阅读说明：本技术 一种海带的酶解方法 (Enzymolysis method of kelp ) 是由 蒋蓉 叶元士 王敏 于 2018-06-27 设计创作，主要内容包括：本发明公开了一种海带的酶解方法,属于海藻加工技术领域,包括原料处理、固液比控制、酶解条件控制三个步骤,原料处理：干海带采用粉碎机粉碎至20-60目；新鲜海带则采用搅碎机绞碎至20-60目；固液比控制：粉碎或绞碎的海带按照“样品重量：水重量”＝1:80-90的比例进行稀释,用于酶解；酶解条件控制,本发明采用生物手段将海带细胞在用果胶酶分解成单个分散细胞的基础上,添加纤维素酶将海带细胞细胞壁破壁,酶解海带产品可以用于作为功能性的饲料原料使用,提高养殖动物对海带有效成分如氨基酸、色素物质、矿物质等的利用率,增强饲料的诱食活性、增强饲料的抗氧化功能、增强饲料对养殖动物的免疫能力。(The invention discloses an enzymolysis method of kelp, belonging to the technical field of seaweed processing, comprising three steps of raw material treatment, solid-liquid ratio control and enzymolysis condition control, wherein the raw material treatment comprises the following steps: pulverizing dried herba Zosterae Marinae into 20-60 mesh powder with pulverizer; mincing fresh herba Zosterae Marinae with a mincing machine to 20-60 mesh; controlling the solid-liquid ratio: the crushed or minced kelp is prepared according to the following steps of: diluting the mixture in a ratio of 1:80-90 by weight of water for enzymolysis; the enzymolysis condition is controlled, the invention adopts a biological means to decompose the kelp cells into single dispersed cells by pectinase, and adds cellulase to break the cell walls of the kelp cells, and the enzymolysis kelp product can be used as a functional feed raw material, thereby improving the utilization rate of the cultured animals on the effective components of the kelp, such as amino acid, pigment substances, mineral substances and the like, enhancing the food calling activity of the feed, enhancing the antioxidant function of the feed and enhancing the immunity of the feed on the cultured animals.)

1. An enzymolysis method of kelp is characterized by comprising three steps of raw material treatment, solid-liquid ratio control and enzymolysis condition control, wherein,

the method comprises the following steps: raw material treatment: comprises 2 states of raw materials of fresh kelp and dried kelp. Pulverizing dried herba Zosterae Marinae into 20-60 mesh powder with pulverizer; mincing fresh herba Zosterae Marinae with a mincing machine to 20-60 mesh;

step two: controlling the solid-liquid ratio: the crushed or minced kelp is prepared according to the following steps of: diluting the mixture in a ratio of 1:80-90 by weight of water for enzymolysis;

step three: the enzymolysis condition control comprises the following technical conditions:

(1) the dry matter content of the kelp in the enzymolysis liquid is 10 to 25 percent;

(2) and (3) hydrolyzing enzyme: the hydrolase is cellulase and pectinase; based on the number of enzyme activity units, the combination ratio of the cellulase to the pectinase is as follows: cellulase: pectinase 1:10-1: 20; the number of the cellulase activity units in the enzymolysis liquid is 150IU/mL-250IU/mL, and the number of the corresponding pectinase activity units is 1500IU/mL-3000 IU/mL;

(3) controlling the pH value of the enzymolysis liquid: adjusting the pH value of the enzymolysis liquid to 4.5 by using analytically pure hydrochloric acid, and maintaining the pH value within the range of 4.5 +/-0.5 in the enzymolysis process;

(4) enzymolysis temperature: maintaining the temperature of enzymolysis liquid at 45 +/-5 ℃ in the enzymolysis process in a steam pipeline heating mode;

(5) and (3) enzymolysis time: keeping the above conditions, and controlling the enzymolysis time at 4-5 hr.

2. The method for enzymatically hydrolyzing kelp according to claim 1, characterized in that: the combination and the combination ratio of the cellulase and the pectinase are 1:10-1:20, and the activity unit number of the cellulase and the pectinase in the enzymatic hydrolysate is as follows: the cellulase activity number is 150IU/mL-250IU/mL, and the corresponding pectinase activity unit number is 1500IU/mL-3000 IU/mL.

