CN105368906A - Microwave-assisted method for preparing cassava leaf protein polypeptides - Google Patents
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a microwave-assisted method for preparing cassava leaf protein polypeptides. The microwave-assisted method for preparing the cassava leaf protein polypeptides includes steps of cleaning, airing, chopping, dilute alkali liquor adding, colloid milling, primary microwave treatment, coarse filtration, primary enzymatic hydrolysis, secondary microwave treatment, secondary enzymatic hydrolysis, tertiary microwave treatment, enzyme inactivation, decolorization, micro-filtration, ultra-filtration, concentration and spray drying. The microwave-assisted method for extracting the cassava leaf protein polypeptides has the advantages of high yield and purity, low solvent consumption and cost and easiness in industrial production. Besides, the microwave-assisted method is short in integral technological process and time, low in energy consumption, little in pollution and free of pollutant emission, toxic solvents can be omitted, and accordingly the purpose of clean production can be achieved.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of method that microwave-assisted prepares cassava leaves protein polypeptide.
Background technology
Cassava (ManihotesculentaCrantz), Euphorbiaceae casava, originate from tropical America, have the laudatory title of " underground granary ", " king of starch " and " energy crop ", being one of large tuber crops in the world three, is the important Fodder and food crop in subtropical and tropical zones and renewable energy source-alcohol main raw material.At present, more than 500,000,000 populations are also had to be that main food is depended on for existence with cassava in the world.
Cassava is mainly distributed in the states such as Brazil, Mexico, Nigeria, Bolivia, Thailand, Colombia, Indonesia.China is in the introducing culture twenties in 19th century, and be now distributed widely in South China, the cultivated area in Guangdong and Guangxi is maximum, takes second place in Fujian and Taiwan, and Yunnan, Guizhou, Sichuan, Hunan, Jiangxi etc. are economized also a small amount of cultivation.
Tapioca root rich in starch 25-35%, in its starch, amylose starch accounts for 17%, and amylopectin accounts for 83%.This special starch is light industry important raw and processed materials, nearly 2000 multiple products can be produced with it, comprising three major types: one is modified starch, is widely used in the industries such as weaving, papermaking, medicine, food, building materials, casting, light industry, oil, the energy, feed, agricultural, environmental protection, biotechnology; Two is Chemicals, alcohol, polyethylene, acetic acid, oxyethane, sorbyl alcohol, thanomin, citric acid etc. are had with organic chemical industry's product that tapioca (flour) is produced, it is the important source material of producing rubber, agricultural chemicals, grease, packaged product, makeup, military supplies, particularly alcohol is the interpolation fuel in gasoline, market potential is considerable, the substitute at present in China and external other countries as gasoline; Three is β-amyloses, tapioca (flour) can produce glucose, high fructose syrup, fructose, maltose, high wheat sugar, and the sugar of the tens of kinds of different sugarinesses such as oligose, oligosaccharides, different oligosaccharides, trehalose is emerging protective foods, become the new lover on international candy market, have the very strong market competitiveness.As can be seen here, the industrial value of tapioca (flour) is very high, but limits throughput, therefore genus-kind of product in short supply in international trade.According to statistics, cassava world year volume of trade accounts for 10% of ultimate production, and major product has dry plate, particle and tapioca (flour).China, Japan, cassava product main body entrance state of the Deng Guoshi world of the U.S., account for the 70-80% of its total trade volume.Thailand is cassava products export state the biggest in the world, the state-owned Indonesia of other main exits and Vietnam.
Guangxi is located in south subtropics, and the tropic of Cancer traverses within the border, high temperature and rainy, and rain heat same season, have hotwork district area 11.4 ten thousand square kilometres, account for 38.5% of the national hotwork district total area, position is the first in the nation, and is the major area that China most appropriate development cassava produces.According to statistics, Guangxi cassava cultivated area accounts for more than 60% of the whole nation, and fresh tuber yield accounts for more than 70%, 2015 of the whole nation, and cassava cultivated area reaches 4,200,000 mu, and predict that fresh potato ultimate production reaches 8,400,000 tons, the output value reaches 5,000,000,000 yuan.
