阅读说明：本技术 一种利用日本曲霉pj01产酶降解西番莲果皮多糖的制备方法及应用 (Preparation method and application for degrading passion fruit peel polysaccharide by utilizing enzyme produced by aspergillus japonicus PJ01 ) 是由 李霞 孙永进 单杨 李培骏 关媛 于 2021-01-08 设计创作，主要内容包括：本发明公开了一种利用日本曲霉PJ01产酶降解西番莲果皮多糖的酶解修饰工艺及其应用,属于药用食品研发领域。本发明取西番莲果皮制备成西番莲果皮多糖,以日本曲霉PJ01为实验菌株,培养产出粗酶液与多糖混匀,进行酶解得到酶解修饰后的西番莲果皮多糖。结果显示经日本曲霉PJ01产酶降解后的西番莲果皮多糖具有更好的抗氧化活性,在抗氧化类保健品和化妆品中具有广阔的应用前景。(The invention discloses an enzymolysis modification process for degrading passion fruit peel polysaccharide by utilizing aspergillus japonicus PJ01 produced enzyme and application thereof, belonging to the field of medicinal food research and development. The invention takes passion fruit peel to prepare passion fruit peel polysaccharide, takes aspergillus japonicus PJ01 as an experimental strain, cultures and produces crude enzyme liquid to be mixed with the polysaccharide evenly, and carries out enzymolysis to obtain the passion fruit peel polysaccharide after enzymolysis modification. The result shows that the passion fruit peel polysaccharide after being degraded by the enzyme produced by the aspergillus japonicus PJ01 has better antioxidant activity and has wide application prospect in antioxidant health care products and cosmetics.)

1. A preparation method for degrading passion fruit peel polysaccharide by utilizing Aspergillus japonicus PJ01 produced enzyme is characterized by comprising the following specific steps:

(1) cleaning fresh passion fruit peel, drying at 50 ℃, crushing, adding 80% ethanol according to the feed-liquid ratio of 1:20g/mL at 85 ℃, condensing, refluxing and extracting for 3 times (2 h each time), filtering, and drying residues to obtain passion fruit peel powder with pigments and phenols removed;

(2) weighing 1g of passion fruit peel powder prepared in the step (1), adding distilled water according to the ratio of material to liquid of 1:27g/mL, performing microwave extraction for 3min, filtering, centrifuging at 3000rpm for 10min, and concentrating the supernatant to obtain a concentrated solution;

(3) adding 4 times volume of absolute ethyl alcohol into the concentrated solution prepared in the step (2), uniformly mixing, standing for 48h, centrifuging at 4000rpm for 15min, taking the precipitate, and freeze-drying to obtain water-extracted passion fruit peel polysaccharide;

(4) inoculating Aspergillus japonicus PJ01 as experimental strain on PDA slant for culture; selecting single colony with good growth vigor, purifying, activating for 2 times, inoculating to slant, and storing to obtain activated strain;

(5) inoculating the activated strain in the step (4) into a PDA solid state fermentation culture medium, culturing for 3 days in a constant temperature incubator at 30 ℃, adding distilled water according to the proportion of 1:20(w: v) to soak a fermentation substrate for producing enzyme by solid state fermentation, and reacting for 2 hours in a shaking incubator at the conditions of 170rpm and 40 ℃ to obtain a crude enzyme solution;

(6) mixing the water-extracted passion fruit peel polysaccharide obtained in the step (3) and the crude enzyme solution obtained in the step (5) according to the ratio of 1:200(w: v), dissolving in a water bath kettle at 45 ℃, then transferring into a shaking incubator, hydrolyzing for 6h under the conditions of 45 ℃ and 170rpm, finally carrying out boiling water bath for 5min to terminate the reaction, concentrating, and freeze-drying to obtain the enzymolysis passion fruit peel polysaccharide.

2. The use of the enzymatically degraded passion fruit peel polysaccharide prepared by the preparation method of claim 1 is characterized in that the enzymatically degraded passion fruit peel polysaccharide is used in antioxidant health products and cosmetics.

Technical Field

The invention relates to an enzymolysis modification process for degrading passion fruit peel polysaccharide by utilizing enzyme produced by aspergillus japonicus PJ01 and application of the enzymolysis modification process in preparation of an antioxidant, belonging to the field of research and development of medicinal foods.

Background

The passion fruit peel polysaccharide has various biological activities, such as blood sugar and blood fat regulation, antioxidation, anti-tumor and the like, and the activity of the polysaccharide is closely related to the structure, monosaccharide content and the like of the polysaccharide, so the structure and the activity before and after the polysaccharide is degraded need to be deeply researched.

