CN106480143B - Maca oligosaccharide polypeptide and preparation method thereof - Google Patents
Maca oligosaccharide polypeptide and preparation method thereof Download PDFInfo
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- CN106480143B CN106480143B CN201610790732.4A CN201610790732A CN106480143B CN 106480143 B CN106480143 B CN 106480143B CN 201610790732 A CN201610790732 A CN 201610790732A CN 106480143 B CN106480143 B CN 106480143B
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Abstract
The invention provides a maca oligosaccharide polypeptide, which is characterized in that: it has a weight average molecular weight of less than 2000 Da; the macamide accounts for 0.031-0.136% of the total weight of the macamide; the weight percentage of arginine in the amino acid is not less than 10 percent, and the weight percentage of proline in the amino acid is not less than 20 percent; the glucosinolate accounts for not less than 1% by weight. The invention also provides a preparation method and application of the maca oligosaccharide polypeptide. The maca oligosaccharide polypeptide provided by the invention can exert biological effects of protein and polysaccharide to the maximum extent, including relieving alcoholism, improving immunity, resisting fatigue, promoting fertility and the like, is easy to digest and absorb in a human body, has high bioavailability, can be used for preparing foods, food additives, health-care foods and medicines, and has a good industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of maca processing, and particularly relates to a maca oligosaccharide polypeptide and a preparation method thereof.
Background
The plant maca (Lepidium meyenii Walp, also known as maca) is a cruciferous plant native to the Andes mountain of Peru, and has a long history as a plant used as both medicine and food in the Andes mountain area. Because maca has rich nutritive value and medicinal efficacy, especially in the aspects of fatigue resistance, fertility improvement and the like, maca is regarded as a precious gift granted by the god of Andes mountain and is called Peru ginseng. The food and agriculture group of the united nations proposed that people eat maca, and the maca is introduced into Chinese planting since 2003.
Modern researches prove that maca is rich in various nutritional and chemical components such as carbohydrates, proteins, unsaturated fatty acids, minerals and the like, and various secondary metabolites such as bioactive components such as macaene (macaene), alkaloids (including macamide), glucosinolates and hydrolysates thereof (Piacenteet al, 2002), sterols, polyphenols and the like. These secondary metabolites are thought to be closely related to the health efficacy of maca.
As people increasingly advocate natural plant medicines and functional foods, the development of maca products with unique effects is widely concerned by researchers in various countries in the world. However, the maca products sold in the market are different, and the maca dry powder or water and alcohol extracts are mostly directly used. The maca product has low content of effective components and unobvious effect, and cannot fully exert the potential value of the maca.
Some researches on maca preparation processes exist at present, for example, a patent of publication No. CN103864949A discloses a method for preparing maca polysaccharide and maca alkaloid by adopting a membrane separation technology in combination with protease and polysaccharide hydrolase enzymolysis, the steps are complex, and maca polypeptide is not obtained; the patent publication No. CN103725741A discloses a method for enzyme digestion of maca by using maca-specific enzymes consisting of incision enzymes, excision enzymes and flavor enzymes to extract maca peptides, but does not mention the molecular weight and the content ratio of maca amides in the obtained maca peptides. Therefore, the technical conditions of the maca are improved, and the maca product rich in functional components and small molecular active polypeptide is obtained, so that the nutritional value of the maca is fully exerted. Is the urgent need for the development of the maca products at present.
Disclosure of Invention
The invention aims to provide a maca oligosaccharide polypeptide containing functional components and small-molecule active polypeptide and a preparation method thereof.
The maca oligosaccharide polypeptide of the invention: the main components are oligosaccharide, polypeptide, flavone, macamide, glucosinolate and other substances.
The invention provides a maca oligosaccharide polypeptide, the weight average molecular weight of which is less than 2000; the macamide accounts for 0.031-0.136% of the total weight of the macamide; the weight percentage of arginine in the amino acid is not less than 10 percent, and the weight percentage of proline in the amino acid is not less than 20 percent; the glucosinolate accounts for not less than 1% by weight.
