CN114250209A

CN114250209A - Fucoidan and application thereof in compound enzymolysis of sea cucumbers

Info

Publication number: CN114250209A
Application number: CN202111530973.2A
Authority: CN
Inventors: 常耀光; 陈广宁; 蒋笑笑; 薛长湖; 刘艳艳; 石菲菲; 张玉莹; 刘冠辰; 梅轩玮; 薛勇
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-03-29
Anticipated expiration: 2041-12-14
Also published as: CN114250209B

Abstract

The invention relates to the technical field of biology, in particular to fucoidan and application thereof in sea cucumber composite enzymolysis. According to the invention, low-temperature protease and fucosidase Fun168B are simultaneously added into sea cucumber homogenate according to a certain proportion for synchronous enzymolysis, and when the collagen is degraded by the protease, the macromolecular fucosan released from the sea cucumber body wall to the enzymolysis liquid can be instantly degraded by the Fun168B, so that the low protease hydrolysis efficiency caused by the increase of the viscosity of the enzymolysis liquid is avoided. The sea cucumber enzymolysis liquid obtained based on the enzymolysis technology can be clarified by low-speed centrifugation treatment at 4000rpm of 1000-. The synchronous enzymolysis technology can be carried out at room temperature, so that energy is saved, fucosan in the sea cucumber can be fully degraded into oligosaccharide, and the nutritive value of the enzymolysis liquid is improved.

Description

Fucoidan and application thereof in compound enzymolysis of sea cucumbers

Technical Field

The invention relates to the technical field of biological enzymolysis, in particular to fucoidan and application thereof in sea cucumber composite enzymolysis.

Background

Sea cucumber is a traditional nourishing food in Asian countries such as Zhongjapanese Korea and the like. In the last two decades, the sea cucumber aquaculture industry in China has rapidly developed, the aquaculture amount is increased, and the sea cucumber aquaculture amount reaches 171700 tons in 2019. The sea cucumber has various nutritional functional components, wherein the collagen is rich in various non-essential amino acids, and has the physiological activities of resisting thrombus, resisting tumor, regulating immunity, reducing blood fat, improving memory and the like. Fucosan (also called as fucoidan, or fucoidan) is another effective component in Stichopus japonicus, and is composed of fucose and sulfate ester group, and accounts for 5-10% of dry weight of Stichopus japonicus wall, and has high viscosity in water solution. Fucoidan has been shown to have a wide range of biological activities, including anticoagulant, osteoclastogenesis inhibition, neural stem/progenitor cell proliferation, protection against ethanol-induced gastric ulceration, antioxidant, insulin resistance regulation, anti-adipogenesis, and intestinal mucositis prevention.

The enzymolysis liquid is a main processing form of the sea cucumber, and can obviously improve the bioavailability of the nutrient components of the sea cucumber. However, the current enzymolysis technology of sea cucumber is single, most of enzymolysis liquid and its deep-processed products (such as sea cucumber wine, sea cucumber milk, etc.) in the market are based on protease enzymolysis, and after enzymolysis, the viscosity is often higher, which seriously affects the efficiency and effect of the subsequent operations of clarification, decoloration, ultrafiltration, concentration and/or sterilization. Research shows that the reason is that during the protease hydrolysis process, macromolecular fucosan in the sea cucumber body wall is gradually released into the enzymolysis liquid along with the degradation of collagen, so that the viscosity of the enzymolysis liquid is increased. More importantly, the high viscosity of the enzymolysis solution caused by the fucosan further affects the mass transfer and hydrolysis efficiency of the protease, so that the collagen is difficult to be further degraded, and the effect of the subsequent clarification operation is poor.

Recently, a published patent of a sea cucumber composite enzymolysis method based on fucoidan and protease (patent number 202010565103.8) establishes a preparation process of sea cucumber composite enzymolysis liquid based on sea cucumber fucoidan and commercially available protease. The sea cucumber is subjected to two-step segmented enzymolysis through protease and fucosidase, namely, the protease is firstly added for enzymolysis at 50-60 ℃, and then the temperature is reduced to about 30 ℃, and the fucosidase is added for enzymolysis. After the hydrolysis of the protease is finished, the influence of the fucosan on the viscosity of the enzymatic hydrolysate is eliminated by adding the fucoidan, obviously, the hydrolysis efficiency of the protease cannot be improved, and the two-step segmented enzymatic hydrolysis method is also energy-consuming. It is also mentioned in the patent that "the optimum reaction conditions of the protease are different from those of the fucoidan, so that the protease is not suitable for being added into the sea cucumber for enzymolysis at the same time". Based on the technical defects, if the viscosity of the sea cucumber enzymolysis liquid is further reduced, fucoidan and protease are required to be synchronously subjected to enzymolysis, fucoidan released from the raw material into the enzymolysis liquid is immediately degraded, the viscosity of the enzymolysis liquid is prevented from being increased, the enzymolysis efficiency of the protease is improved, and the efficiency and the effect of subsequent clarification and other processes are improved.