Current, cassava major country of production of world overwhelming majority in cassava comprehensive utilization exploitation only rests in tapioca (flour), very few to the comprehensive utilization development research of a large amount of byproduct produced in cassava production process as Cassava stalk, fresh cassava leaf, manioc waste, cassava skin the like waste, part can be said and also be in blank, these resources gone out of use some still belong to-kind be worth very high resource, they are arbitrarily lost, not only can cause the waste of resource, also can cause the pollution of ecotope.Fresh cassava blade is exactly one of them.According to laboratory test results, fresh fresh cassava leaf protein content is generally between 4.0-9.6%, and most high-content can reach 11.8%, and general content is at 20.6-36.4% in cured leaf, and most high-content can reach 39.9%.Its whole amino acid whose content is 8.42-9.4%, indispensable amino acid average out to 4.21%, account for about 50%, except methionine(Met) is comparatively lower than except critical level, other primary amino acid are quite abundant, and wherein carotene, robust fibre, crude protein, calcium, VITAMIN and trace element are all abundant, can compare favourably with overwhelming majority torrid zone leguminous forage, even better, its precedence is close with Alfalfa, can raise grass carp, goat and silkworm.Because cassava leaves albumen generally exists with macromolecular structure form, organism digestive utilization ratio of directly searching for food is often not high, and it is not wide without its application surface of deep processing, added value is not high yet, therefore, develop a kind of novel method of the protein polypeptide produced in cassava wastes, not only can turn waste into wealth, also for high-quality feed, food, medicine, health products trade provide and enrich peptide matters, promote the well-being of mankind.
The information being disclosed in this background technology part is only intended to increase the understanding to general background of the present invention, and should not be regarded as admitting or imply in any form that this information structure has been prior art that persons skilled in the art are known.
Summary of the invention
In order to overcome the deficiencies in the prior art, the invention provides a kind of method that microwave-assisted prepares cassava leaves protein polypeptide.
For solving the problems of the technologies described above, the present invention by the following technical solutions:
Microwave-assisted prepares a method for cassava leaves protein polypeptide, comprises the steps:
(1) get fresh cassava leaves, through cleaning, after airing, be chopped into fragment;
(2) get fresh cassava leaf fragment and add sig water by solid-to-liquid ratio 1:2, and add stablizer, adjust pH is 8.5, feed liquid is placed in colloidal mill defibrination, coarse filtration, discards filter residue, be heated to 55 DEG C to microwave extraction device, keep 1h at this temperature, obtain fresh cassava leaf and extract coarse filtration liquid;
(3) enzymolysis 1: above-mentioned fresh cassava leaf is extracted coarse filtration liquid and is down to room temperature, adjust its pH value to be 8.5, add trypsinase by 3% of substrate quality, abundant stirring, start microwave extraction device heating 20min, pH value remains on more than 7.5, obtains first time enzymolysis solution;
(4) enzymolysis 2: treat that enzymolysis solution is down to normal temperature for the first time, then adjust its pH value to 7.6, add neutral enzymatic by substrate quality 2%, fully stir, again start microwave extraction device heating 10min, when pH value is down to 7.2, enzymolysis completes;
(5) after enzymolysis completes, again start microwave extraction device and heat to 90 DEG C, maintain 10min, the enzyme that goes out is lived and is namely completed;
(6) in the fresh cassava leaf protein enzymolysis solution of enzyme work that goes out, gac is added by substrate 3%, stir, 1h is kept at 70 DEG C, then tripod pendulum type batch centrifugal elimination sediment is used, filtrations concentrated solution conjunction And through ceramic micro filter membrane filtration, ultrafiltration, sodium filter, RO Fibrous membrane filtration, concentrated, is obtained cassava leaves protein polypeptide concentrated solution by filtrate again;
(7) by gained cassava leaves protein polypeptide concentrated solution, vacuum spray drying, namely obtains cassava leaves protein polypeptide.