Wherein, the maca oligosaccharide polypeptide is prepared from maca;
and/or the weight average molecular weight of the maca oligosaccharide polypeptide is 500-2000; the weight percentage of arginine in the amino acid is 10-20%, and the weight percentage of proline in the amino acid is 20-30%;
and/or the maca oligosaccharide polypeptide contains polysaccharide with the weight percentage not less than 15%.
The invention also provides a preparation method of the maca oligosaccharide polypeptide, which comprises the following steps:
(a) taking 100 plus 300 parts by weight of maca, adding 800-1200 parts by volume of water, and adding 1-8 parts by weight of carbohydrase to react for 0.5-12 hours at the temperature of 30-65 ℃;
(b) adjusting the pH value to 8-9 at the temperature of 30-65 ℃, adding 1-8 parts by weight of alkaline protease, and reacting for 0.5-12 hours;
(c) adding 1-8 parts of neutral protease to react for 0.5-12 hours at the temperature of 30-65 ℃;
(d) enzyme inactivation;
(e) filtering, and concentrating the filtrate.
The relationship between parts by weight and parts by volume is "1 g to 1 mL". Wherein, in the step (a), the maca is maca root tuber or maca leaf.
Wherein the carbohydrase is at least one of amylase, cellulase, pectinase and glucoamylase; the alkaline protease is at least one of alcalase or pancreatin; the neutral protease is at least one of A1398, Neutrase and Flavourzyme.
Wherein in step (a), (b) and/or (c), the reaction is stirred.
Wherein in the step (a), after the temperature is adjusted to 30-65 ℃, the pH value is adjusted to 5 before the carbohydrase is added.
Wherein, in the step (a), 100 parts of maca are taken, 1000 parts of water are added, the temperature is adjusted to be 30-65 ℃, the pH value is adjusted to be 5, 2 parts of carbohydrase are added, and the reaction is carried out for 2 hours;
or taking 100 parts of maca, adding 800 parts of water, adjusting the temperature to 30 ℃, adding 2 parts of carbohydrase, and reacting for 2 hours;
or adding 1000 parts of water into 250 parts of maca, adjusting the temperature to 30-65 ℃, adjusting the pH value to 5, adding 2 parts of carbohydrase, and reacting for 2 hours;
or taking 300 parts of maca, adding 1200 parts of water, adjusting the temperature to 65 ℃, adding 8 parts of carbohydrase, and reacting for 12 hours.
Wherein in the step (b), the temperature is 30-65 ℃, the pH value is adjusted to 8, 2 parts of alkaline protease is added, and the reaction is carried out for 2 hours;
in the step (c), adding 2 parts of neutral protease at the temperature of 30-65 ℃ and reacting for 2 hours;
in step (d), the enzyme inactivation is performed by: adjusting the temperature to 75-90 ℃, and keeping the temperature for 15-30 minutes; preferably, the enzyme inactivation employs: adjusting the temperature to 90 ℃, and keeping the temperature for 30 minutes;
in the step (e), the concentration adopts vacuum spray drying or freeze vacuum drying.
And/or, after step (e), further comprising a sterilization step.
The invention also provides the application of the maca oligosaccharide polypeptide in preparing foods, food additives, health-care foods and/or medicines;
wherein the health food and the medicine are health food and medicine for relieving alcoholism, improving immunity, resisting fatigue and/or promoting fertility.
The invention also provides application of the maca oligosaccharide polypeptide in preparation of special nutritional foods and medicines.
The maca from different production places has different contents of protein, glucosinolate, maca amide and the like, for example, the content of the glucosinolate is between 1.05 and 1.79 percent, and is closely related to the processing method. The content of protein, glucosinolate, macaene and macamide in the maca oligosaccharide polypeptide is equivalent to the initial content of raw maca.
The maca oligosaccharide polypeptide prepared by the method can exert biological effects of protein and polysaccharide to the maximum extent, including relieving alcoholism, improving immunity, resisting fatigue, promoting fertility and the like, is easy to digest and absorb in a human body, has high bioavailability, and can be used for preparing foods, food additives, health-care foods and medicines.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a chromatography-mass spectrometry detection map of macamide in maca oligosaccharide polypeptide
FIG. 2 is a liquid phase map of the maca oligosaccharide polypeptides (i.e., maca zymolyte) of the invention; where data 1 is the spray-dried product and data 2 is the freeze-dried product (i.e., the maca oligosaccharide polypeptide prepared in a freeze-dried manner).
