A method for efficiently liquefying porphyra by using a compound enzyme and its application

Abstract

The application belongs to the technical field of bioengineering and its application, and particularly relates to a high-efficiency liquefaction method of compound enzymolysis of Porphyra and application. After rehydration of Porphyra powder, proteinase, Porphyra polysaccharidase and mannanase are added for combined enzymolysis, so that the liquefaction rate of Porphyra is improved, and the obtained Porphyra enzymolysis liquid and enzymolysis residue can be used for Porphyra deep processing product development and application. On the basis of the enzymolysis of Porphyra by proteinase and Porphyra polysaccharidase, specific mannanase is added, and the enzymolysis conditions are optimized so as to achieve the highest Porphyra liquefaction rate. At the same time, the Porphyra polysaccharidase and mannanase enzyme preparations are obtained by using a food-grade lactic acid bacteria NICE expression system, and are applied to the food industry to develop edible Porphyra deep processing products.

Description

Technical fields:

[0001] This invention belongs to the field of bioengineering and its application technology, specifically relating to a high-efficiency liquefaction method for complex enzymatic hydrolysis of laver and its application. Background technology:

[0002] Laver (Porphyra) is a general term for seaweed belonging to the genus Porphyra in the family Bangiaceae, class Protoflorideae, phylum Rhodophyta. It is a common edible seaweed. Laver is a highly nutritious seaweed, high in protein and fiber, low in calories and fat, and is one of the most commercially valuable marine algae. Research shows that the laver products on the domestic market are currently limited, mainly consisting of laver cakes and nori. The development and utilization of laver is still in its early stages, with deep-processed products lagging behind. Utilizing low-value laver to achieve high-value utilization is one of the important directions for the future development of the laver industry. Enzymatic liquefaction technology has advantages such as simple operation, mild conditions, high yield, and strong product functionality, which are conducive to industrial production and are an important way to achieve high-value utilization of laver.

[0003] Porphyra polysaccharides comprise approximately 40% of the dry weight of laver and are the main water-soluble polysaccharide component. Found in the cell walls and intercellular spaces, they exhibit a linear structure composed of alternating (1-4)-O-α-L-galactopyranose-6-sulfate (L6S) and (1-3)-O-β-D-galactopyranose (G) groups. Porphyra polysaccharides have been proven to possess various physiological activities, including scavenging reactive oxygen species, inhibiting cancer cell growth, and lowering cholesterol. Porphyra polysaccharide enzymes can specifically degrade laver polysaccharides. Studies have shown that utilizing these enzymes to specifically degrade laver polysaccharides to obtain a laver enzymatic hydrolysate, which, after formulation, yields a laver-based refreshing beverage, solves the problem of high viscosity in existing laver beverage enzymatic hydrolysates and improves the efficiency of processes such as centrifugation, clarification, and filtration during beverage preparation, significantly increasing the laver recovery rate. Therefore, laver polysaccharide enzymes can play a crucial role in the research and development of laver products and in the high-value development and application of laver.

[0004] Experiments revealed that enzymatic hydrolysis of laver using only protease and laver polysaccharide enzymes still resulted in a large amount of insoluble substances in the hydrolysis products, most of which were mannan. Mannan is an insoluble polysaccharide found in laver and is a key factor restricting its liquefaction. Mannanase can degrade mannan into low-molecular-weight polysaccharides and oligosaccharides, making mannan soluble. Therefore, finding a mannanase with high activity and high yield is a crucial breakthrough for improving the liquefaction rate of laver.

[0005] Furthermore, current research on laver polysaccharide enzymes and mannanases has relied on heterologous expression in *E. coli* or *Pichia pastoris*. However, due to the lack of food safety concerns with *E. coli* expression systems and the common use of methanol as an inducer in *Pichia pastoris* expression systems, food-grade, safe laver polysaccharide enzymes and mannanases have not been obtained. Therefore, obtaining food-safe laver polysaccharide enzymes and mannanases remains a key technological challenge in the liquefaction processing of laver. Summary of the Invention:

[0006] The technical problems to be solved by this invention are: first, the enzymatic hydrolysis of laver using only protease and laver polysaccharide enzyme still contains a large amount of insoluble substances, most of which are mannan, and it is necessary to find a mannanase with high activity and high yield; second, the laver polysaccharide enzymes and mannanases currently studied are heterologous expressions of Escherichia coli or Pichia pastoris, which do not have food safety.

