Areca nut softening compound enzyme, preparation as well as preparation method and application of areca nut softening compound enzyme

Abstract

The invention discloses a betel nut softening compound enzyme, a preparation as well as a preparation method and application thereof, and belongs to the technical field of betel nut processing. The compound enzyme is prepared from 2 to 10 percent of cellulase, 5 to 15 percent of xylanase, 1 to 5 percent of lipase, 1 to 8 percent of pectinase, 1 to 5 percent of glucose oxidase, 1 to 5 percent of transglucosidase, 1 to 5 percent of laccase and 1 to 5 percent of alpha-L-arabinofuranosidase. The softening effect is enhanced through the synergistic effect of the laccase, the xylanase and the alpha-L-arabinofuranosidase, the flavor of the areca nuts is improved through the glucose oxidase and the transglucosidase, the softening efficiency of the areca nuts can be improved, and damage of the areca nuts to the oral cavity is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of areca nut processing, and more specifically, relates to an areca nut softening complex enzyme, a preparation thereof, and a preparation method and application thereof. Background Art

[0002] Areca nut processing requires fresh areca nuts to be made into dried fruits after being smoked and dried or air-dried, which are known as smoked areca nuts or green areca nuts in the industry. Due to low water content and dense fiber structure, long-term chewing is likely to wear teeth, stab oral mucosa, and cause oral lesions or even canceration. Therefore, during the processing, it is necessary to soften the areca nut fibers.

[0003] In the primary processing of traditional areca nut products, there are residues of harmful substances, and quicklime and ephedrine are added in subsequent processing, which makes traditional areca nut products have a strong stimulating effect on the oral cavity. At the same time, after the fibers of traditional areca nut products become lignified, they are hard, and long-term chewing will cause oral submucous fibrosis. In recent years, some enterprises have tried to use biological enzymes to process areca nuts, but all of them mainly use cellulase, hemicellulase, and pectinase, and achieve the softening effect by destroying the fiber structure of areca nuts, which will lead to fiber fragmentation, affecting the chewing experience and appearance integrity of the finished product. In addition, areca nuts also contain a large amount of tannins and bitter substances, resulting in a bitter taste during chewing. At present, there is no clear implementation solution in the areca nut processing industry. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides an areca nut softening complex enzyme preparation, a preparation method and application thereof, which enhance the softening effect through the synergistic effect of laccase, xylanase, and α-L-arabinofuranosidase, and improve the appearance and flavor of areca nuts with glucose oxidase and glucose transglucosidase.

[0005] The present invention provides an areca nut softening complex enzyme, which comprises: 2-10% of cellulase, 5-15% of xylanase, 1-5% of lipase, 1-8% of pectinase, 1-5% of glucose oxidase, and 1-5% of α-L-arabinofuranosidase.

[0006] Preferably, it further comprises: 1-5% of glucose transglucosidase and 1-5% of laccase.

[0007] Preferably, the dosage ratio of the glucose oxidase to the glucose transglucosidase is 1:1-2.

[0008] Preferably, the dosage ratio of the laccase, xylanase, and α-L-arabinofuranosidase is 1-2:2-3:1-2.

[0009] In this application, the α-L-arabinofuranosidase is the α-L-arabinofuranosidase reported in CN201911410865.4. Its amino acid sequence is shown in SEQ ID NO.1.

[0010] SEQ ID NO.1

[0011] MTVKQAKMTIDKEYKVGEVDKRLYGSFIEHLGRAVYEGIYEPDHPEADESGFRKDVIKLVKELKVPFIRYPGGNFVSGYNWEDGVGPVEKRPTRLDLAWATTEPNLVGTNEFMDWAKLVGAEVNMAVNLGTRGIDAARNLVEYCNHPSGSYYSDLRKSHGYKEPHKIKTWCLGNEMDGPWQIGHKTAAEYGRIAAEAAKVMKWTDPSIELVACGSSGSGMQTFIDWETTVLDHTYDHVEYISLHSYYGNRDNDLPNYLARSLDMDHFINTVVAVCDYMKAKKRSKKTIHLSYDEWNVWYHSNEKDKLVERWERAPHLLEDIYNFEDALLVGCMLITMLKHADRVKIACLAQLVNVIAPIMTEKGGEAWRQTIFYPFMHASVYGRGTALQTVVSSPKYDSKDFTDVPYLESVSVFNEEAEELTIFAVNRDTEGGLQIEADVRSFEGYAVSEHIVLEHEDNKATNEQDRNNVVPHSGGDAKVCDGRLTAHLPKLSWNVIRLKKR