Disclosure of Invention

The invention aims to solve the technical problem that the sea cucumber enzymolysis liquid prepared based on the existing enzymolysis technology has high viscosity, and seriously affects the efficiency and the effect of subsequent operations such as clarification, decoloration, ultrafiltration, concentration and/or sterilization.

In order to solve the problems, the invention provides fucoidan and application thereof in compound enzymolysis of sea cucumbers, namely, fucoidan Fun168B excavated by the inventor can carry out double-enzyme synchronous enzymolysis on the sea cucumbers with low-temperature protease, immediately degrades fucoidan released from raw materials into enzymolysis liquid while the protease plays a role, synergistically improves the enzymolysis efficiency of the protease, and obtains the sea cucumber enzymolysis liquid with low viscosity, easy clarification and good processing characteristics.

In order to achieve the purpose, the invention is realized by the following technical scheme: provides an endo-1, 3-fucoidan enzyme, named as fucoidan 168B, with the amino acid sequence of SEQ ID NO.1 and an enzyme derived from SEQ ID NO.1 by substituting, deleting or adding one or more amino acids and still having continuous endo-activity to alpha-1, 3-fucoidan. The inventor carries out heterologous expression on the enzyme sequence and carries out systematic study on the enzymology property of the enzyme sequence, and the optimum reaction temperature is 35 ℃ and the optimum pH is 7.5. Experiments show that the fucosidase Fun168B is not degraded by protease in the surface during synchronous enzymolysis, and can be used for enzymolysis of sea cucumber in cooperation with the protease; the enzyme and the protease have overlapping ranges of optimal reaction temperature and pH, and can be used simultaneously.

SEQ ID NO.1：

MNQLKNFYSTYIKCLTVLFIVLSQQSYAQVVGTGDWSSLRLYGHAYNVNGFSSAEYD WIANHYFLFTTEKRHASVVYGNPTSELASDVASQQINTNNSVCRPLFYWNSSKIFDNIYVT VQDAVTNNPSWVRPDNKWDYTNSDFRNWWVDVAQDQVNNAAHEGVFVDAVPNVVGA QGIAALAELENMMDQLPGLVIYNGFYTPVNGGSLLAGLTTLEHADGVFVEKFMNSTCDTK EKGKVLLDDLLLVPANKYIIANSEHESAWNSTNHEFSLACYLIIANNRSFYRYTDQEGFDYS SNALTYWHEDFGKNIGAPLGKAMVNGYVYTRTFENVSVTVDLENKTSSIVWGSGTNLALS GTATQSSTGASGVASRAIDGNTDGIFSNQSVTYANASVSKAWWELDLGAEYNVGDIKIFGR MDSAHQASLSNFTVLIYDNTGRVDFQTFTSFPDPSITYNLNGRTISRVRIRQNDTTKPLALAE VQVFEHSVNSSVSQSQQNILGNDQATFTTELSNGGNSEFNLHPNPVDNELFLNAKNNIEAT YTIVNFLGQTVLSGKLKETITTIDTSGLTSGSYVVVLSNVTGVHTRKMLKK

Further, the nucleotide sequence corresponding to the gene for coding the endo-1, 3-fucosidase Fun168B is shown as SEQ ID NO. 2; and all genes which can be translated into SEQ ID NO. 1.