The enzymatic degradation method is a biological degradation method for obtaining polysaccharide with lower molecular weight by degrading polysaccharide with enzyme, has many advantages, has stronger specificity of enzymatic degradation, is more environment-friendly in the whole degradation process, relatively mild in reaction, lower in energy consumption, higher in degradation efficiency, and can protect the structure and activity of a degraded product to the maximum extent, and the enzyme catalysis has high efficiency and specificity.

The molecular weight of the crude polysaccharide extracted from passion fruit peel is relatively large, the viscosity is also relatively high, and the maximum effect is influenced to a certain extent, so that the polysaccharide with small molecular weight is obtained by adopting a degradation method. Huang Yongchun et al found that it could significantly reduce the molecular weight of chitosan by using alpha-amylase to degrade chitosan. Studies such as Suchang find that the papain can degrade chitosan. In the degradation process of the polysaccharide, certain specific chemical bonds are broken, so that more reducing ends are exposed, the content of reducing sugar is obviously increased, the intrinsic viscosity of the polysaccharide is gradually reduced, and the reducing capacity is gradually improved. By chemically degrading the polysaccharide, both the reducing power and the free radical scavenging power of the degraded polysaccharide are significantly improved compared to before degradation, which may be related to the reduction in molecular weight. The biological activity of polysaccharides is related to many factors, such as solubility and molecular weight. Degradation increases the hydrophilic groups of the polysaccharide, increasing the solubility of the polysaccharide, which in turn further increases the activity level of the polysaccharide.

The invention carries out enzymolysis on passion fruit peel polysaccharide, and compares the change of antioxidant capacity before and after the crude polysaccharide is degraded by enzyme.

Disclosure of Invention

The invention aims to provide a preparation method of enzymolysis passion fruit peel polysaccharide, which is used for researching the antioxidant activity of the polysaccharide before and after enzymolysis.

A method for preparing Passiflora edulis pericarp polysaccharide by using Aspergillus japonicus PJ01 enzyme production degradation. The method specifically comprises the following steps: preparation of passion fruit peel polysaccharide and preparation of passion fruit peel polysaccharide degraded by enzyme produced by aspergillus japonicus PJ 01. Firstly, preparing passion fruit peel into passion fruit peel polysaccharide powder, taking aspergillus japonicus PJ01 as a test strain, culturing by using a PDA culture medium to obtain a crude enzyme solution, mixing the crude enzyme solution with the polysaccharide uniformly, and carrying out enzymolysis to obtain the enzymolysis modified passion fruit peel polysaccharide.

The invention achieves the above purpose by the following technical scheme:

(1) cleaning fresh passion fruit peel, drying at 50 deg.C, and pulverizing. Adding 80% ethanol at a ratio of 1:20g/mL at 85 deg.C, condensing, reflux-extracting for 3 times (2 hr each time), filtering, and drying the residue to obtain passion fruit peel powder with pigment and phenols removed.

(2) Weighing 1g of the above powder, adding distilled water according to the ratio of material to liquid (1:27g/mL), microwave extracting for 3min, filtering, centrifuging at 3000rpm for 10min, and concentrating the supernatant.

(3) Adding anhydrous ethanol at a ratio of 1:4, precipitating for 48h, centrifuging at 4000rpm for 15min, collecting the precipitate, and freeze drying to obtain Water-extracted Passiflora edulis pericarp crude polysaccharide (WPEP).

(4) Inoculating Aspergillus japonicus PJ01 as experimental strain on PDA slant for culture; and selecting a single colony with better growth vigor for purification. Activating for 2 times, inoculating slant for preservation; culturing in PDA solid fermentation culture medium at 30 deg.C for 3 days.

(5) Adding distilled water into the mixture according to the ratio of 1:20(w: v) to soak a fermentation substrate for producing the enzyme by solid state fermentation, and reacting for 2h under the conditions of 170rpm and 40 ℃ to prepare a crude enzyme solution.

(6) Accurately weighing the polysaccharide powder in the step (3), dissolving the polysaccharide powder in distilled water, mixing the polysaccharide powder with passion fruit peel polysaccharide by taking an Enzyme produced by aspergillus japonicus PJ01 as a hydrolase, and carrying out an enzymolysis reaction to obtain the passion fruit peel polysaccharide (EWPPE) after the enzymolysis modification.

Drawings

FIG. 1 illustrates the effect of different degradation reaction times on reducing sugar content. .

FIG. 2 shows the DPPH radical scavenging effect of polysaccharides and Vc before and after the enzyme production and degradation of Aspergillus japonicus PJ 01.

FIG. 3 shows the effect of reducing power of polysaccharide and Vc before and after the enzyme degradation by Aspergillus japonicus PJ 01.

FIG. 4 shows the scavenging effect of polysaccharide and Vc on hydroxyl radicals before and after the enzyme production and degradation of Aspergillus japonicus PJ 01.

FIG. 5 shows the scavenging effect of polysaccharide and Vc on superoxide anion free radicals before and after the enzyme production degradation of Aspergillus japonicus PJ 01.