Detailed Description
The following examples are further illustrative, but the present invention is not limited to these examples.
The process flow of the invention is as follows:
maca root tuber or maca leaf → washing and crushing → enzymolysis → inactivation enzyme → filtration → spray drying (or freeze drying) → sterilization;
or maca root tuber or maca leaf → washing and crushing → enzymolysis → inactivating enzyme → filtering → spray drying (or freeze-drying).
The experimental reagents used in the present invention were as follows:
amylase, cellulase, pectinase, glucoamylase: all purchased from AB company, germany;
alcalase, pancreatin, Neutrase, Flavourzyme: all purchased from novicent;
a1398: purchased from general enzyme preparations, Beijing House county.
The detection methods are as follows:
content determination of maca glucosinolates
1. Materials: shimadzu CBM-10A VP PLUS liquid chromatograph (ultraviolet detector: SPD-10A VP PLUS, infusion pump: LC-10AVP PLUS), KQ-300DE ultrasonic apparatus (Kunshan ultrasonic apparatus Co., Ltd.), FA2004N electronic balance (Shanghai Cyanin apparatus Co., Ltd., d 0.1mg), HH-6 constant temperature water bath (Wako Hua electric apparatus Co., Ltd.), 10mL headspace sample introduction bottle, and C18 chromatographic column.
2. Reagent: acetonitrile (HPLC pure, family reagent), 95% ethanol (family reagent), benzyl isothiocyanate BITC (CAS622-78-6, purity > 98%, Tokyo Kasei Kogyo Co., Ltd.), glucothiolase (CAS 9025-38-1, enzyme activity > 100UN/g, Tokyo Kasei Kogyo Co., Ltd.)
3. Chromatographic conditions are as follows:
a chromatographic column: c18
Mobile phase: acetonitrile (A) -Water (B)
Sample introduction amount: 20 μ L
Wavelength: 246nm
Flow rate: 1.0mL/min
Column temperature: 40 deg.C
Gradient conditions:
4. preparing a glucothiolase solution:
precisely weighing 33mg of glucosinolate, placing into a 10mL measuring flask, dissolving with distilled water, and diluting to constant volume of 10mL
5. Preparing a reference substance solution:
a measuring flask of 250mg to 25mL of BITC is accurately weighed, and 25mL of acetonitrile is dissolved and contained.
6. Drawing a standard curve:
precisely transferring 0, 20, 50, 100, 150 and 200 mu L of reference substance solution into a 10mL measuring flask respectively, adding acetonitrile solution, diluting to scale, shaking up, and preparing reference substance solution with mass concentration of 0, 0.02, 0.05, 0.10, 0.15 and 0.20 g/L; and (3) taking the mass concentration (g/L) of the reference substance solution as an abscissa (X) and the peak area (Y) of the reference substance as an ordinate, and performing linear regression to draw a standard curve.
7. Preparing a test sample:
1) 250mg of maca powder is precisely weighed and placed in a 25mL measuring flask.
2) Adding 15mL of 47.5% ethanol solution, heating in water bath at 50 ℃ for 1h, cooling to room temperature, and diluting to constant volume with 47.5% ethanol solution.
3) Precisely measure 4mL of the above solution, place in a 10mL headspace bottle, add 1mL of the glucosinolate enzyme solution, shake, seal with a cap.
Placing in a 45 deg.C constant temperature water bath for enzymolysis for 30min, taking out, transferring into a 10mL measuring flask, diluting with 47.5% ethanol to scale, and filtering with 0.22 microporous membrane.
Second, maca total flavone content determination method
1. Required reagent test solution:
rutin standard (Chinese Biochemical institute), methanol solution (subject reagent), 5% sodium sulfite solution (subject reagent), 10% aluminum nitrate solution (subject reagent), 5% sodium hydroxide solution (subject reagent)
2. The required instruments are as follows: pujinu TU-1900 double-beam ultraviolet visible spectrophotometer, KQ-300DE ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.), FA2004N electronic balance (Shanghai Cyanin instruments Co., Ltd., d ═ 0.1mg)
3. Preparing a rutin standard solution:
1) accurately weighing 12mg of dry constant-temperature rutin reference substance, placing the reference substance in a 50mL volumetric flask, adding methanol to completely dissolve the reference substance and diluting the reference substance to a scale mark, wherein the concentration is 0.24 mg/mL.
