: the selected slant strain is selected and inoculated into a seed culture medium, and is cultured for 14 hours at 37 ℃ and 180r/min to prepare seed liquid for standby.
(3) Preparation of high-quality peanut shell dietary fiber
Inoculating 50mL (total 100 mL) of each seed solution of two microorganisms into 1kg of solid fermentation medium (peanut shell powder (coarse powder 20-40 mesh) 40.0g, beef peptone 10.0g, yeast extract 6.0g, KH) 2 PO 4 2.0g，K 2 HPO 4 1.5g，MgSO 4 ·7H 2 O 0.5g，FeSO 4 ·7H 2 O0.01 g, natural pH, supplementing water to 1000 mL), culturing at 30deg.C for 4d at 150r/min, filtering the fermentation broth, centrifuging at 10000r/min for 10min, lyophilizing the precipitate, pulverizing to obtain Insoluble Dietary Fiber (IDF) of peanut shell, and collectingCollecting supernatant, adding 95% ethanol (four times of supernatant), standing at 4deg.C for 12 hr, centrifuging at 10000r/min for 10min, lyophilizing the precipitate, pulverizing to obtain high activity Soluble Dietary Fiber (SDF), and calculating SDF yield.
Drawings
Physicochemical Properties of SDF of FIG. 1
FIG. 2SDF adsorption Capacity to glucose
FIG. 3SDF adsorption Capacity to cholesterol
The specific embodiment is as follows:
the invention is further illustrated, but is not limited, by the following specific examples.
Example 1 Process for preparing high quality dietary fiber by solid state fermentation of two-strain composite fermented peanut hulls
Inoculating 100mL of seed liquid into 1kg of fermentation medium, culturing at 30deg.C for 4d at 150r/min, filtering the fermentation liquid, centrifuging at 10000r/min for 10min, freeze-drying the precipitate, pulverizing to obtain Insoluble Dietary Fiber (IDF) of peanut shell, collecting supernatant, adding 95% ethanol (four times of the supernatant), standing at 4deg.C for 12h, centrifuging at 10000r/min for 10min, freeze-drying the precipitate, pulverizing to obtain Soluble Dietary Fiber (SDF) of corn cob, and calculating the SDF yield.
EXAMPLE 2 high Activity verification test of dietary fibers
(1) Taking into consideration the SDF (peanut shell with the same water content, adding high-temperature resistant alpha-amylase, incubating for 30min at 95 ℃, then adding a proper amount of saccharifying enzyme at 60 ℃ for reaction for 30min, then adding protease into the solution for 30 min), and obtaining the solubility, water holding capacity and oil holding capacity of the peanut shell soluble dietary fiber (B-SDF) and the SDF (F-SDF) prepared by composite fermentation by the same method.
Solubility refers to the extent to which a substance can be dissolved, which is an important reference indicator of SDF. Experimental results show (see FIG. 1) that the initial solubility is significantly improved by the fermentation treatment. The solubility of SDF increased from 0.43g/g to 0.580g/g by 34.88% (where B-SDF represents dietary fiber before treatment (blank control) and B-SDF represents dietary fiber after fermentation). The porous structure of SDF is reported to cause it to expand upon water absorption.
The Water Holding Capacity (WHC) is defined as the amount of water that a hydrocolloid of known weight retains after an external force (e.g., centrifugal force) is applied, and is characterized by a strong binding force. The dietary fiber with high WHC can prevent food shrinkage and change the viscosity of certain foods, and is a good functional characteristic of the dietary fiber. As can be seen from FIG. 1, the WHC increased from 6.13g/g to 14.92g/g by fermentation treatment. It has been found that dietary fibers with good WHC are due to the hydrophilic groups of polysaccharides and are also closely related to factors such as SDF content, particle size, surface properties and source.
The ability of dietary fiber to retain grease is important in food applications, for example, dietary fiber with high OHC's can stabilize high-fat foods and dairy products. As can be seen from FIG. 1, the oil holding capacity of F-SDF is higher than that of B-SDF, and OHC increases from 5.21g/g to 7.13g/g after fermentation treatment, which is probably due to the fact that the structure becomes more porous after fermentation, and in addition, arabinoxylans, pectins, arabinogalactans and the like in SDF are also possible, and due to their strong affinity for lipid substances, the adsorption and removal of saturated fats and unsaturated lipid substances by SDF are possibly facilitated. High OHC is an important feature of dietary fiber because this ability may interfere with intestinal absorption of lipids in the diet, thereby helping to control weight and abnormal blood lipid conditions.
(2) Glucose absorption capacity of dietary fiber
High quality dietary fiber can delay or reduce the digestive absorption of glucose in the gastrointestinal tract, which plays an important role in controlling blood glucose. The adsorption capacities of B-SDF and F-SDF for glucose were examined, and the results are shown in FIG. 2. As can be seen by comparison, the adsorption capacity of glucose can be remarkably improved by the SDF after the composite fermentation, and the adsorption quantity of the F-SDF to the glucose is 2910.36 mu mol/g, which is improved by 24.86% compared with that of the B-SDF (2330.84 mu mol/g). The improved glucose adsorption capacity of F-SDF may be due to the fact that cellulase and xylanase produced by fermentation degrade the fiber surface, so that the fiber structure is loose, the pores are enlarged, and glucose molecules are more easily absorbed into the fiber. The unfermented dietary fiber has compact structure, and most of the key functional groups are surrounded by the internal structure of the fiber, so that the fiber cannot play an effective role.
