CN114891643B - Preparation method of buckwheat xylooligosaccharide, flavone extraction and health-care buckwheat aromatic vinegar - Google Patents

Preparation method of buckwheat xylooligosaccharide, flavone extraction and health-care buckwheat aromatic vinegar Download PDF

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CN114891643B
CN114891643B CN202210452290.8A CN202210452290A CN114891643B CN 114891643 B CN114891643 B CN 114891643B CN 202210452290 A CN202210452290 A CN 202210452290A CN 114891643 B CN114891643 B CN 114891643B
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buckwheat
xylooligosaccharide
vinegar
flavone
straw
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CN114891643A (en
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王曙阳
肖国青
董妙音
许富强
王俊凯
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Institute of Modern Physics of CAS
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Abstract

The invention discloses a preparation method of buckwheat aromatic vinegar for extracting and health care of buckwheat xylooligosaccharide and flavone. The invention discloses a glucose-resistant high-yield cellulase strain of trichoderma longibrachiatum, which is named as LC-M4.21 and is preserved in China general microbiological culture Collection center (China center for type culture collection) for 8 months and 11 days in 2021, wherein the preservation number is as follows: CGMCC NO 23206. Xylanase and cellulase activity in fermentation liquor of the high-yield strain with resistance and containment of trichoderma longibrachiatum can reach 350+/-0.39 IU/mL and 5.6+/-0.05 IU/mL respectively, enzyme requirements of preparing xylooligosaccharide by directly carrying out enzymolysis on untreated straws are met, a green process method for preparing the xylooligosaccharide by directly carrying out enzymolysis on the buckwheat straws meeting the health-care requirement of xylooligosaccharide is established by optimizing the adding amount of xylanase and the enzymolysis time, meanwhile, an extraction process of buckwheat flavone is established by optimizing the feed-liquid ratio, the ethanol concentration and the extraction time, vinegar spraying is directly carried out by adopting the xylooligosaccharide of the buckwheat straws and the flavone hydrolysate, and health-care buckwheat aromatic vinegar is prepared by adopting the vinegar spraying process optimization and the nuclear pore membrane filtration clarification technology.

Description

Preparation method of buckwheat xylooligosaccharide, flavone extraction and health-care buckwheat aromatic vinegar
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a preparation method of buckwheat aromatic vinegar for health care by extracting buckwheat xylooligosaccharide and flavone.
Background
Buckwheat is also called triangular wheat and Wu Mai, which are one of the oldest cultivated crops in China, the annual yield of the buckwheat is the second in the world after Russian [ Pu Shenghui, gao Ying, zhao Zhifeng, lin Huasong, criminal day, he Xueqin ] active substances in tartary buckwheat and the research progress of health care efficacy thereof [ J ]. Food industry science and technology, 2019,40 (08): 331-336]. The buckwheat not only contains rich nutrients such as starch, fat, essential amino acids, minerals and the like, but also contains various bioactive components with therapeutic effects such as flavone, polyphenol, vitamin, D-chiral inositol and the like, and has health care effects such as reducing blood sugar and blood fat, softening blood vessels and the like [ Chen Hui, li Jianting, qin Dan ].
The buckwheat kernel contains flavone mainly including rutin, orientin, vitexin, quercetin, isovitexin, isoerythroside, etc. and has excellent auxiliary treatment effect on diabetes, hypertension, hyperlipemia, coronary heart disease, apoplexy and gastropathy caused by the effective degradation of the buckwheat flavone, etc., [ Meng Jingjing, zhang Zhiwei, zhou Wenxi, ni Na, zhao Ru, li Hua, wang Hua ], ultrasonic assisted extraction of buckwheat total flavone, etc., [ J ] food research and development, 2022,43 (04): 82-88 ], and clinical auxiliary treatment effect on diabetes, hypertension, hyperlipemia, coronary heart disease, apoplexy and gastropathy caused by the effective factors and health function of buckwheat [ Zhao Xiuling ], food engineering, 2011 (03): 16-18 ]. Therefore, the flavonoid component is used as one of the buckwheat vinegar drink health care factors through proper process research and development, so that the buckwheat vinegar drink health care factor has various physiological functions of reducing blood sugar and blood fat, resisting oxidation, enhancing human immunity and the like, and has health care effects of treating diabetes, hypertension, coronary heart blood, apoplexy and other diseases.
The xylooligosaccharide is a degradation product of hemicellulose, is the oligosaccharide with the best bifidobacterium proliferation effect and the smallest effective amount, is called super-strong bifidus factor or prebiotic, has unique physiological health care function, can inhibit the growth of harmful bacteria while promoting the proliferation of beneficial bacteria in human intestinal tracts, is beneficial to regulating the microecological balance of the intestinal tracts, can enhance the nutrition absorption and improve the immunity, and is widely applied to the fields of medical health products, foods and the like [ Ma Tingting, du Qian, gansaobo, shao Xianbao, wang Binbin ], the application progress of xylanase in the preparation of xylooligosaccharide [ J ] [ fine and special chemicals, 2021,29 (02): 6-11; ren Chunlin, dong Gongli, wang Fengqin, song An east. Xylo-oligosaccharide production technology and research progress on animal probiotics [ J ]. Food and fermentation industry, 2021,47 (09): 293-298; gu Fengyuan, yao Zixuan, pan Yan, liu Lian, research on the preparation of xylo-oligosaccharides, development [ J ]. Shandong chemical, 2020,49 (21): 59-60; pan Qing, sunzhi, xu Wenbiao, li Xiangyu, shi Junyou. Research progress on the preparation of xylo-oligosaccharides from corn stalks [ J ]. Forest industry, 2020,57 (10): 8-12+22; xie Fang, zhang Haibo, unfortunately qingfeng, li Li, guan Wei, guo Dongsheng, liu Xiaogao. Influence of xylo-oligosaccharides on the intestinal barrier of animals and research progress for their use in animal production [ J ]. J.China animal journal, 2020,56 (10): 7-12 ]. However, the xylo-oligosaccharide produced and sold in the market at present is prepared by adopting corncob and other raw materials through an alkali-dissolution alcohol precipitation method, and the xylo-oligosaccharide component is introduced through exogenous compounding and adding when corresponding xylo-oligosaccharide products are developed, so that the development and processing mode of the xylo-oligosaccharide product is high in cost, complex in operation and complex in process, and other impurity components of exogenous materials are introduced, so that the original flavor and quality of the xylo-oligosaccharide product are seriously influenced [ Xie Fang, zhang Haibo, unfortunately clear phoenix, li Li, guan Wei, guo Dongsheng and Liu Xiaogao ] [ J ]. Research progress of the xylo-oligosaccharide on animal intestinal barriers and application of the xylo-oligosaccharide in animal production [ J ]. Chinese livestock journal 2020,56 (10): 7-12 ]. The buckwheat vinegar has the problems of turbidity, precipitation, wall attachment and the like with different degrees in the production and storage processes, so that the quality of the vinegar is affected, the main reasons of turbidity are that the starch utilization rate is not high, components such as polyphenol, protein, iron ions and the like exist, the precipitation speed is low, the effect is also not ideal, and the traditional natural clarification method cannot adapt to the development requirements of the brewing industry. In addition, the buckwheat aromatic vinegar developed by taking buckwheat as a main raw material in the market at present is a superior flavoring although the effective nutritional ingredients such as rich amino acid, starch, mineral substances, nicotinic acid and the like in the buckwheat are excavated and utilized, but the bioactive ingredients with treatment and health care effects in the buckwheat are not developed and utilized yet [ Puchun red, research and development of buckwheat sprout fermentation health care food, jilin province, jilin agricultural university, 2015-06-18; zhao Xiuling the research of the efficacy factors and health care functions of buckwheat is progressed [ J ]. Food engineering, 2011 (03): 16-18]. Therefore, the buckwheat grain flavone and hemicellulose rich in the buckwheat straw are utilized, the buckwheat xylooligosaccharide and the flavone with high added value are prepared and extracted from the buckwheat by a green enzymolysis preparation process and different extraction methods, and the buckwheat grain flavone and hemicellulose rich in the buckwheat straw are applied to the medical and edible health-care buckwheat aromatic vinegar product developed by taking the buckwheat as the main raw material, so that the buckwheat grain flavone and xylose rich in the buckwheat straw has high economic value and market prospect.