[0007] To address the aforementioned problems, this invention provides a highly efficient liquefaction method for laver using a complex enzymatic hydrolysis. Based on the enzymatic hydrolysis of laver using protease and laver polysaccharide enzymes, a specific mannanase is added, and the hydrolysis conditions are optimized to achieve the highest laver liquefaction rate. Simultaneously, this invention utilizes the food-grade lactic acid bacteria NICE expression system to obtain laver polysaccharide enzyme and mannanase preparations for application in the food industry, developing edible deep-processed laver products.

[0008] To achieve the above objectives, this invention provides a highly efficient liquefaction method for laver compound enzymatic hydrolysis. After rehydrating laver powder, protease, laver polysaccharide enzyme, and mannanase are added and combined for enzymatic hydrolysis to obtain a laver compound enzymatic hydrolysate. A seamless cloning method is used to construct a lactic acid bacteria NICE system, which is then used to heterologously express laver polysaccharide enzyme and mannanase. The protease effectively degrades the protein. Simultaneously, laver polysaccharides are released from the laver and dissolved in the enzymatic hydrolysate. The laver polysaccharide enzyme hydrolyzes the laver polysaccharide, while mannans are released from the laver but cannot dissolve in the hydrolysate. The mannanase then hydrolyzes the mannan, thereby increasing the laver liquefaction rate.

[0009] Specifically, the following steps are included:

[0010] (1) Weigh out the seaweed powder, put it into an enzymatic hydrolysis tank, add water, stir evenly and soak for 30-40 minutes;

[0011] (2) Maintain the temperature of the solution in the enzymatic hydrolysis tank at 25-50℃, adjust the pH to 7-9, and add protease, mannanase and laver polysaccharide enzyme for stirring and enzymatic hydrolysis.

[0012] (3) Heat the obtained enzyme hydrolysate to inactivate the enzyme;

[0013] (4) The enzyme hydrolysate after enzyme inactivation is centrifuged to obtain laver enzyme hydrolysate and enzyme hydrolysate residue.

[0014] In step (1), seaweed powder and water are added at a ratio of 1:20 to 1:30.

[0015] In step (2), the amount of neutral protease added is 1000–20000 U / g, the amount of mannanase added is 0.1–10 U / g, and the amount of laver polysaccharide enzyme added is 0.1–10 U / g, with enzymatic hydrolysis lasting 3–6 hours. Within the above parameter range, the protease, laver polysaccharide enzyme, and mannanase have high enzymatic hydrolysis activity, ensuring rapid enzymatic hydrolysis. Exceeding this range will reduce enzymatic hydrolysis efficiency and increase production costs.

[0016] The protease is a neutral protease with high enzymatic hydrolysis efficiency.

[0017] Step (3) Heat the seaweed hydrolysate in a 100℃ water bath for 5-10 minutes to inactivate the enzyme.

[0018] The centrifugation parameters for step (4) are 5000-10000 rpm for 10-15 min.

[0019] Previous experiments showed that the liquefaction rate of laver hydrolyzed with protease alone was less than 50%. The inventors screened for highly expressed laver polysaccharide enzymes and mannanases using big data genomics. By adding these two enzymes to the protease hydrolysis process, the liquefaction rate of laver was significantly improved. Based on this efficient liquefaction method using combined enzymatic hydrolysis of laver, the inventors developed and applied products from laver hydrolysate and hydrolysis residue, and developed deep-processed laver products. The laver hydrolysate can be used in the production of effervescent tablets and gummies, while the hydrolysis residue can be used in the production of biscuits, feed, and fertilizer, fully utilizing laver processing byproducts.

[0020] Furthermore, the method for heterologous expression of *Porphyra yezoensis* polysaccharide enzyme and mannanase using the NICE lactic acid bacteria system includes the following steps:

[0021] (1) PCR amplification: pexHFHSDNAPolymeraseMix: the volume ratio of the target gene template is 1:4-8, forward primer, reverse primer, and ddH2O is added to 50-150μL; strictly follow the above order to construct the PCR reaction system and perform amplification; use the NE.ZNA gel recovery kit to recover the target gene.

[0022] (2) Extraction of pNZ8149 empty vector plasmid: The plasmid was extracted using the Biomed high-purity plasmid small-volume rapid extraction kit, with a concentration of not less than 100 ng / mL. If the concentration is too low, it will affect the subsequent seamless cloning. Therefore, if the recovered concentration is insufficient, further enrichment and concentration are required.

[0023] (3) Vector linearization: Linearization was achieved by digestion with dual restriction endonucleases; the reaction system was added in strict order as described above: 10X Speedyone Buffer: the volume ratio of the vector was 1:4 to 10, 1 μL of each dual restriction endonuclease, and 20 to 50 μL of ddH2O was added; the vector was inactivated after incubation.