SEQ ID NO.2：

ATGAATCAACTAAAAAACTTTTATTCAACTTACATTAAATGTTTAACTGTTCTATTCA TTGTGTTGTCACAACAAAGTTATGCCCAAGTTGTGGGTACAGGTGATTGGTCTTCGTTA AGGTTATATGGGCATGCTTATAATGTGAATGGTTTTAGTTCAGCTGAATATGATTGGATAG CAAATCATTATTTTTTATTTACAACTGAAAAACGTCATGCAAGTGTTGTTTATGGGAATCC TACTTCTGAGTTAGCATCAGATGTTGCTTCTCAACAAATTAATACAAACAATTCGGTTTG TAGACCTTTATTCTATTGGAATTCATCAAAAATATTTGATAATATTTATGTAACCGTTCAAG ATGCTGTTACTAATAATCCGTCTTGGGTTAGACCTGATAACAAATGGGATTATACAAATTC CGATTTTAGAAATTGGTGGGTAGATGTAGCACAGGATCAAGTAAATAATGCTGCACATGA GGGGGTTTTTGTAGATGCTGTTCCTAATGTAGTTGGTGCACAGGGTATTGCAGCGCTAGC GGAATTAGAAAACATGATGGATCAATTACCAGGACTTGTTATTTATAATGGATTTTATACA CCAGTCAACGGAGGGAGTTTATTGGCGGGTTTAACAACTTTAGAACATGCTGATGGTGT TTTTGTAGAGAAATTTATGAATAGTACTTGTGATACAAAGGAGAAAGGAAAAGTCTTAC TTGATGATTTGCTTTTAGTTCCAGCAAATAAATATATTATAGCAAATTCAGAGCATGAATC AGCTTGGAATTCAACAAATCATGAGTTTAGTTTGGCTTGTTATCTTATTATCGCTAATAAT CGTAGTTTTTATCGTTATACAGATCAAGAAGGATTTGATTATAGTTCTAATGCGCTTACTTA TTGGCATGAAGATTTTGGAAAAAACATAGGAGCACCCTTAGGGAAAGCAATGGTAAATG GTTATGTGTATACAAGAACATTTGAAAATGTTTCAGTGACTGTAGATTTAGAAAATAAAA CATCTTCTATCGTTTGGGGTTCTGGTACCAATCTTGCCTTGTCAGGTACGGCAACTCAAT CAAGTACGGGAGCTAGCGGAGTAGCTTCAAGAGCTATTGATGGAAATACAGATGGAATT TTTTCTAATCAATCTGTTACCTATGCCAATGCATCAGTAAGCAAAGCATGGTGGGAGTTG GATTTAGGAGCAGAATATAATGTAGGAGATATTAAAATATTTGGTAGGATGGATAGCGCTC ATCAAGCTTCTTTATCAAATTTTACAGTCCTAATATATGACAATACTGGTAGGGTTGATTT TCAAACATTCACATCTTTCCCAGACCCATCGATAACCTACAATTTAAATGGTAGAACTAT AAGTAGAGTAAGAATTAGACAAAATGACACCACCAAACCTTTAGCTTTAGCTGAGGTTC AGGTTTTTGAACATTCAGTAAATTCTTCTGTATCACAATCACAGCAAAATATATTAGGCAA TGATCAAGCAACATTTACAACTGAATTATCTAATGGAGGGAACTCAGAGTTTAATCTGCA TCCAAATCCTGTAGATAATGAACTGTTTTTAAATGCTAAAAACAATATAGAAGCAACTTA TACAATTGTTAACTTTTTAGGTCAAACAGTTCTTTCTGGTAAATTAAAAGAAACTATTAC CACTATTGATACAAGTGGTTTAACTTCTGGAAGCTATGTTGTTGTTCTTTCAAATGTTACG GGAGTACATACACGAAAGATGCTAAAAAAATAG

A sea cucumber synchronous enzymolysis method by utilizing fucosidase Fun168B and low-temperature protease comprises the following steps:

1) adding fucosidase Fun168B and low-temperature protease into the sea cucumber homogenate according to a certain proportion for synchronous enzymolysis, and inactivating at high temperature to obtain the sea cucumber composite enzymolysis liquid. Wherein, the protease is used for degrading collagen into oligopeptide; when the collagen is degraded, the fucosan released from the body wall of the sea cucumber can be degraded into low molecular weight polysaccharide to oligosaccharide by the fucoidan, so that the viscosity of the sea cucumber enzymatic hydrolysate is effectively and immediately reduced, the enzymatic hydrolysis efficiency of the protease is improved, and the sea cucumber enzymatic hydrolysate with good processing characteristics is obtained.

2) The enzymolysis liquid which is clear and has the same viscosity with the pure water can be obtained by the low-speed centrifugation at 4000rpm of 1000-. Lower than 1000rpm results in low clarification efficiency, and higher than 4000rpm is difficult to realize in industrial production.

3) According to the requirements of the form and quality of the final product, the processes of decolorization, deodorization, concentration, ultrafiltration, spray drying, blending and the like are carried out subsequently.

Further, the sea cucumber raw material can be common edible sea cucumber such as acaudina molpadioides, stichopus japonicus, American ginseng, thelenota ananas, north atlantic cucurbita and the like. Before enzymolysis, simple pretreatment is carried out on sea cucumber raw materials: homogenizing fresh sea cucumber or grinding dried sea cucumber into powder, adding a proper amount of water, wherein the common proportion is the mass of the sea cucumber: the water mass is 1:10-1: 3.

Further, the types of the low-temperature protease described in the step 1) and the corresponding gene accession numbers are shown in the following table and can be obtained by gene engineering. Experiments show that the fucosidase Fun168B is not degraded by the protease in the table when the simultaneous enzymolysis is carried out, and can be used for enzymolysis of the sea cucumber in cooperation with the protease.