Disclosure of Invention
The invention aims to provide a glucose-resistant high-yield cellulase strain of trichoderma longibrachiatum, which has the significance that the glucose-resistant high-yield cellulase strain obtained by screening effectively reduces the inhibition of substrate glucose while ensuring the high-yield enzyme performance, and mainly solves the key problem that the high-yield cellulase strain is inhibited by glucose to inhibit enzyme production in actual production.
The glucose-resistant high-yield cellulase strain of trichoderma longibrachiatum provided by the invention is named as LC-M4.21, and is preserved in China general microbiological culture Collection center (CGMCC; address: north Xiyi Lu No. 1,3 of the Korean region of Beijing, institute of microorganisms of the national academy of sciences, postal code: 100101) in the year 2021, 8 and 11, and the preservation number is: CGMCC NO 23206.
The invention provides a trichoderma longibrachiatum glucose-resistant high-yield cellulase strain which is prepared by a method comprising the following steps of:
1) For Trichoderma longibrachiatum LC-M4 (Trichoderma longibrachiatum) [ see Dong MY, wang SY, xu FQ et al Effectent utilization ofwaste paper as an inductive feedstock for simultaneous production ofcellulase and xylanase by T. Longibrachiatum. Journal ofCleaner Production, 2021,308:127287; heavy ion beam irradiation mutagenesis was performed on a spore suspension of the strain Dong MY, wang SY, xu FQ et al pretreament ofsweet sorghum straw and its enzymatic digestion: insight into the structural changes and visualization of hydrolysis process for Biotechnology 2019, 12:276;
2) Screening of excellent strain of glucose-resistant high-yield cellulase of trichoderma longibrachiatum:
and (3) carrying out primary screening on the heavy ion beam irradiation induced trichoderma longibrachiatum LC-M4 strain spore suspension by adopting a double-layer primary screening plate through a hydrolysis circle method, and carrying out shake flask secondary screening on the high HC value strain obtained by the primary screening of the plate to obtain the glucose-resistant cellulase high-yield mutant strain LC-M4.21.
In the above method step 1), the spore concentration in the spore suspension of the Trichoderma longibrachiatum LC-M4 strain may be 1×10 4 -1×10 8 The specific volume of the catalyst is 1X 10 6 individual/mL;
the irradiation dose of the heavy ion beam is 120-200Gy, specifically can be 150-180Gy, and more specifically can be 160 Gy;
the irradiation ion is carbon ion, the energy is 60-100MeV/u, specifically 80MeV/u, the dose rate is 10-40Gy/min, specifically 20Gy/min;
the heavy ion beam irradiation experiment is completed at the TR4 irradiation terminal of the biological shallow layer of the heavy ion accelerator;
in the step 2) of the method, the culture medium used for the double-layer preliminary screening plate comprises the following components: (lower 14 mL+upper 6 mL)/plate
The lower layer: enrichment of Mandel's nutrient salt Medium (g/L): k (K) 2 HPO 4 1-5g，(NH 4 ) 2 SO 4 1-4g， MgSO 4 ·7H 2 0.3-0.7g of O, 0.3-0.7g of urea and CaCl 2 0.3-0.7g，FeSO 4 ·7H 2 O 0.003-0.007g， MnSO 4 ·H 2 O 0.001-0.002g，ZnSO 4 ·7H 2 O 0.001-0.002g，CoCl 2 0.001-0.004g, agar 15-20 gThe pH is natural;
Upper layer: adding concentrated Mandel's nutrient salt culture medium, microcrystalline cellulose (MCC) 5-20g/L, glucose 3-10 g/L, sodium deoxycholate 0.1-0.5g/L, agar 15-20g/L, and natural pH.
The primary screening is operated as follows: coating spore liquid on a double-layer primary screening flat plate, culturing for 2-5d at 25-35 ℃, dyeing the flat plate growing with bacterial colonies with gram iodine liquid after the culturing is finished, measuring the diameters of transparent rings and bacterial colonies after dyeing, calculating HC value according to the ratio of the transparent rings and the bacterial colonies, storing mutant bacterial strains with HC value more than or equal to 2.0 on an inclined plane, and continuing shaking, fermenting and enzyme production and rescreening;
the shaking and re-screening operation is as follows: inoculating the spore suspension to shake flask fermentation seed culture medium, culturing at 25-35deg.C and 150-300rpm for 12-36 hr, inoculating seed solution at 2-10% (v/v) ratio to shake flask fermentation enzyme production culture medium, culturing at 25-35deg.C and 150-300rpm for 6-10d, and performing rescreening of high-yield enzyme mutant strain by measuring filter paper enzyme activity after fermentation;
wherein, the composition of the shake flask fermentation seed culture medium (g/L) is as follows:
5-15g of glucose, 3-7g of peptone, and 801-5mL of tween (NH) 4 ) 2 SO 4 1-3g，CaCl 2 0.1-0.5g, urea 0.1-0.5g, mgSO 4 ·7H 2 O 0.1-0.5g，FeSO 4 ·7H 2 O 0.003-0.007g，MnSO 4 ·H 2 O 0.001-0.002g，ZnSO 4 ·7H 2 O 0.001-0.002g，CoCl 2 0.001-0.003g, and the pH is natural;
the formula of the fermentation enzyme production medium comprises the following components: 10-30g of microcrystalline cellulose, 5-15g of bran, 15-25g of corn steep liquor and K 2 HPO 4 1-4g，(NH 4 ) 2 SO 4 1-3g，MgSO 4 ·7H 2 0.1-0.5g of O, 0.1-0.5g of urea and CaCl 2 0.1-0.5g, tween-801-5 mL, feSO 4 ·7H 2 O 0.003-0.007g，MnSO 4 ·H 2 O 0.001-0.002g，ZnSO 4 ·7H 2 O 0.001-0.002g，CoCl 2 0.001-0.003g, pH natural value;
the glucose-resistant high-yield cellulase strain of the trichoderma longibrachiatum is used for producing xylanase and cellulase through liquid fermentation.
The invention also aims to provide a method for simultaneously producing xylanase and cellulase by adopting the liquid fermentation of the anti-inhibition high-yield strain LC-M4.21 of trichoderma longibrachiatum, which meets the enzyme requirement of preparing xylooligosaccharide by direct enzymolysis of untreated straws, and obtains the xylooligosaccharide from buckwheat for preparing the health-care buckwheat aromatic vinegar.
The method for simultaneously producing xylanase and cellulase provided by the invention comprises the following steps:
inoculating the high-yield strain LC-M4.21 of the trichoderma longibrachiatum to a seed culture medium, culturing for 12-36h at 25-35 ℃ and 150-300rpm, inoculating seed liquid to a fermentation enzyme-producing culture medium according to the proportion of 2-10% (v/v), and fermenting for 6-10d at 25-35 ℃ and 150-300rpm to obtain a fermentation liquor containing xylanase and cellulase;
wherein, the formula of the seed culture medium is as follows:
glucose 5-15g, peptone 3-7g, tween-80 1-5mL, (NH) 4 ) 2 SO 4 1-3g，CaCl 2 0.1-0.5g, urea 0.1-0.5g, mgSO 4 ·7H 2 O 0.1-0.5g，FeSO 4 ·7H 2 O 0.003-0.007g，MnSO 4 ·H 2 O 0.001-0.002g，ZnSO 4 ·7H 2 O 0.001-0.002g，CoCl 2 0.001-0.003g, and the pH is natural;
The formula of the fermentation enzyme production medium comprises the following components: 10-30g of microcrystalline cellulose, 5-15g of bran, 15-25g of corn steep liquor and K 2 HPO 4 1-4g，(NH 4 ) 2 SO 4 1-3g，MgSO 4 ·7H 2 0.1-0.5g of O, 0.1-0.5g of urea and CaCl 2 0.1-0.5g, tween-801-5 mL, feSO 4 ·7H 2 O 0.003-0.007g，MnSO 4 ·H 2 O 0.001-0.002g，ZnSO 4 ·7H 2 O 0.001-0.002g，CoCl 2 0.001-0.003g, pH natural value;
the culture medium is autoclaved at 115-121deg.C for 15-30min.
The application of the glucose-resistant high-yield cellulase strain of the trichoderma longibrachiatum or the fermentation liquor containing xylanase and cellulase in preparing the straw xylooligosaccharide by directly hydrolyzing the straw also belongs to the protection scope of the invention.
