CN115216501A - Method for preparing high-quality dietary fiber by solid-state composite fermentation of peanut shells - Google Patents
Method for preparing high-quality dietary fiber by solid-state composite fermentation of peanut shells Download PDFInfo
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Abstract
The invention provides a method for preparing high-quality dietary fiber by solid-state composite fermentation of peanut shells, and relates to a method for preparing high-quality dietary fiber by solid-state composite fermentation of peanut shellsThe technical field of preparing high-quality dietary fiber by enzyme-catalyzed hydrolysis and solid-state fermentation of peanut shells. The invention discloses a technical scheme for screening mould for high yield of cellulase and hemicellulase and bacteria for high yield of novel xylanase and a method for improving the proportion of high-quality dietary fiber in peanut shells through solid-state composite fermentation of two strains. The strain comprises: aspergillus clavatus (A. Clavatus)Aspergillus clavatus) MZ211 and Paenibacillus (B)Paenibacillus sp.) B1709, respectively in 2021 year 4 month 22 days and 2017 year 11 month 8 days in China general microbiological culture Collection center (CGMCC), the preservation numbers are CGMCC NO.22410 and CGMCC NO.14870 respectively. The invention is characterized in that the product attribute of the invention improves the specific quality of a specific object by using high-performance strains screened by a specific region and the specific object. The invention has great significance for developing a method for preparing high-quality dietary fiber by using peanut shells.
Description
Technical Field
The invention relates to a method for preparing high-quality dietary fiber by enzymatic hydrolysis and solid state fermentation of peanut shells.
Background
In recent years, with the rapid development of the food industry, the precision of food processing is continuously improved. The decrease of dietary fiber content in the dietary structure of people leads to the common occurrence of sub-health diseases caused by various unhealthy diets, and the prevention of the diseases becomes a research hotspot in the fields of food, nutrition and epidemiology. Many studies have shown that reasonable dietary fiber intake can prevent the development of sub-health diseases or reduce the risk of such diseases as kidney stones, inflammation, colon and other cancers, obesity and cardiovascular diseases, etc. This finding has led to a rapid development in the study of dietary fiber-related disease epidemiology and underlying physiological mechanisms that reduce the risk of disease, and has also led the public and food industry to a rapid acceptance of dietary fiber as a beneficial special nutritional site in a healthy diet.
At present, the preparation method of dietary fiber is mainly divided into three types: physical, chemical and biological techniques, and the combination of the above methods. Among them, the biotechnology methods are classified into an enzymatic method and a microbial fermentation method. The enzyme method is to adjust the optimum pH value required by the enzymolysis reaction, add biological enzymes such as protease, cellulase, diastase, hemicellulase and the like into the raw materials, degrade non-fiber components and IDF components such as cellulose, hemicellulose and the like in the raw materials by controlling reaction conditions, and generate SDF components such as micromolecular monosaccharide and the like. The enzymatic extraction of DF has high efficiency, and the prepared DF has small loss of physiological activity, light color and high purity, but cannot be widely popularized in the aspect of actual operation due to higher production cost of the enzyme preparation. Microbial fermentation is a relatively safe, efficient and low cost process. It uses amylase, cellulase and other enzyme systems secreted by itself to consume starch and protein in raw materials, loose structure, so that insoluble macromolecules such as cellulose and hemicellulose are more easily degraded, IDF is converted into SDF, and the purpose of improving SDF yield is achieved.
Peanuts are an important edible vegetable oil raw material. Currently, only a small portion of the peanut hulls are used to make artificial boards and animal feed, and most are used as fuel or discarded, resulting in waste of resources. The peanut shell is rich in dietary fiber, generally more than 65%, and has very low price and easy availability. Therefore, the development of a novel preparation method of the high-quality dietary fiber has great significance.