[0024] (4) Seamless cloning: The target gene was ligated to the linearized vector using the Ready-to-Use SeamLess Cloning Kit (CAT.NO.B632219) to construct the recombinant vector.

[0025] (5) Preparation of Lactococcus lactis NZ3900 competent cells and electroporation.

[0026] After 24–36 hours of growth, single colonies successfully grew on Ellike medium. These colonies were picked and verified as positive clones. Active food-grade laver polysaccharide enzyme and food-grade mannanase were obtained by inducing expression in these colonies.

[0027] Furthermore, the laver polysaccharide enzyme is laver polysaccharide enzyme Por16B_Wf, and its protein sequence is shown in SEQ ID NO.1.

[0028] SEQ ID NO.1:

[0029] QQSPTFIDGEDPKPDNTKWKLVKNMSDEFNGTKVDEEKWQISGQGWIGRAPGLFLADNVKVTNGSLQITTTMLPKPIIKNNKEFTHGGGYVGSRNGMTYGYYECEMKANKTFMSSTFWLINEGKNIKGCDKRTTEL DIQECVGQITNDAEWMKNFDQAMNSNTHSRNIPEGCNYIKGSEKSGATIGAKVYNDFHVYGVWWKSKDEILFFLDGKFQSKVKPPSDFDIEMYLRMVVETYDWNPVPADGGMAYSKEDRTTTYNWVRSWTLVNPKK

[0030] Furthermore, the mannanase is mannanase Man26 / 5_8a, and its protein sequence is shown in SEQ ID NO.2.

[0031] SEQ ID NO.2:

[0032] AISTQPVTKDATEGATLLYKFLYDHFKKNTISGVMCGDMDNGASYKTQVDVAYLYSIDGNKYPALVGVDLLNATGAQSDEAWFKTYTQSGISLAKELWKDGGIPSFNWHWKVGSENAFYAKGANDSYTDFDYTKGFKAGTTEWDTSSETYRLLIADIDHVADLFLELQASGVA AIWRPLHEASGGWFWWGTKGAKSYVALYRLVYDRMVNVKGVKNLIWVWNLEKDPTQGYAYNEAWYPGDQYIDVIGVDIYNGANNTQSNINTWNTIISKMGSDKILALTENGPIVDPAEAQKNGDIWSWWMPWYNSWSGGFIDQTSASLWKSAMSSDLIITLDEMPGWGTYQEV

[0033] Current research on mannanases primarily targets the degradation of soluble mannans, and the lack of food-grade mannanases prevents their application in the food industry. The mannanase in this invention is a gene-mined enzyme that degrades insoluble mannans in laver. This mannanase initiates mannan degradation by randomly cleaving the main chain, releasing short mannan oligomers, and generating new chain ends, thereby improving the liquefaction rate of laver.

[0034] The beneficial effects of this invention are:

[0035] (1) This invention describes a high-efficiency liquefaction method and application of laver compound enzymatic hydrolysis. Based on the enzymatic hydrolysis of laver by protease and laver polysaccharide enzyme, mannanase is used to degrade insoluble mannan, thereby increasing the liquefaction rate of laver to 80%.

[0036] (2) This invention utilizes laver polysaccharide enzyme and mannanase to enzymatically hydrolyze laver. The enzymatic hydrolysate contains low molecular weight polysaccharides and oligosaccharides that are more easily absorbed by the human body than large molecular weight polysaccharides, thereby enhancing its nutritional value.

[0037] (3) This invention utilizes the food-grade lactic acid bacteria NICE expression system to obtain consumer-approved laver polysaccharide enzymes and mannanases, and applies them to enzymatic hydrolysis of laver to develop new safe and edible laver products. Attached Figure Description

[0038] Figure 1 Image of pNZ8149-porphyria polysaccharide enzyme plasmid.

[0039] Figure 2 Image of pNZ8149-mannanase plasmid.

[0040] Figure 3 A schematic diagram showing the change in laver liquefaction rate with the amount of neutral protease added.

[0041] Figure 4 A schematic diagram showing the change in laver liquefaction rate with the amount of mannanase added.

[0042] Figure 5 A schematic diagram showing the change in laver liquefaction rate with the amount of laver polysaccharide enzyme added. Detailed implementation method:

[0043] To make the objectives, 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. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Example 1: A method for enzymatic hydrolysis of laver using a combination of protease, mannanase, and laver polysaccharide enzyme, comprising the following steps:

[0045] (1) Weigh 1 kg of seaweed powder, put it into a 50 L enzymatic hydrolysis tank, add 25 L of water, stir evenly and soak for 40 min to allow it to fully swell and rehydrate.