The straw may be a buckwheat straw.
The invention also aims to provide a method for preparing the straw xylooligosaccharide by directly carrying out enzymolysis on the straw by adopting the glucose-resistant high-yield cellulase strain of trichoderma longibrachiatum or the fermentation liquor containing xylanase and cellulase.
The method for preparing the straw xylo-oligosaccharide provided by the invention comprises the following steps:
the method comprises the steps of taking straws as raw materials, and adopting the glucose-resistant high-yield cellulase strain LC-M4.21 of trichoderma longibrachiatum or the fermentation liquor prepared by the method and containing xylanase and cellulase for enzymolysis to obtain enzymolysis liquor containing straw xylooligosaccharide;
In the method, before enzymolysis, crushing, sieving with a 20-mesh sieve, and intercepting with a 60-mesh sieve, wherein straws with intercepting granularity between 20-60-mesh sieves are selected as raw materials;
the straw can be buckwheat straw;
the xylanase is added into the straw at an amount of 260-1600IU/g, specifically 520-1600IU/g, 520-1000 IU/g and 1000-1600 IU/g; more specifically, 1000IU/g straw;
the ratio of feed liquid adopted in the enzymolysis is 1:6-1:12 (w: v), specifically, may be 1:10 (w: v) (the feed-to-liquid ratio herein refers to the mass of the buckwheat straw and the total volume of liquid (wherein the liquid is a mixture of distilled water and the supernatant of the fermentation broth of the glucose-repressing high-yield cellulase strain of Trichoderma longibrachiatum).
The enzymolysis time can be 12-48 h, and can be 30-40h, 34-38h and 35-37h; more specifically 36h;
when the xylanase is used for preparing the buckwheat aromatic vinegar, the adding amount of the xylanase is 260IU/g, the enzymolysis is carried out for 36 hours, and the content of xylooligosaccharide in the prepared enzymolysis liquid is 5.68mg/mL.
The invention also aims to provide an extraction method of the flavonoids in the buckwheat grains, which optimizes the extraction process, and comprises the following steps of (1), (2) and (3):
(1) Extracting buckwheat grains serving as a raw material by using ethanol solution with a certain concentration to obtain buckwheat grain flavone extracting solution;
in the method, the buckwheat grains are firstly crushed, and then are filtered by a 60-mesh sieve, and the filtered buckwheat flour is used as a flavone extraction raw material;
wherein the ratio of flavone extraction liquid is 1:5-1:30 (w/v); the extraction time is 0.5-3h; ethanol concentration for extraction is 50% -80%;
the flavone extraction concentration is 5-8.50mg/ml;
the optimal extraction process of buckwheat flavone comprises the following steps: feed liquid ratio 1: 20. under the condition of extracting with 60% ethanol for 2 hours, the highest concentration of the obtained flavone is 8.50mg/ml.
(2) Extracting flavone from semen Fagopyri Esculenti grain as raw material with xylooligosaccharide hydrolysate (i.e. the enzymatic hydrolysate containing stalk xylooligosaccharide prepared by the above method) to obtain extractive solution of xylooligosaccharide and flavone;
wherein the concentration of the xylooligosaccharide hydrolysis liquid is 5.6-9.6g/L;
the ratio of flavone extraction liquid is 1:5-1:30 (w/v); the extraction time is 0.5-3h;
the flavone extraction concentration is 0.04-0.1mg/ml;
the optimal extraction process of buckwheat flavone comprises the following steps: feed liquid ratio 1: 20. extracting for 3h to obtain flavone with highest concentration of 0.1mg/mL;
(3) Extracting flavonoids from buckwheat grains by using a hydrolysis solution of xylooligosaccharide containing 5% ethanol (namely the hydrolysis solution containing straw xylooligosaccharide prepared by the method) by using the buckwheat grains as a raw material and adopting an enzymatic extraction process;
wherein the enzyme is crude enzyme liquid produced by fermenting Trichoderma longibrachiatum LC-M4 strain, namely, the fermentation liquid containing xylanase and cellulase prepared by the method, and the addition amount of the cellulase is as follows: 2-8IU/ml;
the extraction conditions are as follows: extraction temperature: 50 ℃; rotational speed: 150rpm; feed liquid ratio: 1:20, a step of; extraction time: 1-3h;
the optimal extraction process of buckwheat flavone comprises the following steps: the addition amount of cellulase is 2IU/g substrate, extraction is carried out for 1h, the extraction rate of the obtained flavone is 3.27mg/g seed grain at the highest, the extraction rate of buckwheat flavone is 0.10mg/g (2 h extraction) at the highest, and the extraction time is reduced.
The invention further aims to provide the buckwheat xylooligosaccharide health-care aromatic vinegar and the preparation method of the buckwheat xylooligosaccharide and flavone health-care aromatic vinegar, which adopt buckwheat straw xylooligosaccharide hydrolysate to extract buckwheat kernel flavone, the buckwheat xylooligosaccharide flavone extract is used for spraying vinegar, xylooligosaccharide and flavone components with health-care effects are introduced in the vinegar spraying process, and the complicated operation process of spraying vinegar with tap water and then introducing health-care functional components exogenously in the preparation of other vinegar is replaced. In addition, the nuclear pore membrane filtration clarification technology established in the invention can be popularized and applied to the processing production of products such as soy sauce and the like.
The preparation method of the buckwheat xylooligosaccharide health-care aromatic vinegar provided by the invention comprises the following steps:
the buckwheat xylooligosaccharide hydrolysate prepared by the method is used for leaching vinegar from mature vinegar grains, and the leached xylooligosaccharide health-care buckwheat aromatic vinegar is subjected to filtering and clarifying treatment by adopting a nuclear pore membrane material, so that the buckwheat xylooligosaccharide health-care aromatic vinegar is obtained.
The preparation method of the buckwheat xylooligosaccharide and flavone health-care aromatic vinegar provided by the invention is as follows (1) or (2):
(1) The buckwheat xylooligosaccharide and flavone extracting solution prepared by the method is used for leaching vinegar from mature vinegar grains, and the leached xylooligosaccharide and flavone health-care buckwheat aromatic vinegar is obtained by filtering and clarifying the leaching vinegar by a nuclear pore membrane material;
(2) The enzymolysis liquid containing the straw xylo-oligosaccharide, namely the hydrolysis liquid of the buckwheat straw xylo-oligosaccharide, is prepared by the method, the mature vinegar grains are subjected to vinegar leaching, the buckwheat kernel flavone extracting liquid is added into the leached xylo-oligosaccharide health-care buckwheat aromatic vinegar, and the obtained xylo-oligosaccharide and flavone health-care buckwheat aromatic vinegar is subjected to filtering and clarification treatment by adopting a nuclear pore membrane material, so that the buckwheat vinegar is obtained;
in the step (2), the buckwheat kernel flavone extracting solution is added according to the volume ratio of 0.5-1 percent.
The feed liquid ratio in the vinegar spraying process is 1:1-1:1.7 (w/v) (both endpoints are not preferable), specifically 1: 1.4-1: 1.6 (w/v);
The vinegar spraying time can be 18-30 h, and can be specifically 24h;
the temperature of the vinegar spraying can be 4-40 ℃;
the pore size of the nuclear pore membrane material may be 2 μm to 3 μm, more specifically 3 μm.
The nuclear pore membrane material used here was commercially available, and the manufacturer was Wu Weike, a near new technology liability company, under the trade name nuclear pore membrane (pore size specification of 2 μm-3 μm).
The buckwheat xylo-oligosaccharide health-care aromatic vinegar or the buckwheat xylo-oligosaccharide and flavone health-care aromatic vinegar prepared by the method also belongs to the protection scope of the invention.
