CN113106136B - Chlorella polypeptide and preparation method thereof - Google Patents

Abstract

The invention relates to the field of biological extraction, in particular to a chlorella polypeptide and a preparation method thereof, wherein the preparation method of the chlorella polypeptide comprises the steps of carrying out wall breaking extraction, centrifugal standing, impurity removal, enzymolysis, separation and purification on chlorella suspension obtained by uniformly mixing chlorella dry powder and purified water in sequence to obtain the chlorella polypeptide; the enzymolysis is to centrifugate, stand and remove the water extract of chlorella which is obtained, add neutral protease to hydrolyze; separating and purifying to obtain neutral protease hydrolysate with inactivated enzyme activity, centrifuging, filtering to obtain chlorella protein hydrolysate with supernatant cut-off molecular weight of 3000Da, removing impurities with 200Da nanofiltration membrane, concentrating, and drying to obtain chlorella polypeptide. The chlorella polypeptide obtained by hydrolyzing neutral protease has high lipase inhibiting activity and blood lipid reducing effect; the preparation method adopts wall breaking extraction and enzymolysis, combines membrane filtration purification, and is suitable for industrial production.

Description

Chlorella polypeptide and preparation method thereof

Technical Field

The invention relates to the field of biological extraction, in particular to a chlorella polypeptide and a preparation method thereof.

Background

Obesity and overweight increase the risk of people suffering from chronic diseases such as diabetes, cardiovascular disease and cancer. One of the methods for preventing obesity is to reduce the digestive absorption of fat in the human body. Pancreatic lipase is secreted by pancreatic acinar cells and is a key enzyme for digesting fat. Therefore, the digestion and decomposition of fat in the intestinal tract can be reduced by inhibiting pancreatic lipase, thereby achieving the purposes of reducing fat absorption, and controlling and treating obesity. Currently, orlistat (Orlistat) is the only pancreatic lipase inhibitor approved by the U.S. food and drug administration for treating obesity, but due to the artificial synthetic drugs, the patients may have adverse reactions such as diarrhea, gastrointestinal discomfort, endocrine dyscrasia and the like, and serious damages to the liver and kidney of the human body can be caused by long-term administration. Screening of pancreatic lipase inhibitors from natural products for small side effects is therefore becoming a growing research focus.

For example, the publication number is CN109497470A, the publication date is 22 days of 3 months in 2019, and the patent document named as a Chlorella pyrenoidosa polypeptide relieving and fattening composition discloses that the Chlorella pyrenoidosa polypeptide is taken as one of main effective components and is compatible with soybean polypeptide, corn polypeptide, lentinan, hericium erinaceus polysaccharide, wheat oligomerization polypeptide powder, green tea polyphenol extract and medlar concentrated juice, so that the composition can promote fat metabolism, reduce storage of subcutaneous fat and have the effect of relieving and fattening.

However, the existing preparation technology of the chlorella pyrenoidosa polypeptide has the problem that the prepared product has insufficient lipase inhibition activity, and needs to be further improved.

Disclosure of Invention

In order to solve the problems mentioned in the background art, the invention provides a preparation method of chlorella polypeptide, which comprises the following steps:

s100, wall breaking and extraction: mixing chlorella powder and water, homogenizing, and performing ultrasonic treatment to obtain a crude extract;

homogenizing and ultrasonic treating the chlorella solution to obtain a crude extract; the chlorella is subjected to wall breaking treatment by adopting homogenization and ultrasound, and then the chlorella protein is extracted by taking water as a solvent, so that the protein extraction efficiency is high, the total protein of the chlorella is reserved, rich proteins are provided for the subsequent enzymolysis process, the variety of polypeptides in the enzymolysis liquid is improved, and the water-extracted protein has good re-solubility and is convenient to use;

s200, centrifuging, standing and removing impurities: centrifugally separating the crude extract, standing at 1-5 ℃ for precipitation, and concentrating the supernatant to obtain chlorella water extract; the process mainly removes liposoluble components such as pigment in the crude extract;

s300, enzymolysis: adding neutral protease into the chlorella water extract obtained in the step S200 for enzymolysis, and inactivating the enzyme to obtain neutral protease hydrolysate;

s400, separating and purifying: centrifuging the neutral protease hydrolysate to obtain liquid, intercepting filtrate with molecular weight of 200-3000Da, concentrating, and spray drying to obtain the Chlorella polypeptide.