Further, the addition amount of the low-temperature protease and the fucosidase in the step 1) is 1-1000U, the reaction temperature is 20-40 ℃, and the reaction pH is 6.0-8.0. Within the parameter range, the fucoidan and the low-temperature protease both have higher enzymolysis activity, and can ensure the rapid enzymolysis reaction. The addition amount of the enzyme is properly adjusted according to the raw material amount of the sea cucumber, and is corresponding to the reaction time, and if the addition amount of the enzyme is small, the reaction time is increased to ensure that the enzymolysis of the sample is thorough. Outside this range, the enzymatic efficiency may be reduced and/or the production cost may be increased.

Further, the temperature range of the high-temperature inactivation in the step 1) is 60-80 ℃, the fucosidase and the low-temperature protease can be fully inactivated within the temperature range, and the inactivation time needs to be adjusted according to the volume of the product.

The invention has the beneficial effects that:

(1) based on the synchronous enzymolysis technology, the viscosity of the sea cucumber enzymolysis liquid can be effectively reduced to be the same as that of pure water (figure 1). The reduction of the viscosity brings the following benefits, and the technological level of sea cucumber enzymolysis and the quality of related products can be obviously improved:

the clarification can be completed by simple low-speed centrifugation, and the production cost is reduced (figure 2).

Viscosity reduction is convenient for decoloring and deodorization treatment, and treatment time is shortened;

viscosity reduction is favorable for heat and mass transfer in concentration, concentration efficiency is higher, local scorching is avoided, preparation of high-concentration concentrated liquid is facilitated, and spray drying efficiency is further improved.

(2) The synchronous enzymolysis reaction can be carried out at room temperature, and compared with the traditional enzymolysis temperature of 50-60 ℃, the energy consumption is greatly shortened.

(3) The fucosidase Fun168B and low temperature protease can be completely inactivated at 60-80 deg.C, which is lower than the inactivation temperature (100 deg.C) of common protease, and can prevent chemical reaction of Stichopus japonicus nutritional components due to high temperature, thereby losing its nutritional function.

(4) The fucosan with high molecular weight in the sea cucumber is degraded into polysaccharide and/or oligosaccharide with low molecular weight, which is beneficial to improving the bioavailability of the fucosan.

(5) The fucosidase Fun168B has strong stability, can be kept stable (enzyme activity residual > 80%) at pH 3.0-11.0, and can be stably placed for at least 1d (residual enzyme activity > 70%) at 4 ℃, 25 ℃ and 30 ℃ (FIG. 3). The enzymolysis reaction condition is controllable and easy to be amplified, and can meet the production of different scales.

The invention has novelty, creativity and practicability. Detailed information such as the sequence, the function, the action mode, the action substrate, the enzymological property and the like of Fun168B is verified by the inventor through creative experiments, and the inventor does not have any unit or person to apply for the same invention before the application date, so that the invention has novelty; the enzymolysis liquid obtained based on the synchronous enzymolysis technology can be clarified only by low-speed centrifugation treatment at 1000-4000rpm, the viscosity is as low as 1 mPa.S (the shear rate is more than 1(1/S)), and the method is close to pure water, and compared with the existing sea cucumber enzymolysis technology, the method has outstanding substantive characteristics and remarkable progress and is creative; the Fun168B has good enzymatic properties and strong storage stability, and the synchronous enzymolysis technology can improve the production effect of the sea cucumber enzymolysis liquid and develop the sea cucumber enzymolysis liquid into subsequent products, has great significance for extending the sea cucumber processing industry chain and has practicability.

Drawings

FIG. 1: comparing the viscosity of the enzymolysis liquid produced by the synchronous enzymolysis technology and the sectional enzymolysis technology;

FIG. 2: comparing the clarity of the enzymolysis liquid produced by the synchronous enzymolysis technology and the segmented enzymolysis technology;

FIG. 3: enzymatic properties of the fucosidase Fun 168B.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all 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.

Example 1: enzymatic Properties of the Funanase Fun168B

In order to obtain the optimum reaction conditions of the fucoidan, the temperature, pH and the like are respectively studied.

1) Optimum reaction temperature and temperature stability

Obtaining recombinant enzyme solution by using escherichia coli, properly diluting the recombinant enzyme solution, mixing the recombinant enzyme solution with fucosan substrate solution with pH of 8.0 and 2mg/mL, reacting the mixture for 10min at the temperature of 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 ℃, determining activity by using a reducing sugar increment detection method pHBH method after inactivation, and calculating specific enzyme activity, wherein the result is shown in figure 3, and the optimal reaction temperature is 25 ℃. After a proper amount of enzyme is placed at 4 ℃, 25 ℃, 30 ℃ and 40 ℃ for 0, 1, 2, 4, 6, 16 and 24 hours, a certain amount of enzyme is taken to be mixed with a substrate solution for measuring the activity, the activity when the enzyme is placed at 4 ℃ for 0 hour is 100 percent, the result is expressed by residual enzyme activity (figure 3), and the result shows that the enzyme can be stably placed at least for 1d (the residual enzyme activity is more than 70 percent) at 4 ℃, 25 ℃ and 30 ℃.