[0012] Furthermore, the α-L-arabinofuranosidase is secreted by Bacillus subtilis, and the Bacillus subtilis contains or integrates the target gene encoding α-L-arabinofuranosidase.

[0013] The present invention also provides a betel nut softening complex enzyme preparation, comprising: the above-mentioned complex enzyme, and the balance is a pharmaceutically acceptable carrier.

[0014] In the preparation, the enzyme activity ranges of each enzyme are as follows: cellulase 500 - 20000 u / g, xylanase 2000 - 100000 u / g, lipase 100 - 20000 u / g, pectinase 50 - 5000 u / g, laccase 30 - 3000 u / g, α-L-arabinofuranosidase 300 - 30000 u / g, glucose oxidase 200 - 50000 u / g, glucose transglucosidase 30 - 8000 u / g.

[0015] Preferably, the carrier is cyclodextrin.

[0016] Preferably, the carrier is water, and it further comprises: 5-15% of NaCl, 5-20% of sorbitol, 0.1-1.5% of calcium propionate, and 15.5-79% of water.

[0017] The present invention also provides a preparation method of the above-mentioned compound enzyme preparation for betel nut softening (solid state). The biological enzyme concentrated solutions of cellulase, xylanase, lipase, pectinase, laccase, α-L-arabinofuranosidase, glucose oxidase, and glucosyltransferase are compounded according to corresponding proportions, and then the compounded concentrated solution is granulated by a fluidized bed dryer granulator with maltodextrin as the carrier to form a granular semi-finished product, and finally it becomes a solid-state betel nut enzyme finished product after processes such as sieving, mixing, testing, and packaging.

[0018] The present invention also provides a preparation method of the above-mentioned compound enzyme preparation for betel nut softening (liquid state). The concentrated solutions of various single enzymes such as cellulase, xylanase, lipase, pectinase, laccase, α-L-arabinofuranosidase, glucose oxidase, and glucosyltransferase are respectively formulated with NaCl, sorbitol, and calcium propionate according to corresponding proportions, filtered through a plate and frame fine filter, and then filtered and sterilized through a three-stage filter to form various single enzyme products, and finally the single enzyme products are compounded into a liquid finished product.

[0019] The present invention also provides the application of the above-mentioned compound enzyme for betel nut softening or the above-mentioned compound enzyme preparation for betel nut softening in betel nut processing.

[0020] α-L-arabinofuranosidase can hydrolyze saccharide compounds containing furanoside bonds, remove α-L-arabinose substituents in arabinoxylan, and play a key role in the biodegradation of hemicellulose and the bioconversion of lignocellulose. In betel nut processing, adding α-L-arabinofuranosidase and laccase can promote the degradation of xylan in hemicellulose, disintegrate the complex polysaccharide complexes such as cellulose, hemicellulose, and lignin in the betel nut shell, and destroy its network structure, thereby achieving the softening effect.

[0021] In this application, the α-L-arabinofuranosidase and glucosyltransferase with independent intellectual property rights can act on the saccharide substances in betel nuts together, improve the taste and flavor of betel nuts by changing their polysaccharide backbone structure and glycoside type, enhance the edible comfort of betel nuts, and meet the consumers' demand for taste.

