CN114158621A

CN114158621A - Composition of wolfberry fruit oil and walnut oil and application thereof

Info

Publication number: CN114158621A
Application number: CN202111509884.XA
Authority: CN
Inventors: 张波; 刘兰英
Original assignee: Ningxia Jiubao Ecological Agricultural Technology Development Co ltd
Current assignee: Ningxia Jiubao Ecological Agricultural Technology Development Co ltd
Priority date: 2021-12-10
Filing date: 2021-12-10
Publication date: 2022-03-11
Anticipated expiration: 2041-12-10
Also published as: CN114158621B

Abstract

The invention relates to the technical field of functional food preparation, in particular to a composition of medlar fruit oil and walnut oil and application thereof; the composition comprises medlar oil and walnut oil in any proportion, and protein peptide with anti-fatigue effect is also added in the composition; according to the invention, the medlar oil and the walnut oil are used in a matching manner, and the prepared composition has the function of relieving asthenopia. Meanwhile, the tuna meat is subjected to enzymolysis by using a compound enzyme of Alcalase2.4 enzyme and trypsin to prepare the protein peptide with the anti-fatigue function, and the prepared protein peptide is added into a solvent prepared from the Chinese wolfberry fruit oil and the walnut oil, so that the prepared product has the effect of resisting asthenopia and also has the effect of relieving fatigue.

Description

Composition of wolfberry fruit oil and walnut oil and application thereof

Technical Field

The invention belongs to the technical field of functional food preparation, and particularly relates to a composition of wolfberry fruit oil and walnut oil and application thereof.

Background

Asthenopia refers to eye fatigue caused by excessive use of eyes while working or learning at close distances. This condition is often experienced by people who are engaged in close-range precision work, computer work, or study or work in environments with poor lighting conditions (too bright/too dark light), and who suffer from ametropia such as myopia, hyperopia, presbyopia, and physical weakness. The symptoms of asthenopia include asthenopia, dry eye, foreign body sensation, heavy eyelid, blurred vision, photophobia, lacrimation, eye swelling and pain, hyperemia of eyes, etc., severe patients also have general symptoms such as headache, dizziness, nausea, listlessness, attention deficit, memory loss, inappetence, etc., a few patients have double vision, stereoscopic vision dysfunction, increased intraocular pressure, corneal lesion, etc., and teenagers also have myopia or deepened myopia. The glaucoma, eye surface or anterior segment disease patients can also cause or aggravate the original eye diseases due to the over-fatigue of eyes, so that the vision fatigue is relieved, which is always a research hotspot.

At present, a lot of students do a lot of work on the aspect of relieving asthenopia, but various measures for relieving asthenopia have unsatisfactory effects, and certain side effects exist in the developed traditional Chinese medicines, chemical medicines and the like with the asthenopia resisting effect after long-term administration.

Disclosure of Invention

The invention aims to provide a composition of Chinese wolfberry fruit oil and walnut oil and application thereof, wherein the Chinese wolfberry fruit oil and the walnut oil have the function of relieving asthenopia; the added protein peptide has the function of resisting fatigue, thereby making up the defects of the prior art.

The invention firstly provides a composition of medlar fruit oil and walnut oil, wherein the composition comprises the medlar fruit oil and the walnut oil in any proportion;

furthermore, the composition is also added with protein peptide with anti-fatigue effect;

the protein peptide with the anti-fatigue effect is obtained by centrifuging enzymolysis liquid of Alcalase2.4 enzyme and trypsin of tuna meat as a specific description of an embodiment;

the enzymolysis liquid is subjected to enzymolysis for 5 to 6 hours at the enzymolysis temperature of 35 ℃;

wherein the enzyme activity ratio of Alcalase2.4 enzyme to trypsin is 1: 3;

further, the molecular weight of the protein peptide is 500-3000 Da.

The protein peptide has the amino acid sequence of YYMEGDFAFGDNY.