2) Reaction pH and pH stability

Using recombinant enzyme solution obtained by escherichia coli, taking a proper amount of fucosan substrate solution mixed with buffers with different pH values (sodium dihydrogen phosphate-disodium hydrogen phosphate buffers with pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5 and pH 7.0; sodium dihydrogen phosphate-disodium hydrogen phosphate buffers with pH 6.5, pH7.0, pH7.5, pH 8.0, pH 8.5 and pH 9.0; sodium carbonate-sodium bicarbonate buffers with pH 9.0, pH 9.5, pH 10.0, pH 10.5 and pH 11.0) to ensure that the reaction is in different pH value environments for 10min, using a pHBH method to measure the activity and calculate the specific enzyme activity after inactivation, and the result is shown in figure 3, wherein the fucosan enzyme has the activity under the condition of pH 7.5-9.0; the optimum reaction pH value is 8.0. And (3) placing a proper amount of enzyme at the pH value for 1h, adjusting the pH value to 8.0, mixing with a substrate, and carrying out enzyme activity determination. The enzyme activity of the highest activity is recorded as 100%, and the rest are expressed as residual enzyme activity (figure 3), and the result shows that the enzyme can be kept stable at the pH of 3.0-11.0 (the residual enzyme activity is greater than 80%), which indicates that the enzyme has a wider pH stability range.

Example 2: enzymatic Properties of Low temperature proteases

In order to obtain the optimal reaction conditions for the low temperature protease, the low temperature protease was obtained by genetic engineering and studied for the optimal reaction conditions (table below). The results show that the optimal reaction temperature of the low-temperature protease from different sources is between 20 and 35 ℃, and the optimal pH is between 6.0 and 8.0.

Example 3: based on the synchronous enzymolysis technology, the acaudina molpadioides and the ginseng are used as raw materials to prepare the enzymolysis liquid

1. Removing internal organs of fresh and alive acaudina molpadioides and ginseng, cleaning, taking 100kg of processed acaudina molpadioides, cutting into blocks, homogenizing into meat paste, putting into a 2000L enzymolysis tank, adding 1500L of water, and stirring uniformly;

2. adjusting the temperature of the solution in the enzymolysis tank to 30 ℃, adjusting the pH to 7.5, and adding 1000U of low-temperature alkaline protease lupA and fucosidase Fun168B for enzymolysis for 3 h;

inactivating at 3.60 deg.C for 15min, and centrifuging at 3000rpm for 10min to obtain clarified enzymolysis solution (the effect figure is shown in figure 2 of the specification).

4. The viscosity of the enzymolysis liquid is about 1mPa & S under the condition that the shear rate is 10-100 (1/S) through determination of a rheometer, and is the same as that of pure water, and the viscosity of the sea cucumber ginseng enzymolysis liquid obtained by adopting the traditional enzymolysis technology is 128mPa & S.

Example 4: based on the synchronous enzymolysis technology, American ginseng is used as a raw material to prepare enzymolysis liquid

1. Removing viscera of fresh American ginseng, cleaning, cutting 400kg of processed American ginseng into pieces, homogenizing into meat paste, loading into 5000L enzymolysis tank, adding 3000L of water, and stirring;

2. adjusting the temperature of the solution in the enzymolysis tank to 25 ℃, adjusting the pH to 7.0, adding 500U of low-temperature alkaline protease pro-2127 and fucosidase Fun168B for enzymolysis for 3 h;

inactivating at 3.70 deg.C for 10min, and centrifuging at 4000rpm for 15min to obtain clarified enzymolysis solution.

4. The viscosity of the enzymolysis liquid is about 2mPa & S under the condition that the shear rate is 10-100 (1/S) through determination of a rheometer, and is close to that of pure water, and the viscosity of the sea cucumber ginseng enzymolysis liquid obtained by adopting the traditional enzymolysis technology is 850mPa & S.

Example 5: based on the synchronous enzymolysis technology, the enzymolysis liquid is prepared by taking thelenota ananas as raw material

2. adjusting the temperature of the solution in the enzymolysis tank to 30 ℃, adjusting the pH to 7.5, adding 500U of low-temperature protease subtilase and fucoidan 168B for enzymolysis for 6 h;

inactivating at 3.65 deg.C for 20min, and centrifuging at 3000rpm for 20min to obtain clarified enzymolysis solution.

