CN113045683B - A kind of preparation method of seaweed oligosaccharide and its application in fish feed - Google Patents

Abstract

The invention provides a preparation method of laver oligosaccharide and application of the laver oligosaccharide in fish feed. The laver oligosaccharide is mainly composed of 3-linked beta-D-galactose and 4-linked alpha-L-galactose-6-sulfate linkage, the polymerization degree is 2-20, the galactose content is 60-80%, and the sulfate content is 15-30%. The porphyra oligosaccharide is added into the fish feed, so that the meat quality of the cultured fish can be improved, the fat content of the fish body is reduced, and the protein content of the fish body is increased, so that the meat quality and flavor of the fish are improved. The porphyra oligosaccharide is a brand-new, green and safe fish feed additive, can effectively solve the problems of loose meat quality, low protein content and poor taste of cultured fish, can improve the immunity of the fish, and can broaden the market of the cultured fish.

Description

A kind of preparation method of seaweed oligosaccharide and its application in fish feed

technical field

The invention relates to the field of aquaculture, in particular to a preparation method of laver oligosaccharide and its application in fish feed.

Background technique

With the development of aquaculture, the problems restricting aquaculture production have become increasingly prominent. One of the problems currently seriously affecting the sales of farmed fish is the poor flavor of farmed fish. The severe fishy smell, loose meat and high fat content make consumers stay away. The lower quality of farmed fish has seriously affected its commercial value and development. The poor meat quality of farmed fish is directly related to the harsh farming environment and low feed quality. The polluted water environment will inevitably lead to the accumulation of fish diseases and fishy substances, while the energy of ordinary formula feed is high, resulting in high body fat and low lean meat rate. Therefore, it has become a trend to develop safe, non-toxic and green aquaculture anti-disease preparations. At present, to improve the meat quality of farmed fish, on the one hand, we can adjust the feed formula, and on the other hand, we should also pay attention to the enhancement of the immunity of farmed fish. Studies have shown that microbial polysaccharides, chitin, animal and plant extracts, etc. have different degrees of immune-enhancing effects on aquatic animals. However, considering the preparation cost and technical difficulty, the application of these immune-enhancing agents needs to be further improved at present.

Porphyra polysaccharide is a sulfated polysaccharide, which is a unique polysaccharide component of Porphyra. It exists in the cell wall of Porphyra and accounts for 20-40% of the dry weight of Porphyra. Its main structural unit is →3)β-D-galactose-(1→4)-α-L-6-sulfate-galactose-(1→. Part of α-L-6-sulfate-half Lactose is replaced by L-3,6-lactone galactose, forming the structural unit: →3)-β-D-galactose-(1→4)-α-L-3,6-lactone galactose-(1→ .Porphyra polysaccharide has various biological activities such as anti-coagulation, lowering blood sugar, regulating blood lipid, antithrombotic, inhibiting tumor and enhancing cellular immune function, and is widely used in food and medicine industries.Porphyra oligosaccharide is directly prepared from Porphyra or Porphyra Polysaccharides are prepared by degradation, and the degree of polymerization is 2-20. However, the current preparation methods of laver oligosaccharides mainly include acid degradation, oxidative degradation, microwave, ultrasonic-assisted degradation and enzymatic degradation. Chemical degradation In the method, hydrogen peroxide has the best degradation prospect. This method has low cost and is easy to realize industrial production. However, the oxidant does not fully participate in the reaction process, and at the same time, the temperature increases, and the product is more prone to browning; biological enzymes are used to assist the reaction. , can effectively avoid side effects, and the external reaction conditions are also easy to control, however, the use of enzymatic reactions leads to increased costs; the degradation reaction is incomplete through physical methods. The molecular structure determines its biological function, and the oligosaccharide chain of laver is in molecular size, The heterogeneity of microstructure such as sequence arrangement, configuration and conformation may cause huge differences in its biological functions. Therefore, the establishment of the preparation method of Porphyra oligosaccharides is the premise of the application of Porphyra oligosaccharides.