As preferably, the sig water described in step (2) is NaOH solution, and its add-on can make the pH value of feed liquid be adjusted to 8.5-9.0 weakly alkaline; Described stablizer is sodium bisulfite, and its add-on accounts for the 0.1-0.2% of cassava leaves quality.
As preferably, the hydrolysis temperature in step (3) and step (4) is 55 DEG C.
As preferably, step (3) neutral and alkali enzymolysis pH value is 8.0-7.5.
As preferably, neutral enzymolysis pH value >=7.5 in step (4).
As preferably, the neutral enzymatic described in step (4) is papain.
As preferably, the frequency of step (2), (3), (4) and the microwave extraction device described in (5) is 2450MHZ, and power is 20KW.
As preferably, the filter cloth of the tripod pendulum type batch centrifugal described in step (6) is more than 200 orders.
As preferably, described in step (6), ceramic microfiltration membrane is the ceramic membrane that can retain 15000-20000 molecular weight; Described ultrafiltration, sodium filter, RO tunica fibrosa are the tunica fibrosa that can retain 10000,2000,1000, more than 200 molecular weight respectively.
Compared with prior art, the present invention has following beneficial effect:
1. the method for the cassava leaves protein polypeptide of the present invention's extraction has the advantage that yield is high, purity is high, solvent consumption is few, cost is low and be easy to suitability for industrialized production.
2. the whole technical process of the present invention is short, the time is short, energy consumption is low, pollution less, does not use toxic reagent, non-pollutant discharge, reaches cleaner production target.
Accompanying drawing explanation
Fig. 1 is the process flow sheet that microwave-assisted of the present invention prepares cassava leaves protein polypeptide.
Embodiment
Below in conjunction with specific embodiment, elaboration detailed is further done to the present invention, but embodiments of the present invention are not limited to the scope that embodiment represents.These embodiments only for illustration of the present invention, but not for limiting the scope of the invention.In addition, after reading content of the present invention, those skilled in the art can do various amendment to the present invention, and these equivalent variations fall within appended claims limited range of the present invention equally.
The experimental technique used in following embodiment if no special instructions, is ordinary method.The material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
embodiment 1:
Microwave-assisted prepares a method for cassava leaves protein polypeptide, comprises the steps:
(1) get fresh cassava leaves, through cleaning, after airing, be chopped into fragment;
(2) get fresh cassava leaf fragment and add sig water by solid-to-liquid ratio 1:2, and add stablizer, adjust pH is 8.5, feed liquid is placed in colloidal mill defibrination, coarse filtration, discards filter residue, be heated to 55 DEG C to microwave extraction device, keep 1h at this temperature, obtain fresh cassava leaf and extract coarse filtration liquid; Described sig water is NaOH solution, and its add-on can make the pH value of feed liquid be adjusted to 8.5 weakly alkalines; Described stablizer is sodium bisulfite, and its add-on accounts for 0.1% of cassava leaves quality; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(3) enzymolysis 1: above-mentioned fresh cassava leaf is extracted coarse filtration liquid and is down to room temperature, adjust its pH value to be 8.5, add trypsinase by 3% of substrate quality, abundant stirring, start microwave extraction device heating 20min, pH value remains on more than 7.5, obtains first time enzymolysis solution; Hydrolysis temperature is 55 DEG C; Enzymolysis pH value is 8.0; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(4) enzymolysis 2: treat that enzymolysis solution is down to normal temperature for the first time, then adjust its pH value to 7.6, add neutral enzymatic by substrate quality 2%, fully stir, again start microwave extraction device heating 10min, when pH value is down to 7.2, enzymolysis completes; Hydrolysis temperature is 55 DEG C; Enzymolysis pH value >=7.5; Described neutral enzymatic is papain; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(5) after enzymolysis completes, again start microwave extraction device and heat to 90 DEG C, maintain 10min, the enzyme that goes out is lived and is namely completed; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(6) in the fresh cassava leaf protein enzymolysis solution of enzyme work that goes out, gac is added by substrate 3%, stir, 1h is kept at 70 DEG C, then tripod pendulum type batch centrifugal elimination sediment is used, filtrations concentrated solution conjunction And through ceramic micro filter membrane filtration, ultrafiltration, sodium filter, RO Fibrous membrane filtration, concentrated, is obtained cassava leaves protein polypeptide concentrated solution by filtrate again; The filter cloth of described tripod pendulum type batch centrifugal is more than 200 orders; Described ceramic microfiltration membrane is the ceramic membrane that can retain 15000-20000 molecular weight; Described ultrafiltration, sodium filter, RO tunica fibrosa are the tunica fibrosa that can retain 10000,2000,1000, more than 200 molecular weight respectively;
(7) by gained cassava leaves protein polypeptide concentrated solution, vacuum spray drying, namely obtains cassava leaves protein polypeptide.