[0046] (2) Keep the temperature of the solution in the enzymatic hydrolysis tank at 40℃, adjust the pH to 7, add 15000U / g of neutral protease, 0.5U / g of mannanase and 0.5U / g of laver polysaccharide enzyme and stir for 4h for enzymatic hydrolysis.

[0047] (3) The obtained enzyme hydrolysate was heated at 100°C for 15 min to inactivate the enzyme.

[0048] (4) The enzyme hydrolysate after enzyme inactivation is centrifuged at 5000 r / min for 15 min to obtain the seaweed hydrolysate and the hydrolysate residue.

[0049] Example 2: A method for enzymatic hydrolysis of laver using a combination of protease, mannanase, and laver polysaccharide enzyme, comprising the following steps:

[0050] (1) Weigh 1 kg of seaweed powder, put it into a 50 L enzymatic hydrolysis tank, add 30 L of water, stir evenly and soak for 40 min to allow it to fully swell and rehydrate.

[0051] (2) Maintain the temperature of the solution in the enzymatic hydrolysis tank at 45℃, adjust the pH to 8, add 10000U / g of neutral protease, 0.6U / g of mannanase and 0.6U / g of laver polysaccharide enzyme and stir for 6 hours for enzymatic hydrolysis.

[0052] (3) The obtained enzyme hydrolysate was heated at 100°C for 15 min to inactivate the enzyme.

[0053] (4) The enzyme hydrolysate after enzyme inactivation is centrifuged at 5000 r / min for 15 min to obtain the seaweed hydrolysate and the hydrolysate residue.

[0054] Example 3: A method for enzymatic hydrolysis of laver using a combination of protease, mannanase, and laver polysaccharide enzyme, comprising the following steps:

[0055] (1) Weigh 1 kg of seaweed powder, put it into a 50 L enzymatic hydrolysis tank, add 20 L of water, stir evenly and soak for 40 min to allow it to fully swell and rehydrate.

[0056] (2) Keep the temperature of the solution in the enzymatic hydrolysis tank at 30℃, adjust the pH to 7, add 20000U / g of neutral protease, 0.3U / g of mannanase and 0.3U / g of laver polysaccharide enzyme and stir for 4 hours for enzymatic hydrolysis.

[0057] (3) The obtained enzyme hydrolysate was heated at 100°C for 15 min to inactivate the enzyme.

[0058] (4) The enzyme hydrolysate after enzyme inactivation is centrifuged at 5000 r / min for 15 min to obtain the seaweed hydrolysate and the hydrolysate residue.

[0059] Example 4: Study on the effects of the addition amounts of neutral protease, mannanase and laver polysaccharide enzyme on the liquefaction rate of laver.

[0060] (1) Accurately weigh 1g of laver powder and place it in a 100mL beaker. Before enzymatic hydrolysis, soak the laver powder in water for 40min, ensuring the laver content is 3%, allowing it to fully swell and rehydrate. Neutral protease was added at concentrations of 0, 2000, 5000, 10000, 15000, and 20000 U / g; mannan was added at 1U / g; and laver polysaccharide enzyme was added at 1U / g. Enzymatic hydrolysis was performed at 40℃ for 4h, and the resulting hydrolysate was then inactivated. The solution was then centrifuged at 5000rpm for 15min. The centrifuged residue was dried, weighed, and the laver liquefaction rate was determined to identify the optimal amount of neutral protease.

[0061] Depend on Figure 3It was observed that with the increase of neutral protease addition, the liquefaction rate of laver initially increased, then slowed down. This pattern of increasing liquefaction rate occurred between 15000-20000 U / g. When the enzyme in the reaction system reaches a certain amount, the enzyme-substrate ratio approaches the optimal binding state, and the reaction tends towards saturation. Further increasing the enzyme dosage does not help the enzymatic reaction. Therefore, controlling the enzyme dosage within a certain range can improve the enzyme efficiency during enzymatic hydrolysis. Considering both economic benefits and enzymatic hydrolysis efficiency, a neutral protease addition of 15000 U / g is preferred.

[0062] (2) Accurately weigh 1g of laver powder and place it in a 100mL beaker. Before enzymatic hydrolysis, soak the laver powder in water for 40min (laver content 3%) to allow it to fully swell and rehydrate. The neutral protease dosage is 15000U / g, the mannanase dosage is 0, 0.1, 0.5, 1.0, 1.5, and 2.0U / g, and the laver polysaccharide enzyme dosage is 1U / g. Hydrolyze at 40℃ for 4h, and then inactivate the enzymes in the hydrolysate. Then centrifuge at 5000rpm for 15min. Dry and weigh the centrifuged residue, determine the laver liquefaction rate, and identify the optimal amount of mannan.

