CN114686553A - Maca and okra peptide composite polysaccharide polypeptide and extraction method and application thereof - Google Patents

Maca and okra peptide composite polysaccharide polypeptide and extraction method and application thereof Download PDF

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CN114686553A
CN114686553A CN202210415768.XA CN202210415768A CN114686553A CN 114686553 A CN114686553 A CN 114686553A CN 202210415768 A CN202210415768 A CN 202210415768A CN 114686553 A CN114686553 A CN 114686553A
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maca
okra
fruits
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CN114686553B (en
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尹晓清
吴海
刘雅颀
孙梦
王希搏
靳祯亮
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Hubei Reborn Biotech Co ltd
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    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract

The invention discloses a maca okra peptide composite polysaccharide polypeptide and an extraction method and application thereof. The extraction method of the maca okra peptide compound polysaccharide polypeptide comprises the following steps: adding water into maca fruits and okra fruits, mixing, breaking the walls, and homogenizing to obtain maca and okra dispersion liquid; carrying out appropriate denaturation treatment on the maca okra dispersion liquid; carrying out primary enzymolysis on the properly modified maca okra dispersion liquid through cellulase and amylase, and then separating to obtain a supernatant A and a residue precipitate B; adding water into the residue precipitate B, mixing, performing secondary enzymolysis by using alkaline protease, and separating to obtain a supernatant C and a residue precipitate D; and (3) uniformly mixing the supernatant A and the supernatant C, and then performing membrane filtration, membrane concentration, secondary filtration and drying to obtain the maca okra peptide composite polysaccharide polypeptide. The method adopts a biological enzyme enzymolysis mode, can extract effective active ingredients such as polysaccharide polypeptide and the like in the product under mild conditions, and can obtain higher yield.

Description

Maca and okra peptide composite polysaccharide polypeptide and extraction method and application thereof
Technical Field
The invention relates to the technical field of food processing, in particular to a maca okra peptide composite polysaccharide polypeptide and an extraction method and application thereof.
Background
Maca is a cruciferous plant originally produced in the andes mountain of south america, is a pure natural food, is rich in nutrients and is reputed as "south american ginseng". Is widely and artificially planted in Yunnan and Xinjiang provinces in China. The chemical composition of maca dry root is obtained by Italian scientist Dini A in 1994: carbohydrate with protein content of more than 10% and 59%; 8.5% of fiber contains abundant mineral substances such as zinc, calcium, iron, titanium, rubidium, potassium, sodium, copper, manganese, magnesium, strontium, phosphorus, iodine, etc., contains vitamins C, B1, B2, B6, A, E, B12 and B5, has low fat content, mostly contains unsaturated fatty acid, linoleic acid and linolenic acid with content of more than 53%, and contains natural active components such as alkaloid, glucosinolate and its decomposition product benzyl isothiocyanate, sterol, polyphenol, etc. In 1999, american scientists discovered that maca contains two new plant active ingredients, macamide and macaene, and determined that these two substances have a significant effect on balancing the secretion of hormones in the human body, so maca is also known as a natural hormone engine.
Okra is an annual herb plant of the family malvaceae, is native to india, and is widely planted in north river, east Shandong, Jiangsu, Zhejiang, Hunan, Hubei, Yunnan, Guangdong and other provinces in China. The okra is rich in nutrition, and young fruits contain a large amount of slimy juice and have special fragrance. The juice contains pectin, bovine lactoglycan, araban, etc. The pectin is soluble fiber, and is very important in modern health care new concept. It has the functions of strengthening stomach and intestine, nourishing Yin and Yang.
Maca and okra are mainly eaten, picked fresh fruits are popular in the market, but picking is greatly influenced by seasons and weather, and the picked fresh fruits are seriously wasted due to the fact that consumers have a large part of defective products on the appearance of the fruits in the market. At present, a plurality of maca peptides, okra peptides and other related products exist in the market, and the inventor finds that the products are directly extracted by water, namely the products are prepared by separating liquid, concentrating and drying after being boiled by water, and are also extracted by organic reagents, and small molecular peptides prepared by an enzymolysis mode exist in the methods, and the methods have the problems of low extraction rate, single extraction component and incapability of completely extracting effective components.