4. The viscosity of the enzymolysis liquid is about 1mPa & S under the condition that the shear rate is 10-100 (1/S) through determination of a rheometer, the viscosity of the enzymolysis liquid is the same as that of pure water, and the viscosity of the enzymolysis liquid of the acaudina molpadioides is 180mPa & S through the traditional enzymolysis technology.

Example 6: based on the synchronous enzymolysis technology, the stichopus japonicus is used as a raw material to prepare an enzymolysis liquid

2. adjusting the temperature of the solution in the enzymolysis tank to 30 ℃, adjusting the pH to 7.5, and adding 500U of low-temperature protease SapSh and fucosidase Fun168B for enzymolysis for 2 h;

inactivating at 3.70 deg.C for 15min, and centrifuging at 4000rpm for 10min to obtain clarified enzymolysis solution.

4. Through determination of a rheometer, the viscosity of the enzymolysis liquid is about 3mPa & S under the condition that the shear rate is 10-100 (1/S), and is close to that of pure water, and the viscosity of the sea cucumber ginseng enzymolysis liquid obtained by adopting the traditional enzymolysis technology is 840mPa & S.

Example 7: sea cucumber nutritional oral liquid prepared based on synchronous enzymolysis technology

1. Removing internal organs of fresh and alive prickled bodies, cleaning, taking 100kg of processed acaudina molpadioides, cutting into blocks, homogenizing into meat paste, putting into a 2000L enzymolysis tank, adding 1500L of water, and stirring uniformly;

4. Adding 8% white sugar into the enzymolysis solution, and adjusting pH to 5.5 with citric acid.

5. Filling and sealing by an automatic filling machine, placing the sea cucumber into a sterilization kettle, and sterilizing for 30min under the conditions of 0.20MPa and 121 ℃ to obtain the sea cucumber nutritional oral liquid.

Example 8: preparation of sea cucumber enzymolysis liquid tablet

1. Removing viscera from fresh and alive stinging body, cleaning, taking 100kg of processed raw materials, cutting into blocks, homogenizing into meat paste, putting into a 2000L enzymolysis tank, adding 1500L of water, and stirring uniformly;

2. adjusting the temperature of the solution in the enzymolysis tank to 30 ℃, adjusting the pH to 7.5, adding 500U of low-temperature protease SapSh and fucosidase Fun168B, and carrying out stirring enzymolysis treatment for 2 h;

3. heating the obtained enzymolysis liquid at 70 deg.C for 15min for enzyme deactivation;

4. clarifying the enzyme-inactivated enzymolysis liquid, and centrifuging at 3000r/min for 15 min;

5. concentrating the enzymolysis solution to 20%, and drying in a spray drying tower to obtain sea cucumber enzymolysis solution dry powder;

6. sieving the dried powder of the sea cucumber enzymolysis liquid with a 50-mesh sieve, adding magnesium stearate, honey powder and sorbitol, and uniformly stirring;

7. and (3) putting the mixed dry powder into a tablet press, adjusting parameters of the tablet press, and performing tabletting treatment to obtain the enzymatic hydrolysate tablets with the tablet weight of 0.5 g.

Example 9: preparation of sea cucumber and fruit juice composite beverage

1. Removing internal organs of fresh and alive stinging body, cleaning, taking 50kg of processed raw materials, cutting into blocks, homogenizing into meat paste, putting into a 1000L enzymolysis tank, adding 500L of water, and stirring uniformly;

2. adjusting the temperature of the solution in the enzymolysis tank to 25 ℃, adjusting the pH to 7, adding 100U of low-temperature protease SapSh and fucosidase Fun168B, and stirring for enzymolysis for 1 h;

3. heating the obtained enzymolysis liquid at 60 deg.C for 10min for enzyme deactivation;

5. adding grape juice, lemon juice and grapefruit juice into the sea cucumber enzymolysis liquid, blending, homogenizing, performing UHT ultrahigh temperature instantaneous sterilization, and aseptically canning to obtain a finished beverage.

Example 10: preparation of sea cucumber milk

5. taking raw milk as a base material, heating to 70 ℃, and adding the sea cucumber enzymolysis liquid, the lemon juice and the stabilizer for blending;

6. the mixed solution is kept at 65-70 ℃ for emulsification and homogenization, the first-stage pressure is 15-17MPa, and the second-stage pressure is 1-3 MPa;

7. the sea cucumber milk is prepared by UHT ultrahigh temperature instant sterilization at 135 deg.C for 4s, sterilizing, cooling, aseptically canning.

Finally, it should be noted that the above-mentioned embodiments, although describing the specific embodiments of the present invention, are only the preferred embodiments of the present invention, and are not intended to limit the present invention in any way. It will be understood by those skilled in the art that these are by way of example only and that the scope of the invention is defined by the appended claims. And all modifications and equivalents may be resorted to, falling within the scope of the invention.