The invention has the following advantages:
(1) Establishes a double-layer plate screening method for the glucose-resistant high-yield cellulase strain after the heavy ion irradiation mutagenesis of trichoderma longibrachiatum, and screens to obtain an anti-resistant high-yield strain LC-M4.21. The traditional enzyme producing strain improvement uses high-yield enzyme as the only target to screen, but the high-yield enzyme strain is extremely easy to be blocked by glucose in a substrate to inhibit enzyme production in fermentation production, the method has the significance that the screening of the enzyme producing strain which can be truly used for industrial fermentation ensures the high-yield enzyme performance and simultaneously effectively reduces the substrate glucose blocking, and mainly solves the key scientific problem that the high-yield enzyme strain is blocked by glucose to inhibit enzyme production in actual production;
(2) Xylanase and cellulase activity in the fermentation liquor of the anti-blocking high-yield strain of the trichoderma longibrachiatum can reach 350+/-0.39 IU/mL and 5.6+/-0.05 IU/mL respectively, so that the enzyme requirement of preparing the xylooligosaccharide by directly carrying out enzymolysis on untreated straws is met, the fermentation enzyme liquor of the anti-blocking high-yield strain of the trichoderma longibrachiatum is adopted to prepare the xylooligosaccharide of the buckwheat straws through a green enzymolysis process, and the xylooligosaccharide of the buckwheat sources is obtained to prepare the health-care buckwheat aromatic vinegar;
(3) The green technological method for preparing the buckwheat xylooligosaccharide by directly hydrolyzing the buckwheat straw meeting the health-care requirement of xylooligosaccharide is established by optimizing the adding amount of xylanase and the enzymolysis time, and the xylooligosaccharide content in the straw enzymolysis liquid can reach 5.68mg/mL under the condition that the adding amount of xylanase is 260 IU/g for 36 hours. The preparation method of the xylo-oligosaccharide in the industrial field is that the traditional alkali-dissolution alcohol precipitation method is used for extracting xylan components and then xylanase is used for enzymolysis preparation of the xylo-oligosaccharide, so the green preparation technology of the direct enzymolysis method of the xylo-oligosaccharide by the buckwheat straw in the research has the innovation points that: the use of chemical reagents such as strong acid, strong alkali, industrial ethanol and the like is avoided, the product quality is improved, the processing production cost is reduced, the environmental pollution is reduced, and meanwhile, the green processing production of green food raw materials is realized;
(4) The buckwheat straw xylooligosaccharide and flavone extracting solution is adopted for vinegar leaching, the xylooligosaccharide and flavone bioactive components with health care effects are introduced in the vinegar leaching process, the complex operation process of firstly leaching vinegar by tap water and then exogenously introducing and adding health care functional components in the preparation of other health care vinegar is replaced, the feed liquid ratio, the temperature and the time parameters of the buckwheat straw xylooligosaccharide hydrolysate vinegar leaching are optimized, and the buckwheat straw xylooligosaccharide hydrolysate is adopted in the study in the formula 1:1.5 (w/v) the ratio of feed to liquid and the temperature of 4-30 ℃ for 24 hours, the total acid content of the obtained health-care buckwheat raw vinegar is more than or equal to 3.72 percent, and a vinegar leaching process by utilizing the buckwheat straw xylooligosaccharide hydrolysate is established;
(5) The research establishes a xylooligosaccharide health-care buckwheat original vinegar nuclear pore membrane filtration clarification technology for the first time. In the research, the nuclear pore membrane material with the aperture of 3 mu m is selected, and the buckwheat aromatic vinegar stock solution is transparent and clear after being filtered, has no particles and suspended matters which are visible to naked eyes to precipitate, is still transparent and clear after being stood for 30 days, and has no precipitation. The health-care buckwheat raw vinegar which is not subjected to filtering treatment contains a large amount of suspended cereal particles and other fermentation impurities, and flocculent precipitate which is visible to the naked eye is generated after the buckwheat raw vinegar is left for 24 hours. The nuclear pore membrane filtration clarification technology established in the research can be popularized and applied to the processing production of soy sauce and other similar products.
Preservation description
Classification naming: trichoderma longibrachiatum LC-M4.21;
preservation mechanism: china general microbiological culture Collection center;
the preservation organization is abbreviated as: CGMCC;
address: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing;
preservation date: 2021, 8, 11;
accession numbers of the preservation center: CGMCC NO 23206
Drawings
FIG. 1 is a graph showing the results of shake-flask rescreening of the high-yield cellulase mutant strain of Trichoderma longibrachiatum in example 1.
FIG. 2 is a graph showing the results of fermentation and enzyme production of the high-yield strain LC-M4.21 of Trichoderma longibrachiatum in example 2.
FIG. 3 is a graph showing the results of HPLC detection of the xylooligosaccharide content in the enzymatic hydrolysate of example 3, wherein A is a standard curve of xylobiose, xylotriose and xylotetraose; and B, a liquid chromatogram of xylobiose, xylotriose and xylotetraose.
FIG. 4 is a rutin standard curve in example 4, abscissa: rutin concentration (μg/mL); ordinate: absorbance (OD value).
FIG. 5 is a graph showing the comparative filtration effect of the xylo-oligosaccharide buckwheat aromatic vinegar nuclear pore membrane in example 7.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1, construction of method for double-layer plate screening of Trichoderma longibrachiatum glucose-resistant high-yield cellulase Excellent Strain and Strain screening
(1) Trichoderma longibrachiatum LC-M4 strain [ Dong MY, wang SY, xu FQ et al Effectent utilization of waste paper as an inductive feedstock for simultaneous production of cellulase and xylanase by T.longifflorem. Journal of Cleaner Production,2021,308:127287 ], dong MY, wang SY, xu FQ et al pretreament of sweet sorghum straw and its enzymatic digestion: insight into the structural changes and visualization of hydrolysis process.biotechnology for Biofuels.2019,12:276 ] spore suspension heavy ion beam irradiation mutagenesis:
washing spores on fresh Trichoderma longibrachiatum LC-M4 inclined plane with physiological saline, filtering with 4 layers of sterile gauze to remove mycelium, shaking the obtained spore filtrate in a triangular flask containing glass beads at 200rpm for 10min, and adjusting spore concentration to 1×10 with a blood cell counting plate 6 And each mL. 2mL of the prepared spore suspension is sucked and placed into a 35mm irradiation vessel, sealing treatment is carried out by using a sealing film, the irradiation dose of heavy ion beams is 160Gy, and three samples are arranged in parallel and are totally 3 samples. The irradiation experiment of the spore suspension is completed at the irradiation terminal of the biological shallow layer TR4 of the ion accelerator with the weight close to the object, the irradiation ion type is carbon ion, the energy is 80MeV/u, and the dosage rate is 20Gy/min. The spore suspension sample after irradiation treatment is immediately subjected to glycerol cryopreservation for subsequent mutant strain screening.
(2) Screening of excellent strain of glucose-resistant repressed high-yield cellulase of trichoderma longibrachiatum
(1) Double-deck plate screening medium: (lower 14 mL+upper 14 mL)/plate
The lower layer: enrichment of Mandel's nutrient salt Medium (g/L): k (K) 2 HPO 4 3.0g，(NH 4 ) 2 SO 4 2.0g， MgSO 4 ·7H 2 O0.5 g, urea 0.5g, caCl 2 0.5g，FeSO 4 ·7H 2 O 0.005g，MnSO 4 ·H 2 O 0.0016 g，ZnSO 4 ·7H 2 O 0.0014g，CoCl 2 0.002g, agar 17g, pH natural.
Upper layer: the nutrient salt culture medium of Mandel's is enriched, microcrystalline cellulose (MCC) is 10.0g/L, glucose is 5.0g/L, sodium deoxycholate is 0.3g/L, agar is 17g/L, and the pH is natural. (all the chemical reagents used in the culture medium are domestic analytically pure)
(2) Primary screening by a double-layer plate method: the spore suspension after heavy ion beam irradiation mutagenesis is subjected to primary screening of the anti-repression high-yield enzyme mutant strain by adopting the double-layer screening plate through a hydrolysis circle method. Diluted to 1X 10 2 -1×10 3 100 mu L of spore liquid per mL is sucked up and coated on a double-layer preliminary screening plate, the plate with the colony growing is dyed with gram iodine liquid after the culture is finished in a constant temperature incubator at 30 ℃, and the iodine liquid can react with MCC to generate blue-violet compound but does not react with glucose, so that hydrolysis rings with different sizes are formed around the colony due to enzymolysis of the MCC. And measuring the diameters of the transparent ring and the colony by using a vernier caliper after dyeing, calculating an HC value according to the ratio of the transparent ring and the colony, and storing the mutant strain with the HC value more than or equal to 2.0 on an inclined plane for continuous shake flask fermentation enzyme production screening.