Disclosure of Invention
The invention aims to overcome the technical defects, provides a maca and okra peptide composite polysaccharide polypeptide, and an extraction method and application thereof, and solves the technical problems that the extraction rate of maca peptides and okra peptides is low, the extraction components are single, and the effective components cannot be completely extracted in the prior art.
The invention provides a method for extracting maca okra peptide compound polysaccharide polypeptide, which comprises the following steps:
adding water into maca fruits and okra fruits, mixing, breaking the walls, and homogenizing to obtain maca and okra dispersion liquid;
carrying out appropriate denaturation treatment on the maca okra dispersion liquid;
carrying out primary enzymolysis on the properly modified maca okra dispersion liquid through cellulase and amylase, and then separating to obtain a supernatant A and a residue precipitate B;
adding water into the residue precipitate B, mixing, performing secondary enzymolysis by using alkaline protease, and separating to obtain a supernatant C and a residue precipitate D;
and (3) uniformly mixing the supernatant A and the supernatant C, and then performing membrane filtration, membrane concentration, secondary filtration and drying to obtain the maca okra peptide composite polysaccharide polypeptide.
The second aspect of the invention provides a maca okra peptide complex polysaccharide polypeptide, which is obtained by the extraction method of the maca okra peptide complex polysaccharide polypeptide provided by the first aspect of the invention.
The third aspect of the invention provides an application of maca okra peptide compound polysaccharide polypeptide as an anti-fatigue food.
Compared with the prior art, the invention has the beneficial effects that:
the method adopts a biological enzyme enzymolysis mode, can extract effective active ingredients such as polysaccharide polypeptide and the like in the product under mild conditions, and can obtain higher yield.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a method for extracting maca okra peptide compound polysaccharide polypeptide, which comprises the following steps:
s1, adding water into the maca fruits and the okra fruits, mixing, breaking the walls, and homogenizing to obtain maca and okra dispersion liquid;
s2, performing appropriate denaturation treatment on the maca okra dispersion liquid;
s3, carrying out primary enzymolysis on the properly modified maca okra dispersion liquid through cellulase and amylase, and then separating to obtain a supernatant A and a residue precipitate B;
s4, adding water into the residue precipitate B, mixing, performing secondary enzymolysis through alkaline protease, and separating to obtain a supernatant C and a residue precipitate D;
s5, mixing the supernatant A and the supernatant C uniformly, and then performing membrane filtration, membrane concentration, secondary filtration and drying to obtain the maca okra peptide compound polysaccharide polypeptide.
The invention has the advantages that through the step-by-step extraction of various enzymes (cellulase, amylase and alkaline protease) and the combination of physical wall breaking, physical hydrolysis and biological hydrolysis (ultrasonic wave and protease enzymolysis), the extraction rate of maca and okra is obviously improved by controlling the parameters (time, pH, enzyme addition amount and temperature) of the enzymolysis process; in the treatment process of enzyme deactivation and concentration, the conventional steps of high-temperature enzyme deactivation and high-temperature concentration are abandoned, and the mode of an ultrafiltration membrane is changed to remove macromolecular protease, water, inorganic salt and the like, so that the product is purer.
In step S1 of the present invention, the mass ratio of maca fruits to okra fruits is 1: (0.1 to 5), preferably 1: (0.5-2), preferably 1:1, wherein the obtained maca okra peptide compound polysaccharide polypeptide has optimal performance within the mass ratio range; the adding amount of the water is 10-30 times, further 15-20 times and further 15 times of the total amount of the raw materials of the maca and the okra; breaking cell wall with a cell wall breaking machine, and homogenizing with a homogenizer.