3. The method for enzymatically hydrolyzing kelp according to claim 1, characterized in that: controlling enzymolysis conditions, including that the dry matter content of the kelp in the enzymolysis liquid is 10 to 25 percent; the temperature of the enzymolysis liquid is 45 +/-5 ℃; the pH value of the enzymolysis liquid is 4.5 +/-0.5; the enzymolysis time is 4-5 hours.

4. The method for enzymatically hydrolyzing kelp according to claim 1, characterized in that: the content of reducing sugar in the enzymatic kelp product obtained after enzymolysis is 8.0-12.5g/100 g; the content of free amino acid is 9.0-15.0g/100 g.

5. The method for enzymatically hydrolyzing kelp according to claim 1, characterized in that: the enzymatic kelp product can be used as a functional feed raw material.

Technical Field

The invention relates to the technical field of seaweed processing, in particular to an enzymolysis method of kelp.

Background

Algae, a general term for marine algae, is a single plant or a long string of simple plants consisting of basic cells, usually supported on the sea floor or some solid structure, and is an important component of marine biological resources. In the dry matter content of the seaweed, the crude protein content of most seaweed is between 1 percent and 48 percent, more than 60 mineral elements, more than 20 free amino acids and more than 12 unsaturated fatty acids and vitamins, besides non-nitrogen compounds (such as seaweed gel, mannitol, starch and the like), the content of iodine and calcium can reach 3 percent to 6 percent, and the seaweed has been used as a main raw material for extracting iodine and occupies an important position on a dining table of a human; at present, the seaweed meal can be prepared into seaweed meal to be added into feed due to the fact that the seaweed meal is rich in various minerals, vitamins, active substances for stimulating the growth of animals, flavor development substances and pigments.

There are more than 15000 seaweeds in the ocean. The algae can be classified into microalgae and macroalgae, wherein the macroalgae is more than 100, and mainly classified into three types, namely Phaeophyta (kelp, Undaria pinnatifida, gulfweed, Cyrtymenia Sparsa, etc.), Rhodophyta (laver, gracilaria, Eucheuma Gelatinosum, etc.) and Chlorophyta (Ulva lactuca, Enteromorpha prolifera, Ulva pertusa, chlorella, etc.). The seaweed can be eaten by human as food, can also be used as feed, additive and other substances to be applied to other aspects, and has excellent edible value and economic value. However, the special biological structure limits the application of the seaweed enzymolysis product in a large amount, the existence of cell walls determines the application range of the seaweed enzymolysis product, but the reasonable application of resources is more important in the modern society with increasingly tense resources, and the seaweed enzymolysis product has important significance on the development of the seaweed enzymolysis product under the large background.

The kelp is a large marine brown algae plant growing in low-temperature seawater, belongs to the marine algae plant, the class Phaeophyceae, the family Laminariaceae, has large sporophyte, is brown and flat belt-shaped, the maximum length can reach 20m, and the holdfast is divided into leaves, stems and holdfast, and is in the shape of false roots. The leaf consists of epidermis, cortex and medulla tissue, and has sporangia, mucous cavity and capable of secreting synovia. The holdfast tree branches are used for attaching submarine rocks. The method is suitable for large-scale cultivation in the northern coastal region of China, Zhejiang and Fujian coastal regions, and the yield is the first in the world. It is rich in algin and iodine, and can be used as industrial raw material for edible and extraction of iodine, algin and mannitol, and its thallus can be used as medicine, and at the same time the kelp is a vegetable with high nutrient value, and contains rich mineral elements of iodine, etc.. The kelp has low calorie, moderate protein content and rich mineral substances, and researches show that the kelp has multiple biological functions of reducing blood fat, reducing blood sugar, regulating immunity, resisting coagulation, resisting tumors, expelling lead, detoxifying, resisting oxidation and the like.