(3) Liquid shaking and fermenting to produce enzyme and re-screening: and (5) carrying out shake flask rescreening on the strain with high HC value obtained by the plate primary screening. Aspiration of 2mL of 1X 10 6 Inoculating the spore suspension in shake flask fermentation seed culture medium, culturing at 30deg.C and 200rpm for 24 hr, inoculating seed solution in shake flask fermentation enzyme production culture medium at 5% (v/v), culturing at 30deg.C and 200rpm for 8d, and performing rescreening of high-yield enzyme mutant strain by measuring filter paper enzyme activity after fermentation.
Shake flask fermentation seed medium (g/L): glucose 10g, peptone 5g, tween-802 mL, (NH) 4 ) 2 SO 4 1.4g，CaCl 2 0.3g, urea 0.3g, mgSO 4 ·7H 2 O 0.3g，FeSO 4 ·7H 2 O 0.005g，MnSO 4 ·H 2 O 0.0016g，ZnSO 4 ·7H 2 O 0.0014g，CoCl 2 0.002g, pH is natural.
Shake flask fermentation enzyme production medium (g/L): microcrystalline cellulose20g, 10g of bran, 17g of corn steep liquor and K 2 HPO 4 2.0g，(NH 4 ) 2 SO 4 1.4g，MgSO 4 ·7H 2 O0.3 g, urea 0.3g, caCl 2 0.3g, tween-80 2mL, feSO 4 ·7H 2 O 0.005g，MnSO 4 ·H 2 O 0.0016g，ZnSO 4 ·7H 2 O 0.0014g，CoCl 2 0.002g, pH natural value. (all the chemical reagents used in the culture medium are domestic analytically pure)
(3) Screening results of anti-repression high-yield cellulase strain
(1) Double-deck plate method primary screening results: through preliminary screening by a double-layer flat plate method, 31 candidate strains with HC value more than or equal to 2.0 and high yield of the trichoderma longibrachiatum resistance are obtained through total screening, and the statistical results of the preliminary screening are shown in table 1.
TABLE 1 preliminary screening statistical result of double-layer flat plate method (HC value is more than or equal to 2.0)
(2) Primary screening strains shake flask fermentation to produce enzyme re-screening results: and (3) carrying out liquid shake flask fermentation to produce enzyme re-screening on 31 anti-repressing high-yield candidate strains with HC value more than or equal to 2.0 obtained by the preliminary screening of the double-layer flat plate in the step (1), and completing screening of excellent anti-repressing high-yield cellulase mutant strains by measuring the enzyme activity of filter paper in fermentation liquor, wherein the shake flask re-screening result is shown in figure 1. Finally, a high-yield mutant strain of the excellent glucose-resistant repressed cellulase, which has obviously improved filter paper enzyme activity compared with the original strain LC-M4, is screened out and named as LC-M4.21. Meanwhile, the trichoderma longibrachiatum LC-M4.21 strain is subjected to patent preservation in China general microbiological culture collection center (CGMCC), and the preservation number is as follows: CGMCC NO 23206 with preservation date of 2021, 8 and 11.
EXAMPLE 2 production of xylanase and cellulase by liquid fermentation of Trichoderma longibrachiatum anti-repression high-yield Strain LC-M4.21
The formula of the seed culture medium comprises: 10g of glucose, 5g of peptone, 2mL of Tween-80, (NH) 4 ) 2 SO 4 1.4g，CaCl 2 0.3g, urea 0.3g, mgSO 4 ·7H 2 O 0.3g，FeSO 4 ·7H 2 O 0.005g，MnSO 4 ·H 2 O 0.0016g， ZnSO 4 ·7H 2 O 0.0014g，CoCl 2 0.002g, pH is natural.
The formula of the fermentation enzyme production medium comprises: microcrystalline cellulose 20g, bran 10g, corn steep liquor 17g, K 2 HPO 4 2.0g， (NH 4 ) 2 SO 4 1.4g，MgSO 4 ·7H 2 O0.3 g, urea 0.3g, caCl 2 0.3g, tween-802 mL, feSO 4 · 7H 2 O 0.005g，MnSO 4 ·H 2 O 0.0016g，ZnSO 4 ·7H 2 O 0.0014g，CoCl 2 0.002g, pH natural value. The above culture media were autoclaved at 121℃for 30min. (all the chemical reagents used in the culture medium are domestic analytically pure)
Washing spores on the slope of freshly cultured high-yield strain LC-M4.21 of Trichoderma longibrachiatum with physiological saline and adjusting the concentration to 1×10 6 2mL of the seed liquid is inoculated into 50mL (250 mL triangular flask) of seed culture medium, the seed liquid is inoculated into 50mL (250 mL triangular flask) of fermentation enzyme production culture medium according to the proportion of 5% (v/v) after the seed liquid is cultured for 24h at the temperature of 30 ℃ and the rpm of 200, the fermentation is carried out for 8d at the temperature of 30 ℃ and the rpm of 200, and the supernatant liquid is taken after the fermentation is finished to determine the filter paper enzyme activity (FPA), the endoenzyme activity (CMC) and the xylanase activity. Definition of enzyme Activity Unit (IU/mL): the amount of enzyme required to produce glucose or xylose per micromole per minute under assay conditions.
The results of the fermentation and enzyme production of the high-yield strain LC-M4.21 of the trichoderma longibrachiatum are shown in figure 2, the xylanase activity in the fermentation liquor can reach 350+/-0.39 IU/mL, and simultaneously, the filter paper enzyme activity and the endonuclease activity also respectively reach 5.6+/-0.05 IU/mL and 10.84+/-0.82 IU/mL, which shows that the high-yield strain of the trichoderma longibrachiatum can express and secrete a large amount of xylanase and cellulase at the same time and can be used for the direct enzymolysis preparation of the buckwheat straw xylooligosaccharide by a green process method.
Example 3, direct enzymatic hydrolysis of buckwheat straw xylo-oligosaccharide (the sample of buckwheat straw used in this part of experiments is provided by Gansu Wanjia modern agriculture and animal husbandry development services Co., ltd.) was carried out by the green process.
Drying the buckwheat straw raw material in a 105 ℃ oven until the weight is constant, crushing the buckwheat straw raw material by a crusher, sieving the crushed buckwheat straw raw material by a 20-mesh sieve and intercepting the crushed buckwheat straw raw material by a 60-mesh sieve, and carrying out enzymolysis on the buckwheat xylooligosaccharide by the buckwheat straw with the intercepting granularity between the 20-60-mesh sieve. Accurately weighing 1.0g of buckwheat straw sample, directly carrying out enzymolysis preparation by adopting different xylanase loadings produced by fermenting the glucose-resistant high-yield strain of trichoderma longibrachiatum under the conditions of 50 ℃ and 200rpm in a 50ml centrifuge tube, wherein the feed liquid ratio adopted in the enzymolysis preparation of xylooligosaccharide is 1:10 (w: v), xylanase loading gradients are 260, 520, 1000 and 1600IU/g straw, and respectively taking enzymolysis supernatant liquid for carrying out detection on the content of xylooligosaccharide by HPLC (high performance liquid chromatography) during 12 hours, 24 hours, 36 hours and 48 hours of enzymolysis, and optimizing the optimal enzyme dosage and enzymolysis time according to the content of xylooligosaccharide.
And (3) detecting the content of the xylo-oligosaccharide: centrifuging the reaction solution after enzymolysis at 8000rpm for 5min, collecting supernatant, and detecting the content of xylobiose, xylotriose and xylotetraose in the enzymolysis solution. The detection of the xylooligosaccharide content was carried out by HPLC (UltiMate 3000, termo, USA). The detector used was a differential refractive index detector (RefactoMa. Times.520), and the column was a Shodex SUGAR KS-802 gel column (inner diameter 8mm, column length 300mm, particle size 6 μm). The detection conditions are as follows:
mobile phase: water;
flow rate: 0.6mL/min;
column temperature: 80 ℃;
detector temperature: 45 ℃;
sample injection amount: 20. Mu.L;
retention time: 20min.
FIG. 3 is a high performance liquid chromatography assay for xylooligosaccharide content, wherein A is a standard curve of xylobiose, xylotriose, and xylotetraose; and B, a liquid chromatogram of xylobiose, xylotriose and xylotetraose.