Compared with seaweed polysaccharide, seaweed oligosaccharide has better antioxidant, immune stimulating activity and better absorption, and has broader application prospects. Our research results show that using laver oligosaccharide as a meat quality modifier on fish can not only significantly improve the meat quality of farmed fish, but also improve the immunity of farmed fish.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides a preparation process of laver oligosaccharide on the one hand, which has high yield, maximizes the retention of the sulfate group content in the oligosaccharide, and improves the activity of laver oligosaccharide. On the other hand, from the perspective of improving fish meat flavor, starting from a fish meat quality improving agent, an application of laver oligosaccharide in fish feed is provided.

For achieving the above object, the technical scheme adopted in the present invention is as follows:

The preparation method of the laver oligosaccharide, the specific preparation steps are:

Step 1: After crushing the seaweed, add 20-40 times of water, preferably 30 times; add immobilized cellulase to the solution to a final concentration of 0.1-2%, preferably 0.8-1%; adjust the pH with dilute HCl to 4.5-6.5, preferably 5.0; hydrolyzed at 30-60°C for 1-5h under stirring, preferably 50°C, hydrolyzed for 3-4h;

In step 2, the molecular sieve-loaded Fenton catalyst is added to the solution after enzymatic hydrolysis in step 1, so that the mass concentration of the catalyst is 0.1-1%, preferably 0.5-0.7%, and hydrogen peroxide with a mass concentration of 28-30% is added to the end. The concentration is 10-50 mM, preferably 30-40 mM; the precipitate is removed by centrifugation, the obtained supernatant is desalted by nanofiltration, concentrated and dried to obtain laver oligosaccharide, and the yield of the oligosaccharide is 10-22%.

The preparation method of the immobilized cellulase is as follows: immersing the dry molecular sieve in a cellulase solution with a mass concentration of 0.5-5%, preferably 2-3%; making the final concentration of the molecular sieve 0.5-5%, Preferably 2-3%; stir at room temperature for 6-24h, preferably 12-18h, centrifuge and wash with distilled water, precipitate and vacuum dry to obtain immobilized enzyme catalyst; the molecular sieves are M41S series mesoporous molecular sieves MCM-41, MCM-48 , one or more of MCM-50.

The preparation method of the molecular sieve-loaded Fenton catalyst is as follows: immersing the molecular sieve in a 0.5-5M ferric sulfate aqueous solution of 10-20 times the volume, preferably 12-15 times, 2-3M; stirring at room temperature for 4-12h, preferably 8h , 110 ℃ drying, muffle furnace 500-600 ℃ roasting for 2-5h after use.

The molecular sieve is one or more of M41S series mesoporous molecular sieves MCM-41, MCM-48 and MCM-50.

The molecular sieves prepared by the preparation method of the molecular sieve-supported Fenton catalyst and the immobilized cellulase can be the same or different.

The laver oligosaccharide is alternately connected by 3-connected β-D-galactose-6-sulfate group and 4-connected α-L-galactose, the oligosaccharide polymerization degree is 2-20, and the galactose content is 60 -80%, sulfuric acid group content is 15-30%.

The seaweed oligosaccharide is directly added to the basic fish feed as a feed additive, and the additive amount is 0.05-5%, preferably 0.1-1%.

Compared with the prior art, the present invention has the following advantages:

1. Improve the level of high-value utilization of seaweed. my country is a big country of seaweed farming, and the taste of seaweed in the later stage is poor and the added value is low. The application of seaweed oligosaccharide in fish feed in this project can greatly improve the added value of seaweed.

2. The preparation process of seaweed oligosaccharide is simple and efficient. For the first time, a new technology for efficient preparation of laver oligosaccharides by biochemical synergistic heterogeneous catalysis is proposed. The technology is simple in process, low in cost, suitable for industrial production, and lays a foundation for the application of laver oligosaccharides.

3. The immobilized cellulase and supported catalyst technology improves the use efficiency of enzymes and catalysts and saves costs compared with ordinary cellulase and catalysts.