2) Accurately sucking 10mL of the solution into a 25mL volumetric flask, adding methanol to the scale mark, and shaking up to obtain a control solution with the concentration of 0.096 mg/mL.
4. The maximum absorption wavelength determines:
1) accurately sucking 4.0mL of rutin standard solution of 0.096mg/mL into a 10mL colorimetric tube, adding 0.3mL of 5% sodium sulfite solution, and performing ultrasonic treatment for 6 min.
2) 0.3mL of 10% aluminum nitrate solution was added and sonicated for 6 min.
3) 4mL of 5% sodium hydroxide solution was added, sonicated for 15min, and allowed to stand at room temperature.
4) Methanol is added to the scale, and the maximum absorption wavelength is determined by full-wavelength scanning of a spectrophotometer.
5. Drawing a standard curve:
1) accurately sucking 0.096mg/mL rutin standard solution 0mL, 1.0mL, 2.0mL, 4.0mL, 6.0mL, 8.0mL and 10.0mL into a20 mL volumetric flask respectively.
2) 0.3mL of 5% sodium sulfite solution was added and sonicated for 6 min.
3) 0.3mL of 10% aluminum nitrate solution was added and sonicated for 6 min.
4) 4mL of 5% sodium hydroxide solution was added, sonicated for 15min, and allowed to stand at room temperature.
5) Adding methanol to the scale, measuring the absorbance respectively, and making a standard curve by using a regression equation.
6. And (3) extracting and determining total flavonoids of maca:
1) 0.1g of a maca sample (including raw materials and maca zymolyte) is weighed and moistened with 8mL of purified water.
2) Ultrasonically extracting with 30mL methanol for 25min, transferring supernatant into 50mL volumetric flask, extracting residue with methanol for 2 times, mixing extractive solutions, diluting with methanol to 50mL, and filtering with 0.45 filter membrane.
3) Sucking 4.0mL of the extract into a 10mL colorimetric tube, adding 0.3mL of 5% sodium sulfite solution, and performing ultrasonic treatment for 6 min.
4) 0.3mL of 10% aluminum nitrate solution was added and sonicated for 6 min.
5) Adding 4mL of 5% sodium hydroxide solution, performing ultrasonic treatment for 15min, standing until the temperature is reduced to room temperature, and adding methanol to the scale.
6) Measuring absorbance, and converting flavone content.
Determination of macamide content
1. The required instruments are as follows: shimadzu CBM-10A VP PLUS liquid chromatograph (ultraviolet detector: SPD-10A VP PLUS, infusion pump: LC-10AVP PLUS), KQ-300DE ultrasonic cleaner (Kunshan ultrasonic apparatus Co., Ltd.), FA2004N electronic balance (Shanghai Cyanin apparatus Co., Ltd., d 0.1mg)
2. The required materials are as follows: c18Chromatographic column, maca powder, acetonitrile (HPLC pure, reagent for department test, 0.05% phosphoric acid water solution (reagent for department test), absolute ethyl alcohol (reagent for department test),
macamides standards (N-benzylhexadecamamide, N- (m-methoxybenzyl) -linolenamide, N-benzyllinolenamide, N- (m-methoxybenzyl) -linoleamide, N-benzyllinoleamide)
3. Preparation of a reference solution:
1) taking an appropriate amount of N-benzyl hexadecane amide, N- (m-methoxybenzyl) -linolenamide, N-benzyl linolenamide, N- (m-methoxybenzyl) -linoleamide and N-benzyl linoleamide, precisely weighing, and adding methanol into a 10mL volumetric flask to dilute to a scale mark.
2) 1mL of each control solution was diluted to a standard solution with a constant concentration gradient.
3) And (4) according to the liquid phase data, making a standard curve of each reference substance according to the peak area and the concentration.
