CN114250209B

CN114250209B - Fucoidan and application thereof in sea cucumber complex enzymolysis

Info

Publication number: CN114250209B
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: 2023-05-05
Anticipated expiration: 2041-12-14
Also published as: CN114250209A

Abstract

The invention relates to the technical field of biology, in particular to a fucosidase and application thereof in complex enzymolysis of sea cucumbers. According to the invention, the low-temperature protease and the fucoidan Fun168B are simultaneously added into the sea cucumber homogenate according to a certain proportion for synchronous enzymolysis, and when the protease degrades collagen, the Fun168B can instantly degrade macromolecular fucoidan released from the sea cucumber body wall to the enzymolysis liquid, so that the low protease hydrolysis efficiency caused by the increase of the viscosity of the enzymolysis liquid is avoided. The sea cucumber enzymatic hydrolysate obtained based on the enzymatic hydrolysis technology can be clarified by low-speed centrifugation at 1000-4000rpm, has viscosity close to that of pure water, and is favorable for subsequent operations such as decolorization, ultrafiltration, concentration, sterilization and the like. The synchronous enzymolysis technology can be carried out at room temperature, so that energy is saved, and the fucoidan in the sea cucumber can be fully degraded into oligosaccharide, so that the nutritive value of the enzymolysis liquid is improved.

Description

Fucoidan and application thereof in sea cucumber complex enzymolysis

Technical Field

The invention relates to the technical field of biological enzymolysis, in particular to a fucosidase and application thereof in complex enzymolysis of sea cucumbers.

Background

Sea cucumber is a traditional tonic food in asian countries such as japanese and korean. In the last twenty years, the Chinese sea cucumber aquaculture rapidly develops, the cultivation amount is increased, and the sea cucumber cultivation amount in 2019 reaches 171700 tons. The sea cucumber has various nutritional functional components, wherein the collagen is rich in various non-essential amino acids, and has physiological activities of resisting thrombus, resisting tumor, regulating immunity, reducing blood lipid, improving memory and the like. Fucan (also called as "fucoidan", "fucoidan sulfate", "fucoidan") is another major functional component in sea cucumber, and consists of fucose and sulfate groups, accounting for about 5-10% of the dry weight of sea cucumber body wall, and its aqueous solution has high viscosity. Fucans have been shown to have a wide range of biological activities including anticoagulation, inhibition of osteoclast generation, proliferation of neural stem/progenitor cells, protection of ethanol-induced gastric ulcers, antioxidation, regulation of insulin resistance, anti-adipogenesis and prevention of intestinal mucositis.

The enzymolysis liquid is a main processing form of sea cucumber, and can obviously improve the bioavailability of the nutrient components of the sea cucumber. However, the enzymolysis technology of sea cucumber is single, most of enzymolysis liquid and its deep processing products (such as sea cucumber wine, sea cucumber milk, etc.) in the market are based on protease enzymolysis, and the enzymolysis often has higher viscosity, and the efficiency and effect of subsequent operations such as clarification, decolorization, ultrafiltration, concentration, sterilization, etc. are seriously affected. Research shows that the macromolecular fucoidan in the sea cucumber body wall can be gradually released into the enzymolysis liquid along with the degradation of collagen in the protease hydrolysis process, so that the viscosity of the enzymolysis liquid is increased. More importantly, the high viscosity of the enzymatic hydrolysate caused by the fucoidan can further influence 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 (patent number 202010565103.8) discloses a sea cucumber compound enzymolysis method based on fucanase and protease, and a preparation process of sea cucumber compound enzymolysis liquid is established based on sea cucumber fucanase FunA and commercial protease. The process carries out two-step sectional enzymolysis on sea cucumber by protease and fucosidase, namely, firstly adding protease for enzymolysis at 50-60 ℃, then reducing the temperature to about 30 ℃ and adding fucosidase for enzymolysis. After the protease hydrolysis is completed, the influence of the fucan on the viscosity of the enzymolysis liquid is eliminated by adding the fucoidan, so that the hydrolysis efficiency of the protease cannot be obviously improved, and the two-step sectional enzymolysis method is relatively energy-consuming. The patent also mentions that "the optimal reaction conditions of protease are different from those of fucosidase, so that it is not suitable for simultaneous addition to sea cucumber for enzymolysis". Based on the technical defects, if the viscosity of the sea cucumber enzymatic hydrolysate is further reduced, synchronous enzymolysis is needed by utilizing the fucosidase and the protease, and the fucosan released from the raw materials into the enzymatic hydrolysate is degraded in time, so that the viscosity of the enzymatic hydrolysate is prevented from being increased, the enzymatic hydrolysis efficiency of the protease is improved, and the efficiency and the effect of the subsequent clarification and other processes are improved.