Furthermore, the composition also comprises common flavoring agents, preferably, sweetening agents or souring agents;

the invention also provides an anti-fatigue product prepared by the preparation method of the technical scheme.

According to the invention, the medlar oil and the walnut oil are used in a matching manner, and the prepared composition has the function of relieving asthenopia. Meanwhile, the tuna meat is subjected to enzymolysis by using a compound enzyme of Alcalase2.4 enzyme and trypsin to prepare the protein peptide with the anti-fatigue function, and the prepared protein peptide is added into a solvent prepared from the Chinese wolfberry fruit oil and the walnut oil, so that the prepared product has the effect of resisting asthenopia and also has the effect of relieving fatigue.

Drawings

FIG. 1: HPLC profile of 50% ethanol eluted fraction;

FIG. 2: purifying and detecting the eluate for 30-32 min;

FIG. 3: graph of the effect of different doses of PA1 polypeptide on the weight-bearing swimming time of mice.

Detailed Description

The invention adds the protein peptide with the anti-fatigue function into the solvent prepared from the medlar fruit oil and the walnut oil, so that the prepared edible product has the efficacy of resisting asthenopia and also has the effect of relieving fatigue.

The present invention will be described in detail with reference to the following examples and accompanying drawings

Example 1: method for preparing medlar fruit oil

The preparation method of the medlar fruit oil comprises the following steps:

(1) selecting high-quality medlar without broken particles and full fruits, placing the medlar in a low-temperature pulverizer for pulverizing, sieving the medlar by a 40-mesh sieve, weighing 5kg of the obtained medlar powder, placing the medlar powder into an extraction cloth bag, tying a mouth, placing the medlar powder into a closed extraction tank of subcritical extraction equipment, closing a feeding channel, vacuumizing to-0.075 MPa, pumping 20L of butane, heating to 35 ℃, extracting under the extraction pressure of 0.5MPa and the ultrasonic frequency of 15KHz for 25min, and extracting for 3 times;

(2) and after extraction is finished, pumping the extraction solvent into a separation tank, starting a vacuum pump, reducing the pressure of the separation tank to-0.06 MPa, keeping the temperature at 35 ℃, evaporating butane in the material, compressing the gasified butane into a liquid state by a diaphragm compressor, refluxing into the solvent tank, closing the vacuum pump to enable the pressure of the separation tank to reach 0MPa, and discharging the wolfberry fruit oil from a purification port to obtain the wolfberry fruit oil.

The medlar fruit oil used by the invention can adopt any commercially available medlar fruit oil product.

Example 2 method for preparing walnut oil

(1) Cleaning fresh oil walnut, pulverizing to 20 mesh, adding purified water at 40 deg.C 4 times of fresh fruit, and stirring;

(2) further crushing and extracting the cell tissues in the pulp in the step (1) by high-speed shearing, wherein the high-speed shearing rotation speed is 12000r/min, the extraction time is 8min, and the interval is 0.5min every 2min of extraction;

(3) fusing the pulp aqueous solution after high-speed shearing and crushing by using a stirrer to form pulp, and heating, preserving heat and stirring at the temperature of 35 ℃;

(4) then squeezing the fruit pulp in the step (3) at normal temperature;

(5) performing solid-liquid separation on the squeezed pulp by using a high-speed centrifuge (the rotating speed of the high-speed centrifuge is 3000r/min, the time is 25min), separating pomace, and further filtering the oil-water mixed solution by using a 200-mesh filter membrane;

(6) adding sodium chloride accounting for 2 percent of the weight of the oil-water mixture into the oil-water mixed solution, and stirring to completely dissolve the sodium chloride;

(7) standing for 25min, and performing oil-water separation to obtain high-content hydroxytyrosol-rich walnut crude oil and fruit water;

(8) standing the walnut crude oil rich in the high-content hydroxytyrosol to obtain the walnut oil rich in the hydroxytyrosol.