In step S2 of the present invention, the maca okra dispersion liquid is properly denatured to allow better reaction with protease, thereby increasing the extraction rate. Further, the temperature of the moderate denaturation treatment is 55-65 ℃, and the time is 2.5-3.5 hours, and further 3 hours.
In the step S3, the total adding amount of the cellulase and the amylase is 1-3% of the total mass of the raw materials of the maca and the okra, preferably 1.5-2.5%, and further 1.8-2.5%; the mass ratio of the cellulase to the amylase is 1: (0.8-1.2), preferably 1: 1; the temperature of the primary enzymolysis is 45-55 ℃, further 48-52 ℃, and the time is 3.5-4.5 h, further 4 h; in the primary enzymolysis process, controlling the pH to be 5-6, and assisting ultrasonic waves in the whole process, wherein the frequency is 50-70 KHz, further 55-65 KHz, and further 60 KHz; and after the primary enzymolysis is finished, separating by using a tubular centrifuge to obtain supernatant A and residue precipitate B, wherein the rotating speed of the centrifuge is more than 20000r/min, the centrifugation temperature is 20-30 ℃, and the centrifugation time is 10-20 min.
In step S4, the amount of water added is 3 to 10 times, further 4 to 7 times, and further 5 times the mass of the residue precipitate B; the adding amount of the alkaline protease is 0.1-0.3% of the mass of the residue precipitate B, and preferably 0.18-0.22%; the temperature of the secondary enzymolysis is 55-65 ℃, further 58-62 ℃, and the time is 1.5-2.5 h, further 2 h; in the secondary enzymolysis process, the pH is controlled to be 8.5-9.5, ultrasonic waves are assisted in the whole process, the frequency is 50-70 KHz, further 55-65 KHz, and further 60 KHz; and after the secondary enzymolysis is finished, separating by using a tubular centrifuge to obtain supernatant C and residue precipitate D, wherein the rotating speed of the centrifuge is more than 20000r/min, the centrifugation temperature is 20-30 ℃, and the centrifugation time is 10-20 min.
In step S5, residual enzyme in the mixed supernatant is removed through membrane filtration, and impurities such as macromolecular pigments and the like can be removed to obtain dilute feed liquid; in the membrane filtration process, the membrane pore size is more than 3000D, for example, 5000D or 10000D; concentrating by membrane equipment until the solid content is more than 20%, removing water and small molecular inorganic salt, and the like to obtain a concentrated solution; in the membrane concentration process, the membrane aperture is 100-300D; removing microorganisms by filtering again, and passing the concentrated solution through PES filter element with the diameter of 0.22 μm; the drying mode is spray drying, and in the spray drying process, the air inlet temperature is 180-220 ℃, further 190-210 ℃, further 200 ℃, the air outlet temperature is 80-100 ℃, further 85-95 ℃, further 90 ℃.
In the invention, the selected maca fruits and okra fruits are not beautiful in appearance or dried in the sun, so that the market of fresh fruits can be ensured, the fresh fruits with poor appearance can be utilized, and the technical application is further provided for the deep processing of maca okra.
In the invention, the cellulase, amylase and alkaline protease are all obtained from bacillus subtilis.
The second aspect of the invention provides a maca okra peptide composite polysaccharide polypeptide, which is obtained by the extraction method of the maca okra peptide composite polysaccharide polypeptide provided by the first aspect of the invention.
The third aspect of the invention provides an application of maca okra peptide compound polysaccharide polypeptide as an anti-fatigue food.