On the basis of the scheme, further, the chlorella powder and water are mixed in a proportion of 10 mL/g.

Based on the scheme, further, the homogenization is carried out by adopting a ball mill for 30-60min.

Based on the scheme, further, the ultrasonic wave adopts an ultrasonic wave extractor with a cooling function, and the ultrasonic wave is carried out for 20-25min under the condition of 570-580W at the temperature of 2-6 ℃. The chlorella solution is subjected to ultrasonic treatment at 2-6deg.C to prevent protein denaturation due to heat generated during ultrasonic treatment.

Based on the scheme, further, the centrifugation in S200 and S400 is tubular centrifugation, and the rotation speed is 10000-14000r/min.

Based on the scheme, in the step S400, the liquid obtained by centrifugal separation of the neutral protease hydrolysate is filtered by diatomite to obtain clear liquid, and then the clear liquid is filtered by a 50nm ceramic membrane and a 3000Da ultrafiltration membrane, and after a ultrafiltration cup is closed, the pressure is increased by nitrogen, and the ultrafiltration pressure is 0.25MPa; and then removing impurities through a nanofiltration membrane with the molecular weight of 200Da to 3000Da to obtain filtrate.

Based on the scheme, the protein content of the chlorella water extract is 2% -4%.

Based on the scheme, the enzymolysis condition of the neutral protease is that the enzyme bottom ratio is 3% -5%, the pH is 6.8-7.2, and the neutral protease is subjected to constant temperature enzymolysis for 4-6h in a water bath at 50-60 ℃. Under the condition, the neutral protease has high stability, uneasiness, high speed of converting the substrate into the product and high enzymolysis efficiency, and the product mainly uses low-molecular short peptide and free amino acid, so that the chlorella resource is fully utilized.

Based on the scheme, the enzyme deactivation condition of the neutral protease is water bath at 80-90 ℃ for 10-20min.

The invention also provides a chlorella polypeptide prepared by the preparation method of the chlorella polypeptide.

Compared with the prior art, the preparation method of the chlorella polypeptide provided by the invention has the following technical effects:

1. the chlorella polypeptide obtained by hydrolyzing neutral protease has high inhibition activity on lipase and has lipid-lowering effect;

2. the ultrafiltration method has the advantages of low energy consumption, less consumption of chemical reagents, short process flow and the like, and is suitable for industrial production;

3. the chlorella polypeptide prepared by the method also has the advantages of low gel property and low viscosity, and has less loss in the production process; has good stability, is not easy to be denatured, and is convenient for production and preservation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a diagram of chlorella separation and purification analysis provided by the invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The present invention provides the following example 1:

s100, wall breaking and extraction: accurately weighing 5 parts of chlorella powder, wherein the volume usage of purified water is 10mL/g based on the mass of chlorella dry powder, stirring uniformly at normal temperature, then sending into a ball mill for homogenization for 30min, then placing chlorella powder suspension into an ultrasonic extractor with a cooling function, setting the temperature to 2-6 ℃, and carrying out ultrasonic treatment for 20min under the ultrasonic power of 580W to obtain a crude extract;

s200, centrifuging, standing and removing impurities: centrifuging the crude extract at 4deg.C and rotation speed of 10000r/min for 30min, filtering, standing at 4deg.C for precipitation, collecting supernatant, detecting protein concentration, and concentrating with nanofiltration membrane to protein content of 2% to obtain chlorella water extract;

s300, enzymolysis: adding 1398 neutral protease into the chlorella water extract obtained in the step S200, setting the enzyme-to-base ratio to be 4%, adjusting the pH to be 7, carrying out constant-temperature enzymolysis for 6 hours at the water bath of 55 ℃, and carrying out enzyme deactivation operation for 15 minutes at the water bath of 85 ℃ to terminate the hydrolysis reaction; centrifuging the hydrolysate at 10000r/min for 30min after cooling at room temperature, filtering with diatomite to obtain supernatant, and concentrating under reduced pressure to obtain enzymolysis product with vacuum degree of 0.08-0.1Mpa, concentration temperature of 40-60deg.C and concentration time of 2 hr;