embodiment 2:
Microwave-assisted prepares a method for cassava leaves protein polypeptide, comprises the steps:
(1) get fresh cassava leaves, through cleaning, after airing, be chopped into fragment;
(2) get fresh cassava leaf fragment and add sig water by solid-to-liquid ratio 1:2, and add stablizer, adjust pH is 8.5, feed liquid is placed in colloidal mill defibrination, coarse filtration, discards filter residue, be heated to 55 DEG C to microwave extraction device, keep 1h at this temperature, obtain fresh cassava leaf and extract coarse filtration liquid; Described sig water is NaOH solution, and its add-on can make the pH value of feed liquid be adjusted to 9.0 weakly alkalines; Described stablizer is sodium bisulfite, and its add-on accounts for 0.2% of cassava leaves quality; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(3) enzymolysis 1: above-mentioned fresh cassava leaf is extracted coarse filtration liquid and is down to room temperature, adjust its pH value to be 8.5, add trypsinase by 3% of substrate quality, abundant stirring, start microwave extraction device heating 20min, pH value remains on more than 7.5, obtains first time enzymolysis solution; Hydrolysis temperature is 55 DEG C; Enzymolysis pH value is 7.5; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(4) enzymolysis 2: treat that enzymolysis solution is down to normal temperature for the first time, then adjust its pH value to 7.6, add neutral enzymatic by substrate quality 2%, fully stir, again start microwave extraction device heating 10min, when pH value is down to 7.2, enzymolysis completes; Hydrolysis temperature is 55 DEG C; Enzymolysis pH value >=7.5; Described neutral enzymatic is papain; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(5) after enzymolysis completes, again start microwave extraction device and heat to 90 DEG C, maintain 10min, the enzyme that goes out is lived and is namely completed; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(6) in the fresh cassava leaf protein enzymolysis solution of enzyme work that goes out, gac is added by substrate 3%, stir, 1h is kept at 70 DEG C, then tripod pendulum type batch centrifugal elimination sediment is used, filtrations concentrated solution conjunction And through ceramic micro filter membrane filtration, ultrafiltration, sodium filter, RO Fibrous membrane filtration, concentrated, is obtained cassava leaves protein polypeptide concentrated solution by filtrate again; The filter cloth of described tripod pendulum type batch centrifugal is more than 200 orders; Described ceramic microfiltration membrane is the ceramic membrane that can retain 15000-20000 molecular weight; Described ultrafiltration, sodium filter, RO tunica fibrosa are the tunica fibrosa that can retain 10000,2000,1000, more than 200 molecular weight respectively;
(7) by gained cassava leaves protein polypeptide concentrated solution, vacuum spray drying, namely obtains cassava leaves protein polypeptide.