[0022] Glucosyltransferase is a class of enzymes that can catalyze glycosidic bond transfer. It can transfer a sugar group from one aglycone to another aglycone or receptor molecule, thereby changing the original glycoside structure. In areca nut processing, glucosyltransferase can act on the carbohydrate substances in areca nut and improve the taste and flavor of areca nut by changing its glycoside structure. The areca nut treated with glucosyltransferase may have a softer and more delicate taste, reducing the original roughness and irritation of areca nut. At the same time, optimizing the carbohydrate structure of areca nut can also improve the stability and storage resistance of areca nut and extend its shelf life. Glucose oxidase can consume the dissolved oxygen in food, so it may help to extend the shelf life of areca nut and reduce the quality degradation caused by oxidation. Adding glucose oxidase and glucosyltransferase simultaneously can make the flavor of areca nut more rich, improve the taste, prevent browning and halide return phenomenon, and extend the shelf life.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) By using the bacterial-type neutral heat-resistant α-L-arabinofuranosidase, xylanase and laccase with independent intellectual property rights, the present invention greatly improves the softening effect of areca nut, reduces the abrasion of the oral cavity, and reduces the occurrence of oral diseases; at the same time, adding glucose oxidase and glucosyltransferase improves the flavor and taste of areca nut and extends the shelf life. The composite enzyme formula of the present invention can not only improve the softening effect and taste of areca nut, but also reduce the damage to the oral cavity. In addition, the composite enzyme preparation can also greatly improve the softening efficiency of areca nut and reduce the production cost of areca nut.

[0025] (2) The present invention adopts modern enzyme preparation processes to obtain high-quality composite enzyme solid and liquid products. For the solid enzyme preparation process, the high-concentration concentrates of various enzymes are first compounded, and then spray-dried and granulated simultaneously. The production efficiency is high, the uniformity is better, and there is no dust; for the liquid enzyme preparation process, various single enzymes are first made into products, and then compounded, and sterilized by three-stage filter. Its production is more flexible, there is no inventory backlog, the product is more stable, and only salt and calcium propionate are added as preservatives in the product, which is more green and safe. Description of the Drawings

[0026] Figure 1 It is the product appearance of a kind of areca nut softening composite enzyme (solid) preparation in the present invention.

[0027] Figure 2 It is the product appearance of a kind of areca nut softening composite enzyme (liquid) preparation in the present invention. Detailed Embodiments

[0028] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the spirit of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. The terms used in the specification of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. It should be noted that the reagents and the like used in this embodiment are all ordinary commercially available products.

[0030] Example 1

[0031] A compound enzyme preparation for softening betel nuts, wherein the compound enzyme is: cellulase 10%, xylanase 5%, lipase 3%, pectinase 6%, glucose oxidase 3%, glucose transglucosidase 3%, laccase 3%, α-L-arabinofuranosidase 3%; the balance is cyclodextrin.

[0032] Its preparation method is as follows:

[0033] The biological enzyme concentrates of cellulase, xylanase, lipase, pectinase, laccase, α-L-arabinofuranosidase, glucose oxidase, and glucose transglucosidase are compounded in corresponding proportions, and then the compounded concentrate is granulated by a boiling dry granulator using maltodextrin as a carrier to form a granular semi-finished product. Finally, after processes such as sieving, mixing, testing, and packaging, it becomes a solid-state betel nut enzyme preparation finished product.

[0034] Example 2

[0035] A compound enzyme preparation for softening betel nuts, wherein the compound enzyme is: cellulase 8%, xylanase 10%, lipase 5%, pectinase 8%, glucose oxidase 2%, glucose transglucosidase 4%, laccase 2%, α-L-arabinofuranosidase 4%; NaCl 10%, sorbitol 17%, calcium propionate 1%, and the balance is water.

[0036] Its preparation method is: The concentrated solutions of various single enzymes such as cellulase, xylanase, lipase, pectinase, laccase, α-L-arabinofuranosidase, glucose oxidase, and glucose transglucosidase are respectively formulated with NaCl, sorbitol, and calcium propionate in corresponding proportions, filtered through a plate and frame fine filter, and then filtered and sterilized through a three-stage filter to form various single enzyme products. Finally, the single enzyme products are compounded into a liquid finished product.

[0037] Example 3

[0038] A compound enzyme preparation for betel nut softening, wherein the compound enzyme is: 2% cellulase, 15% xylanase, 1% lipase, 3% pectinase, 5% glucose oxidase, 5% glucose transglucosidase, 5% laccase, 5% α-L-arabinofuranosidase; the balance is cyclodextrin.