4. Broaden the application field of laver oligosaccharide. It is the first time that seaweed oligosaccharide is added to fish feed, which can improve meat quality, is suitable for a variety of farmed fish, and has the convenience of operation without changing the original feed formula.

5. Seaweed oligosaccharide is natural, safe and non-toxic. Porphyra oligosaccharide has no toxic and side effects on the object of use and the environment, no drug resistance, and no risk of damage to human health caused by residues in fish.

6. Seaweed oligosaccharide has strong biological activity. Porphyra oligosaccharide not only has the effect of improving meat quality and enhancing immunity, but also has pharmacological activities such as antioxidant, anti-aging, and anti-tumor. Due to the increasingly serious environmental pollution, the tumor diseases of fish are becoming more and more common. The use of laver oligosaccharide has a certain effect on preventing fish tumors.

In addition, laver oligosaccharides have immunomodulatory effects on farmed fish, thereby accelerating the metabolism of fish and reducing the accumulation of fishy substances. The seaweed oligosaccharide is a brand-new, green and safe fish feed additive, which can effectively solve the problems of loose meat, low protein content and poor taste in farmed fish, at the same time, it can improve the immunity of fish, and can provide food for farmed fish. Broaden the market.

Description of drawings

Figure 1 Effects of seaweed oligosaccharide diet PS on crude protein and crude fat of grouper fish, *P<0.05 compared with the control group, **P<0.01 compared with the control group

Figure 2 The effect of seaweed oligosaccharide diet PS on the production of reactive oxygen species in the head kidney of grouper, *P<0.05 compared with the control group, **P<0.01 compared with the control group

Figure 3 The effect of seaweed oligosaccharide diet PS on the weight gain and survival rate of grouper, *P<0.05 compared with the control group, **P<0.01 compared with the control group

Figure 4 Effects of seaweed oligosaccharide diet PS on crude protein and crude fat of sea bass, *P<0.05 compared with the control group, **P<0.01 compared with the control group

Figure 5 Effects of different seaweed oligosaccharide diets on the production of reactive oxygen species in the head kidney of sea bass, *P<0.05 compared with the control group, **P<0.01 compared with the control group

Figure 6 Effects of different seaweed oligosaccharide diets on crude protein and crude fat of sea bass, *P<0.05 compared with the control group, **P<0.01 compared with the control group

Figure 7 Effects of laver oligosaccharide and laver polysaccharide diet on grouper body crude protein and crude fat, *P<0.05 compared with the control group, **P<0.01 compared with the control group

Figure 8 The effect of adding different doses of laver oligosaccharide OP on the crude protein and crude fat of grouper, *P<0.05 compared with the control group, **P<0.01 compared with the control group

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the limited scope of the present invention.

Embodiment 1, the preparation of seaweed oligosaccharide

Method 1. High-efficiency preparation technology of biochemical synergistic biocatalysis of laver oligosaccharide (the method used in the patent of the present invention)

① Preparation of immobilized enzyme catalyst (enzyme-MCM-48)

Weigh 3 g of cellulase and add it to 50 mL of acetic acid-sodium acetate buffer solution (pH=4.5), stir for 1 hour, collect the supernatant by centrifugation, continue to stir the precipitate for 1 hour and centrifuge until it is completely dissolved, and make up the solution to 100 mL. Cellulase enzyme solution. 10 g of dry MCM-48 molecular sieves were weighed and immersed in 100 mL of 3% cellulase solution, stirred at room temperature for 12 h, centrifuged and washed with distilled water. The immobilized enzyme catalyst was obtained after the precipitate was vacuum-dried.

② Preparation of molecular sieve supported Fenton catalyst (Fe-MCM-48)

Prepare 100 mL of 2M ferric sulfate aqueous solution, weigh 10 g of dry MCM-48 molecular sieve and immerse it in it, stir at room temperature for 8 hours, dry at 110 °C, and roast in a muffle furnace at 500 °C for 3 hours before use.

