CN115956646A - Method for enhancing permeability of fruit peel by biological enzyme method treatment - Google Patents
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Abstract
The invention discloses a method for enhancing the permeability of fruit peel by using a biological enzyme method, belonging to the technical field of enzyme engineering. The invention aims to develop a method for enhancing the permeability of fruit peel under mild reaction conditions, which is energy-saving and environment-friendly, adopts a single or mixed enzyme preparation which is efficient and stable and can degrade the components of cuticle, such as cutin, cellulose, pectin and the like, the reaction conditions are mild, the permeability of the fruit peel can be improved by more than two times, the method is beneficial to more effectively conveying substances such as seasonings, sweeteners, preservatives, stabilizers and the like to the interior of the fruit, and compared with physical treatment methods such as pinhole cutting and the like and chemical treatment methods of soaking with acid, alkali or organic reagents and the like, the biological enzyme treatment ensures the complete retention of fruit juice, and is more energy-saving and environment-friendly. The used biological enzyme liquid can still have better treatment effect after being recycled, and the problem of high cost of the enzyme preparation is solved.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for enhancing the permeability of fruit peel by biological enzyme treatment.
Background
With the continuous improvement of the living standard and the cultural quality of people, the requirements of people on food safety, health, nutrition and flavor are higher and higher. Nowadays, the 'low-sugar preserved fruit' gradually becomes the mainstream of preserved fruit, and on the basis, the preserved fruit must be developed towards the directions of nutrition, flavor and variety to adapt to the continuous change of the dietary structure of people.
In the production and processing of preserved fruits, the solid yield and the product quality of the preserved fruits are usually improved by adding external nutrient solution such as sucrose, fructose, trehalose, sodium chloride, vitamins, organic acids and the like or additives such as a sweetening agent, a preservative, a color fixative, an antioxidant and the like in a soaking pretreatment mode. The surfaces of peel tissues and cell walls of some fruits, which are in contact with the outside, are thick and are covered with a cuticle membrane to form a cuticle, the cuticle contains a plurality of mutually embedded and intertwined water-insoluble components such as cutin, pectin, cellulose and the like, the function of protecting the cell walls is realized, the evaporation of liquid in the fruits is greatly reduced, and meanwhile, the lack of remarkable water permeability on the peel can hinder the permeation and transportation of natural substances or artificially synthesized substances to the inside of the fruits, so that a great deal of time is spent in the production process, and the application of the fruits in the production of preserved fruits is hindered.
The common treatment method aiming at the problems comprises some physical methods, such as a pinhole method, which is realized by destroying the structure of fruit peel, and the reaction process is too violent, so that the loss of fruit juice is easily caused, and the product quality is influenced. As another example, the microwave method, the process has high requirements on the control of microwave power and osmotic temperature, otherwise the high-temperature thermal effect of the microwave can cause irreversible damage to fruit histiocyte. In addition, some chemical treatments, such as adding chemical agents, including sodium hydroxide, ethanol, propylene glycol, surfactants, etc., can enhance the permeability of the fruit peel, however, traces of the used chemical agents are finally retained in the product, and the subsequent intake of the chemical agents can cause potential harmful side effects to human bodies, and the subsequent treatment of waste can cause environmental pollution. Therefore, it is necessary to develop a method for enhancing the permeability of fruit peel with mild reaction conditions, energy conservation and environmental protection.
Disclosure of Invention
In order to solve the problems, the invention provides a method for enhancing the permeability of fruit peel by using a biological enzyme method, which is energy-saving, environment-friendly, mild in reaction process, simple and controllable, and the treated fruit has higher peel permeability, so that the requirement of subsequently processing the fruit peel into higher-quality preserved fruit can be met, and the processing time can be greatly shortened. The specific technical scheme of the invention is as follows:
it is a first object of the present invention to provide a method for enhancing the permeability of fruit peel by a bio-enzymatic treatment by soaking the fruit in a volume of an enzyme solution capable of degrading various cuticle components for a period of time, said enzyme solution comprising any one or more of cutinase, pectinase, cellulase. This method is easier to deliver natural or synthetic substances to the interior of fruits having a pericarp cuticle than untreated fruits.
In one embodiment of the invention, the fruit is a fruit having a pericarp cuticle, including but not limited to cherry tomato, grape, prune, plum.
In one embodiment of the invention, the pericarp cuticle component is any one or a combination of two or more of cutin, cellulose, pectin, triglyceride, and wax ester.
In one embodiment of the present invention, the cutinase is any one of Humicola insolens-derived cutinase, thermobifidafa-derived cutinase mutant D204C/E253C and Thermobifidafa-derived cutinase mutant Q92G/H184S/D204C/F209I/I213K/E253C.
In one embodiment of the invention, the enzyme solution may be simultaneously added with other natural or synthetic substances to achieve combined soaking.
In one embodiment of the invention, the natural substance is a substance that is inherently present in nature, including sucrose, vitamins, sodium chloride, organic acids, and the like.