The invention relates to a method for effectively increasing the proportion of high-quality dietary fiber in a treated object by matching solid-state composite fermentation of two strains in self-screened mould for producing cellulase and hemicellulase at high yield, bacteria for producing novel xylanase and the like. The product attribute related by the invention is basically characterized in that the specific quality of a specific object is improved by high-performance strains screened in a specific area and the specific object, and the product attribute is also a main characteristic which is different from other similar products.
Disclosure of Invention
The invention aims to provide a method for preparing high-quality dietary fiber by enzyme-catalyzed hydrolysis and solid state fermentation of peanut shells.
In order to achieve the purpose, the technical scheme of the invention provides the mould and the bacteria which can better utilize peanut shells to ferment and produce high-quality dietary fibers. Identified as Aspergillus clavatus MZ211 (Aspergillus clavatus MZ 211) respectively by the institute of microbiology of the Chinese academy of sciences, which is deposited in the China center for general microbiological cultures in 22.4.22.2021 at the CGMCC No. 3 of the northern Chen Xilu 1 of the area facing the sun in Beijing, with the deposition number of CGMCC NO. 22410), and Paenibacillus B1709 (Paenibacillus sp.B1709) which is deposited in the center for general microbiological cultures in 8.11.2017 at the CGMCC No. 3 of the national institute of sciences in the northern Chen of the area facing the sun in Beijing, with the deposition number of CGMCC NO.14870.
The specific preparation method related by the invention mainly comprises the following steps:
(1) High-yield screening of various macromolecular degrading enzymes and fermentation-adaptive microorganisms
And respectively carrying out plate primary screening and fermentation secondary screening on the screened strains. Wherein the plate prescreening culture medium is respectively as follows: (1) xylanase screening culture medium: beech xylan 1.0%, beef peptone 0.3%, yeast extract 0.2%, KH 2 PO 4 0.6%，K 2 HPO 4 0.15%，MgSO 4 ·7H 2 O 0.05%，FeSO 4 ·7H 2 0.001% of O, 2.0% of agar and 6.0 of pH. Sterilizing at 121 deg.C for 20min. (2) Amylase screening culture medium: 0.3% of beef extract, 0.5% of sodium chloride, 1.0% of tryptone, 2.0% of agar powder and 2.0% of soluble starch, wherein the beef extract is sterilized at the temperature of 121 ℃ for 20min under natural pH. (3) Protease screening medium: 0.3% of beef extract, 0.5% of sodium chloride, 1.0% of tryptone, 2.0% of agar powder and 1.5% of skimmed milk powder, wherein the beef extract is sterilized at the temperature of 121 ℃ for 20min under natural pH. Shaking flask re-screening culture medium: peanut shell powder (dried and sieved by a 65-mesh sieve) 4.0 percent, tryptone 1.0 percent, yeast extract powder 0.6 percent and NaNO 3 0.4%，KH 2 PO 4 0.2%，K 2 HPO 4 0.1%，MgSO 4 ·7H 2 O 0.05%，FeSO 4 ·7H 2 O0.001%, pH6.0. Sterilizing at 121 deg.C for 20min. And (3) taking the fermentation liquor to respectively measure amylase, xylanase and protease.
(2) Fermentative microbial preparation
Preparation of a mould spore bacterium suspension: collecting fungal spores with platinum ring, suspending in sterile water, adjusting concentration to about 10 6 ~10 7 spores/mL. 10 mL (about 10%) suspension (10%) is added 6 ~10 7 spore/mL)Inoculating into 100mL sterile seed culture medium, culturing 2 d at 28 deg.C in 150 r/min, filtering to remove mycelium, and making into seed solution;
preparing bacterial seed fermentation liquor: selecting slant strain, inoculating to seed culture medium, culturing at 37 deg.C and 180r/min for 14h, and making into seed solution.