[0063] Depend on Figure 4 It can be seen that with the increase of mannanase addition, the liquefaction rate of laver first increases and then slightly decreases. Controlling the enzyme dosage within a certain range can improve the enzyme efficiency during enzymatic hydrolysis. Considering both economic benefits and enzymatic hydrolysis efficiency, a mannanase addition dosage of 0.5 U / g is preferred.

[0064] (3) Accurately weigh 1g of laver powder and place it in a 100mL beaker. Before enzymatic hydrolysis, soak the laver powder in water for 40min, ensuring the laver content is 3%, allowing it to fully swell and rehydrate. The dosage of neutral protease is 15000U / g, the dosage of mannanase is 1.0U / g, and the dosage of laver polysaccharide enzyme is 0, 0.1, 0.5, 1.0, 1.5, and 2.0U / g. Enzymatic hydrolysis is performed at 40℃ for 4h, and the resulting hydrolysate is then inactivated. The solution is then centrifuged at 5000rpm for 15min. The residue after centrifugation is dried and weighed, and the laver liquefaction rate is determined to ascertain the optimal dosage of laver polysaccharide enzyme.

[0065] Depend on Figure 5 It was found that with the increase of the amount of laver polysaccharide enzyme added, the liquefaction rate of laver initially increased rapidly, then the increase slowed down. This change in the rate of increase of laver liquefaction rate occurred between 0.1-0.5 U / g. Therefore, controlling the amount of enzyme added within a certain range can improve the enzyme's efficiency during enzymatic hydrolysis. Considering both economic benefits and enzymatic hydrolysis efficiency, a laver polysaccharide enzyme addition of 0.5 U / g is preferred.

[0066] Example 5: Calculation of Laver Liquefaction Rate under Optimal Conditions

[0067] Based on the quality assessment of laver before and after enzymatic hydrolysis, the extraction efficiency of laver contents by the water and enzymatic hydrolysis process is measured, i.e., the laver liquefaction rate. The laver enzymatic hydrolysate is centrifuged, the resulting precipitate is dried and weighed, and the weight of the dried laver powder is calculated by subtracting the precipitate from the dry laver powder. This value is defined as the laver liquefaction rate, used to measure the degree of enzymatic hydrolysis of laver.

[0068]

[0069] m1: Weight of dried laver powder; m2: Weight of sediment dried.

[0070] (1) Weigh 1 kg of seaweed powder, put it into a 50 L enzymatic hydrolysis tank, add 30 L of water, stir evenly and soak for 40 min to allow it to fully swell and rehydrate.

[0071] (2) Keep the temperature of the solution in the enzymatic hydrolysis tank at 45℃, adjust the pH to 7, add 15000U / g of neutral protease, 0.50U / g of mannanase and 0.50U / g of laver polysaccharide enzyme and stir for 4 hours for enzymatic hydrolysis.

[0072] (3) The obtained enzyme hydrolysate was heated at 100°C for 15 min to inactivate the enzyme.

[0073] (4) The enzyme hydrolysate after enzyme inactivation is centrifuged at 5000 r / min for 15 min to obtain the seaweed hydrolysate and the hydrolysate residue. Under these conditions, the seaweed liquefaction rate is 82%.

[0074] Example 6: Heterologous expression of *Porphyra yezoensis* polysaccharide enzymes using the NICE lactic acid bacteria system

[0075] (1) PCR amplification: pexHFHSDNAPolymeraseMix: the volume ratio of the target gene template was 1:4-5, 1 μL (10 pM) of forward primer, 1 μL (10 pM) of reverse primer, and ddH2O was added to 50 μL; the PCR reaction system was constructed strictly according to the above order, and the PCR conditions were: 95℃ for 5 min; 34 cycles: 95℃ for 0.5 min, 67℃ for 5 s, 72℃ for 1 min; 72℃ for 10 min, and incubated at 4℃ for amplification. The target gene was recovered using the NE.ZNA gel extraction kit.

[0076] Upstream primer:

[0077] GAGGCACTCACCATGGCGCAACAGTCACCAACTTTTATTGATGG

[0078] Downstream primer:

[0079] GCTCTCTAGAACTAGTTTTATTTTTTAGGATTTACCAATGTCCAAGACCTAACC

[0080] (2) Extraction of pNZ8149 empty vector plasmid: Plasmid was extracted using the Biomed high-purity plasmid small-volume rapid extraction kit.