[0039] Its preparation method is as follows:

[0040] The biological enzyme concentrates of cellulase, xylanase, lipase, pectinase, laccase, α-L-arabinofuranosidase, glucose oxidase and glucose transglucosidase are compounded according to the corresponding proportions, and then the compounded concentrate is granulated by a boiling dryer granulator with maltodextrin as the carrier to form a granular semi-finished product. Finally, after processes such as sieving, mixing, testing and packaging, it becomes the finished solid betel nut enzyme preparation.

[0041] Comparative Example 1

[0042] The difference from Example 1 is that the glucose oxidase is 6% and the glucose transglucosidase is not added.

[0043] Comparative Example 2

[0044] The difference from Example 1 is that the glucose transglucosidase is 6% and the glucose oxidase is not added.

[0045] Comparative Example 3

[0046] The difference from Example 1 is that the laccase is 11%, and the α-L-arabinofuranosidase and xylanase are not added.

[0047] Comparative Example 4

[0048] The difference from Example 1 is that the α-L-arabinofuranosidase is 11%, and the laccase and xylanase are not added.

[0049] Comparative Example 5

[0050] The difference from Example 1 is that the xylanase is 11%, and the α-L-arabinofuranosidase and laccase are not added.

[0051] Comparative Example 6

[0052] The difference from Example 1 is that ordinary commercially available α-L-arabinofuranosidase (manufacturer: Megazyme) is used

[0053] Product application and effect detection

[0054] I. Softening method

[0055] Weigh 0.3 - 0.7% of the compound enzyme preparation of the example or comparative example based on the weight of the betel nut dry fruit, add purified water which is 5 - 10 times the weight of the enzyme preparation at 40 - 50°C to make a mixed solution, and place it in a special seed - germinating tank. At the same time, add 0.08 - 0.15% of the auxiliary agent based on the weight of the betel nut dry fruit. Add the pretreated betel nuts and water to the special seed - germinating tank at a ratio of 3 - 6:1, mix them evenly with the enzyme preparation and the auxiliary agent, and adjust the pH value to 4.5 - 6.5. At 45 - 60°C and a pressure of 3 - 7 atm, germinate for 6 - 12 h.

[0056] II. Detection methods

[0057] 1. Hardness determination: Measured by a texture analyzer;

[0058] Take samples about 3 cm high in the middle section, stand them upright under the probe of the texture analyzer for TPA testing. Take 10 samples each time, and repeat the test for each sample 3 times.

[0059] 2. Total alkaloid content determination: Measured by acid - dye colorimetry;

[0060] Absorb 1 mL of the test solution, add 4 mL of pH 5.0 acetic acid - sodium acetate buffer solution and 2 mL of bromocresol green solution, and mix well. Add 2 mL, 3 mL, and 5 mL of chloroform successively for 3 extractions. Shake for 2 min each time, let it stand for 30 min, separate the chloroform layer, place it in a dry volumetric flask pre - placed with 0.2 g of anhydrous sodium sulfate, shake, let it stand for 30 min, absorb 5 mL of the supernatant, add 1 mL of 0.01 mol / L potassium hydroxide anhydrous ethanol solution, shake well, and measure the absorbance at a wavelength of 618 nm.

[0061] 3. Crude fiber content determination:

[0062] 1) Measure the weight of the fiber bag after drying and cooling, put 1 g of the sample into the fiber bag, and seal it.

[0063] 2) Put the fiber bag into a 600 - ml tall - stemmed beaker, add 0.128 mol / L sulfuric acid solution (add 100 ml for each fiber bag), press a heavy object on the fiber bag, cover it with a condenser ball, connect to tap water, heat the sulfuric acid solution to a gentle boil with an electric hot plate, requiring the solution to boil gently within 2 min, and treat for 30 min; spin - dry the treated fiber bag for 2 min, put it into a beaker of boiling water at 100°C for 3 min, and add a small amount of 0.313 mol / L sodium hydroxide to neutralize. Repeat 3 times until neutral (the blue litmus paper does not change color), and then spin - dry the fiber bag for 2 min.