4. Preparing a test solution:
1) a maca sample 1g is precisely weighed and placed in a 50mL conical flask with a plug.
2) Accurately adding 10mL of absolute ethyl alcohol, weighing for mass determination, performing ultrasonic treatment for 45min, weighing for mass determination again, and supplementing with absolute ethyl alcohol to reduce mass.
3) Shaking up, passing through 0.45 microporous filter membrane, and testing.
5. Chromatographic conditions are as follows:
mobile phase: acetonitrile (A) to 0.05% phosphoric acid aqueous solution (B)
Gradient elution conditions: 0-15 min, 70% -90% A
15～35min，90％A
Detection wavelength: 208nm
Volume flow rate: 1.0mL/min
Column temperature: 30 deg.C
Fourthly, determining the molecular weight of the maca polypeptide: indantrione method for measuring hydrolysis degree and molecular weight
1. Instrument & reagent
Beijing Pujingyu TU-1900 type ultraviolet-visible spectrophotometer or similar instrument
Pipette (2ml)
Liquid-transfering gun
Volumetric flask (100mL, 50mL)
Colorimetric tube with plug (10ml)
Ninhydrin, glycine, fructose, disodium hydrogen phosphate, potassium dihydrogen phosphate (analytically pure)
Ethanol
2. Experimental procedure
1) 20. mu.g/mL glycine standard solution: 2mg glycine was metered into a 100mL volumetric flask.
2) Ninhydrin display preparation: 0.5g of ninhydrin, 0.3g of fructose, 11.2g of disodium hydrogen phosphate and 6g of potassium dihydrogen phosphate, wherein the volume is 100mL, the ninhydrin is dissolved in heat, and the ninhydrin can be stored at low temperature for 1 week.
3) Drawing a standard curve: taking 9 test tubes with plugs, precisely absorbing standard solutions according to the following table to 10mL test tubes with plugs, adding pure water to 2.0mL each, adding ninhydrin color developing agent to 1.0mL, uniformly mixing, heating in boiling water bath for 15min, rapidly cooling to room temperature with cold water, adding 40% ethanol to 5mL, shaking up, standing for 15min, and measuring absorbance (A) at 570nm wavelength as ordinate and content as abscissa to prepare a standard curve.
4) And sample measurement
4.1 sample solution: taking 0.50mL of hydrolysate to fix the volume to 50 mL.
4.2 sample determination: sucking 0.5mL of sample solution, placing the sample solution into a colorimetric tube with a plug (each sample is repeated for 2 times), adding pure water to 2.0mL, adding ninhydrin color developing agent to 1.0mL, uniformly mixing, heating in a boiling water bath for 15min, then rapidly cooling to room temperature with cold water, adding 40% ethanol to 5mL, shaking up, standing for 15min, carrying out color comparison at 570nm, using a test tube No. 0 as a reference, measuring absorbance, and checking the content through a standard curve.
Total millimoles of peptide bonds (i.e. the sum of millimoles of peptide bonds)
4.3 results calculation:
hydrolysate-NH2The content (mu mol/mL) is [ C/(75X V)]*100
In the formula, N-hydrolysate protein content (Kjeldahl nitrogen determination)
htotMillimoles of peptide bonds per gram of starting protein
C-content of standard curve found (μ g)
Molecular weight of 75-Glycine
V-volume measured of sample solution
100-dilution factor of sample
4.4 determination of the average Peptide Chain Length (PCL) and average molecular weight (Mw):
PCL＝1/DH
Mw＝110*PCL+18
example 1 preparation of maca oligosaccharide polypeptides of the invention
1. Raw materials: drying the maca root tuber.
2. The preparation method comprises the following steps:
crushing 100 g of clean maca dry root tuber, adding 1000 ml of deionized water, adjusting the temperature to be 30-65 ℃, adjusting the pH value to be 5, and adding 2g of amylase to react for 2 hours; then, under the same temperature, the pH value is adjusted to 8, and 2g of alcalase is added with stirring for reaction for 2 hours; then adding 2g of A1398 enzyme under the same temperature condition for reaction for 2 hours; then heating to 90 deg.C, reacting at constant temperature for half an hour to inactivate enzyme, and filtering. And concentrating the supernatant under reduced pressure at the vacuum degree of 0.06-0.10 Mpa and the temperature of 40-65 ℃, and finally freeze-drying to obtain the maca oligosaccharide polypeptide.