Example 1
(1) Mixing maca fruits and okra fruits according to a mass ratio of 1:1, adding 15 times of water by mass, breaking the walls by using a wall breaking machine, and then homogenizing by using a homogenizer to obtain a uniformly dispersed maca and okra dispersion liquid;
(2) placing the maca okra dispersion liquid at 60 +/-5 ℃, preserving heat for 3 hours, and performing moderate denaturation to enable the maca okra dispersion liquid to better react with alkaline protease;
(3) adding cellulase and amylase (obtained from Bacillus subtilis) respectively accounting for 1% of the raw materials by mass into the properly modified Abelmoschus esculentus dispersion liquid for primary enzymolysis to obtain primary enzymolysis liquid, and centrifuging the primary enzymolysis liquid for 10min by using a tubular centrifuge to obtain supernatant A and residue precipitate B; in the primary enzymolysis process, the temperature of the system is maintained at 50 +/-2 ℃, the pH is 5-6, the time is 4 hours, ultrasonic waves are assisted in the whole process, and the frequency is 60 KHz; in the centrifugation process, the rotation speed of the centrifuge is 25000r/min, and the centrifugation temperature is 25 +/-5 ℃;
(4) adding water into the residue precipitate B according to the mass ratio of 1:5, mixing, adding alkaline protease (obtained from Bacillus subtilis) accounting for 0.2% of the mass of the residue precipitate B, performing secondary enzymolysis to obtain secondary enzymolysis liquid, and centrifuging the secondary enzymolysis liquid for 10min by using a tubular centrifuge to obtain supernatant C and residue precipitate D; maintaining the temperature of the system at 60 + -2 deg.C, pH at 9.0 + -0.5, time at 2h, and ultrasonic wave frequency at 60KHz during the second enzymolysis; in the centrifugation process, the rotation speed of the centrifuge is 25000r/min, and the centrifugation temperature is 25 +/-5 ℃;
(5) mixing the supernatant A and the supernatant C, filtering by using a membrane (the membrane aperture is 5000D) to obtain a dilute liquid product with the solid content of about 7 +/-2%, then concentrating the dilute liquid by using membrane equipment until the solid content is more than 20% to obtain a concentrated solution, passing the concentrated solution through a 0.22 mu mPES filter core to remove microorganisms, and finally performing spray drying to obtain solid powder; in the spray drying process, the air inlet temperature is 200 ℃ and the air outlet temperature is 90 ℃. The yield of the maca okra peptide complex polysaccharide polypeptide obtained in the embodiment is 79.2%.
The molecular weight distribution of the product obtained in example 1 above was tested and the results are shown in Table 1.
TABLE 1
Molecular weight range Percent peak area (%, lambda =220 nm) Number average molecular weight
＞5000 2.90 6841
5000-3000 6.40 3857
3000-2000 9.11 2438
2000-1000 20.45 1419
1000-500 20.56 741
500-180 19.63 329
＜180 20.95 77
As can be seen from Table 1, the molecular weight distribution of the maca okra peptide complex polysaccharide polypeptide obtained in example 1 of the invention is concentrated, wherein 61.14% accounts for less than 1000 dal, and 97.1% accounts for less than 5000 dal.
Example 2
Compared with example 1, the difference is only that: the mass ratio of the maca fruits to the okra fruits is 1: 0.5.