[0064] 3) Then put the fiber bag into a 600 ml tall beaker, add 0.313 mol / L sodium hydroxide solution (100 ml for 1 fiber bag), press a heavy object on the fiber bag, cover it with a condenser ball, turn on the tap water, and heat it to a gentle boil with a hot plate. It is required that the solution boils gently within 2 minutes of heating, and process for 30 minutes; spin-dry the processed fiber bag for 2 minutes, put it into a tall beaker with boiling water at 100 °C for 3 minutes, and add a small amount of 0.128 mol / L sulfuric acid to neutralize. Repeat 3 times until neutral (the red litmus paper does not change color), and spin-dry the fiber bag for 2 minutes.

[0065] 4) Immerse the fiber bag in ether and ethanol respectively in a beaker for 10 minutes. After immersion, take it out, spin-dry the fiber bag for 5 minutes, and put it into a half-open oven to dry at 130 °C for 20 minutes.

[0066] 5) Put the fiber bag into a crucible and dry it in an oven at 130 °C for 2 hours until it reaches a constant weight, and record the weight.

[0067] 6) Put the empty crucible and the fiber bag together into a muffle furnace and carbonize at 300 °C until there is no smoke (the furnace door is slightly open), and burn at 550 °C for 3 hours until it reaches a constant weight, and record the weight.

[0068] 7) Calculate the crude fiber content:

[0069] CF% = [(weight of the sample bag after drying - weight of the empty bag) - (weight of the crucible after burning - weight of the empty crucible - weight of the empty bag × ash content percentage of the empty bag)] / weight of the sample × 100%

[0070] Crude fiber = (M1 - M2) / M × 100%

[0071] Among them, M1 is the weight of the porcelain crucible and the fiber bag after drying at 105 °C (unit: g); M2 is the weight of the porcelain crucible and the fiber bag after burning at 550 °C (unit: g); M is the weight of the sample (unit: g).

[0072] 4. Determination of moisture content: Use the direct drying method

[0073] Take a flat weighing bottle made of glass, place it in a drying oven at 103 °C, prop the bottle cap obliquely on the edge of the bottle, heat for 1.0 hour, take it out and cover it, place it in a desiccator to cool for 0.5 hour and then weigh it, and record the weight of the weighing bottle. Accurately weigh 2 g of the sample and put it into this weighing bottle. The thickness of the sample is about 5 mm. Cover it and weigh it precisely, and record the total weight of the weighing bottle and the sample. Then place the weighing bottle containing the sample in a drying oven at 103 °C, prop the bottle cap obliquely on the edge of the bottle, dry for 4 hours, cover it and take it out, place it in a desiccator to cool for 0.5 hour and then weigh it, and record the total weight of the weighing bottle and the sample. Then put it back into the drying oven at 103 °C to dry for 1 hour, take it out, place it in a desiccator to cool for 0.5 hour and then weigh it again, and record the total weight of the weighing bottle and the sample. Until the difference in mass between the two times does not exceed 0.002 g, it is considered to have reached a constant weight.

[0074] Calculation: X = (m2 - m3) / (m2 - m1) * 100%

[0075] Wherein, X is the moisture content in the sample (unit: %)

[0076] m1 is the weight of the weighing bottle and the sample (unit: g); m2 is the weight of the weighing bottle and the sample after drying (unit: g); m3 is the weight of the weighing bottle (unit: g).

[0077] Test the products prepared in Examples 1-3 and Comparative Groups 1-6 respectively according to the above steps, and the results are shown in Table 1

[0078] Table 1

[0079]

[0080] The results show that: the hardness, cellulose, and water content of Examples 1-3 are much smaller than those of Comparative Examples 1-6; it shows that the softening effect of the corresponding enzyme preparation formula in the examples is better. And the effects of Comparative Examples 1-3 are obviously lower than those of Comparative Examples 3-6. It can be seen that α-L-arabinofuranosidase, laccase, and xylanase in the present invention have obvious synergistic effects in degrading areca nut polysaccharides, can improve the softening effect, and can improve the fiber structure.