Disclosure of Invention

The invention aims to solve the technical problems that the viscosity of the sea cucumber enzymatic hydrolysate prepared based on the existing enzymatic hydrolysis technology is high, and the efficiency and the effect of subsequent operations such as clarification, decoloration, ultrafiltration, concentration, sterilization and the like are seriously affected.

In order to solve the problems, the invention provides the fucoidan and the application thereof in the complex enzymolysis of the sea cucumber, namely the fucoidan Fun168B excavated by the inventor can be used for carrying out double-enzyme synchronous enzymolysis on the sea cucumber with low-temperature protease, and the fucoidan released into the enzymolysis liquid by the raw materials is degraded in time while the protease plays a role, so that the enzymolysis efficiency of the protease is synergistically improved, and the sea cucumber enzymolysis liquid with low viscosity, easy clarification and good processing characteristics is obtained.

In order to achieve the above purpose, the invention is realized by the following technical scheme: an endo-1, 3-fucosidase, designated fucosidase Fun168B, having the amino acid sequence SEQ ID NO.1 and enzymes derived from SEQ ID NO.1 having one or more amino acids substituted, deleted or added and still having continuous endo-activity on alpha-1, 3-fucosan is provided. The inventor carries out heterologous expression on the enzyme sequence and carries out systematic study on the enzymatic property of the enzyme sequence, and the optimal reaction temperature is 35 ℃ and the optimal pH is 7.5. Experiments show that when synchronous enzymolysis is carried out, the fucoidan Fun168B can not be degraded by proteases in the table, and can be used for carrying out synergistic enzymolysis on sea cucumbers with proteases; the enzyme and the protease have overlapping optimal reaction temperature and pH ranges, 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 encoding the endo-1, 3-fucosidase Fun168B is shown as SEQ ID NO. 2; and all genes of SEQ ID NO.1 can be translated.

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 synchronous enzymolysis method of sea cucumber by using fucoidan enzyme Fun168B and low-temperature protease comprises the following steps:

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

2) And (3) centrifuging at a low speed of 1000-4000rpm to obtain a clear enzymolysis liquid with the same viscosity as pure water. A lower than 1000rpm results in a low clarification efficiency, and a higher than 4000rpm is difficult to realize in industrial production.

3) And (3) according to the requirements of the final product form and quality, the following procedures of decoloring, deodorizing, concentrating, ultrafiltering, spray drying, preparing and the like are carried out.

Further, the sea cucumber raw material can be common edible sea cucumber such as sea cucumber, stichopus japonicus, american ginseng, etc. Before enzymolysis, the sea cucumber raw material needs to be subjected to simple pretreatment: homogenizing fresh sea cucumber or grinding dried sea cucumber, adding a proper amount of water, and the common proportion is sea cucumber mass: water mass = 1:10-1:3.

Furthermore, the types of the low-temperature proteases described in step 1) and the corresponding gene accession numbers are shown in the following table, and can be obtained by genetic engineering. Experiments show that when synchronous enzymolysis is carried out, the fucoidan Fun168B can not be degraded by proteases in the table, and can be used for carrying out synergistic enzymolysis on sea cucumbers with proteases.