The main components of higher plant cell walls are polysaccharides and proteins, including cellulose, hemicellulose and pectin; the cell wall is divided into three layers, namely an intercellular layer, a primary wall and a secondary wall. The intercellular layer (also called middle layer) is located on the outermost side of the cell wall, mainly consists of pectin substances, has strong hydrophilicity and plasticity, and the multicellular plants are used for adhering adjacent cells together, and pectin is easily decomposed by acid or enzyme, thereby causing cell separation; primary walls (primary walls) are cell wall layers secreted by protoplasts during cell growth or before cell growth has ceased, and contain, in addition to cellulose, hemicellulose and pectin, a number of enzymes and glycoproteins which cross-link microfibrils of cellulose, the microfibrils being in the form of a network which are distributed in a matrix of non-cellulosic polysaccharides, the pectin providing cell wall ductility which allows the cell wall to expand as the cell grows; the secondary wall (secondary wall) is a wall layer formed by depositing wall substances generated by the metabolism of protoplasts on the inner side of the primary wall after the growth of cells is stopped and the surface area of the primary wall is not increased any more, is adjacent to a plasma membrane, has high cellulose content and has a certain directionality because the microfibril arrangement is denser than that of the primary wall. Little pectin is present, the substrate is mainly hemicellulose, and glycoprotein and various enzymes are not contained.

According to the components and characteristics of the cell walls of higher plants, the kelp cells are firstly dispersed into single cells on the biological basis by using cellulase and pectinase, and then the walls of the kelp cells are broken on the basis. Pectin is a heteropolysaccharide, and is a straight-chain high-molecular compound mainly formed by connecting D-galacturonic acid through alpha-1, 4 glycosidic bonds. Pectinase is an enzyme that can break down pectin, which is hydrolyzed into reducing sugars under the catalysis of pectinase.

Seaweed is an important component of the marine biological resources of the earth, and as a large-scale seaweed, the seaweed plays an important role not only on the dining table of human beings, but also has more and more extensive application in the fields of feed and additives due to the fact that the seaweed contains a large amount of amino acids, minerals, proteins, various functional polysaccharides and the like with the continuous development of biotechnology in recent years.

The utilization rate of the enzymolysis products of the seaweed is low at present and needs to be improved.

Disclosure of Invention

The invention aims to provide an enzymolysis method of kelp, which adopts a biological means to decompose kelp cells into single dispersed cells by pectinase, adds cellulase to break the cell wall of the kelp cells, and the enzymolysis kelp product can be used as a functional feed raw material, thereby improving the utilization rate of the cultured animals on the effective components of the kelp, such as amino acid, pigment substances, mineral substances and the like, enhancing the feeding attraction activity of the feed, enhancing the anti-oxidation function of the feed, and enhancing the immunity of the feed on the cultured animals, and solving the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

an enzymolysis method of kelp comprises three steps of raw material treatment, solid-liquid ratio control and enzymolysis condition control, wherein,

the method comprises the following steps: raw material treatment: comprises 2 states of raw materials of fresh kelp and dried kelp. Pulverizing dried herba Zosterae Marinae into 20-60 mesh powder with pulverizer; mincing fresh herba Zosterae Marinae with a mincing machine to 20-60 mesh;

step two: controlling the solid-liquid ratio: the crushed or minced kelp is prepared according to the following steps of: diluting the mixture in a ratio of 1:80-90 by weight of water for enzymolysis;

step three: the enzymolysis condition control comprises the following technical conditions:

(1) the dry matter content of the kelp in the enzymolysis liquid is 10 to 25 percent;

(2) and (3) hydrolyzing enzyme: the hydrolase is cellulase and pectinase; based on the number of enzyme activity units, the combination ratio of the cellulase to the pectinase is as follows: cellulase: pectinase 1:10-1: 20; the number of the cellulase activity units in the enzymolysis liquid is 150IU/mL-250IU/mL, and the number of the corresponding pectinase activity units is 1500IU/mL-3000 IU/mL;

(3) controlling the pH value of the enzymolysis liquid: adjusting the pH value of the enzymolysis liquid to 4.5 by using analytically pure hydrochloric acid, and maintaining the pH value within the range of 4.5 +/-0.5 in the enzymolysis process;

(4) enzymolysis temperature: maintaining the temperature of enzymolysis liquid at 45 +/-5 ℃ in the enzymolysis process in a steam pipeline heating mode;

(5) and (3) enzymolysis time: keeping the above conditions, and controlling the enzymolysis time at 4-5 hr.