[0081] III. Sensory evaluation:

[0082] The sensory evaluation was carried out by 30 evaluators aged between 25 and 55 years old on the samples of the products prepared in Examples 1-2 and Comparative Groups 1-6 respectively. The evaluators judged and scored each evaluation index, and the score was any integer between 0 and 9, and filled the score in the blank in the questionnaire form.

[0083] The scoring criteria for each index are as follows: 1) Appearance: very dislike = 1, dislike very much = 2, dislike = 3, dislike a little = 4, average = 5, like a little = 6, like = 7, like very much = 8, like very much = 9; 2) Chewability: 9-7 means it disperses immediately in the mouth and is chewable, 5-6 means generally chewable, and less than 5 points means not chewable; 3) Mouth binding: 9-7 means not mouth binding, 5-6 means generally mouth binding, and less than 5 points means very mouth binding; 4) Flavor: 9-7 means the aroma is persistent and gentle, 5-6 means the aroma is average and slightly irritating, and less than 5 points means basically no aroma and irritating.

[0084] Finally, take the average score of each index. It is stipulated that the average score of 9 is excellent, 7-8 is good, 5-6 is general, and less than 5 is poor. The results are shown in Table 2

[0085] Table 2

[0086]

[0087]

[0088] The results show that the enzyme preparation formula in the examples has good softening effect and taste improvement. From Comparative Example 1 and Comparative Example 2, it can be seen that glucose oxidase and glucosyltransferase in the present invention have an obvious synergistic effect, which can improve the flavor of areca nut. From Comparative Examples 3 - 5, it is known that α-L-arabinofuranosidase, laccase and xylanase in the present invention produce a synergistic effect, which can improve the softening effect of areca nut. From Comparative Example 6, it can be seen that the α-L-arabinofuranosidase with independent intellectual property rights has a better effect than the common α-L-arabinofuranosidase.

[0089] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A betel nut softening complex enzyme, characterized in that: The complex enzyme comprises: 2-10% of cellulase, 5-15% of xylanase, 1-5% of lipase, 1-8% of pectinase, 1-5% of laccase and 1-5% of α-L-arabinofuranosidase.

2. The betel nut softening complex enzyme according to claim 1, characterized in that Also includes: Glucose oxidase 1-5%, glucose transaminase 1-5%.

3. The betel nut softening complex enzyme according to claim 2, characterized in that The usage ratio of the glucose oxidase to the glucose transaminase is 1:1-2.

4. The complex enzyme according to claim 1, characterized in that The dosage ratio of the laccase, xylanase and α-L-arabinofuranosidase is 1-2:2-3:1-2.

5. A betel nut softening complex enzyme preparation comprising: The complex enzyme according to any one of claims 1 to 4, and the remainder is a carrier acceptable in the food industry.

6. The betel nut softening complex enzyme preparation according to claim 5, characterized in that: The carrier is cyclodextrin.

7. The betel nut softening complex enzyme preparation according to claim 5, characterized in that: The carrier is water, and further comprises: 5-15% NaCl, 5-20% sorbitol, 0.1-1.5% calcium propionate, and 15.5-79% water.

8. The method for preparing the betel nut softening complex enzyme preparation according to claim 4, characterized in that: The biological enzyme concentrates of cellulase, xylanase, lipase, pectinase, laccase, α-L-arabinofuranosidase, glucose oxidase and glucose transaminase are compounded in corresponding proportions, and then the compounded concentrate is granulated with maltodextrin as a carrier through a boiling drying granulator to form a granular semi-finished product, and finally becomes a solid betel nut enzyme finished product after screening, mixing, testing, packaging and other processes.

9. The method for preparing the betel nut softening complex enzyme preparation according to claim 7, characterized in that: Various single enzyme concentrates such as cellulase, xylanase, lipase, pectinase, laccase, α-L-arabinofuranosidase, glucose oxidase, glucose transaminase, etc. are respectively prepared with NaCl, sorbitol, and calcium propionate in corresponding proportions, filtered through plate and frame, and then filtered and sterilized through a three-stage filter to form various single enzyme products, and finally the single enzyme products are compounded into liquid finished products.

10. Use of the betel nut softening complex enzyme according to any one of claims 1 to 4 or the betel nut softening complex enzyme preparation according to claim 5 in betel nut processing.

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