[0081] (3) Vector linearization: Linearization was performed by double digestion with NcoI / XbaI; the reaction system was added in strict order as described above: 10X Speedyone Buffer: the volume ratio of the vector was 1:4-5, NcoI 1 μL, XbaI 1 μL, and ddH2O 20 μL was added; the reaction conditions were incubation at 37℃ for 15 min and inactivation at 80℃ for 20 min.

[0082] (4) Seamless cloning: The target gene was ligated to the linearized vector using the Ready-to-Use SeamLess Cloning Kit (CAT.NO.B632219) to construct the recombinant vector. The sample was mixed and incubated at 50°C for 20 min, then placed on ice for 2 min to obtain the seamless cloning product, which was stored at -20°C.

[0083] (5) Preparation of Lactococcus lactis NZ3900 competent cells and electroporation.

[0084] After 24–36 hours of growth, single colonies successfully grew on Ellike medium. These colonies were picked and verified as positive clones. Active food-grade laver polysaccharide enzymes were obtained by inducing expression of these colonies in M17 medium.

[0085] Example 7: Heterologous expression of mannanase using the NICE lactic acid bacteria system

[0086] (1) PCR amplification: pexHFHSDNAPolymeraseMix: the volume ratio of the target gene template was 1:4-5, with 1 μL (10 pM) of forward primer and 1 μL (10 pM) of reverse primer, and ddH2O added to 50 μL; the PCR reaction system was constructed strictly according to the above order, and the PCR conditions were: 95℃ for 5 min; 34 cycles: 95℃ for 0.5 min, 65℃ for 5 s, 72℃ for 1 min; 72℃ for 10 min, and incubated at 4℃ for amplification. The target gene was recovered using the NE.ZNA gel extraction kit.

[0087] Upstream primer:

[0088] GCAGGCATGCGGTACCGGGCGATTTCCACCCAAC

[0089] Downstream primer:

[0090] GCTCTCTAGAACTAGTCACCTCTTGGTAAGTGCCCCA

[0091] (2) Extraction of pNZ8149 empty vector plasmid: Plasmid was extracted using the Biomed high-purity plasmid small-volume rapid extraction kit.

[0092] (3) Vector linearization: Linearization was performed by double digestion with BanI / SpeI; the reaction system was added in strict order as described above: 10X Speedyone Buffer: the volume ratio of the vector was 1:4-5, BanI 1 μL, SpeI 1 μL, and ddH2O 20 μL was added; the reaction conditions were incubation at 37℃ for 15 min and inactivation at 80℃ for 20 min.

[0093] (4) Seamless cloning: The target gene was ligated to the linearized vector using the Ready-to-Use SeamLess Cloning Kit (CAT.NO.B632219) to construct the recombinant vector. The sample was mixed and incubated at 50°C for 20 min, then placed on ice for 2 min to obtain the seamless cloning product, which was stored at -20°C.

[0094] (5) Preparation of Lactococcus lactis NZ3900 competent cells and electroporation.

[0095] After 24–36 hours of growth, single colonies successfully grew on Ellike medium. These colonies were picked and verified as positive clones. Active food-grade mannanase was obtained by inducing expression of these colonies in M17 medium.

[0096] Example 8: Application of the heterologous expression of porphyra polysaccharide enzyme and mannanase from the lactic acid bacteria NICE system in Examples 6 and 7 in the preparation of porphyra enzymatic hydrolysate effervescent tablets.

[0097] (1) Preparation of seaweed enzymatic hydrolysate

[0098] Weigh 1 kg of laver powder and place it in a 50 L enzymatic hydrolysis tank. Add 25 L of water, stir and soak for 40 min to allow it to fully swell and rehydrate. Maintain the solution temperature in the enzymatic hydrolysis tank at 40 °C and adjust the pH to 7. Add 15000 U / g of neutral protease, 0.5 U / g of mannanase and 0.5 U / g of laver polysaccharide enzyme and stir for 4 h of enzymatic hydrolysis. Heat the resulting hydrolysate at 100 °C for 15 min to inactivate the enzyme. Centrifuge the hydrolysate at 5000 r / min for 15 min. Freeze-dry and pulverize the supernatant of the centrifuged laver hydrolysate to obtain laver enzymatic hydrolysate powder.

[0099] (2) Formulation of seaweed enzymatic hydrolysate effervescent tablets (by weight): 48 parts seaweed enzymatic hydrolysate, 35 parts disintegrant citric acid: sodium bicarbonate (0.8:1), 10 parts filler mannitol: lactose (1:1), 2.5 parts lubricant PEG6000 by weight, 1.5 parts sweetener aspartame, 1 part flavoring agent, and an appropriate amount of 2% PVP ethanol solution as binder.