3. And (3) detection results:
the yield of the maca oligosaccharide polypeptide prepared by the method is 58.2%.
Wherein: crude protein content 20.12%, total amino acid content: 17.61%; glucosinolates 1.35 g/100 g; macamide 136 mg/100 g maca oligosaccharide polypeptide, and the weight-average molecular weight is 547 Da.
The chromaticness-mass spectrometry detection map of macamide in the maca oligosaccharide polypeptide is shown in figure 1.
The amino acid content of the maca oligosaccharide polypeptides of the invention is compared with that of the maca raw material in table 1.
Table 1 comparison of amino acid content of the maca oligosaccharide polypeptides of the invention with maca raw material
Example 2 preparation of maca oligosaccharide polypeptides of the invention
1. Raw materials: dried maca root tuber (produced in Tibet).
2. The preparation method comprises the following steps:
crushing 100 g of clean maca dry root tubers, adding 800 ml of deionized water, keeping the temperature at 30 ℃, adding 2g of cellulase, and reacting for 2 hours; then, under the same temperature, the pH value is adjusted to 8, and 2g of pancreatin is added at constant temperature for reaction for 2 hours; then adding 2g of Neutrase enzyme under the same temperature condition for reaction for 2 hours; then heating to 90 deg.C, reacting at constant temperature for half an hour to inactivate enzyme, and filtering. And concentrating the supernatant under reduced pressure at the vacuum degree of 0.06-0.10 Mpa and the temperature of 40-65 ℃, and finally freeze-drying to obtain the maca oligosaccharide polypeptide.
3. And (3) detection results:
the yield of the maca oligosaccharide polypeptide prepared by the method is 51.6%.
Wherein: crude protein content 17.53%, total amino acid content: 15.17 percent; glucosinolates 0.85 g/100 g; the macamide content is 31 mg/100 g of maca oligosaccharide polypeptide, and the weight average molecular weight is 623 Da.
The liquid phase map of the maca oligosaccharide polypeptide is shown in figure 2 (the final drying in the preparation method of the invention can be carried out without freeze-drying, but the spray drying method is adopted, and the results are compared with the results shown in figure 2).
The amino acid content of the maca oligosaccharide polypeptides of the invention is shown in table 2.
TABLE 2 amino acid content of the maca oligosaccharide polypeptides of the invention
Example 3 preparation of the maca oligosaccharide polypeptides of the invention
1. Raw materials: dried maca root tuber (produced in Yunnan).
2. The preparation method comprises the following steps:
crushing 300 g of clean maca dry root tuber, adding 1200 ml of deionized water, keeping the temperature at 65 ℃, stirring, adding 8 g of pectinase, and reacting for 12 hours; then, under the same temperature, the pH value is adjusted to 9, 8 g of alcalase is added under stirring at constant temperature for reaction for 12 hours; then adding 8 g Flavourzyme under the condition of stirring at the same temperature for reaction for 12 hours; then heating to 90 deg.C, reacting at constant temperature for half an hour to inactivate enzyme, and filtering. And (3) carrying out vacuum freeze-drying and sterilization on the supernatant to obtain the maca oligosaccharide polypeptide.
3. And (3) detection results:
the yield of the maca oligosaccharide polypeptide prepared by the method is 48.6%.
Wherein: crude protein content 18.25%, total amino acid content: 14.31 percent; glucosinolates 1.02 g/100 g; the macamide has the advantages that the macamide content is 120 mg/100 g of maca oligosaccharide polypeptide, and the weight-average molecular weight is 613 Da.
The amino acid content of the maca oligosaccharide polypeptides of the invention is shown in table 3.