Further, the addition amounts of the low-temperature protease and the fucosidase in the step 1) are 1-1000U, the reaction temperature is 20-40 ℃, and the reaction pH is 6.0-8.0. In the above parameter range, the fucosidase and the low-temperature protease have high enzymolysis activity, and can ensure the rapid progress of enzymolysis reaction. The addition amount of the enzyme is properly adjusted according to the raw material amount of the sea cucumber, and the enzyme is mutually corresponding to the reaction time, and the reaction time is increased when the addition amount of the enzyme is small so as to ensure thorough enzymolysis of the sample. Exceeding this range may decrease the enzymatic hydrolysis efficiency and/or increase the production cost.

Further, the high temperature inactivation temperature in the step 1) is 60-80 ℃, and the fucosidase and the low temperature protease can be sufficiently inactivated in the high temperature inactivation temperature range, and the inactivation time is required 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 viscosity brings the following benefits, and the process level of sea cucumber enzymolysis and the quality of related products can be obviously improved:

(1) clarification can be accomplished by simple low-speed centrifugation, reducing production costs (figure 2).

(2) The viscosity is reduced, so that the decolorization and deodorization treatment is facilitated, and the treatment time is shortened;

(3) the viscosity reduction is beneficial to heat and mass transfer in concentration, the concentration efficiency is higher, local scorching is avoided, the preparation of high-concentration concentrated liquid is facilitated, and the spray drying efficiency is further improved.

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

(3) The fucoidan enzyme Fun168B and low-temperature protease can be completely inactivated at 60-80deg.C, and is lower than the inactivation temperature (100deg.C) of common protease, so as to avoid chemical reaction of sea cucumber nutritional components due to high temperature, and thus lose nutritional function.

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

(5) The fucoidan Fun168B has high stability, and can be kept stable at pH 3.0-11.0 (residual enzyme activity > 80%), and can be kept stable at 25deg.C for at least 1d (residual enzyme activity > 70%) at 30deg.C (FIG. 3). The enzymolysis reaction condition is controllable, is easy to amplify, and can meet the production of different scales.

The invention has novelty, creativity and practicability. The detailed information of the sequence, the function, the action mode, the action substrate, the enzymatic property and the like of Fun168B is verified by creative experiments, and the invention is applied on the premise of the application date without any unit or person, so that the novel Fun168B has novelty; the enzymolysis liquid obtained based on the synchronous enzymolysis technology can be clarified and has the viscosity as low as 1 mPa.S (the shear rate is more than 1 (1/S)) only through low-speed centrifugation at 1000-4000rpm, is similar to pure water, has outstanding substantive characteristics and obvious progress compared with the existing sea cucumber enzymolysis technology, and has creativity; fun168B has good enzymology property and strong storage stability, and the synchronous enzymolysis technology can improve the production effect of 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: the viscosity of the enzymolysis liquid produced by the synchronous enzymolysis technology is compared with that of the enzymolysis liquid produced by the sectional enzymolysis technology;

fig. 2: the clarity of the enzymolysis liquid produced by the synchronous enzymolysis technology is compared with that of the enzymolysis liquid produced by the sectional 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 more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the attached drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: enzymatic Properties of Fun168B, a fucosidase

In order to obtain the optimal reaction conditions of the fucosidase, the reaction conditions are respectively explored in the aspects of temperature, pH and the like.

1) Optimum reaction temperature and temperature stability

The recombinant enzyme solution is obtained by using escherichia coli, after proper dilution, the recombinant enzyme solution is mixed with fucan substrate solution with the pH of 8.0 and 2mg/mL, and the mixture is reacted for 10min at the temperature of 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 ℃, and after inactivation, the activity is measured by using a reducing sugar incremental detection method pHBH method, and the specific enzyme activity is calculated, and the optimal reaction temperature is 25 ℃ as shown in the result of figure 3. After a proper amount of enzyme is placed at 4 ℃, 25 ℃,30 ℃, 0, 1, 2, 4, 6, 16 and 24 hours, a certain amount of enzyme is mixed with a substrate solution to measure the activity, the activity when the enzyme is placed at 4 ℃ for 0 hour is 100%, the result is expressed as residual enzyme activity (figure 3), and the result shows that the enzyme can be stably placed at least for 1d at 4 ℃, 25 ℃ and 30 ℃ (the residual enzyme activity is more than 70%).