Preferably, the combination ratio of the cellulase and the pectinase is 1:10-1:20, and the activity unit number of the cellulase and the pectinase in the enzymolysis liquid is as follows: the cellulase activity number is 150IU/mL-250IU/mL, and the corresponding pectinase activity unit number is 1500IU/mL-3000 IU/mL.

Preferably, the control of the enzymolysis conditions comprises that the dry matter content of the kelp in the enzymolysis liquid is 10 to 25 percent; the temperature of the enzymolysis liquid is 45 +/-5 ℃; the pH value of the enzymolysis liquid is 4.5 +/-0.5; the enzymolysis time is 4-5 hours.

Preferably, the content of reducing sugar in the enzymatic hydrolysis kelp product obtained after enzymolysis is 8.0-12.5g/100 g; the content of free amino acid is 9.0-15.0g/100 g.

Preferably, the enzymatic hydrolyzed kelp product can be used as a functional feed raw material.

Compared with the prior art, the invention has the beneficial effects that: according to the enzymolysis method of the kelp provided by the invention, the kelp cells are decomposed into single dispersed cells by pectinase by adopting a biological means, the cellulase is added to break the cell walls of the kelp cells, and the enzymolysis kelp product can be used as a functional feed raw material, so that the utilization rate of effective ingredients of the kelp, such as amino acid, pigment substances, mineral substances and the like, of a cultured animal is improved, the feeding attraction activity of the feed is enhanced, the antioxidant function of the feed is enhanced, and the immunity of the feed to the cultured animal is enhanced.

Drawings

FIG. 1 is a schematic diagram showing the influence of the time of the present invention on the enzymatic process;

FIG. 2 is a comparative graph of all the enzymatic hydrolysates of the present invention;

FIG. 3 is a first schematic diagram of optical microscopic observation under the optimal enzymolysis conditions according to the present invention;

FIG. 4 is a second schematic view of optical microscopy under the optimal enzymatic hydrolysis conditions of the present invention;

FIG. 5 is a third schematic view of optical microscopy under the optimal enzymatic hydrolysis conditions of the present invention;

FIG. 6 is a fourth schematic diagram of optical microscopic observation under the optimal enzymolysis conditions according to the present invention;

FIG. 7 is a schematic diagram of optical microscopic observation under the optimal enzymolysis condition according to the present invention;

FIG. 8 is a sixth schematic view of optical microscopy under the optimal enzymatic hydrolysis conditions of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

An enzymolysis method of kelp comprises three steps of raw material treatment, solid-liquid ratio control and enzymolysis condition control, wherein,

the method comprises the following steps: raw material treatment: comprises 2 states of raw materials of fresh kelp and dried kelp. Pulverizing dried herba Zosterae Marinae into 20-60 mesh powder with pulverizer; mincing fresh herba Zosterae Marinae with a mincing machine to 20-60 mesh;

step two: controlling the solid-liquid ratio: the crushed or minced kelp is prepared according to the following steps of: diluting the mixture in a ratio of 1:80-90 by weight of water for enzymolysis;

step three: the enzymolysis condition control comprises the following technical conditions:

(1) the dry matter (mass) content of the kelp in the enzymolysis liquid is 10 to 25 percent; controlling enzymolysis conditions, wherein the content of dry substances (mass) of the kelp in the enzymolysis liquid is 10-25%; the temperature of the zymolytic fluid is 45 +/-5 ℃; the pH value of the enzymolysis liquid is 4.5 +/-0.5; the enzymolysis time is 4-5 hours;

(2) and (3) hydrolyzing enzyme: the hydrolase is cellulase and pectinase; based on the number of enzyme activity units, the combination ratio of the cellulase to the pectinase is as follows: cellulase: pectinase 1:10-1: 20; the number of the cellulase activity units in the enzymolysis liquid is 150IU/mL-250IU/mL, and the number of the corresponding pectinase activity units is 1500IU/mL-3000 IU/mL; the combination and the combination ratio of the cellulase and the pectinase are 1:10-1:20, and the activity unit number of the cellulase and the pectinase in the enzymatic hydrolysate is as follows: the cellulase activity number is 150IU/mL-250IU/mL, and the corresponding pectinase activity unit number is 1500IU/mL-3000 IU/mL;

(3) controlling the pH value of the enzymolysis liquid: adjusting the pH value of the enzymolysis liquid to 4.5 by using analytically pure hydrochloric acid, and maintaining the pH value within the range of 4.5 +/-0.5 in the enzymolysis process;

(4) enzymolysis temperature: maintaining the temperature of enzymolysis liquid at 45 +/-5 ℃ in the enzymolysis process in a steam pipeline heating mode;

(5) and (3) enzymolysis time: keeping the above conditions, and controlling the enzymolysis time at 4-5 hr.