(3) Preparation of high-quality peanut shell dietary fiber
Inoculating 50mL (total 100 mL) of seed liquid of two microorganisms to 1kg of solid fermentation medium (peanut shell powder (20-40 mesh coarse powder) 40.0 g, beef peptone 10.0 g, yeast extract 6.0 g 2 PO 4 2.0 g，K 2 HPO 4 1.5 g，MgSO 4 ·7H 2 O 0.5 g，FeSO 4 ·7H 2 O0.01 g, natural pH, replenishing water to 1000 mL), culturing 4 d at 30 ℃ and 150 r/min, filtering fermentation liquor, centrifuging at 10000 r/min for 10 min, freeze-drying precipitate, pulverizing to obtain peanut shell Insoluble Dietary Fiber (IDF), collecting supernatant, adding 95% ethanol (four times of the amount of the supernatant), standing at 4 ℃ for 12 h, centrifuging at 10000 r/min for 10 min, freeze-drying and pulverizing the precipitate to obtain high-activity peanut shell Soluble Dietary Fiber (SDF), and calculating the yield of the SDF.
Drawings
The attached drawings of the specification comprise three parts:
(1) The physicochemical properties of the SDF of fig. 1;
(2) FIG. 2 SDF adsorption capacity for glucose;
(3) Figure 3 SDF capacity for cholesterol adsorption.
Detailed Description
The invention is further illustrated by, but is not limited to, the following examples.
Example 1 Process for preparing high-quality dietary fiber by solid state fermentation of peanut shells through two-strain composite fermentation
Inoculating 100mL seed liquid into 1kg fermentation medium, culturing 4 d at 30 ℃ and 150 r/min, filtering the fermentation liquid, centrifuging at 10000 r/min for 10 min, freeze-drying the precipitate, pulverizing to obtain peanut shell Insoluble Dietary Fiber (IDF), collecting the supernatant, adding 95% ethanol (four times of the amount of the supernatant), standing at 4 ℃ for 12 h, centrifuging at 10000 r/min for 10 min, freeze-drying the precipitate, pulverizing to obtain corn cob Soluble Dietary Fiber (SDF), and calculating the yield of the SDF.
Example 2 high Activity confirmation test for dietary fiber
(1) The solubility, water holding capacity and oil holding capacity of the soluble dietary fiber (B-SDF) of the peanut shell and the SDF (F-SDF) prepared by composite fermentation are obtained by the same method.
Solubility refers to the extent to which a substance can be dissolved, which is an important reference indicator for SDF. The experimental results show (see figure 1 in the attached figure of the specification) that the initial solubility is obviously improved by the fermentation treatment. The solubility of SDF increased from 0.43 g/g to 0.580g/g, an increase of 34.88% (where B-SDF stands for dietary fiber before treatment (blank control); B-SDF stands for dietary fiber after fermentation). The porous structure of SDF is reported to cause it to swell with water.
Water Holding Capacity (WHC) is defined as the amount of water retained by a known weight of hydrocolloid after application of an external force (e.g., centrifugal force) and is characterized by strong binding forces. The high WHC dietary fiber can prevent food from shrinking and change the viscosity of certain foods, and is the embodiment of the good functional property of the dietary fiber. As can be seen from the figure 1 of the accompanying drawings, the WHC increased from 6.13g/g to 14.92g/g by the fermentation treatment. It has been found that dietary fiber with good WHC is due to the hydrophilic groups of the polysaccharide and is also closely related to factors such as SDF content, particle size, surface properties and source.
The ability of dietary fiber to retain oil is important in food applications, for example, dietary fiber with high OHC can stabilize high fat foods and dairy products. As can be seen from FIG. 1 of the accompanying drawings, the oil-holding capacity of F-SDF is higher than that of B-SDF, and OHC is increased from 5.21g/g to 7.13g/g by fermentation treatment, which may be due to the fact that the structure becomes more porous after fermentation, and further, it may be due to the fact that arabinoxylan, pectin, arabinogalactan, etc. in SDF may help SDF adsorb and remove saturated fat and unsaturated lipid substances due to their strong affinity for lipid substances. High OHC is an important feature of dietary fiber as this ability may interfere with the intestinal absorption of lipids in the diet, thereby helping to control body weight and abnormal blood lipid conditions.