s400, separating and purifying: purifying the enzymolysis product by gel chromatography, collecting each separated component according to the peak condition, and performing gel column chromatography: the packing is Toyopearl HW-40F gel, the column volume is 135mL, the loading amount is 500mg (3 mL), the mobile phase is purified water and ethanol, the flow rate is 1mL/min, one tube is collected every 6min, 40 tubes are collected in total, and the detection wavelength is 214nm, 260nm, 280nm and 405nm.

As the absorbance of the product at the detection wavelength of 280nm is most sensitive, as shown in figure 1, the separated product is collected according to the detection result of 280nm to obtain 11 parts, which are respectively named as A1-A11, and after decompression concentration, the inhibition activity of each component on lipase is identified, wherein the lipase activity inhibition rate is determined by the following method:

measuring the lipase activity inhibition rate of each component by adopting a p-nitrophenol method, adding 175 mu L of Tris buffer solution, 20 mu L of PL working solution and 4 mu L of sample into a 96-well plate, incubating for 5min at 37 ℃, then adding 1 mu L of pNPB solution, measuring the OD value at 405nm by using an enzyme-labeling instrument after 2.5min, and repeating the steps for three times;

orlistat (Orlistat) as a positive control and DMSO as an inhibitor-free group;

inhibition (%) = (AE-AT)/AE 100%

Wherein AT is the OD value of the inhibitor adding group, AE is the OD value of the inhibitor not adding group; the test results are shown in table 1:

TABLE 1

	Average molecular weight	Inhibition of Lipase activity%
			Orlistat	495.73	95.1
A1	1262.71	0.31
			A2	1051.25	0
A3	861.63	0
			A4	819.07	0
A5	742.95	0
			A6	720.47	0
A7	679.85	24.27
			A8	629.18	1.73
A9	584.44	0
			A10	565.43	0
A11	494.17	0

As shown in Table 1, the inhibition rate of A7 was highest, and it was 24.27% (4 mg/mL), and the IC50 value was 8.2459.+ -. 0.0694mg/mL.

The invention also provides the following comparative examples:

comparative example 1.1

Papain is adopted to replace the enzymolysis process in the embodiment 1, the enzymolysis condition is 50 ℃, the pH=7, the enzyme bottom ratio is 4%, the enzymolysis time is 5 hours, and the rest is the same as the embodiment 1;

comparative example 1.2

2709 alkaline protease is adopted to replace the enzymolysis process in the example 1, the enzymolysis condition is 55 ℃, the pH=6.5, the enzyme bottom ratio is 4%, the enzymolysis time is 8 hours, and the rest is the same as the example 1;

comparative example 1.3

537 acid protease is adopted to replace the enzymolysis process in the embodiment 1, the enzymolysis condition is 55 ℃, the pH=7, the enzyme bottom ratio is 4%, the enzymolysis time is 5 hours, and the rest is the same as the embodiment 1;

comparative example 1.4

The pancreatin is adopted to replace the enzymolysis process in the example 1, the enzymolysis condition is 37 ℃, the pH=2, the enzyme bottom ratio is 6%, the enzymolysis time is 8 hours, and the rest is the same as the example 1;

comparative example 1.5

The enzymolysis process in the example 1 is replaced by flavourzyme, the enzymolysis condition is 42 ℃, the pH=8, the enzyme bottom ratio is 3%, the enzymolysis time is 8 hours, and the rest is the same as the example 1;

comparative example 1.6

The enzymolysis process in example 1 is changed into the enzymolysis process that composite protease (2709 alkaline protease: trypsin=5:1) is added, the enzyme bottom ratio is set to be 2.2%, the substrate concentration is 4.0%, the pH is adjusted to be 8.8, the enzymolysis is carried out for 30min under the constant temperature of 60 ℃ in water bath, and the rest is unchanged;