(3) Cholesterol adsorption capacity
The pH value is an important factor influencing the cholesterol adsorption capacity of dietary fiber, so that the adsorption amounts of B-SDF and F-SDF to cholesterol are respectively examined under the conditions of pH2.0 (simulating the stomach environment) and pH 7.0 (simulating the small intestine environment), and the result is shown in figure 3. Under the condition of pH2.0, the cholesterol absorption amount of B-SDF is 3.14mg/g, and compared with B-SDF, the cholesterol absorption amount of F-SDF is 7.25mg/g, which is improved by 1.31 times; under the condition of pH 7.0, the cholesterol adsorption amounts of B-SDF and F-SDF are respectively 8.45mg/g and 13.57mg/g, and 60.59% is improved. Clearly, a CAC value of pH 7.0 higher than that of pH2.0 indicates that the CAC of F-SDF is stronger in a simulated small intestine environment than in a simulated stomach environment. In combination with scanning electron microscopy, F-SDF has a higher cholesterol absorption capacity than B-SDF, probably due to loose dietary fiber structure and increased specific surface area, resulting in easier penetration of fat-soluble substances into the dietary fiber.
Claims (10)
1. Aspergillus clavatus (Aspergillus clavatus) MZ211 for producing high-quality dietary fibers by fermenting peanut shells, wherein the strain is preserved in China general microbiological culture Collection center (China center for type culture Collection) at the year 2021, month 4 and day 22: the collection number of the national institute of microbiology, national academy of sciences, no. 3, north Chen West Lu 1, chao yang, beijing, is: CGMCC No.22410.
2. A microbial agent for producing high quality dietary fiber by fermentation of peanut shells, comprising aspergillus clavatus and Paenibacillus B1709 (Paenibacillus sp.) of claim 1, wherein the Paenibacillus is deposited at the chinese common microorganism strain deposit management center at 11 months 8 of 2017 at the address: the collection number of the national institute of microbiology, national academy of sciences, no. 3, north Chen West Lu 1, chao yang, beijing, is: CGMCC No.14870.
3. A method for preparing high quality dietary fiber using the microbial agent of claim 2, said method comprising:
1) Preparation of fermenting microorganism
Preparation of aspergillus clavatus MZ211 mould spore bacterial suspension: collecting fungus spores by a platinum ring, suspending in sterile water, and adjusting the concentration to about 106-107 spores/mL; inoculating 10mL of suspension into 100mL of sterile seed culture medium, culturing the culture medium inoculated with the suspension at 150r/min and 28 ℃ for 2d, filtering mycelium from the fermented liquid culture medium, and preparing seed liquid for later use; paenibacillus B1709 seed fermentation broth preparation: selecting the selected inclined plane strain, inoculating into a seed culture medium, and culturing at 37 ℃ for 14h at 180r/min to prepare seed liquid for later use;
2) Preparation of high-quality peanut shell dietary fiber
Inoculating 50mL of each of the two microorganism seed solutions into 1kg of solid fermentation medium, culturing at 30deg.C for 4d at 150r/min, filtering the fermentation liquid, centrifuging at 10000r/min for 10min, lyophilizing the precipitate, pulverizing to obtain Insoluble Dietary Fiber (IDF) of peanut shell, collecting supernatant, adding 95% ethanol, standing at 4deg.C for 12h, centrifuging at 10000r/min for 10min, lyophilizing the precipitate, and pulverizing to obtain Soluble Dietary Fiber (SDF) of peanut shell.
4. The method of claim 3, wherein the solid culture medium comprises 40.0g of peanut shell powder, 10.0g of beef peptone, 0.5g of yeast extract 6.0g,KH2PO4 2.0g,K2HPO41.5g,MgSO4.7H2O and 0.01g of FeSO4.7H2O, and the solid culture medium is naturally pH and is supplemented with water to 1000mL.
5. The method of claim 4 wherein the peanut shell meal is 20-40 mesh in size.
6. The method of claim 4, wherein the volume of 95% ethanol added is four times the volume of supernatant.
7. High quality dietary fiber obtainable by the process according to claims 3-6.
8. Use of the strain of claim 1 for fermentation of peanut hulls to produce high quality dietary fiber.
9. Use of the microbial agent of claim 2 in the production of high quality dietary fiber by fermentation with peanut hulls.
10. Use of the method of claims 3-6 for producing high quality dietary fiber by fermentation with peanut hulls.
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| US20120083019A1 (en) * | 2010-08-20 | 2012-04-05 | Codexis, Inc. | Use of Glycoside Hydrolase 61 Family Proteins in Processing of Cellulose |
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| CN112515179A (en) * | 2020-11-30 | 2021-03-19 | 安徽大学 | Method for preparing tartary buckwheat soluble dietary fiber by using aspergillus niger liquid fermentation |
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