embodiment 3:
Microwave-assisted prepares a method for cassava leaves protein polypeptide, comprises the steps:
(1) get fresh cassava leaves, through cleaning, after airing, be chopped into fragment;
(2) get fresh cassava leaf fragment and add sig water by solid-to-liquid ratio 1:2, and add stablizer, adjust pH is 8.5, feed liquid is placed in colloidal mill defibrination, coarse filtration, discards filter residue, be heated to 55 DEG C to microwave extraction device, keep 1h at this temperature, obtain fresh cassava leaf and extract coarse filtration liquid; Described sig water is NaOH solution, and its add-on can make the pH value of feed liquid be adjusted to 8.8 weakly alkalines; Described stablizer is sodium bisulfite, and its add-on accounts for 0.15% of cassava leaves quality; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(3) enzymolysis 1: above-mentioned fresh cassava leaf is extracted coarse filtration liquid and is down to room temperature, adjust its pH value to be 8.5, add trypsinase by 3% of substrate quality, abundant stirring, start microwave extraction device heating 20min, pH value remains on more than 7.5, obtains first time enzymolysis solution; Hydrolysis temperature is 55 DEG C; Enzymolysis pH value is 7.8; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(4) enzymolysis 2: treat that enzymolysis solution is down to normal temperature for the first time, then adjust its pH value to 7.6, add neutral enzymatic by substrate quality 2%, fully stir, again start microwave extraction device heating 10min, when pH value is down to 7.2, enzymolysis completes; Hydrolysis temperature is 55 DEG C; Enzymolysis pH value >=7.5; Described neutral enzymatic is papain; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(5) after enzymolysis completes, again start microwave extraction device and heat to 90 DEG C, maintain 10min, the enzyme that goes out is lived and is namely completed; The frequency of described microwave extraction device is 2450MHZ, and power is 20KW;
(6) in the fresh cassava leaf protein enzymolysis solution of enzyme work that goes out, gac is added by substrate 3%, stir, 1h is kept at 70 DEG C, then tripod pendulum type batch centrifugal elimination sediment is used, filtrations concentrated solution conjunction And through ceramic micro filter membrane filtration, ultrafiltration, sodium filter, RO Fibrous membrane filtration, concentrated, is obtained cassava leaves protein polypeptide concentrated solution by filtrate again; The filter cloth of described tripod pendulum type batch centrifugal is more than 200 orders; Described ceramic microfiltration membrane is the ceramic membrane that can retain 15000-20000 molecular weight; Described ultrafiltration, sodium filter, RO tunica fibrosa are the tunica fibrosa that can retain 10000,2000,1000, more than 200 molecular weight respectively;
(7) by gained cassava leaves protein polypeptide concentrated solution, vacuum spray drying, namely obtains cassava leaves protein polypeptide.
embodiment4: the extracting method of conventional leaf albumen
Get the plant leaf of about 0.3g, by liquid nitrogen grinding, what pigment was many can add PVP by 10% of material fresh weight, and proceeds in centrifuge tube by fully ground sample, adds the Extraction buffer of 4mL, shakes up and extracts 1h, abundant soluble protein under being placed on 4 DEG C of conditions.Sample after placement fully shakes up, and 4 DEG C of centrifugal 30min of 10000r/min, abandon precipitation, spends the night, allow albumen fully precipitate in supernatant liquor under adding-20 DEG C, the village condition of 2.5-3 times of volume.Afterwards, 4 DEG C of centrifugal 30min of 10000r/min, its supernatant, at precipitation is placed in-20 DEG C, makes acetone volatilize completely, adds sample-loading buffer dissolution precipitation if necessary.Damping fluid consumption 300ul, precipitation is transferred in 1.5mL centrifuge tube, 4 DEG C of centrifugal 15min of 12000r/min, gets supernatant liquor and be extracted leaf protein solution after fully dissolving.
The beneficial effect of table 1 extracting method of the present invention
As known from Table 1, above-mentioned traditional method unavoidably uses relatively large soda acid and solvent, and complex procedures, length consuming time, weakness and the deficiencies such as extraction yield is low, and impurity is many; And Bian microwave radiation exaraction of the present invention, do not need solvent, soda acid consumption is few, and enzymolysis time is short, and transformation efficiency is high, and prepares polypeptide for next step and create better condition.
The aforementioned description to concrete exemplary of the present invention is to illustrate and the object of illustration.These descriptions not want the present invention to be defined as disclosed precise forms, and obviously, according to above-mentioned instruction, can much change and change.The object selected exemplary embodiment and describe is to explain certain principles of the present invention and practical application thereof, thus those skilled in the art can be realized and utilize various different exemplary of the present invention and various different selection and change.Scope of the present invention is intended to limited by claims and equivalents thereof.