Example 3
Compared with example 1, the difference is only that: the mass ratio of the maca fruits to the okra fruits is 1: 2.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method takes the defective products as raw materials to carry out technical treatment, extracts the maca and okra polysaccharide polypeptides, analyzes the efficacy of the maca and okra polysaccharide polypeptides, can ensure that fresh fruits are sold in the market, can utilize the fresh fruits with poor appearance, and further provides technical application for deep processing of maca okra;
(2) according to the production process of maca peptides and okra peptides commonly adopted in the existing industry, the production yield is 40-50%, the yield of maca okra polysaccharide polypeptides produced by the invention can reach 70-80%, and is far higher than the production yield of common maca peptides and okra peptides, so that the cost can be saved and the production value can be created;
(3) the maca okra polysaccharide polypeptide obtained by the invention retains the nutritional ingredients in maca okra to a great extent, has extremely high solubility and sweet mouthfeel, is more easily accepted by consumers, and is also more easily compatible and combinable with other products;
(4) the invention further proves that compared with a control group (a mouse which is administrated by intragastric administration by using normal saline with the same volume), the survival time of the mouse which is administrated by intragastric administration in a high, medium and low dose group (the sample of the embodiment 1 is 100ml each time and 2 times a day) in a swimming box is higher than that of the control group, and the high dose group is more obvious, so that the maca okra peptide compound polysaccharide polypeptide has strong antifatigue effect and high bioactivity.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. The extraction method of the maca okra peptide compound polysaccharide polypeptide is characterized by comprising the following steps of:
adding water to maca fruits and okra fruits, mixing, breaking the walls, and homogenizing to obtain maca and okra dispersion liquid;
carrying out appropriate denaturation treatment on the maca okra dispersion liquid;
carrying out primary enzymolysis on the properly modified maca okra dispersion liquid through cellulase and amylase, and then separating to obtain a supernatant A and a residue precipitate B;
adding water into the residue precipitate B, mixing, performing secondary enzymolysis by using alkaline protease, and separating to obtain a supernatant C and a residue precipitate D;
and (3) uniformly mixing the supernatant A and the supernatant C, and then performing membrane filtration, membrane concentration, secondary filtration and drying to obtain the maca okra peptide composite polysaccharide polypeptide.
2. The extraction method of maca okra peptide complex polysaccharide polypeptide according to claim 1, wherein the mass ratio of maca fruits to okra fruits is 1: (0.1-5).
3. The extraction method of maca okra peptide complex polysaccharide polypeptide according to claim 1, wherein the moderate denaturation treatment is carried out at 55-65 ℃ for 2.5-3.5 h.
4. The extraction method of maca and okra peptide composite polysaccharide polypeptides according to claim 1, wherein the total addition amount of the cellulase and the amylase is 1% -3% of the total mass of maca and okra raw materials, and the mass ratio of the cellulase to the amylase is 1: (0.8 to 1.2); the temperature of the primary enzymolysis is 45-55 ℃, the time is 3.5-4.5 h, the pH is controlled to be 5-6, and ultrasonic waves are assisted in the whole process.
5. The extraction method of the maca okra peptide complex polysaccharide polypeptide according to claim 1, wherein the addition amount of the alkaline protease is 0.1-0.3% of the mass of the residue precipitate B, the temperature of the secondary enzymolysis is 55-65 ℃, and the time is 1.5-2.5 h; in the secondary enzymolysis process, the pH is controlled to be 8.5-9.5, and ultrasonic waves are assisted in the whole process.
6. The extraction method of maca okra peptide composite polysaccharide polypeptide according to claim 1, characterized in that a tubular centrifuge is adopted for separation, the rotation speed of the centrifuge is more than 20000r/min, the centrifugation temperature is 20-30 ℃, and the centrifugation time is 10-20 min.
7. The extraction method of maca okra peptide composite polysaccharide polypeptide according to claim 1, wherein in the membrane filtration process, the membrane pore size is greater than 3000D; in the membrane concentration process, the membrane aperture is 100-300D; in the secondary filtering process, the concentrated solution passes through a PES filter element with the diameter of 0.22 mu m; the drying mode is spray drying, and in the spray drying process, the air inlet temperature is 180-220 ℃ and the air outlet temperature is 80-100 ℃.
8. The extraction method of maca and okra peptide compound polysaccharide polypeptide according to claim 1, wherein the maca fruits and the okra fruits are fruits with an unattractive appearance or dried fruits after being dried in the sun.
9. A maca okra peptide complex polysaccharide polypeptide, which is obtained by the extraction method of the maca okra peptide complex polysaccharide polypeptide according to any one of claims 1 to 8.
10. The use of the maca okra peptide complex polysaccharide polypeptide of claim 9 as an anti-fatigue food.
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Denomination of invention: A Maka Okra Peptide Composite Polysaccharide Peptide and Its Extraction Method and Application

Granted publication date: 20231121

Pledgee: China Construction Bank Corporation Jingzhou Yuqiao Branch

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