comparative example 1.7

Xia Cheng aminopeptidase is adopted to replace the enzymolysis process in the example 1, the enzymolysis condition is 57 ℃, the pH=8.5, the enzyme bottom ratio is 5%, the enzymolysis time is 5 hours, and the rest is the same as the example 1;

the enzymatic hydrolysate obtained in comparative examples 1.1 to 1.7 and the enzymatic hydrolysate obtained in example 1 were formulated to 4mg/mL, and were screened for lipase inhibitory activity, and compared with Orlistat (Orlistat), a positive drug, and the inhibition ratios of the enzymatic hydrolysate to lipase activity are shown in Table 3:

TABLE 3 Table 3

	Lipase inhibition ratio%
		Orlistat	95.1
Example 1	8.58
		Comparative example 1.1	0
Comparative example 1.2	0
		Comparative example 1.3	3.62
Comparative example 1.4	2.83
		Comparative example 1.5	0
Comparative example 1.6	0
		Comparative example 1.7	0

As can be seen from the results in Table 3, the enzyme hydrolysis product of 1398 neutral protease has good lipase inhibitory activity, and the rest enzyme hydrolysis products have no lipase inhibitory activity.

The invention also provides the following example 2:

s100, wall breaking and extraction: accurately weighing 1 kg of chlorella powder, adding purified water, mixing uniformly, wherein the volume dosage of the purified water is 10mL/g based on the mass of chlorella dry powder, stirring uniformly at normal temperature, then sending into a ball mill for homogenizing for 60min, then placing chlorella suspension into an ultrasonic extractor with a cooling function, setting the temperature to 2-6 ℃, and carrying out ultrasonic treatment for 20min under the power of 580W to obtain crude extract;

s200, centrifuging, standing and removing impurities: performing tubular centrifugal separation on the crude extract at the rotating speed of 12000r/min, standing at the temperature of 4 ℃ for 30min, precipitating, taking supernatant to detect the protein concentration, and concentrating the supernatant to the protein content of 4% through a nanofiltration membrane to obtain chlorella water extract;

s300, enzymolysis: adding 1398 neutral protease into the chlorella water extract obtained in the step S200, setting the enzyme-to-base ratio to be 4%, adjusting the pH value to be 7, carrying out constant temperature enzymolysis for 5 hours at the water bath of 55 ℃, and inactivating enzyme in the water bath of 85 ℃ for 15 minutes;

s400, separating and purifying: centrifuging the neutral protease hydrolysate subjected to enzyme deactivation at 12000r/min, filtering the separated liquid with diatomite to obtain supernatant, filtering with 50nm ceramic membrane, filtering with 3000Da ultrafiltration membrane, sealing the ultrafiltration cup, pressurizing with nitrogen, and ultrafiltering at 0.25MPa; removing impurities through a 200Da nanofiltration membrane, concentrating under reduced pressure, and spray drying to obtain the chlorella polypeptide.

The chlorella polypeptide obtained in example 2 was prepared as a 4mg/mL polypeptide solution, and the lipase inhibitory activity was examined and compared with the positive drug Orlistat, and the test results are shown in Table 4:

TABLE 4 Table 4

Polypeptide separation liquid sample	Orlistat	Examples 1 to A7	Example 2
				Average molecular weight	495.73	679.85	689.31
Lipase inhibition ratio%	95.1	24.27	20.36

As can be seen from the test results in Table 4, the lipase inhibition rate of the chlorella bioactive peptide prepared in example 2 is 20.36% (4 mg/mL), and the chlorella bioactive peptide has the effects of reducing blood fat, regulating fat metabolism in vivo and the like, and the preparation method has the advantages of low energy consumption by adopting an ultrafiltration method, being used for separating and purifying protein products, no chemical reagent, short process flow and the like;

meanwhile, the molecular weights of the components separated in the examples 1 and 2 and the lipase inhibitory activities are very close to each other, which means that the components obtained in the examples 1 and 2 are basically the same, so that those skilled in the art can select the small-batch trial production process of the example 1 or the large-batch industrial production process of the example 2 according to actual needs.