③Study on the process of synergistic degradation of Porphyra oligosaccharides by enzyme-MCM-48 and Fe-MCM-48

Firstly, the enzyme-MCM-48 was used for enzymatic hydrolysis of seaweed. Add 30 times water to the pulverized seaweed, add enzyme-MCM-48 to a final concentration of 1%, adjust the pH to 5.0, stir and hydrolyze at 50°C for 3 hours, and filter out the catalyst after the reaction. Fe-MCM-48 was added to the solution after enzymolysis to a final concentration of 0.5%, and 30% hydrogen peroxide was added to a final concentration of 30 mM. Under stirring, hydrolyzed at 60° C. for 3 hours, centrifuged to remove the precipitate, the obtained supernatant was desalted by nanofiltration, concentrated and dried to obtain laver oligosaccharide OP with a yield of 15.4%. It was determined that the content of galactose in OP was 66.2%, the content of sulfate group was 24.3%, and the degree of polymerization was 2-20.

Method 2. Preparation technology of sulfuric acid hydrolysis of laver oligosaccharide (preparation method of laver oligosaccharide in literature)

①Preparation of seaweed polysaccharide

Add 20 times water to the pulverized laver, 120 degrees, extract in a pressure cooker for 3 hours, centrifuge at 4000 r/min for 15 minutes, and dialyze the supernatant directly. After the dialysis, collect the dialysate, concentrate, and dry to obtain laver polysaccharide (P). The yield is 18%.

②Preparation of seaweed oligosaccharides

Add 5 g of seaweed polysaccharide to 150 mL of water, stir and dissolve at room temperature overnight. Take 5.435 mL of concentrated sulfuric acid and dilute it to 50 mL, add laver polysaccharide solution, stir and mix while adding, and stir and react at 80 degrees for 3 hours (add reflux). At the end of the reaction, 31.546 g of Ba(OH) ₂ ·8H ₂ O was weighed and made into a saturated solution for neutralization (continuous stirring). Centrifuge at 4000 r/min for 8 min, take the supernatant for filtration, concentrate, dialyze the dialysis bag with a molecular weight cut-off of 1 kDa, and freeze-dry to obtain OM with a yield of 55%, which is 9.9% according to the conversion of laver. It was determined that the content of galactose in OM was 55.6%, the content of sulfate groups was 14.3%, and the degree of polymerization was 2-20.

Comparing method 1 and method 2, the yield of laver oligosaccharide OP, galactose and sulfate group content prepared by method 1 are higher than those of laver oligosaccharide OM obtained by method 2, and method 1 has simple steps and is more suitable for industrial production. At the same time, we also tried to prepare laver oligosaccharides only by enzymatic hydrolysis methods such as cellulase and agarase, and by only using H ₂ O ₂ oxidative degradation methods. Oligosaccharide samples with low degree of polymerization. Comprehensive comparison, the preparation method of laver oligosaccharide established by the patent of the present invention is simple and efficient, the obtained oligosaccharide sample is of good quality, suitable for industrial scale-up production, and has good application potential.

Example 2, preparation of laver oligosaccharide feed (PS, PO), laver polysaccharide feed (PP) and laver powder feed (PM)

The seaweed oligosaccharide extract (OP) obtained by the first method in Example 1 is added to the feed at 0.05-5% of the total weight of the feed. Any concentration within this range can significantly improve the meat quality and flavor of the farmed fish, and enhance the body. Cell immunity, improve its ability to resist disease, only a few different concentrations are used as representative samples in the examples. The preparation method is as follows: firstly, the laver oligosaccharide extract is made into a solution of 8-12%, preferably 10%; then anhydrous ethanol is added under stirring so that the total concentration of the ethanol is 10%-20%, preferably 15%; The obtained solution was evenly sprayed on the feed to obtain a feed with laver oligosaccharides accounting for 0.1% of the total weight of the feed, which was dried or dried and stored for later use, which was recorded as PS. The same method was used to prepare feed PO containing 0.1% laver oligosaccharide OM obtained in method 2 of Example 1, feed PP containing 3% laver polysaccharide obtained in method 2 of Example 1, and feed PM containing 20% laver powder.