Example 3: preparation of protein peptide with anti-fatigue function

1. Preparation of protein peptides

Adding 5L of purified water into fresh tuna meat, shearing for 30min by using a high-speed shearing machine, transferring to an extraction tank, adding 2g of Alcalase2.4 enzyme and 6g of trypsin, and carrying out enzymolysis at 35 ℃; stirring for enzymolysis reaction for 6 h; then, the mixture was centrifuged by a tubular centrifuge at 10000rpm to obtain an enzymatic hydrolysate.

And (3) firstly pumping the supernatant obtained after the centrifugation of the enzymolysis product into an ultrafiltration membrane with the molecular weight of 3000Da for ultrafiltration, controlling the membrane-passing temperature to be below 25 ℃, controlling the flow rate to be 50mL/min and the pressure to be 15bar, and collecting the enzymolysis liquid penetrating through the membrane. And (3) allowing the enzymatic hydrolysate passing through the first filter membrane (ultrafiltration membrane) to pass through a second filter membrane (nanofiltration membrane) nanofiltration membrane with the molecular weight of 500Da, controlling the membrane passing temperature at 30 ℃ and the pressure at 25bar, directly spray-drying the enzymatic hydrolysate intercepted by the membrane system to obtain milk white powder as protein peptide solid, subpackaging the obtained protein peptide solid with aluminum foil bags, and sterilizing the protein peptide solid in a Co-60 radiation sterilization device for 3 hours to obtain the finished product of the protein peptide.

2. Protein peptide purification

Preparing the prepared protein peptide into a 100mg/ml solution, adsorbing by macroporous adsorption resin, and eluting by pure water, 10% ethanol, 25% ethanol, 50% ethanol, 75% ethanol and 95% ethanol respectively to obtain 6 elution components. The 6 eluted fractions were each tested for Nitric Oxide Synthase Activity (Nitric Oxide Synthase Activity Assay kit, Colorimetric). The test result shows that the 50% ethanol elution component has the best activity.

Separating the 50% ethanol elution component by using HPLC again, wherein the separation conditions are as follows: the instrument comprises the following steps: agilent1260 type high performance liquid chromatograph, chromatographic column: eilit SimoChrom ODS-BP5 μm (4.6 mm. times.250 mm), mobile phase: methanol and 0.1% TFA water, flow rate: 1ml/min, detection wavelength: 220nm, collecting eluate at different time intervals (FIG. 1), and detecting the activity of the eluate for 30-32 min.

3. HPLC analysis of functional protein peptides

Subjecting 30-32min eluate to high performance liquid chromatography C₁₈The analytical column again purified the 30% methanol elution peak with the best results, resulting in a purer single peak (fig. 2), allowing the isolated sample to be of a purity that allows sequencing.

4. Protein peptide structure identification

And (3) carrying out polypeptide structure identification on the purified protein peptide, wherein the chromatographic conditions are as follows: instrument type: LCMS-9030Q-TOF quadrupole-time of flight mass spectrometer, chromatography column: inertsil HILIC C18 reverse phase chromatography column 150 mm. times.3.0 mm, 3 μm, Shimadzu, Japan), column temperature: 30 ℃, injection volume: 1.0 μ L; mobile phase: a is 0.1% (v/v) formic acid water solution, mobile phase B is 0.1% (v/v) formic acid acetonitrile solution, gradient elution procedure: 0-5.0min, 5%, 5.0-10.0min, 10%, 10.0-15.0min, 20%, 15.0-20.0min, 40%; flow rate 0.3mL/min, mass spectrometry ion mode: ESI source, in positive ion mode, ion source interface voltage: 4.0kV, gas flow rate: 10.0L/min, drying gas flow rate: 10.0L/min, heating gas flow rate: 10.0L/min; heating module temperature: 400 ℃, interface temperature: 300 ℃, scan mode: full scan mode (m/z 50-2000).

The amino acid sequence of the protein peptide obtained by sequencing is YYMEGDFAFGDNY, the molecular weight of the protein peptide is 1535.7Da, and the polypeptide is named as PA 1.