The invention also provides the following comparative examples

Neutral protease NoveXin 0.8L and Xia Cheng neutral protease were used as comparative example 2.1 and comparative example 2.2, respectively, in place of neutral protease 1398 in example 2, the remainder being identical to example 2;

concentrating the neutral protease hydrolysate of the example 2, the comparative example 2.1 and the comparative example 2.2 under reduced pressure, wherein the parameter of the reduced pressure concentration is that the vacuum degree is 0.08-0.1Mpa, the concentration temperature is 40-60 ℃, and the concentration time is 2h;

the concentrated enzymolysis products are prepared into 4mg/L, lipase inhibition activity screening is carried out respectively, and compared with Orlistat, the inhibition rate of each enzymolysis product on lipase activity is shown in table 5:

TABLE 5

Polypeptide separation liquid sample	Orlistat	Example 2	Comparative example 2.1	Comparative example 2.2
					Lipase inhibition ratio%	95.1	8.47	3.41	0

From the test results in table 5, although the same is neutral protease, the lipase inhibition activity of the enzymatic hydrolysis product of 0.8L of novelin neutral protease is far lower than that of example 1 and example 2, and the enzymatic hydrolysis product of Xia Cheng neutral protease does not even have lipase inhibition activity, so that the polypeptide prepared by the process using 1398 neutral protease has higher lipase inhibition rate, and meanwhile, the process is simple, economical and reasonable, and is suitable for industrial production.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (8)

1. The preparation method of the chlorella polypeptide is characterized by comprising the following steps:

s100, wall breaking and extraction: mixing chlorella powder and water, homogenizing, and performing ultrasonic treatment to obtain a crude extract;

s200, centrifuging, standing and removing impurities: centrifugally separating the crude extract, standing at 1-5 ℃ for precipitation, and concentrating the supernatant to obtain chlorella water extract;

s300, enzymolysis: adding neutral protease into the chlorella water extract obtained in the step S200 for enzymolysis, and inactivating the enzyme to obtain neutral protease hydrolysate;

s400, separating and purifying: centrifuging neutral protease hydrolysate to obtain liquid, intercepting filtrate with molecular weight of 200-3000Da, concentrating, and spray drying to obtain Chlorella polypeptide;

in the step S400, the liquid obtained by centrifugal separation of the neutral protease hydrolysate is filtered by diatomite to obtain clear liquid, and then filtered by a 50nm ceramic membrane and a 3000Da ultrafiltration membrane, and after a ultrafiltration cup is closed, the pressure is increased by nitrogen, and the ultrafiltration pressure is 0.25MPa; removing impurities through a nanofiltration membrane with the molecular weight of 200Da to 3000Da to obtain filtrate;

the enzymolysis condition of the neutral protease is that the enzyme bottom ratio is 3% -5%, the pH is 6.8-7.2, and the neutral protease is subjected to constant-temperature enzymolysis for 4-6h in a water bath at 50-60 ℃;

the neutral protease is 1398 neutral protease.

2. The method for producing a chlorella polypeptide according to claim 1, wherein: the chlorella powder and water are mixed in a proportion of 10 mL/g.

3. The method for producing a chlorella polypeptide according to claim 1, wherein: homogenizing for 30-60min by adopting a ball mill.

4. The method for producing a chlorella polypeptide according to claim 1, wherein: the ultrasonic treatment adopts an ultrasonic extractor with a cooling function, and ultrasonic treatment is carried out for 20-25min under the condition of 570-580W at the temperature of 2-6 ℃.

5. The method for producing a chlorella polypeptide according to claim 1, wherein: and the centrifugation in S200 and S400 is tubular centrifugation, and the rotation speed is 10000-14000r/min.

6. The method for producing a chlorella polypeptide according to claim 1, wherein: the content of protein in the chlorella water extract is 2% -4%.

7. The method for producing a chlorella polypeptide according to claim 1, wherein: the enzyme deactivation condition of the neutral protease is water bath at 80-90 ℃ for 10-20min.

8. A chlorella polypeptide, characterized in that: a method for producing the chlorella polypeptide of any one of claims 1 to 7.

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