Example 3, the effect of feeding the aquatic feed containing seaweed oligosaccharide on the improvement of grouper meat quality and immune regulation

In this experiment, aquafeed containing seaweed oligosaccharide was used to feed grouper, and the changes in body composition, muscle texture, and physiological function-related indicators of grouper were used to illustrate its effects on the improvement of grouper meat quality and immune regulation.

Test method: Select healthy and uniform groupers and randomly divide them into 3 groups, use different bait respectively, and carry out according to the normal feeding and management procedures. The first group was the commercial aquatic feed F control group, the second group was the PM control group, and the third group was the PS experimental group. The test period was 42 days. Blood was collected from 9 fish every 14 days, and the head and kidney were aseptically removed. Used to measure head kidney macrophage respiratory burst. Take out the head kidney of the fish from the ultra-clean workbench, put it into a 1.5ml centrifuge tube containing 1ml PBS, grind it on ice, use a 100-mesh cell sieve to remove tissue clumps, centrifuge at 500g to remove the supernatant, and wash with PBS for 2 Next, resuspend to prepare a single cell suspension, and the cell count is not less than 10 ⁶ cells. The single cell suspension was incubated with DCFH-DA fluorescent probe. Follow-up operations were performed in accordance with the Nanjing Jiancheng reactive oxygen species (ROS) test kit instructions. After the experiment, the weight gain rate and survival rate were determined, and the back muscles (2cm×2cm×1.5cm) were taken for the analysis of muscle texture (TPA) characteristics, and the fish body was not taken for the determination of crude protein and crude fat. The activities of total complement CH50 of antioxidant enzymes (SOD, CAT) and alkaline phosphatase AKP in serum were determined by Nanjing Jiancheng kit.

result:

During the test, the weight gain rate and survival rate of grouper were shown in Figure 1. The weight gain rate of group 3 was significantly higher than that of the basal diet group, and its weight gain rate was also higher than that of the 20% seaweed powder addition group, which proved that the oligarch of seaweed Sugar can obviously promote the weight gain of grouper, and its weight gain effect is better than adding high-dose seaweed powder directly. The survival rate of grouper in group 3 was also higher than that in the basal diet group and the 20% laver powder-supplemented group during the test period.

It can be seen from Table 1 that the superoxide dismutase SOD activity of the third group fed with laver oligosaccharide was significantly improved compared with the control group. During the first 28 days of feeding, the third group of superoxide dismutase The SOD activity always showed an upward trend, but decreased after 28 days, which was also consistent with the trend of the control group. For the total complement CH ₅₀ value, the third group also increased significantly compared with the basal diet group, but the overall trend was similar to the control group, and decreased with the prolongation of the feeding experiment. For catalase CAT, the experimental group with added laver oligosaccharide was higher than the control group, which decreased at first and then increased over time. For alkaline phosphatase AKP, the three groups all decreased with time, but the third group had higher activity than the control group and the laver powder-added group. It is worth mentioning that all the indexes of the seaweed oligosaccharide-added group were better than those of the seaweed powder-added group. SOD, CH ₅₀ , CAT and AKP are closely related to the immune ability of grouper, and the increase of these indicators proves that the feed supplemented with seaweed extract has a positive immune regulation effect.

Table 1 Effects of specific indicators in grouper serum

^* P<0.05 compared to the control group, ^** P<0.01 compared to the control group

It can be seen from Figure 2 that the level of reactive oxygen species ROS generated by the respiratory burst of macrophages in the head kidney in the laver oligosaccharide-added group was significantly higher than that in the control group and the laver powder-added group, indicating that its non-specific immunity was significantly improved.

From Figure 3, it can be seen that compared with the control group, the seaweed oligosaccharide addition group effectively increased the crude protein content of the fish and reduced the crude fat content of the fish.

It can be seen from Table 2 that the PS group is superior to the control group and the laver powder-added group in muscle hardness, elasticity, adhesiveness, chewiness, and recovery.