The xylo-oligosaccharide component has standard curves of xylobiose, xylotriose and xylotetraose as shown in figure 3, wherein the standard curve of xylobiose is y= 4.5009x-0.0127 (R) 2 ＝0.9999) The xylotriose standard curve is y= 5.1045x-0.0227 (R 2 The xylotetraose standard curve is y= 4.4081x-0.0055 (R) 2 =0.9999). In the high performance liquid chromatography detection of the xylo-oligosaccharide, the peak-outlet sequence is xylotetraose (11.483 min), xylotriose (12.147 min) and xylotetraose (13.123 min) in sequence, and the result shows that the high performance liquid chromatography detection condition and detection method of the xylo-oligosaccharide established by the method are accurate and reliable, and can be used for quantitative analysis and detection of the content of the xylo-oligosaccharide of the buckwheat and quality control of related xylo-oligosaccharide products.
TABLE 2 Green preparation Process optimization results of buckwheat xylo-oligosaccharide
As can be seen from the green preparation process optimization results of the buckwheat xylo-oligosaccharide, when different xylanases produced by fermenting the glucose-resistant high-yield strain of trichoderma longibrachiatum are directly added for the enzymolysis preparation of the buckwheat straw xylo-oligosaccharide, the yield of the xylo-oligosaccharide is gradually increased along with the increase of the enzyme dosage, and the yield is highest when the enzyme dosage is 1000IU/g straw, but the yield of the xylo-oligosaccharide is reduced when the enzyme dosage is continuously increased to 1600IU/g, which indicates that the excessive xylanase liquid possibly degrades part of xylo-oligosaccharide components which are generated in the hydrolysate, and the result indicates that the dosage of the xylanase is not easy to be too large when the xylo-oligosaccharide is prepared by taking the buckwheat straw as the raw material. As can be seen from the optimized results of different enzymolysis time, in the enzymolysis for the first 36 hours, the yield of the xylo-oligosaccharide rises with the increase of the enzymolysis time, and after 36 hours, the yield of the xylo-oligosaccharide gradually decreases with the increase of the enzymolysis time, and presumably the reason is that the xylanase liquid degrades part of xylo-oligosaccharide components which are generated in the hydrolysate.
In addition, according to the fact that the daily intake of the xylo-oligosaccharide serving as a food raw material is less than or equal to 1.2 g/day as indicated in the No. 12 bulletin of the health department of the people's republic of China bulletin 2008, research results show that the xylo-oligosaccharide has good health care efficacy when the daily effective minimum intake dose of the xylo-oligosaccharide is 0.7-1.4 g [ Cui Yan, liu Licun ] xylo-oligosaccharide characteristics and application [ J ]. Food safety journal, 2017 (19): 60-61; yin Liduan, wang Lizhi, dong Congchao, application of xylo-oligosaccharide in health food [ J ]. Agricultural engineering (agricultural product processing industry), 2008 (06): 29-31; teng Chao, cha Peina, qu Lingyu, sholin, jiao Zhangjun, li Xiuting, functional oligosaccharide research and its use in food [ J ]. Programming for food safety quality detection, 2014,5 (01): 123-130]. Therefore, the optimal preparation process technical parameters of the green process of the buckwheat straw xylooligosaccharide can be obtained by integrating the influence of the enzyme dosage and the enzymolysis time on the preparation yield and the effective dosage requirement of related health-care foods in the green preparation process optimization of the buckwheat xylooligosaccharide, wherein the optimal preparation process parameters are as follows: and (3) performing enzymolysis for 36 hours under the condition that the enzyme quantity is 260IU/g, wherein the content of xylooligosaccharide in the straw enzymolysis liquid is 5.68mg/mL, and the concentration of xylooligosaccharide in the enzymolysis liquid meets the preparation requirement of the follow-up health-care buckwheat aromatic vinegar.
Example 4, buckwheat flavone extraction preparation (buckwheat grain samples used in this part of the experiments were supplied by the company of developing services of modern agriculture and animal husbandry, wanja, gansu).
Crushing buckwheat seeds, and filtering the crushed buckwheat seeds by a 60-mesh sieve, wherein the filtered buckwheat flour is used as a flavone extraction raw material; accurately weighing 1.0g of buckwheat sample in a 150ml triangular flask, extracting flavone by adopting ethanol solutions with different concentrations at 30 ℃ and 150rpm, wherein the ratio of feed liquid adopted in flavone extraction is 1: 5. 1: 10. 1: 20. 1:30 (w: v) ethanol concentrations were 50%, 60%, 70%, 80%, respectively, and the extract was subjected to detection of flavone content by rutin method at 0.5h, 1h, 2h, and 3h, respectively, and the optimum extraction process was optimized according to the flavone content.
And (3) preparing a rutin standard curve: precisely sucking rutin control (0.2 mg/mL70% ethanol) solution 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, placing into 25mL measuring flask, and adding 1mL 5% NaNO 2 Shaking the solution, standing for 6min, and adding 1mL of 10% Al (NO) 3 ) 3 Shaking the solution, standing for 6min, adding 10mL 4% NaOH solution, adding distilled water to a certain volume, shaking, standing for 15min, taking the follower reagent as blank, and measuring at 510nm wavelength And (3) absorbance, so as to obtain an absorbance Y and rutin concentration X (mug/mL), and drawing a standard curve (rutin has a good linear relationship in the range of 8-48 mug/mL).
Measuring the content of flavone in the extract sample: the same method was used to determine the total flavonoids in the samples from 0.5mL of the buckwheat extract. The yield of buckwheat flavone (calculated by rutin),
calculated according to the formula:
Y1(mg/g)＝(C*V*V 0 )/mV1
wherein: c is the flavone concentration of the test solution, mg/mLV is the total volume of the test solution, and mL; v (V) 0 The total volume of the extracting solution is mL; m is the mass of the sample, g; v (V) 1 The volume of the extract was sampled, mL.
Rutin standard curve is y= 0.2324x-0.0093, R 2 The correlation coefficient is 0.9963, which shows that the linear fitting relation between the concentration of the rutin standard substance and the absorbance value is good, and the standard curve (shown in figure 4) can be used for quantitative calculation of flavonoid compounds in a sample.
TABLE 3 optimization of extraction Process of flavonoids from buckwheat grains
The optimized results of the flavone extraction process are shown in the table above, and among three factors of selecting the optimized feed-liquid ratio, the extraction time and the ethanol concentration, the extraction time > the ethanol concentration > the feed-liquid ratio have the greatest influence on the flavone extraction rate. The optimal extraction process of the flavone in the buckwheat grains is obtained by optimizing the process, wherein the feed-liquid ratio is 1: 20. extracting with 60% ethanol for 2 hr to obtain flavone content of 8.50 mg/mL.
Example 5 preparation of buckwheat flavone by using xylooligosaccharide hydrolysate (buckwheat grain sample used in the experiment was supplied by Gansu Wanjia modern agriculture and animal husbandry development service Co., ltd.).
Crushing buckwheat seeds, and filtering the crushed buckwheat seeds by a 60-mesh sieve, wherein the filtered buckwheat flour is used as a flavone extraction raw material; accurately weighing 1.0g of buckwheat sample in a 150ml triangular flask, extracting flavone by adopting xylooligosaccharide hydrolysate under the conditions of 30 ℃ and 150rpm and preparing in example 3), wherein the ratio of feed liquid adopted in flavone extraction is 1: 5. 1: 10. 1: 20. 1:30 (w: v) respectively taking the extracting solutions during 0.5h, 1h, 2h and 3h, detecting the flavone content by adopting a rutin method, and optimizing the optimal extraction process according to the flavone content.
And (3) preparing a rutin standard curve: precisely sucking rutin control (0.2 mg/mL70% ethanol) solution 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, placing into 25mL measuring flask, and adding 1mL 5% NaNO 2 Shaking the solution, standing for 6min, and adding 1mL of 10% Al (NO) 3 ) 3 Shaking the solution, standing for 6min, adding 10mL of 4% NaOH solution, adding distilled water to a certain volume to scale, shaking, standing for 15min, taking the accompanying reagent as a blank, measuring the absorbance at the wavelength of 510nm to obtain the absorbance Y and the rutin concentration X (mug/mL), and drawing a standard curve (rutin has a good linear relationship in the range of 8-48 mug/mL).