Sequence listing

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<120> fucoidan and application thereof in sea cucumber composite enzymolysis

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Met Asn Gln Leu Lys Asn Phe Tyr Ser Thr Tyr Ile Lys Cys Leu Thr

1 5 10 15

Val Leu Phe Ile Val Leu Ser Gln Gln Ser Tyr Ala Gln Val Val Gly

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Thr Gly Asp Trp Ser Ser Leu Arg Leu Tyr Gly His Ala Tyr Asn Val

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Asn Gly Phe Ser Ser Ala Glu Tyr Asp Trp Ile Ala Asn His Tyr Phe

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Leu Phe Thr Thr Glu Lys Arg His Ala Ser Val Val Tyr Gly Asn Pro

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Asn Ser Val Cys Arg Pro Leu Phe Tyr Trp Asn Ser Ser Lys Ile Phe

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Asp Asn Ile Tyr Val Thr Val Gln Asp Ala Val Thr Asn Asn Pro Ser

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Trp Val Arg Pro Asp Asn Lys Trp Asp Tyr Thr Asn Ser Asp Phe Arg

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Asn Trp Trp Val Asp Val Ala Gln Asp Gln Val Asn Asn Ala Ala His

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Glu Gly Val Phe Val Asp Ala Val Pro Asn Val Val Gly Ala Gln Gly

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Ile Ala Ala Leu Ala Glu Leu Glu Asn Met Met Asp Gln Leu Pro Gly

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Leu Val Ile Tyr Asn Gly Phe Tyr Thr Pro Val Asn Gly Gly Ser Leu

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Leu Ala Gly Leu Thr Thr Leu Glu His Ala Asp Gly Val Phe Val Glu

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Lys Phe Met Asn Ser Thr Cys Asp Thr Lys Glu Lys Gly Lys Val Leu

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Leu Asp Asp Leu Leu Leu Val Pro Ala Asn Lys Tyr Ile Ile Ala Asn

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Ser Glu His Glu Ser Ala Trp Asn Ser Thr Asn His Glu Phe Ser Leu

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Ala Cys Tyr Leu Ile Ile Ala Asn Asn Arg Ser Phe Tyr Arg Tyr Thr

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Asp Gln Glu Gly Phe Asp Tyr Ser Ser Asn Ala Leu Thr Tyr Trp His

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Glu Asp Phe Gly Lys Asn Ile Gly Ala Pro Leu Gly Lys Ala Met Val

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Asn Gly Tyr Val Tyr Thr Arg Thr Phe Glu Asn Val Ser Val Thr Val

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Asp Leu Glu Asn Lys Thr Ser Ser Ile Val Trp Gly Ser Gly Thr Asn

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Leu Ala Leu Ser Gly Thr Ala Thr Gln Ser Ser Thr Gly Ala Ser Gly

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Val Ala Ser Arg Ala Ile Asp Gly Asn Thr Asp Gly Ile Phe Ser Asn

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Gln Ser Val Thr Tyr Ala Asn Ala Ser Val Ser Lys Ala Trp Trp Glu

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Leu Asp Leu Gly Ala Glu Tyr Asn Val Gly Asp Ile Lys Ile Phe Gly

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Arg Met Asp Ser Ala His Gln Ala Ser Leu Ser Asn Phe Thr Val Leu

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Ile Tyr Asp Asn Thr Gly Arg Val Asp Phe Gln Thr Phe Thr Ser Phe

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Gln Asn Ile Leu Gly Asn Asp Gln Ala Thr Phe Thr Thr Glu Leu Ser

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Asn Gly Gly Asn Ser Glu Phe Asn Leu His Pro Asn Pro Val Asp Asn

515 520 525

Glu Leu Phe Leu Asn Ala Lys Asn Asn Ile Glu Ala Thr Tyr Thr Ile

530 535 540

Val Asn Phe Leu Gly Gln Thr Val Leu Ser Gly Lys Leu Lys Glu Thr

545 550 555 560

Ile Thr Thr Ile Asp Thr Ser Gly Leu Thr Ser Gly Ser Tyr Val Val

565 570 575

Val Leu Ser Asn Val Thr Gly Val His Thr Arg Lys Met Leu Lys Lys

580 585 590

<210> 2

<211> 1779

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgaatcaac taaaaaactt ttattcaact tacattaaat gtttaactgt tctattcatt 60