In one embodiment of the invention, the synthetic material is a natural substance not belonging to the fruit, but which is delivered to the interior of the fruit to have a beneficial effect, including sweeteners, preservatives, color fixatives, antioxidants, and the like.
In one embodiment of the present invention, the volume of the enzyme solution is 1.5 to 10mL/g of the starting material, and when cutinase is contained in the enzyme solution, the concentration of the cutinase is not less than 0.01g/L.
In one embodiment of the invention, the soaking time is 1-48 h, the temperature is controlled to be 25-37 ℃, and the pH value is 5.0-8.0.
Preferably, the soaking temperature is controlled to be 30 ℃, and the pH value of the solution in the soaking process is 6.5-8.0.
The second purpose of the invention is to provide the application of the method in processing dehydrated fruits and preserved fruits.
The invention has the beneficial effects that:
the invention adopts single or mixed enzyme preparation which is efficient and stable and can degrade the components of the cuticle, such as cutin, cellulose, pectin and the like, to degrade the cuticle, the cellulose, the pectin and the like of the fruit peel cuticle, the reaction condition is mild, the peel permeability can be improved by more than two times, the seasoning, the sweetener, the preservative, the stabilizer and other substances can be more effectively conveyed into the fruit, and compared with physical treatment methods such as pinhole cutting and the like and chemical treatment methods such as acid-base or organic reagent soaking, the biological enzyme treatment ensures the complete retention of fruit juice, and the invention is more energy-saving and environment-friendly. The used biological enzyme liquid can still have better treatment effect after being recycled, and the problem of high cost of the enzyme preparation is solved.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as specifically described herein, and it will be appreciated by those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the present invention and that the present invention is not limited by the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
In the following examples, a batch of cherry tomatoes was purchased from a local market, of millennium variety, similar in color and shape, uniform in weight, and about 10g in weight per fruit. The processing technology comprises the steps of soaking cherry tomatoes in an enzyme solution for 0-48 h, controlling the temperature of enzyme soaking to be 25-37 ℃, controlling the pH value of the enzyme soaking to be 5.0-8.0, then taking out the cherry tomatoes, cleaning the enzyme solution, wiping, weighing, then soaking in 20% sugar solution for 24h, cleaning, wiping, weighing and calculating the solid content (%) of the cherry tomatoes after soaking. The soaking proportion is 5mL of enzyme solution/g of cherry tomatoes, and 200mM of phosphate buffer solution is used as a solvent of the enzyme solution for constant volume. 0.1% sodium metabisulfite was added to both the enzyme and sugar soaking solutions. The recommended addition amount of the cellulase and the pectinase is 0.01-5% of the mass of the raw materials. The 20% sugar solution and 0.1% sodium metabisulfite are in mass percent. The solid content (%) is the percentage of dry matter obtained by drying cherry tomatoes to a constant weight at 105 ℃, and represents the permeability of the peel, the higher the solid content, the stronger the permeability. Each example was replicated in triplicate, and a blank was set for each group.
The cellulase (liquid) and pectinase (powder) selected by the invention are produced by Xia Cheng Biotechnology (Beijing) Co., ltd.
Example 1: effect of different enzymatic regimens on cherry tomato pericarp Permeability
One cherry tomato was soaked in a solution containing one or more of Humicola Insolens-derived cutinase (PDB accession No. 4 OYY), cellulase and pectinase in a volume of 50mL, wherein the cutinase concentration was 0.1g/L, the cellulase concentration was 0.1% (v/v), and the pectinase concentration was 0.01% (m/v).
The control conditions are as follows: 30 ℃, pH =6.5, treatment 24h. Then soaking the mixture in 20% sucrose solution at 30 ℃ for 24h, and weighing. And finally, drying the cherry tomatoes to constant weight by using an air-blast drying oven, and calculating the solid content. And judging the influence of the enzyme treatment on the permeability of the cherry tomato peel according to the calculated solid content of the cherry tomato. As shown in table 1, the addition of one or two or more of cutinase, cellulase and pectinase to the solution increased the pericarp permeability of cherry tomato, and in particular, the mixture of cutinase, pectinase and cellulase was used to increase the solid content (pericarp permeability) by about one time.
TABLE 1 Effect of enzyme treatment with different enzyme addition protocols on cherry tomato pericarp Permeability
Example 2: effect of different sources and different kinds of cutinases on Permeability of cherry tomato pericarp
One cherry tomato was soaked in 50mL of a solution containing 0.1g/L cutinase, 0.1% (v/v) cellulase and 0.01% (m/v) pectinase from various sources and species. Controlling conditions: 30 ℃, pH =6.5, treatment 24h. Then soaking the mixture in 20% sucrose solution at 30 ℃ for 24h, and weighing. And finally, drying the cherry tomatoes to constant weight by using an air-blast drying oven, and calculating the solid content. And judging the influence of the enzyme treatment on the permeability of the cherry tomato peel according to the calculated solid content of the cherry tomato. As shown in Table 2, the addition of Humicola insolens-derived cutinase (PDB accession No. 4 OYY) in solution increased the solid content (peel permeability) of cherry tomato nearly twice, the addition of Zhu Fangjian et al reported that the effect of increasing peel permeability of Thermobifidafa-derived cutinase (NCBI accession No. AAZ 54921) in genomics and applied biology (2021,40 (02): 678-685.) was slightly lower than that of Humicola insolens-derived cutinase, which was substantially identical to that of Thermobifidafa-derived cutinase, while the addition of Chen Xiaoqian et al reported that the effect of Thermobifidafa-derived cutinase on fruit Materials (3924 zxft 3534) in 2022 month almost did not produce the increase of peel permeability of NCb β K3534/E253/K (NCH 3534/F) in Journal of Hamilto Materials (3924 zxft 3534).