Measuring the content of flavone in the extract sample: the same method was used to determine the total flavonoids in the samples from 0.5mL of the buckwheat extract. The yield of buckwheat flavone (calculated by rutin),
calculated according to the formula:
Y1(mg/g)＝(C*V*V 0 )/mV1
wherein: c is the flavone concentration of the test solution, mg/mLV is the total volume of the test solution, and mL; v (V) 0 The total volume of the extracting solution is mL; m is the mass of the sample, g; v (V) 1 The volume of the extract was sampled, mL.
Rutin standard curve is y= 0.2324x-0.0093, R 2 The correlation coefficient is 0.9963, which shows that the linear fitting relation between the concentration of the rutin standard substance and the light absorption value is good, and the standard curve can be used for quantitative calculation of flavonoid compounds in a sample.
TABLE 4 optimization of the Process for extracting the flavonoids from the xylooligosaccharide hydrolysis solution in buckwheat grains
The optimized extraction result of the xylooligosaccharide hydrolysate of the buckwheat flavone is shown in the table, and when the feed liquid ratio is from 1:5 to 1:30, the flavone extraction rate is reduced from 0.101mg/mL to 0.056mg/mL. The optimal extraction process of the xylooligosaccharide hydrolysate of the buckwheat flavone is obtained through process optimization: in the feed liquid ratio of 1:20 for 3h, a flavone content of 0.10mg/mL was obtained.
Example 6 enzymatic extraction of Fagopyrum Esculenti flavone
1. Optimization of buckwheat flavone enzymatic extraction process
The method is characterized in that crushed buckwheat grains are used as raw materials, and an enzymatic extraction process is adopted to optimize the extraction process of flavonoid compounds in the buckwheat grains. The extraction conditions under different cellulase addition amounts are as follows: extraction temperature: 50 ℃; rotational speed: 150rpm; feed liquid ratio: 1:20 (xylooligosaccharide hydrolysate containing 5% ethanol); extraction time: 1h,2h,3h. The cellulase is crude enzyme liquid (prepared in example 2) produced by fermenting Trichoderma longibrachiatum LC-M4 strain, and the addition amount of the cellulase is as follows: 2. 4, 6, 8IU/ml.
2. Experimental results
Table 5 enzymatic extraction of buckwheat flavonoids
When the enzyme extraction process is used for extracting the buckwheat flavone, under the conditions that the addition amount of the cellulase is 2IU/g of substrate and the extraction time is 1h, the flavone extraction rate is highest and is 3.27mg/g of seed, and the extraction time is reduced while the extraction time is increased by 30 times when the extraction time is higher than that of the buckwheat flavone of the xylooligosaccharide hydrolysate which is 0.10mg/g (2 h).
Example 7 preparation of buckwheat xylo-oligosaccharide health aromatic Vinegar and buckwheat xylo-oligosaccharide and flavone health aromatic Vinegar and Nuclear pore Membrane filtration Process
The traditional vinegar spraying process adopts tap water to directly spray vinegar to mature vinegar grains, and adopts the buckwheat xylo-oligosaccharide extracting solution and the buckwheat xylo-oligosaccharide and flavone extracting solution to spray vinegar, so that the buckwheat health-care aromatic vinegar spraying process is optimized at first to improve the vinegar spraying efficiency and the product quality.
In the invention, mature vinegar grains are directly used for vinegar leaching by using the buckwheat straw xylooligosaccharide extract (prepared under the optimal condition of the embodiment 3) and the buckwheat xylooligosaccharide and flavone extract (prepared under the optimal condition of the embodiment 6), and the material-liquid ratio and the time parameters in the vinegar leaching process are optimized. The optimization gradient of the material liquid ratio parameter in the vinegar spraying process is 1:1. 1:1.3, 1: 1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.9, 1:2 and 1:2.5 And (w/v), optimizing the gradient of the vinegar pouring time to be 12h,18h,24h and 30h, and evaluating the optimizing parameters by measuring the pH and total acid indexes of the raw vinegar obtained under different vinegar pouring process conditions.
TABLE 6 influence of the specific parameters of the vinegar-leaching liquor on the total acid content and pH of raw vinegar
The effect of different vinegar leaching process parameters on the raw vinegar pH and total acid evaluation index is shown in Table 6. From the optimization result of the feed liquid ratio, it can be seen that when the feed liquid ratio is 1: the total acid is highest (5.08%) at 1, the total acid gradually decreases with the increase of the liquid consumption, and the pH value also increases, but when the liquid-to-liquid ratio is more than 1:1.7, the total acid content is 3.43%, the total acid value is lower than the requirement that the total acid is more than or equal to 3.5% in national standard (GB/T18187-2000 brewing vinegar), so when the feed-liquid ratio is more than 1:1.7, the raw vinegar obtained by leaching does not reach the standard, and the feed liquid ratio is less than 1: the added liquid in the step 1 can not fully submerge the vinegar grains and influence the quality and flavor of the original vinegar, so that the optimal feed-liquid ratio when the buckwheat straw xylooligosaccharide hydrolysate is directly used for vinegar leaching is 1:1.4-1:1.6 (w/v).
TABLE 7 influence of the parameters of the time of spraying vinegar on the total acid content and pH of raw vinegar
The effect of different vinegar leaching time parameters on the total acid content and pH value of the raw vinegar is shown in Table 7. When the time parameter is optimized, the total acid content is 3.21% and the total acid value is lower than the requirement that the total acid is more than or equal to 3.5% specified in national standard (GB/T18187-2000 brewing vinegar) when the vinegar spraying time is 12h, so that the raw vinegar obtained when the vinegar spraying time is less than 12h does not reach the standard, when the vinegar spraying time is 18-30h, the total acid content is more than 4%, and the total acid content is the highest (4.26%) under the vinegar spraying time of 24h, so that the optimal vinegar spraying time is 24h.
TABLE 8 influence of the temperature parameters of the vinegar leaching on the total acid content and pH value of the raw vinegar
The effect of different temperature parameters of the vinegar is shown in Table 8. For temperature parameter optimization, when the temperature of the vinegar is in the range of 4 ℃ to 40 ℃, the total acid content is above 3.5%, and the pH is below 3.71, which shows that the vinegar can be sprayed by the hydrolysis liquid of the buckwheat straw xylooligosaccharide at the temperature of 4 ℃ to 40 ℃ to develop the production and preparation of the xylooligosaccharide buckwheat health-care aromatic vinegar.
The xylooligosaccharide health-care buckwheat aromatic vinegar which is leached under the optimal vinegar leaching process condition is subjected to filtering clarification treatment by adopting nuclear pore membrane materials with different pore diameters (the nuclear pore membrane materials used in the process are commercially available, the manufacturer is Wu Weike near new technology responsibility company, the trade name is nuclear pore membrane (the pore size specification is 2-3 μm)), and the filtering effect is shown in figure 5. The raw solution of the xylo-oligosaccharide buckwheat aromatic vinegar which is not subjected to filtration treatment contains a large amount of suspended cereal particles and other fermentation impurities, and macroscopic flocculent precipitates are generated after the raw solution is stationary for a period of time (Yang Fulian, xiaoyan. The buckwheat vinegar is clarified research [ J ]. Chinese seasoning, 2015,40 (07): 96-99+103.), but the raw solution of the buckwheat aromatic vinegar which is subjected to nuclear pore membrane filtration treatment is transparent and clear, has no macroscopic particles and suspended matter precipitates, and is still transparent and clear after the raw solution is stationary for 30 days, and no precipitates are generated. The nuclear pore membrane materials with the diameters of 2 mu m and 3 mu m selected in the invention have equivalent filtering effects, but the nuclear pore membrane material with the diameter of 2 mu m has lower filtering flux and efficiency due to smaller pore diameter. Therefore, the invention selects the nuclear pore membrane material with the aperture of 3 mu m to carry out the filtration treatment of the xylo-oligosaccharide health-care buckwheat aromatic vinegar product.
Finally, performing pasteurization (heating to 85-90 ℃ for 30 min), canning, sealing, code spraying, labeling and the like on the core-pore membrane filtration and qualified xylo-oligosaccharide health-care buckwheat aromatic vinegar stock solution to prepare the buckwheat aromatic vinegar with the packaging specification of 125mL, thereby obtaining the xylo-oligosaccharide buckwheat aromatic vinegar product with health-care effect (wherein the xylo-oligosaccharide content is ensured to be 0.7-1.2g/125 mL).