TABLE 3 amino acid content of the maca oligosaccharide polypeptides of the invention
| Amino acids | Maca oligosaccharide polypeptide (%) | Calculated as 100% |
| Aspartic acid | 0.85 | 5.94 |
| Threonine | 0.4 | 2.80 |
| Serine | 0.38 | 2.66 |
| Glutamic acid | 1.04 | 7.27 |
| Glycine | 0.47 | 3.28 |
| Alanine | 0.46 | 3.21 |
| Cystine | 0.17 | 1.19 |
| Valine | 0.54 | 3.77 |
| Methionine | 0.19 | 1.33 |
| Isoleucine | 0.4 | 2.80 |
| Leucine | 0.62 | 4.33 |
| Tyrosine | 0.13 | 0.91 |
| Phenylalanine | 0.35 | 2.45 |
| Lysine | 0.5 | 3.49 |
| Histidine | 0.28 | 1.96 |
| Arginine | 2.69 | 18.80 |
| Proline | 4.7 | 32.84 |
| Total amount of amino acids | 14.31 | 100.00 |
Example 4 preparation of the maca oligosaccharide polypeptides of the invention
1. Raw materials: fresh maca root tuber (produced by agaba, sichuan).
2. The preparation method comprises the following steps:
crushing 250 g of clean fresh maca root tuber (with the water content of 65%), adding 1000 ml of deionized water, adjusting the temperature to be 30-65 ℃, adjusting the pH value to be 5, and adding 2g of saccharifying enzyme for reacting for 2 hours; then, under the same temperature, the pH value is adjusted to 8, and 2g of pancreatin is added under stirring for reaction for 2 hours; then adding 2g of A1398 enzyme under the same temperature condition for reaction for 2 hours; then heating to 90 deg.C, reacting at constant temperature for half an hour to inactivate enzyme, and filtering. And concentrating the supernatant under reduced pressure at the vacuum degree of 0.06-0.10 Mpa and the temperature of 40-65 ℃, and finally freeze-drying to obtain the maca oligosaccharide polypeptide.
3. And (3) detection results:
the yield of the maca oligosaccharide polypeptide prepared by the method is 51.6%.
Wherein: crude protein content 13.62%, total amino acid content: 11.13 percent; glucosinolates 0.89 g/100 g; macamide 106 mg/100 g maca oligosaccharide polypeptide, weight average molecular weight 753 Da.
The amino acid content of the maca oligosaccharide polypeptides of the invention is shown in table 4.
TABLE 4 amino acid content of the maca oligosaccharide polypeptides of the invention
| Amino acids | Maca oligosaccharide polypeptide (%) | Calculated as 100% |
| Aspartic acid | 0.70 | 6.29 |
| Threonine | 0.32 | 2.88 |
| Serine | 0.29 | 2.61 |
| Glutamic acid | 0.82 | 7.37 |
| Glycine | 0.31 | 2.79 |
| Alanine | 0.36 | 3.23 |
| Cystine | 0.16 | 1.44 |
| Valine | 0.49 | 4.40 |
| Methionine | 0.15 | 1.35 |
| Isoleucine | 0.26 | 2.34 |
| Leucine | 0.39 | 3.50 |
| Tyrosine | 0.11 | 0.99 |
| Phenylalanine | 0.26 | 2.34 |
| Lysine | 0.31 | 2.79 |
| Histidine | 0.15 | 1.35 |
| Arginine | 2.46 | 22.10 |
| Proline | 3.48 | 31.27 |
| Total amount of amino acids | 11.13 | 100.00 |
The beneficial effects of the invention are illustrated by way of test examples as follows:
test example 1 Effect test of maca oligosaccharide polypeptide of the invention
The maca oligosaccharide polypeptides prepared in example 1 were subjected to an anti-hangover effect experiment, and the results are shown in table 5:
TABLE 5 Effect of maca oligosaccharide polypeptides of the invention on alleviating hangover
As can be seen from Table 5, the maca oligosaccharide polypeptide can quickly treat alcoholism and has a good anti-alcoholism effect.
In conclusion, the maca oligosaccharide polypeptide provided by the invention can exert biological effects of protein and polysaccharide to the maximum extent, including relieving alcoholism, improving immunity, resisting fatigue, promoting fertility and the like, is easy to digest and absorb in a human body, has high bioavailability, and can be used for preparing foods, food additives, health-care foods and medicines. The process for preparing the maca oligosaccharide polypeptide is simple and convenient, the production cost is low, and the prepared product has obvious distinguishing characteristics and obvious superiority compared with the existing product, and has a very good industrial application prospect.