(2) Ability of dietary fiber to absorb glucose
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. Therefore, the adsorption capacity of B-SDF and F-SDF on glucose is respectively examined, and the results are shown in the attached figure 2 of the specification. As can be seen by comparison, the glucose adsorption capacity of the SDF subjected to the composite fermentation can be obviously improved, the glucose adsorption amount of the F-SDF is 2910.36 mu mol/g, and the glucose adsorption amount is improved by 24.86% compared with that of B-SDF (2330.84 mu mol/g). The structural analysis result shows that the improved glucose adsorption capacity of the F-SDF is probably because the cellulase and xylanase generated by fermentation degrade the surface of the fiber, so that the fiber structure is loose, the pores are enlarged, and glucose molecules are absorbed into the fiber more easily. The unfermented dietary fiber has a compact structure, and most key functional groups are surrounded by the internal structure of the fiber, so that the dietary fiber cannot play an effective role.
(3) Cholesterol adsorption capacity
The pH value is an important factor influencing the cholesterol adsorption capacity of the dietary fiber, so that the adsorption amounts of B-SDF and F-SDF to cholesterol are respectively considered under the conditions of pH 2.0 (simulated gastric environment) and pH 7.0 (simulated small intestine environment), and the result is shown in figure 3 of the attached figure of the specification. Under the condition of pH 2.0, the cholesterol adsorption capacity of the B-SDF is 3.14 mg/g, and compared with the B-SDF, the cholesterol adsorption capacity of the 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 the B-SDF and the F-SDF are respectively 8.45mg/g and 13.57mg/g, and the cholesterol adsorption amount is improved by 60.59%. Clearly, the CAC value at pH 7.0 was higher than that at pH 2.0, indicating that the CAC of F-SDF is stronger in a simulated small intestine environment than in a simulated stomach environment. In combination with sem images, F-SDF has a higher capacity to absorb cholesterol than B-SDF, probably due to the loose structure and increased specific surface area of dietary fibers, resulting in easier penetration of fat-soluble substances into the interior of dietary fibers.
Claims (5)
1. A method for preparing high-quality dietary fiber by solid-state composite fermentation of peanut shells is characterized in that high-performance strains are automatically screened out and subjected to solid-state composite fermentation to prepare the high-quality dietary fiber in the peanut shells.
2. The two high performance strains according to claim 1, wherein each strain is Aspergillus clavatus (Aspergillus awamori)Aspergillus clavatus) MZ211 and Paenibacillus (B)Paenibacillus sp.) B1709, respectively in 2021 year 4 month 22 days and 2017 year 11 month 8 days in China general microbiological culture Collection center (CGMCC), the preservation numbers are CGMCC NO.22410 and CGMCC NO.14870 respectively.