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims (10)

1. Trichoderma longibrachiatum glucose-resistant high-yield cellulase strain, latin name:Trichoderma longibrachiatum，the strain is named as LC-M4.21 and is preserved in China general microbiological culture Collection center (China) with a preservation number of 2021, 8 and 11: CGMCC NO 23206.
2. A method of producing xylanase and cellulase comprising the steps of:
fermenting the glucose-repressed high-yield cellulase strain LC-M4.21 of Trichoderma longibrachiatum of claim 1 to obtain a fermentation broth containing xylanase and cellulase.
3. The method according to claim 2, characterized in that: the method for producing xylanase and cellulase comprises the following steps: inoculating the glucose-resistant high-yield cellulase strain LC-M4.21 of trichoderma longibrachiatum of claim 1 to a seed culture medium, culturing, inoculating the seed solution to a fermentation enzyme production culture medium after culturing, and fermenting to obtain a fermentation broth containing xylanase and cellulase.
4. A method according to claim 3, characterized in that: the formula of the seed culture medium is as follows: 5-15g of glucose, 3-7g of peptone, 1-5 mL of Tween-80, (NH) 4 ) 2 SO 4 1-3 g，CaCl 2 0.1-0.5g, urea 0.1-0.5g, mgSO 4 . 7H 2 O 0.1-0.5 g，FeSO 4 . 7H2O0.003-0.007 g，MnSO 4 . H 2 O 0.001-0.002 g，ZnSO 4 . 7H 2 O 0.001-0.002 g，CoCl 2 0.001-0.003 g, and the pH is natural;
the formula of the fermentation enzyme production medium comprises the following components: microcrystalline cellulose 10-30-g, bran 5-15-g, corn steep liquor 15-25 g, K 2 HPO 4 1-4 g，(NH 4 ) 2 SO 4 1-3 g，MgSO 4 . 7H 2 0.1-0.5. 0.5g g of O, 0.1-0.5g of urea and CaCl 2 0.1-0.5. 0.5g, tween-80-5 mL, feSO 4 . 7H2O 0.003-0.007 g，MnSO 4 . H 2 O 0.001-0.002 g，ZnSO 4 . 7H 2 O 0.001-0.002 g，CoCl 2 0.001-0.003 g, pH natural value;
the culture conditions are as follows: culturing at 25-35deg.C and 150-300 rpm for 12-36 h;
Inoculating the seed solution into a fermentation enzyme production culture medium according to the proportion of 2-10%;
the fermentation conditions are as follows: fermenting at 25-35deg.C and 150-300 rpm for 6-10 d.
5. The use of the trichoderma longibrachiatum glucose-resistant high-yield cellulase strain LC-M4.21 or the xylanase and cellulase-containing fermentation broth prepared by the method of any one of claims 2-4 in the preparation of straw xylooligosaccharide by direct enzymolysis of straw.
6. A method for preparing buckwheat straw xylo-oligosaccharide comprises the following steps:
taking buckwheat straw as a raw material, and carrying out enzymolysis on a fermentation broth containing xylanase and cellulase prepared by adopting the glucose-resistant high-yield cellulase strain LC-M4.21 or the method of any one of claims 2-4 by using the trichoderma longibrachiatum to obtain an enzymolysis solution containing the buckwheat straw xylooligosaccharide.
7. The method according to claim 6, wherein:
the addition amount of xylanase is 260-1600 IU/g straw;
the ratio of feed liquid adopted in the enzymolysis is 1:6-1:12, w: v;
the enzymolysis time is 12-48 hours;
the concentration of the xylooligosaccharide in the enzymatic hydrolysate containing the buckwheat straw xylooligosaccharide is 5.6-9.6g/L.
8. A method for extracting buckwheat flavone comprises the following steps (1) or (2):
(1) Extracting flavone from the buckwheat grains serving as a raw material by using the xylooligosaccharide hydrolysate, namely the enzymatic hydrolysate containing the straw xylooligosaccharide prepared by the method of claim 6 or 7, so as to obtain a buckwheat xylooligosaccharide and flavone extracting solution;
wherein the concentration of the xylooligosaccharide hydrolysis liquid is 5.6-9.6g/L;
the ratio of flavone extraction liquid is 1:5-1:30w/v; the extraction time is 0.5-3h;
the flavone extraction concentration is 0.04-0.1mg/ml;
(2) Taking buckwheat grains as a raw material, and using ethanol-containing xylooligosaccharide hydrolysate, wherein the xylooligosaccharide hydrolysate is the enzyme hydrolysate containing straw xylooligosaccharide prepared in the method of claim 6 or 7, and extracting flavonoid compounds in the buckwheat grains by adopting an enzymatic extraction process to obtain buckwheat xylooligosaccharide and flavone extracting solution;
wherein the enzyme is crude enzyme liquid produced by fermenting Trichoderma longibrachiatum LC-M4 strain, namely, the fermentation liquid containing xylanase and cellulase prepared in the method of claim 2 or 3, and the addition amount of the cellulase is as follows: 2-8IU/ml;
the extraction conditions are as follows: extraction temperature: 50 ℃; rotational speed: 150rpm; feed liquid ratio: 1:20, a step of; extraction time: and 1-3h.
9. A preparation method of buckwheat xylo-oligosaccharide or xylo-oligosaccharide and flavone health aromatic vinegar comprises the following steps (1) or (2) or (3):
(1) Spraying vinegar on mature vinegar grains by adopting the enzymolysis liquid containing the straw xylo-oligosaccharide, namely the hydrolysis liquid of the buckwheat straw xylo-oligosaccharide, which is obtained in the claim 6 or 7, and filtering and clarifying the sprayed xylo-oligosaccharide health-care buckwheat aromatic vinegar by adopting a nuclear pore membrane material to obtain the xylo-oligosaccharide health-care aromatic vinegar;
(2) Spraying vinegar on mature vinegar grains by adopting the buckwheat xylooligosaccharide and flavone extracting solution prepared in the step (1) or (2) in the step (8), and filtering and clarifying the sprayed xylooligosaccharide and flavone health-care buckwheat aromatic vinegar by adopting a nuclear pore membrane material to obtain the buckwheat xylooligosaccharide and flavone health-care aromatic vinegar;
(3) Spraying vinegar on mature vinegar grains by adopting the enzymatic hydrolysate containing the straw xylo-oligosaccharide, namely the buckwheat straw xylo-oligosaccharide hydrolysate, prepared by the method of claim 6 or 7, adding buckwheat kernel flavone extract into the sprayed xylo-oligosaccharide health-care buckwheat aromatic vinegar, and filtering and clarifying the obtained xylo-oligosaccharide and flavone health-care buckwheat aromatic vinegar by adopting a nuclear pore membrane material to obtain the buckwheat vinegar;
in the step (3), the buckwheat kernel flavone extracting solution is prepared by ethanol extraction;
the buckwheat kernel flavone extract is added according to the volume ratio of 0.5-1%.
10. The method according to claim 9, wherein: the feed liquid ratio in the vinegar spraying process is 1:1-1:1.7, w/v;
The vinegar spraying time is 18 h-30 h;
the temperature of vinegar spraying is 4-40 ℃;
the aperture of the nuclear pore membrane material is 2 mu m-3 mu m.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102787078A (en) * 2012-08-06 2012-11-21 广西大学 Trichoderma koningiopsis strain and application of trichoderma koningiopsis to preparation of cellulase
CN103131639A (en) * 2011-11-23 2013-06-05 中国农业科学院作物科学研究所 Trichoderma longibrachiatum strain and application thereof
RU2654564C1 (en) * 2017-03-10 2018-05-21 Аркадий Пантелеймонович Синицын Strain of trichoderma longibrachiatum tw-14-220 filamentous fungus - producer of cellulases, beta-glucanases and xylanases for feed production and a method for obtaining a feed complex enzyme preparation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103131639A (en) * 2011-11-23 2013-06-05 中国农业科学院作物科学研究所 Trichoderma longibrachiatum strain and application thereof
CN102787078A (en) * 2012-08-06 2012-11-21 广西大学 Trichoderma koningiopsis strain and application of trichoderma koningiopsis to preparation of cellulase
RU2654564C1 (en) * 2017-03-10 2018-05-21 Аркадий Пантелеймонович Синицын Strain of trichoderma longibrachiatum tw-14-220 filamentous fungus - producer of cellulases, beta-glucanases and xylanases for feed production and a method for obtaining a feed complex enzyme preparation

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