Table 2. Results of determination of grouper muscle texture (TPA) characteristics

^* P<0.05 compared to the control group, ^** P<0.01 compared to the control group

Example 4, the effect of feeding the aquatic feed containing seaweed oligosaccharide on the improvement of sea bass meat quality and immune regulation

In this experiment, the aquatic feed containing seaweed oligosaccharide was fed to sea bass, and the changes in body composition, muscle texture, and physiological function-related indicators of sea bass were used to illustrate its effects on the improvement of sea bass meat quality and immune regulation.

experiment method:

Select healthy and uniform sea bass and randomly divide them into 3 groups, using different bait respectively, according to the normal feeding and management procedures. The first group was the commercial aquatic feed F control group, the second group was the 20% seaweed powder added feed (PM) control group, and the third group was the PS experimental group. The test period was 28 days. 10 experimental fish from each group were randomly selected for 28 days, and blood was collected from the tail vein, 0.5 ml per tail. After the whole blood was allowed to stand at room temperature for 3 hours, it was allowed to stand at 4 °C for 5 hours, and the serum was collected by centrifugation. The spare samples were stored at -20 °C for serum. Determination of non-specific immune indicators (SOD, AKP, LZM). After the feeding experiment, the dorsal muscle (2cm×2cm×1.5cm) was taken for the analysis of muscle texture (TPA) characteristics, and the fish body was not taken for the determination of crude protein and crude fat.

result:

It can be seen from Figure 4 that the crude protein content of the sea bass in the seaweed oligosaccharide addition group was significantly increased compared with the control group, and the crude fat content was significantly reduced.

As can be seen from Table 3, compared with the control group, the non-specific immune indexes of the PS group increased and the increase was more obvious than that of the PM group, indicating that the addition of seaweed oligosaccharides can effectively improve the non-specific immunity of aquatic animals.

Table 3 Effects on non-specific immune indexes of sea bass

^* P<0.05 compared to the control group, ^** P<0.01 compared to the control group

It can be seen from Table 4 that the laver powder-added group and the laver oligosaccharide-added group are better than the control group in four aspects: hardness, stickiness, adhesiveness, and chewiness, and the muscle properties of the laver oligosaccharide-added group are improved more significantly. There was no significant difference among the three groups in terms of muscle adhesion of sea bass.

Table 4. Results of determination of texture of perch muscle texture (TPA) characteristics

group Hardness (N) Elasticity(mm) Adhesion (g) Adhesion (N) Chewability (N) control group 47.87±0.62 0.33±0.02 0.21±0.01 6.38±0.07 1.72±0.23 PM group 46.23±1.03 0.35±0.04 0.23±0.02 7.72±0.12^* 2.14±0.03^* PS group 50.21±0.33^* 0.38±0.01^* 0.22±0.03 8.92±0.08^** 3.32±0.16^**

^* P<0.05 compared to the control group, ^** P<0.01 compared to the control group

Example 5, the comparison of the effects of several additions of different marine oligosaccharide aquatic feeds on the improvement of sea bass meat quality and immune regulation

In this experiment, the aquatic feed containing 0.1% chitosan oligosaccharide, fucoid oligosaccharide, alginate oligosaccharide and seaweed oligosaccharide was used to feed perch, and the changes in body composition, muscle texture and physiological function of perch were used to explain the different oligosaccharides. Effects of sugar on meat quality improvement and immune regulation in sea bass.

Select healthy and uniform sea bass and randomly divide them into 5 groups, using different bait respectively, according to the normal feeding and management procedures. The first group is the commercial aquatic feed F control group, the second group is the PS experimental group, the third group is the 0.1% chitosan oligosaccharide addition experimental group, the fourth group is the 0.1% fucoid oligosaccharide addition experimental group, and the fifth group For the experimental group added with 0.1% alginate oligosaccharide. The trial period was 28 days. The head kidney was taken to measure the respiratory burst, and the fish body was not taken for the determination of crude protein and crude fat.

result:

It can be seen from Figure 5 that the oligosaccharide supplementation group has a promoting effect on the reactive oxygen species ROS level produced by the macrophage respiratory burst in the perch head kidney compared with the basic diet control group. Secondly, this experiment shows that seaweed oligosaccharide can effectively improve the non-specific immunity of sea bass, and has a better promoting effect than chitosan oligosaccharide, fucoid oligosaccharide, and algin oligosaccharide.