2) Reaction pH and pH stability

The recombinant enzyme solution obtained by using escherichia coli is taken and mixed with 2mg/mL of fucoidan substrate solution prepared by different pH buffers (pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5 and pH 7.0), namely citric acid-disodium hydrogen phosphate buffer solution, pH 6.5, pH7.0, pH7.5, pH 8.0, pH 8.5 and pH 9.0, namely sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, and sodium carbonate-sodium bicarbonate buffer solution with pH 9.0, pH 9.5, pH 10.0, pH 10.5 and pH 11.0, so that the reaction is reacted for 10min under different pH environments, the activity is measured by using a pHBH method after inactivation, and the specific enzyme activity is calculated, and the result is shown in figure 3, and the fucoidan enzyme is active under the conditions of pH 7.5-9.0; the optimal reaction pH value is 8.0. And (3) placing a proper amount of enzyme at the pH value for 1h, and then adjusting the pH value to 8.0 to mix with a substrate for enzyme activity measurement. The enzyme activity at the highest activity point is recorded as 100%, and the other enzyme activities are expressed as residual enzyme activities (figure 3), and the results show that the enzyme can be kept stable at pH 3.0-11.0 (enzyme activity residual > 80%), which indicates that the enzyme has a wider pH stability range.

Example 2: enzymatic Properties of Low temperature proteases

To obtain the optimal reaction conditions for the low-temperature protease, the low-temperature protease was obtained by genetic engineering and the optimal reaction conditions thereof were studied (table below). The results show that the optimal reaction temperature of the low-temperature proteases with 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 method takes the sea cucumber as the raw material to prepare the enzymolysis liquid

1. Removing viscera of fresh radix Pachyrhizi Erosi, cleaning, cutting 100kg of processed radix Pachyrhizi Erosi into pieces, homogenizing to obtain meat paste, placing into 2000L enzymolysis tank, adding 1500L water, and stirring;

2. the temperature of the solution in the enzymolysis tank is adjusted to 30 ℃, the pH is adjusted to 7.5, 1000U of low-temperature alkaline protease lupA and fucoidan 168B are added for enzymolysis treatment for 3 hours;

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

4. The viscosity of the enzymolysis liquid is about 1 mPa.S under the condition of the shearing rate of 10-100 (1/S) and is the same as that of pure water, and the viscosity of the prepared sea cucumber enzymolysis liquid by adopting the traditional enzymolysis technology is 128 mPa.S.

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

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

2. the temperature of the solution in the enzymolysis tank is adjusted to 25 ℃, the pH is adjusted to 7.0, and 500U of low-temperature alkaline protease pro-2127 and fucoidan Fun168B are added for enzymolysis treatment for 3 hours;

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

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

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

2. the temperature of the solution in the enzymolysis tank is adjusted to 30 ℃, the pH is adjusted to 7.5, and 500U of low-temperature protease subtase and fucoidan Fun168B are added for enzymolysis treatment for 6 hours;

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

4. The viscosity of the enzymolysis liquid is about 1 mPa.S under the condition of the shearing rate of 10-100 (1/S) and is the same as that of pure water, and the viscosity of the prepared sea cucumber enzymolysis liquid by adopting the traditional enzymolysis technology is 180 mPa.S.

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

2. the temperature of the solution in the enzymolysis tank is adjusted to 30 ℃, the pH is adjusted to 7.5, and 500U of low-temperature protease SapSh and fucylase Fun168B are added for enzymolysis treatment for 2 hours;

inactivating for 15min at 3.70 ℃, and centrifuging for 10min at 4000rpm to obtain a clear enzymolysis liquid.

4. The viscosity of the enzymolysis liquid is about 3 mPa.S under the condition of the shearing rate of 10-100 (1/S) and is close to that of pure water by the rheometer, and the viscosity of the prepared sea cucumber enzymolysis liquid by adopting the traditional enzymolysis technology is 840 mPa.S.

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

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

4. Adding 8% white granulated sugar into the enzymolysis liquid, and regulating the pH value to 5.5 by using citric acid.