The content of reducing sugar in the enzymatic kelp product obtained after enzymolysis is 8.0-12.5g/100 g; the content of free amino acid is 9.0-15.0g/100 g.

The content of the reducing sugar in the kelp sample is 0.003-0.005g/100g (dry basis) by taking the content of the reducing sugar and free amino acid as evaluation indexes, and the content of the reducing sugar in the enzymatic hydrolysis kelp product obtained after enzymatic hydrolysis is 8.0-12.5g/100g (dry basis) (the content of the reducing sugar is measured by a DNS method); the content of free amino acid in the kelp sample is 0.007-0.01g/100g (dry basis), and the content of free amino acid in the enzymatic hydrolysis kelp product obtained after enzymatic hydrolysis is 9.0-15.0g/100g (dry basis) (the content of free amino acid is determined by automatic analysis of amino acid).

Recording of results of first and second orthogonal experiments

(N＝3,

)

Table 1 orthogonal experimental results table

Calculation of results of two-dimensional and orthogonal experiments

(1) Results of orthogonal test of fresh reducing sugar

Table 2 table for calculating results of reducing sugar orthogonal test of fresh sample

Wherein T represents summing each factor, X represents averaging each factor, and R represents an extreme value

The processing method using orthogonal experiment-the pole difference method can be calculated, and the results can be seen: d > C > A > B, it can be concluded that: cellulase is the major influencing factor in fresh-like reducing sugar levels; the optimum enzymolysis conditions are 45 ℃, pH 4.5, pectinase 0.3% and cellulase 0.3%.

(2) Freeze-dried powder reducing sugar orthogonal experimental result

TABLE 3 calculation table of reducing sugar orthogonal experiment results of lyophilized powder

The processing method of the orthogonal experiment, namely the range method, is used for processing experimental data, and the following can be seen: d > C > A > B, it can be concluded that cellulase is the most important factor in the level of reduced sugar in dry samples, and the optimal conditions for enzymatic hydrolysis are 45 deg.C, 4.5 pH, 0.3% pectinase, and 0.3% cellulase.

(3) Fresh-sample amino acid orthogonal result processing

TABLE 4 calculation table of amino acid orthogonality results of fresh samples

The data calculated by the method of processing using orthogonal experiments, namely the pole difference method can be seen as follows: c > B > a > D, it can be concluded that: at the level of fresh amino acids, pectinase is the most important factor, and the optimal enzymolysis conditions are 55 ℃, pH 6.5, 0.3% of pectinase and 0.2% of cellulase.

(4) Dry sample amino acid orthogonal results processing

TABLE 5 calculation table of amino acid orthogonality results for dry samples

The factor index relation diagram is made by using the processing method-the pole difference method of the orthogonal experiment, and the factor index relation diagram can be seen from the above: b > D > C > A, pH is the main influencing factor at the level of dry amino acid, and the optimal enzymolysis conditions are 55 ℃, pH 6.5, 0.3% of pectinase and 0.3% of cellulase.

(5) Effect of time on the enzymatic Process

TABLE 6 Effect of time on the enzymatic Process

From fig. 1 and the table above, it can be seen that: the enzymolysis effect is better than that of 1, 2 and 3h in 4h of reducing sugar level; the enzymolysis effect of 3h on the amino acid level is better than that of 1, 2 and 4h, which shows that the enzymolysis time required for the determination of different level factors is different under the same enzymolysis condition, and different enzymolysis times are selected according to different purposes of experiments in actual operation.

Third, the change of physical properties in the enzymolysis process

(1) And (4) viscosity.