3. The method for screening high performance bacterial species according to claim 1 or 2, comprising the steps of:
(1) High-yield screening of various macromolecular degrading enzymes and fermentation-adaptive microorganisms
Primary screening by a flat plate:
(1) xylanase screening culture medium: beech xylan 1.0%, beef peptone 0.3%, yeast extract 0.2%, KH 2 PO 4 0.6%，K 2 HPO 4 0.15%，MgSO 4 ·7H 2 O 0.05%，FeSO 4 ·7H 2 0.001% of O, 2.0% of agar, 6.0% of pH, and sterilizing at 121 ℃ for 20min;
(2) amylase screening culture medium: 0.3% of beef extract, 0.5% of sodium chloride, 1.0% of tryptone, 2.0% of agar powder and 2.0% of soluble starch, wherein the beef extract is sterilized at the temperature of 121 ℃ for 20min under natural pH;
(3) protease screening culture medium: 0.3% of beef extract, 0.5% of sodium chloride, 1.0% of tryptone, 2.0% of agar powder and 1.5% of skimmed milk powder, wherein the beef extract is sterilized at 121 ℃ for 20min under natural pH;
and (3) shaking a flask for re-screening:
shake flask rescreening culture medium: peanut shell powder(dried and sieved by a 65-mesh sieve) 4.0 percent, tryptone 1.0 percent, yeast extract powder 0.6 percent and NaNO 3 0.4%，KH 2 PO 4 0.2%，K 2 HPO 4 0.1%，MgSO 4 ·7H 2 O 0.05%，FeSO 4 ·7H 2 O0.001%, pH6.0, sterilizing at 121 deg.C for 20min;
(2) Preparation of fermentative microorganism
Preparation of a mould spore bacterium suspension: collecting fungal spores with platinum ring, suspending in sterile water, adjusting concentration to about 10 6 ~10 7 spore/mL; 10 mL (about 10%) suspension (10%) is added 6 ~10 7 spores/mL) is inoculated into 100mL sterile seed culture medium, then the culture medium inoculated by the suspension is cultured for 2 d at the temperature of 28 ℃ and at the speed of 150 r/min, the liquid culture medium after fermentation is finished, mycelium is filtered out, and seed liquid is prepared for standby;
preparing bacterial seed fermentation liquor: selecting slant strain, inoculating to seed culture medium, culturing at 37 deg.C and 180r/min for 14h, and making into seed solution.
4. The solid-state composite fermentation preparation of high-quality dietary fiber in peanut shells according to claim 1, characterized in that 1:1 is inoculated with two strain seed liquids (total 100 mL) to 1kg of solid fermentation medium prepared from peanut shells, 4 d is cultured at 30 ℃ and 150 r/min, after the fermentation liquid is filtered, 10000 r/min is centrifuged for 10 min, precipitates are freeze-dried and crushed to obtain peanut shell Insoluble Dietary Fiber (IDF), the supernatant is collected, 95% ethanol (four times of the amount of the supernatant) is added, the mixture is stood at 4 ℃ for 12 h and 10000 r/min is centrifuged for 10 min, and the precipitates are freeze-dried and crushed to obtain corn cob Soluble Dietary Fiber (SDF).
5. High quality dietary fiber in peanut shells according to claim 1 or 4, characterized in that the dietary fiber high activity validation test comprises the following aspects:
(1) Inspecting the solubility, water holding capacity and oil holding capacity of SDF (peanut shell with the same water content, added with high-temperature-resistant alpha-amylase, incubated at 95 ℃ for 30 min, added with a proper amount of saccharifying enzyme at 60 ℃ for reaction for 30 min, and added with protease into the solution for 30 min) directly extracted from the raw materials, and comparing the solubility, water holding capacity and oil holding capacity of the SDF prepared by directly obtaining peanut shell soluble dietary fiber (B-SDF) and composite fermentation from the raw materials by using the same method;
(2) Ability of dietary fiber to absorb glucose
The adsorption capacity of the B-SDF and the F-SDF to glucose is respectively inspected, and the result shows that the adsorption capacity of the SDF subjected to composite fermentation can be obviously improved, the adsorption quantity of the F-SDF to the glucose is 2910.36 mu mol/g, and is improved by 24.86% compared with the B-SDF (2330.84 mu mol/g);
(3) Cholesterol adsorption capacity
The Cholesterol Adsorption Capacity (CAC) of B-SDF and F-SDF is respectively considered under the conditions of pH 2.0 (simulated gastric environment) and pH 7.0 (simulated small intestine environment), and the results show that the cholesterol adsorption capacity is 3.14 mg/g and the cholesterol adsorption capacity of F-SDF is 7.25mg/g which are improved by 1.31 times compared with the cholesterol adsorption capacity of B-SDF under the condition of pH 2.0; 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, which is improved by 60.59%; the CAC value at pH 7.0 was higher than that at pH 2.0, indicating that the CAC of F-SDF is stronger in a simulated small intestine environment than in a simulated stomach environment.
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