It can be seen from Figure 6 that the crude protein content of the fish body in the seaweed oligosaccharide addition group was significantly increased compared with the control group, and the crude fat content of the fish body was significantly reduced compared with the control group. The composition of other oligosaccharides did not change significantly. Compared with the control group, the crude protein of fish in the oligosaccharide-added group decreased slightly.

Example 6, the comparison of the effect of adding seaweed oligosaccharide and seaweed polysaccharide on the improvement of grouper meat quality

In this experiment, the aquatic feeds (PS and PO) containing laver oligosaccharides prepared in Example 1 and the aquafeeds (PP) containing undegraded laver polysaccharides were used to feed groupers, and the changes in body composition and muscle texture of groupers were observed. , to illustrate the difference in the improvement of seaweed oligosaccharides and seaweed polysaccharides on grouper meat quality.

Select healthy and uniform groupers and randomly divide them into 4 groups, use different bait respectively, and carry out the normal feeding and management procedures. The first group was the commercial aquatic feed F control group, the second group was the PS experimental group, the third group was the PO experimental group, and the fourth group was the PP experimental group. The trial period was 28 days. The dorsal muscle (2cm×2cm×1.5cm) was taken for the analysis of muscle texture (TPA) characteristics, and the fish body was not taken for the determination of crude protein and crude fat.

It can be seen from Figure 7 that compared with the basal feed group, the PS group had higher crude protein content and lower crude fat content, and the PO group and the laver polysaccharide added group showed no significant difference in fish body composition compared with the basal feed group. It shows that the activities of laver oligosaccharides prepared by different processes are different, and the process adopted in the present invention can retain the biological activity of laver oligosaccharides to the greatest extent. This may be because this process has less damage to the sulfate group, which is an important group for the activity of the laver oligosaccharide.

It can be seen from Table 5 that the PO and PP groups did not show significant differences in muscle texture characteristics compared with the control group, which may be due to the large molecular weight of laver polysaccharides, which are not easily absorbed by aquatic animals, and the oligosaccharide PO. The activity of the group is not significant because of the lower sulfate group content. The laver oligosaccharide PS group showed significant improvement in elasticity, adhesiveness, chewiness, and recovery.

Table 5. Results of determination of grouper muscle texture (TPA) characteristics

^* P<0.05 compared to the control group, ^** P<0.01 compared to the control group

Example 7, the comparison of the effect of adding different doses of seaweed oligosaccharide OP on the improvement of grouper meat quality

In this experiment, aquafeeds PS1, PS2, PS3, PS4, PS5 and PS6 containing 0.05%, 0.1%, 1%, 5%, 10% and 20% of seaweed oligosaccharides were prepared according to the method of Example 2 and fed to groupers. The changes of body composition and muscle texture of grouper were used to illustrate the difference of the improvement effects of seaweed oligosaccharides and seaweed polysaccharides on grouper meat quality.

Select healthy and uniform groupers and randomly divide them into 7 groups, use different bait respectively, and carry out according to the normal feeding and management procedures. The first group was the commercial aquatic feed F control group, and the second to seventh groups were the PS1-PS6 experimental groups respectively, and the experimental period was 28 days. The dorsal muscle (2cm×2cm×1.5cm) was taken for the analysis of muscle texture (TPA) characteristics, and the fish body tissue was taken for the determination of crude protein and crude fat.

It can be seen from Figure 8 that the PS group containing different doses of oligosaccharides has higher crude protein content and lower crude fat content than the basic feed group. and crude fat content have little effect. When the added amount of seaweed oligosaccharide was less than 5%, with the increase of the added amount of seaweed oligosaccharide, each group could gradually increase the content of crude protein in fish body and reduce the content of crude fat in fish body gradually. This shows that when the added amount of seaweed oligosaccharide exceeds a certain dose, the crude protein and crude fat components of fish will not be affected with the increase of the added dose, and the addition amount of 0.05%-5% is more suitable.