[0100] (3) Preparation method of seaweed enzymatic hydrolysate effervescent tablets

[0101] The specific process for preparing laver enzymatic hydrolysate effervescent tablets using a separate acid-base wet granulation method is as follows:

[0102] ① Mix the freeze-dried powder of seaweed enzymatic hydrolysate, citric acid, lactose and mannitol evenly, add 2% PVP ethanol solution to prepare soft material, obtain wet granules through a 20-mesh sieve, and dry granules at 50℃ to obtain dry acid granules.

[0103] ② Mix sodium bicarbonate, lactose and mannitol evenly, add 2% PVP ethanol solution to prepare soft material, obtain wet granules through a 20-mesh sieve, and dry granules at 50℃ to obtain dry alkaline granules.

[0104] ③ The dry granules of acid, granules of alkali, PEG6000 and aspartame were mixed together and compressed into effervescent tablets with a specification of 2.0g / tablet using a tablet press. Finally, the quality was tested.

[0105] Example 9: Application of the heterologous expression of porphyra polysaccharide enzyme and mannanase in the lactic acid bacteria NICE system of Examples 6 and 7 in the preparation of porphyra enzymatic hydrolysate soft candy.

[0106] (1) Preparation of supernatant from seaweed enzymatic hydrolysate

[0107] Weigh 1 kg of laver powder and place it in a 50 L enzymatic hydrolysis tank. Add 25 L of water, stir and soak for 40 min to allow it to fully swell and rehydrate. Maintain the solution temperature in the enzymatic hydrolysis tank at 40 °C and adjust the pH to 7. Add 15000 U / g of neutral protease, 0.5 U / g of mannanase and 0.5 U / g of laver polysaccharide enzyme and stir for 4 h of enzymatic hydrolysis. Heat the resulting hydrolysate at 100 °C for 15 min to inactivate the enzyme. Centrifuge the hydrolysate at 5000 r / min for 15 min to obtain the laver enzymatic hydrolysate supernatant.

[0108] (2) Laver hydrolysate soft candy recipe (by weight): 20 parts water, 45 parts glucose syrup, 55 parts granulated sugar, 15 parts gelatin, 2 parts citric acid, and 50 parts laver hydrolysate.

[0109] (3) Preparation method of seaweed enzymatic hydrolysate soft candy

[0110] ① Heat water, glucose syrup, and granulated sugar to 125℃.

[0111] ② Remove from heat and cool to 80℃, then add gelatin and mix well.

[0112] ③ Add citric acid and seaweed enzymatic hydrolysate, let stand to defoam, and then pour into molds to form the shape.

[0113] Example 10: Application of the heterologous expression of porphyra polysaccharide enzyme and mannanase in the lactic acid bacteria NICE system of Examples 6 and 7 in the preparation of cookies.

[0114] (1) Preparation of seaweed insoluble dietary fiber powder

[0115] Weigh 1 kg of seaweed powder and place it in a 50 L enzymatic hydrolysis tank. Add 25 L of water, stir and soak for 40 min to allow it to fully swell and rehydrate. Maintain the solution temperature in the enzymatic hydrolysis tank at 40 °C and adjust the pH to 7. Add 15000 U / g of neutral protease, 0.5 U / g of mannanase, and 0.5 U / g of seaweed polysaccharide enzyme and stir for 4 h of enzymatic hydrolysis. Heat the resulting hydrolysate at 100 °C for 15 min to inactivate the enzyme. Centrifuge the hydrolysate at 5000 r / min for 15 min. Dry and pulverize the centrifuged seaweed residue to obtain seaweed enzymatic hydrolysate residue powder, also known as seaweed insoluble dietary fiber powder.

[0116] (2) Recipe for seaweed insoluble dietary fiber cookies (by weight): 110 parts low-gluten flour, 80 parts butter, 40 parts powdered sugar, 30 parts egg liquid, and 2.5 parts seaweed insoluble dietary fiber powder (1% added).

[0117] (3) Preparation method of seaweed insoluble dietary fiber cookies: ① Weigh the ingredients. Accurately weigh all the ingredients. ② Mix evenly. Cut the butter into small pieces, place it in a container and soften it at room temperature until it is semi-melted. Add the powdered sugar and beat until the butter turns white and its volume increases significantly. Add the whole egg liquid that has returned to room temperature in two batches, and mix well after each addition. Sift in the weighed and well-mixed low-gluten flour and seaweed insoluble dietary fiber powder, and mix evenly by cutting and folding. ③ Pipe the cookies. ④ Bake. Bake at 170℃ for 10 minutes to set the shape, then bake at 150℃ for 10-15 minutes until cooked and fragrant. ⑤ Cool and finish. Remove the cookies from the oven and cool to room temperature.