5. Detecting the anti-fatigue function of protein peptide

Selecting a male mouse with the weight of 18-22g, dividing the mouse into a blank control group and an experimental group, wherein the experimental group comprises a low-dose group, a medium-dose group and a high-dose group, the blank control group, the low-dose group, the medium-dose group and the high-dose group are all 10 mice, and feeding the mice with synthetic PA1 polypeptide respectively; the anti-fatigue swimming test of the mice is carried out. After a lead wire with the weight of 5% is loaded at the tail of the mouse, the lead wire is put into a water tank for swimming, the time that the mouse can not float out of the water surface (in a exhaustion state) from the time of entering the water to the time of sinking the mouse into the water is observed and recorded, the experimental water temperature is 25 ℃, and the water depth is 25 cm. The results of the experiment are shown in table 1 and fig. 3. The relevant data are recorded in table 1 and bar chart statistics are performed for each set of data, see fig. 3.

Table 1: table of the Effect of different doses of PA1 polypeptide on mouse weight-bearing swimming time (x. + -. s)

Group of	n	Dosage (mg/kg)	Exhaustion State time(s)
				Control group	10	-	365.67±51.2
Low dose group	10	50	554.59±62.4
				Middle dose group	10	100	677.32±45.7
High dose group	10	150	720.75±74.1

The experimental results showed that the time of the mice in the low dose group, the medium dose group and the high dose group was significantly prolonged in the exhaustion state (p <0.01) as compared with the control group, and thus it was found that the PA1 protein peptide was able to exert an anti-fatigue effect on the mice, and the medium dose group and the high dose group were significantly prolonged as compared with the low dose group, indicating that the anti-fatigue effect of the PA1 polypeptide had concentration dependency, but the 150mg/kg group did not significantly prolong the weight swimming time as compared with the 100mg/kg group.

Example 4: composition comprising protein peptide added to Chinese wolfberry fruit oil and walnut oil

Adding the protein peptide prepared in the example 3 into a solvent to prepare a product with an anti-fatigue function, wherein the solvent comprises medlar fruit oil and walnut oil;

one specific article is prepared as follows:

1) mixing 2 parts of medlar oil, 3 parts of walnut oil and 10 parts of water, and stirring to prepare a solvent;

2) adding the protein peptide into the solvent at a concentration of 20-65 μ g/ml.

Furthermore, the solvent is also added with a flavoring agent for improving taste, which is a sweetening agent or an acidulant.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present 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 solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the present invention.

Claims

1. The composition of the medlar fruit oil and the walnut oil is characterized by comprising the medlar fruit oil and the walnut oil in any proportion.

2. The composition of wolfberry fruit oil and walnut oil as claimed in claim 1, wherein the composition further comprises an anti-fatigue protein peptide.

3. The composition of wolfberry fruit oil and walnut oil as claimed in claim 2, wherein the said anti-fatigue protein peptide is obtained by centrifuging the enzymatic hydrolysate of alcalase2.4 and trypsin of tuna meat.

4. The composition of wolfberry fruit oil and walnut oil as claimed in claim 3, wherein the enzymolysis temperature of the enzymolysis solution is 35 ℃ for 5-6 h.

5. The composition of Lycium barbarum fruit oil and walnut oil of claim 3, wherein the ratio of Alcalase2.4 enzyme to trypsin enzyme activity is 1: 3.

6. The composition of Lycium barbarum fruit oil and walnut oil as claimed in claim 2, wherein the molecular weight of the protein peptide is 500-3000 Da.

7. The composition of wolfberry fruit oil and walnut oil as claimed in claim 6, wherein the protein peptide has the amino acid sequence of YYMEGDFAFGDNY.

8. The composition of Lycium barbarum fruit oil and walnut oil and its use of claim 1, wherein said composition further comprises conventional flavoring agents.

9. The composition of lycium barbarum fruit oil and walnut oil and its use according to claim 8, wherein the seasoning is a sweetener or an acidulant.

10. Use of a composition prepared by the process of any one of claims 1 to 9 in an anti-fatigue article.