Claims (11)
1. A maca oligosaccharide polypeptide, which is characterized in that: the weight average molecular weight of the composite material is 500-2000 Da; the macamide accounts for 0.031-0.136% of the total weight of the macamide; the weight percentage of arginine in the amino acid is 10-20%, and the weight percentage of proline in the amino acid is 20-30%; the weight percentage content of the glucosinolate is not less than 1 percent; the weight percentage content of the polysaccharide is not lower than 15 percent; the preparation method of the maca oligosaccharide polypeptide comprises the following steps:
(a) taking 100 plus 300 parts by weight of maca, adding 800-1200 parts by volume of water, and adding 1-8 parts by weight of carbohydrase to react for 0.5-12 hours at the temperature of 30-65 ℃;
(b) adjusting the pH value to 8-9 at the temperature of 30-65 ℃, adding 1-8 parts by weight of alkaline protease, and reacting for 0.5-12 hours;
(c) adding 1-8 parts of neutral protease to react for 0.5-12 hours at the temperature of 30-65 ℃;
(d) enzyme inactivation;
(e) filtering, and concentrating the filtrate.
2. A method of making a maca oligosaccharide polypeptide according to claim 1, which comprises the steps of:
(a) taking 100 plus 300 parts by weight of maca, adding 800-1200 parts by volume of water, and adding 1-8 parts by weight of carbohydrase to react for 0.5-12 hours at the temperature of 30-65 ℃;
(b) adjusting the pH value to 8-9 at the temperature of 30-65 ℃, adding 1-8 parts by weight of alkaline protease, and reacting for 0.5-12 hours;
(c) adding 1-8 parts of neutral protease to react for 0.5-12 hours at the temperature of 30-65 ℃;
(d) enzyme inactivation;
(e) filtering, and concentrating the filtrate.
3. The method of claim 2, wherein: in the step (a), the maca is maca root tuber or maca leaf.
4. The method of claim 2, wherein: the carbohydrase is at least one of amylase, cellulase, pectinase and glucoamylase; the alkaline protease is at least one of alcalase or pancreatin; the neutral protease is at least one of A1398, Neutrase and Flavourzyme.
5. The method of claim 2, wherein: in the step (a), (b) and/or (c), the reaction is stirred.
6. The method of claim 2, wherein: in the step (a), after the temperature is adjusted to 30-65 ℃, the pH value is adjusted to 5 before the carbohydrase is added.
7. The method of claim 2, wherein: in the step (a), 100 parts of maca are taken, 1000 parts of water are added, the temperature is adjusted to be 30-65 ℃, the pH value is adjusted to be 5, 2 parts of carbohydrase are added, and the reaction is carried out for 2 hours;
or taking 100 parts of maca, adding 800 parts of water, adjusting the temperature to 30 ℃, adding 2 parts of carbohydrase, and reacting for 2 hours;
or adding 1000 parts of water into 250 parts of maca, adjusting the temperature to 30-65 ℃, adjusting the pH value to 5, adding 2 parts of carbohydrase, and reacting for 2 hours;
or taking 300 parts of maca, adding 1200 parts of water, adjusting the temperature to 65 ℃, adding 8 parts of carbohydrase, and reacting for 12 hours.
8. The method of claim 2, wherein: in the step (b), the temperature is 30-65 ℃, the pH value is adjusted to 8, 2 parts of alkaline protease is added, and the reaction is carried out for 2 hours;
in the step (c), adding 2 parts of neutral protease at the temperature of 30-65 ℃ and reacting for 2 hours;
in step (d), the enzyme inactivation is performed by: adjusting the temperature to 75-90 ℃, and keeping the temperature for 15-30 minutes;
in the step (e), the concentration adopts vacuum spray drying or freeze vacuum drying.
9. The method of claim 8, wherein: the enzyme inactivation adopts the following steps: the temperature was adjusted to 90 ℃ and the incubation was continued for 30 minutes.
10. Use of the maca oligosaccharide polypeptide of claim 1 in the preparation of a food product, food additive.
11. Use according to claim 10, characterized in that:
the food is health food.
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