gtgttgtcac aacaaagtta tgcccaagtt gtgggtacag gtgattggtc ttcgttaagg 120

ttatatgggc atgcttataa tgtgaatggt tttagttcag ctgaatatga ttggatagca 180

aatcattatt ttttatttac aactgaaaaa cgtcatgcaa gtgttgttta tgggaatcct 240

acttctgagt tagcatcaga tgttgcttct caacaaatta atacaaacaa ttcggtttgt 300

agacctttat tctattggaa ttcatcaaaa atatttgata atatttatgt aaccgttcaa 360

gatgctgtta ctaataatcc gtcttgggtt agacctgata acaaatggga ttatacaaat 420

tccgatttta gaaattggtg ggtagatgta gcacaggatc aagtaaataa tgctgcacat 480

gagggggttt ttgtagatgc tgttcctaat gtagttggtg cacagggtat tgcagcgcta 540

gcggaattag aaaacatgat ggatcaatta ccaggacttg ttatttataa tggattttat 600

acaccagtca acggagggag tttattggcg ggtttaacaa ctttagaaca tgctgatggt 660

gtttttgtag agaaatttat gaatagtact tgtgatacaa aggagaaagg aaaagtctta 720

cttgatgatt tgcttttagt tccagcaaat aaatatatta tagcaaattc agagcatgaa 780

tcagcttgga attcaacaaa tcatgagttt agtttggctt gttatcttat tatcgctaat 840

aatcgtagtt tttatcgtta tacagatcaa gaaggatttg attatagttc taatgcgctt 900

acttattggc atgaagattt tggaaaaaac ataggagcac ccttagggaa agcaatggta 960

aatggttatg tgtatacaag aacatttgaa aatgtttcag tgactgtaga tttagaaaat 1020

aaaacatctt ctatcgtttg gggttctggt accaatcttg ccttgtcagg tacggcaact 1080

caatcaagta cgggagctag cggagtagct tcaagagcta ttgatggaaa tacagatgga 1140

attttttcta atcaatctgt tacctatgcc aatgcatcag taagcaaagc atggtgggag 1200

ttggatttag gagcagaata taatgtagga gatattaaaa tatttggtag gatggatagc 1260

gctcatcaag cttctttatc aaattttaca gtcctaatat atgacaatac tggtagggtt 1320

gattttcaaa cattcacatc tttcccagac ccatcgataa cctacaattt aaatggtaga 1380

actataagta gagtaagaat tagacaaaat gacaccacca aacctttagc tttagctgag 1440

gttcaggttt ttgaacattc agtaaattct tctgtatcac aatcacagca aaatatatta 1500

ggcaatgatc aagcaacatt tacaactgaa ttatctaatg gagggaactc agagtttaat 1560

ctgcatccaa atcctgtaga taatgaactg tttttaaatg ctaaaaacaa tatagaagca 1620

acttatacaa ttgttaactt tttaggtcaa acagttcttt ctggtaaatt aaaagaaact 1680

attaccacta ttgatacaag tggtttaact tctggaagct atgttgttgt tctttcaaat 1740

gttacgggag tacatacacg aaagatgcta aaaaaatag 1779

Claims

1. An endo-1, 3-fucoidan having the amino acid sequence of SEQ ID No.1 and an enzyme derived from SEQ ID No.1 by substitution, deletion or addition of one or more amino acids and still having continuous endo-activity to alpha-1, 3-fucoidan.

2. The gene encoding endo-1, 3-fucoidan according to claim 1, wherein: the nucleotide sequence is shown as SEQ ID NO. 2; and all genes which can be translated into SEQ ID NO. 1.

3. The use of the endo-1, 3-fucoidan as defined in claim 1 in the complex enzymatic hydrolysis of sea cucumber.

4. A sea cucumber composite enzymolysis method using the endo-1, 3-fucoidan and protease in claim 1 is characterized in that: and synchronously adding fucoidan and low-temperature protease into the sea cucumber homogenate for synchronous enzymolysis, and inactivating at high temperature to obtain the sea cucumber composite enzymatic hydrolysate.

5. The fucosidase and protease-based sea cucumber complex enzymolysis method according to claim 4, wherein: the addition amount of the low-temperature protease and the fucoidan is 1-1000U, the reaction temperature is 20-40 ℃, and the reaction pH is 6.0-8.0.

6. The fucosidase and protease-based sea cucumber complex enzymolysis method according to claim 4, wherein: the temperature range of high-temperature inactivation is 60-80 ℃.

7. The fucosidase and protease-based sea cucumber complex enzymolysis method according to claim 4, wherein: the obtained sea cucumber complex enzymatic hydrolysate is centrifuged at low speed of 1000-.

8. The compound enzymolysis method for sea cucumber based on fucoidan and protease as claimed in any one of claims 4-7, wherein: and (3) carrying out decoloration, deodorization, concentration, ultrafiltration, spray drying and blending treatment on the obtained product.