5. Filling with an automatic filling machine, sealing, placing into a sterilizing kettle, sterilizing at 121 deg.C under 0.20MPa for 30min, and obtaining the sea cucumber nutritional oral liquid.

Example 8: preparation of sea cucumber enzymolysis liquid tablet

1. Removing viscera from living body, cleaning, taking 100kg of processed raw material, cutting into pieces, homogenizing to obtain meat emulsion, placing into 2000L enzymatic hydrolysis tank, adding 1500L water, and stirring;

2. the temperature of the solution in the enzymolysis tank is adjusted to 30 ℃, the pH is adjusted to 7.5, and 500U of low-temperature protease SapSh and fucylase Fun168B are added for stirring enzymolysis treatment for 2 hours;

3. heating the obtained enzymolysis liquid at 70 ℃ for 15min to perform enzyme deactivation treatment;

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

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

6. sieving the sea cucumber enzymolysis liquid dry powder 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 tablet pressing treatment to obtain an enzymatic hydrolysis liquid tablet with the tablet weight of 0.5 g.

Example 9: sea cucumber juice compound beverage

1. Removing viscera from living body, cleaning, taking 50kg of processed raw material, cutting into pieces, homogenizing to obtain meat emulsion, placing into 1000L enzymatic hydrolysis tank, adding 500L water, and stirring;

2. the temperature of the solution in the enzymolysis tank is adjusted to 25 ℃, the pH is adjusted to 7, and 100U of low-temperature protease SapSh and fucoidan Fun168B are added for stirring enzymolysis treatment for 1h;

3. heating the obtained enzymolysis liquid at 60 ℃ for 10min to perform enzyme deactivation treatment;

5. mixing the sea cucumber enzymolysis liquid with grape juice, lemon juice and grapefruit juice, homogenizing, performing UHT ultra-high temperature instantaneous sterilization, and aseptic canning to obtain the final beverage product.

Example 10: preparation of sea cucumber milk

5. heating raw milk as base material to 70deg.C, adding the above sea cucumber enzymolysis liquid, lemon juice and stabilizer, and concocting;

6. emulsifying and homogenizing the mixed solution at 65-70deg.C under 15-17MPa for the first stage and 1-3MPa for the second stage;

7. the UHT ultra-high temperature instantaneous sterilization is adopted for 135 ℃ for 4s, and the sea cucumber milk is prepared by aseptic canning after sterilization and cooling.

Finally, it should be noted that the above examples describe specific embodiments of the present invention, but are merely preferred embodiments of the present invention, and are not intended to limit the present invention in any way. It is to be understood by those skilled in the art that these are merely illustrative and that the scope of the invention is defined by the appended claims. All modifications and equivalents should be included within the scope of the invention.

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Sequence listing

<110> university of ocean in China

<120> a fucosidase and its application in complex enzymolysis of sea cucumber

<130> university of ocean in China

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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

20 25 30

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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Thr Ser Glu Leu Ala Ser Asp Val Ala Ser Gln Gln Ile Asn Thr Asn

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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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

355 360 365

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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Pro Asp Pro Ser Ile Thr Tyr Asn Leu Asn Gly Arg Thr Ile Ser Arg

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Val Arg Ile Arg Gln Asn Asp Thr Thr Lys Pro Leu Ala Leu Ala Glu

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Val Gln Val Phe Glu His Ser Val Asn Ser Ser Val Ser Gln Ser Gln

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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 fucosidase has an amino acid sequence of SEQ ID NO.1.

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

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

4. A sea cucumber complex enzymatic hydrolysis method using endo-1, 3-fucosidase and protease as defined in claim 1, characterized by: 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 compound enzymolysis liquid.

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

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

7. The method for complex enzymolysis of sea cucumber according to claim 4, wherein: and (3) centrifuging the obtained sea cucumber compound enzymatic hydrolysate at a low speed of 1000-4000rpm to obtain the enzymatic hydrolysate.

8. The method for complex enzymatic hydrolysis of sea cucumber according to any one of claims 4-7, characterized in that: decolorizing, removing fishy smell, concentrating, ultrafiltering, spray drying, and concocting.