As can be seen from Table 6, compared with the control group, PS groups containing different doses of oligosaccharides showed significant differences in muscle texture characteristics. The elasticity, stickiness, chewiness and recovery have all been significantly improved. When the amount of laver oligosaccharide added was more than 5%, the changes of each index tended to be relatively stable, which indicated that the addition of 0.05-5% of laver oligosaccharide was more appropriate.

Table 6. Results of determination of grouper muscle texture (TPA) characteristics

^* P<0.05 compared to the control group, ^** P<0.01 compared to the control group

The above is a description of the embodiments of the present invention. The above description of the disclosed embodiments enables those skilled in the art to realize or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (7)

1. A preparation method of laver oligosaccharide is characterized in that:

the preparation method comprises the following specific steps:

step 1, pulverizing laver and adding 20-40 times of water; adding immobilized cellulase into the solution to a final concentration of 0.1-2%; adjusting pH to 4.5-6.5 with HCl with concentration of 0.1-1M; hydrolyzing at 30-60 deg.C for 1-5h under stirring;

step 2, adding a Fenton catalyst loaded by a molecular sieve into the solution subjected to enzymolysis in the step 1 to ensure that the mass concentration of the catalyst is 0.1-1%, and adding hydrogen peroxide with the mass concentration of 28-30% to the final concentration of 10-50 mM; centrifuging to remove precipitate, nano-filtering the obtained supernatant for desalting, concentrating, and drying to obtain laver oligosaccharide; the preparation method of the immobilized cellulase comprises the following steps: soaking the dried molecular sieve in cellulase liquid with the mass concentration of 0.5-5%; the final concentration of the molecular sieve is 0.5-5%; stirring for 6-24h at room temperature, performing centrifugal separation, washing with distilled water, and performing vacuum drying on the precipitate to obtain an immobilized enzyme catalyst; wherein the molecular sieve is one or more of M41S series mesoporous molecular sieves MCM-41, MCM-48 and MCM-50;

the preparation method of the molecular sieve supported Fenton catalyst comprises the following steps: soaking the molecular sieve into 10-20 times volume of 0.5-5M ferric sulfate aqueous solution; stirring at room temperature for 4-12h, drying at 110 deg.C, and roasting in muffle furnace at 500-600 deg.C for 2-5 h.

2. The method of claim 1,

in the step 1, adding immobilized cellulase into the solution until the final concentration is 0.8-1%; hydrolyzing at 50 deg.C for 3-4h under stirring; and 2, adding a Fenton catalyst loaded by a molecular sieve into the solution subjected to enzymolysis in the step 1 to ensure that the mass concentration of the catalyst is 0.5-0.7%, and adding hydrogen peroxide with the mass concentration of 28-30% to ensure that the final concentration is 30-40 mM.

3. The method of claim 1,

the preparation method of the immobilized cellulase comprises the following steps: soaking the dried molecular sieve in cellulase liquid with the mass concentration of 2-3%; the final concentration of the molecular sieve is 2-3%; stirring for 12-18h at room temperature, performing centrifugal separation, washing with distilled water, and performing vacuum drying on the precipitate to obtain the immobilized enzyme catalyst.

4. The method according to claim 1, wherein the molecular sieve is one or more of M41S series mesoporous molecular sieves MCM-41, MCM-48, MCM-50.

5. Use of the porphyra oligosaccharide prepared by the preparation method of any one of claims 1 to 4 in fish feed.

6. The use of the porphyra oligosaccharide in fish feed according to claim 5, wherein the porphyra oligosaccharide is directly added to the fish basal feed as a feed additive in an amount of 0.05-5%.

7. The use of the porphyra oligosaccharide in fish feed according to claim 6, wherein the porphyra oligosaccharide is directly added into the basic feed of fish as a feed additive, and the addition amount is 0.1-1%.

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