Claims (9)
1. microwave-assisted prepares a method for cassava leaves protein polypeptide, it is characterized in that, comprises the steps:
(1) get fresh cassava leaves, through cleaning, after airing, be chopped into fragment;
(2) get fresh cassava leaf fragment and add sig water by solid-to-liquid ratio 1:2, and add stablizer, adjust pH is 8.5, feed liquid is placed in colloidal mill defibrination, coarse filtration, discards filter residue, be heated to 55 DEG C to microwave extraction device, keep 1h at this temperature, obtain fresh cassava leaf and extract coarse filtration liquid;
(3) enzymolysis 1: above-mentioned fresh cassava leaf is extracted coarse filtration liquid and is down to room temperature, adjust its pH value to be 8.5, add trypsinase by 3% of substrate quality, abundant stirring, start microwave extraction device heating 20min, pH value remains on more than 7.5, obtains first time enzymolysis solution;
(4) enzymolysis 2: treat that enzymolysis solution is down to normal temperature for the first time, then adjust its pH value to 7.6, add neutral enzymatic by substrate quality 2%, fully stir, again start microwave extraction device heating 10min, when pH value is down to 7.2, enzymolysis completes;
(5) after enzymolysis completes, again start microwave extraction device and heat to 90 DEG C, maintain 10min, the enzyme that goes out is lived and is namely completed;
(6) in the fresh cassava leaf protein enzymolysis solution of enzyme work that goes out, gac is added by substrate 3%, stir, 1h is kept at 70 DEG C, then tripod pendulum type batch centrifugal elimination sediment is used, filtrations concentrated solution conjunction And through ceramic micro filter membrane filtration, ultrafiltration, sodium filter, RO Fibrous membrane filtration, concentrated, is obtained cassava leaves protein polypeptide concentrated solution by filtrate again;
(7) by gained cassava leaves protein polypeptide concentrated solution, vacuum spray drying, namely obtains cassava leaves protein polypeptide.
2. microwave-assisted according to claim 1 prepares the method for cassava leaves protein polypeptide, it is characterized in that, the sig water described in step (2) is NaOH solution, and its add-on can make the pH value of feed liquid be adjusted to 8.5-9.0 weakly alkaline; Described stablizer is sodium bisulfite, and its add-on accounts for the 0.1-0.2% of cassava leaves quality.
3. microwave-assisted according to claim 1 prepares the method for cassava leaves protein polypeptide, it is characterized in that, the hydrolysis temperature in step (3) and step (4) is 55 DEG C.
4. microwave-assisted according to claim 1 prepares the method for cassava leaves protein polypeptide, it is characterized in that, step (3) neutral and alkali enzymolysis pH value is 8.0-7.5.
5. microwave-assisted according to claim 1 prepares the method for cassava leaves protein polypeptide, it is characterized in that, neutral enzymolysis pH value >=7.5 in step (4).
6. microwave-assisted according to claim 1 prepares the method for cassava leaves protein polypeptide, it is characterized in that, the neutral enzymatic described in step (4) is papain.
7. microwave-assisted according to claim 1 prepares the method for cassava leaves protein polypeptide, it is characterized in that, the frequency of step (2), (3), (4) and the microwave extraction device described in (5) is 2450MHZ, and power is 20KW.
8. microwave-assisted according to claim 1 prepares the method for cassava leaves protein polypeptide, it is characterized in that, the filter cloth of the tripod pendulum type batch centrifugal described in step (6) is more than 200 orders.
9. microwave-assisted according to claim 1 prepares the method for cassava leaves protein polypeptide, it is characterized in that, described in step (6), ceramic microfiltration membrane is the ceramic membrane that can retain 15000-20000 molecular weight; Described ultrafiltration, sodium filter, RO tunica fibrosa are the tunica fibrosa that can retain 10000,2000,1000, more than 200 molecular weight respectively.
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| CN114107419A (en) * | 2021-12-06 | 2022-03-01 | 中国热带农业科学院热带作物品种资源研究所 | Cassava leaf polypeptide and preparation method and application thereof |
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