[0118] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for efficient liquefaction of laver through complex enzymatic hydrolysis, characterized in that: After rehydrating the seaweed powder, protease, seaweed polysaccharide enzyme, and mannanase were added and combined for enzymatic hydrolysis to obtain a seaweed complex enzymatic hydrolysate and enzymatic hydrolysis residue. The mannanase was mannanase Man26 / 5_8a, and its protein sequence is shown in SEQ ID NO.

2. Among them, a lactic acid bacteria NICE system was constructed using a seamless cloning method, and the seaweed polysaccharide enzyme and mannanase were heterologously expressed using the lactic acid bacteria NICE system.

2. The high-efficiency liquefaction method for laver compound enzymatic hydrolysis as described in claim 1, characterized in that... Includes the following steps: (1) Weigh out the seaweed powder, put it into an enzymatic hydrolysis tank, add water, stir evenly and soak for 30-40 minutes; (2) Maintain the temperature of the solution in the enzymatic hydrolysis tank at 25-50℃, adjust the pH to 7-9, and add neutral protease, laver polysaccharide enzyme and mannanase for stirring and enzymatic hydrolysis. (3) Heat the obtained enzyme hydrolysate to inactivate the enzyme; (4) The enzyme hydrolysate after enzyme inactivation is centrifuged to obtain laver enzyme hydrolysate and enzyme hydrolysate residue.

3. The high-efficiency liquefaction method for laver compound enzymatic hydrolysis as described in claim 2, characterized in that: In step (2), the amount of neutral protease added is 1000-20000 U / g, the amount of mannanase added is 0.1-10 U / g, the amount of laver polysaccharide enzyme added is 0.1-10 U / g, and the enzymatic hydrolysis is carried out for 3-6 hours.

4. The high-efficiency liquefaction method for laver compound enzymatic hydrolysis as described in claim 2, characterized in that: Step (3) Heat the seaweed enzymatic hydrolysate in a 100℃ water bath for 5-10 minutes to inactivate the enzyme.

5. The high-efficiency liquefaction method for laver compound enzymatic hydrolysis as described in claim 2, characterized in that: After enzyme inactivation in step (4), centrifuge at 5000-10000 rpm for 10-15 min to obtain seaweed hydrolysate and hydrolysate residue.

6. The high-efficiency liquefaction method for laver compound enzymatic hydrolysis as described in claim 1, characterized in that... A method for heterologous expression of *Porphyra yezoensis* polysaccharide enzyme and mannanase using the NICE system of lactic acid bacteria includes the following steps: (1) PCR amplification: The PCR reaction system was constructed by strictly following the order of addition: pexHF HSDNA Polymerase Mix: the volume ratio of the target gene template was 1:4-8, forward primer, reverse primer, and dd H2O was added to 50-150 μL; amplification was performed; the target gene was recovered using the NE.ZNA gel recovery kit. (2) Extraction of pNZ8149 empty vector plasmid: The plasmid was extracted using the Biomed high-purity plasmid small-volume rapid extraction kit, with a concentration of not less than 100 ng / mL; if the concentration is too low, it will affect the subsequent seamless cloning, so if the recovered concentration is insufficient, further enrichment and concentration are required. (3) Vector linearization: Linearization was achieved by digestion with dual restriction endonucleases; the reaction system was added in the following order: 10X Speedyone Buffer: the volume ratio of the vector was 1:4 to 10, 1 to 2 μL of each dual restriction endonuclease, and 20 to 50 μL of ddH2O was added; the enzyme was inactivated after incubation. (4) Seamless cloning: The target gene was ligated to the linearized vector using the Ready-to-Use SeamLess Cloning Kit: CAT .NO .B632219 to construct a recombinant vector; (5) Preparation of Lactococcus lactis NZ3900 competent cells and electroporation.

7. The high-efficiency liquefaction method for laver compound enzymatic hydrolysis as described in claim 1 or 6, characterized in that: The laver polysaccharide enzyme is laver polysaccharide enzyme Por16B_Wf, and its protein sequence is shown in SEQ ID NO.

1.

8. The application of the laver compound enzymatic hydrolysate and enzymatic hydrolysate obtained as claimed in claim 1 in the production of effervescent tablets, soft candies, biscuits, feed and fertilizer.

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