Detailed Description

The technical solution of the present invention is further illustrated by the following examples.

(1) Preparation of passion fruit peel polysaccharide

Extracting passion fruit peel with ethanol, collecting dried fruit residue at a ratio of 1:27g/mL, microwave extracting for 3min, filtering, centrifuging at 3000rpm for 10min, and concentrating the supernatant. Adding absolute ethyl alcohol according to the volume ratio of 1:4 for precipitation for 48h, centrifuging at 4000r/min for 15min, taking the precipitate, and freeze-drying to obtain the water-extracted passion fruit peel crude polysaccharide.

(2) Preparation method of crude enzyme solution

Activating strains: inoculating Aspergillus japonicus PJ01 as experimental strain on PDA slant for culture; selecting single colony with good growth condition, purifying, activating for 2 times, inoculating to slant, and storing.

Culturing in PDA solid fermentation culture medium at 30 deg.C for 3 days. Adding distilled water into the fermentation substrate for solid state fermentation to produce enzyme at a ratio of 1:20(w: v), and incubating for 2h at the temperature of 40 ℃ at 170rpm to obtain a crude enzyme solution.

(3) Determination of enzyme Activity

And (3) measuring the enzyme activity of the crude enzyme liquid obtained in the step (2), wherein the crude enzyme liquid produced by the solid-state fermentation of the aspergillus japonicus PJ01 contains pectinase, cellulase and xylanase, and the enzyme activities are 966U/gds, 58U/gds and 1004U/gds respectively.

(4) Determination of the time of the enzymatic hydrolysis

Adding distilled water into the fermentation substrate for solid state fermentation to produce enzyme at a ratio of 1:20(w: v), and reacting for 2h at the temperature of 40 ℃ and at 170rpm to obtain a crude enzyme solution. Mixing WPEP and crude enzyme solution at a ratio of 1:200(w: v), dissolving in 45 deg.C water bath, rotating to 45 deg.C, continuing hydrolysis at 170rpm, terminating reaction with 100.00 μ L hydrolysate every 1 hr in 100 deg.C water bath for 5min, and determining reducing sugar content. The result is shown in figure 1, the content of reducing sugar is not changed significantly after 6h of degradation, which shows that the polysaccharide can be completely hydrolyzed to obtain the enzymolysis liquid after 6 h.

(5) Aspergillus japonicus PJ01 enzyme-producing degradation passion fruit peel

Mixing the passion fruit peel polysaccharide and the crude enzyme solution according to the ratio of 1:200(w: v), dissolving in a water bath at 45 ℃, then transferring into a shaking incubator, hydrolyzing for 6h under the conditions of 45 ℃ and 170rpm, finally carrying out boiling water bath for 5min to stop the reaction, concentrating, and freeze-drying to obtain the enzymolysis passion fruit peel polysaccharide.

The enzymolysis modified passion fruit peel polysaccharide and unmodified polysaccharide prepared by the embodiment are applied to antioxidant health care products and cosmetics, and the specific comparison effect is as follows:

(1) determination of DPPH radical scavenging Capacity

The result of the DPPH radical scavenging ability measurement is shown in fig. 2, and at the same concentration, the order of the strength of the DPPH radical scavenging ability is: vc > crude polysaccharide > zymolytic polysaccharide, and zymolytic modification is beneficial to improvement of DPPH free radical scavenging capacity of passion fruit peel.

(2) Measurement of reducing ability

The results of the reduction ability measurement using ascorbic acid as a positive control are shown in FIG. 3. Under the same concentration, the reducing power of Vc polysaccharide is higher than that of polysaccharide enzymolysis polysaccharide, and under the same concentration, the reducing power of the polysaccharide enzymolysis polysaccharide is always higher than that of crude polysaccharide, and the enzymolysis modification is beneficial to the improvement of the reducing power of passion fruit peel polysaccharide.

(3) Determination of hydroxyl radical scavenging Capacity

The hydroxyl radical clearance rate measurement result is shown in fig. 4, under the same concentration, the clearance rate of the enzymolysis polysaccharide to the hydroxyl radical is obviously higher than that of the crude polysaccharide to the hydroxyl radical, when the concentration is 3.2mg/mL, the clearance rates of the enzymolysis polysaccharide and the crude polysaccharide are 65.47% and 29.55% respectively, which shows that the clearance rate of the enzymolysis crude polysaccharide to the hydroxyl radical can be improved.

(4) Determination of superoxide anion scavenging ability

The results of measuring the superoxide anion scavenging ability are shown in fig. 5, and under the same concentration, the strength of the polysaccharide and the Vc solution before and after degradation to the superoxide anion scavenging ability is in the following order: vc is polysaccharide subjected to enzymolysis, which shows that the crude polysaccharide subjected to enzymolysis can improve the capacity of removing superoxide anions.