TABLE 2 Effect of adding different sources of different types of cutinases on Permeability of cherry tomato pericarp
Example 3: effect of different pH conditions on cherry tomato pericarp Permeability
One cherry tomato was soaked in a solution containing 0.1g/LHumicola Insolens-derived cutinase (PDB accession No.: 4 OYY), 0.1% (v/v) cellulase, and 0.01% (m/v) pectinase, and the volume of the solution was 50mL. The control conditions are as follows: and (3) treating for 24 hours at the temperature of 30 ℃ and the pH = 5.0-8.0. Then soaking the mixture in 20% sucrose solution at 30 ℃ for 24h, and weighing. And finally, drying the cherry tomatoes to constant weight by using an air-blast drying oven, and calculating the solid content. And judging the influence of the enzyme treatment on the permeability of the cherry tomato peel according to the calculated solid content of the cherry tomato. The results are shown in table 3, and the peel permeability of the cherry tomatoes is enhanced by the biological enzyme method treatment under the condition of pH = 5.0-8.0. The preferable condition is that the pH is = 6.5-8.0, and the solid content (peel permeability) can be improved by more than 90%.
TABLE 3 Effect of bioenzyme treatment on cherry tomato pericarp Permeability at different pH conditions
Example 4: influence of biological enzyme method treatment on permeability of cherry tomato peel under different temperature conditions
One cherry tomato was immersed in a solution containing 0.1g/LHumicola Insolens-derived cutinase (PDB accession No.: 4 OYY), 0.1% (v/v) cellulase and 0.01% (m/v) pectinase, and the volume of the solution was 50mL. The control conditions are as follows: temperature 25/30/37 ℃, pH =6.5, treatment 24h. Then soaking the mixture in 20% sucrose solution at 30 ℃ for 24h, and weighing. And finally, drying the cherry tomatoes to constant weight by using an air-blast drying oven, and calculating the solid content. And judging the influence of the enzyme treatment on the permeability of the cherry tomato peel according to the calculated solid content of the cherry tomato. As shown in Table 4, the biological enzyme method treatment can enhance the peel permeability of the cherry tomatoes at the enzyme immersion temperature of 25-37 ℃, preferably 30 ℃, and can improve the solid content (peel permeability) by more than 90% while ensuring the good appearance quality of the soaked cherry tomatoes.
TABLE 4 Effect of bioenzyme treatment on cherry tomato pericarp Permeability at different temperatures
Example 5: influence of biological enzyme method treatment on cherry tomato peel permeability under different cutinase mass concentrations
One cherry tomato was immersed in a solution containing 0 to 3g/L of LHumicola insolens-derived cutinase (PDB accession No. 4 OYY), 0.1% (v/v) cellulase and 0.01% (m/v) pectinase, and the volume of the solution was 50mL. Controlling conditions: temperature 30 ℃, pH =6.5, treatment 24h. Then soaking the mixture in 20% sucrose solution at 30 ℃ for 24h, and weighing. And finally, drying the cherry tomatoes to constant weight by using an air-blast drying oven, and calculating the solid content. And judging the influence of the enzyme treatment on the permeability of the cherry tomato peel according to the calculated solid content of the cherry tomato. The results are shown in Table 5, where the effect of the bioenzyme process treatment to enhance the pericarp permeability of cherry tomatoes is enhanced with increasing cutinase addition. When the cutinase is added in an amount of 3g/L, the solid content (peel permeability) is increased by more than two times compared with the blank.
TABLE 5 Effect of Bio-enzymatic treatment on Permeability of cherry tomato pericarp at different cutinase Mass concentrations
Example 6: influence of biological enzyme method treatment on permeability of cherry tomato peel under different enzyme treatment time conditions
One cherry tomato was soaked in a solution containing 0.1g/LHumicola Insolens-derived cutinase (PDB accession No.: 4 OYY), 0.1% (v/v) cellulase, and 0.01% (m/v) pectinase, and the volume of the solution was 50mL. Controlling conditions: the temperature is 30 ℃, the pH =6.5, and the treatment time is 0-24 h. Then soaking the mixture in 20% sucrose solution at 30 ℃ for 24h, and weighing. And finally, drying the cherry tomatoes to a constant weight by using an air-blast drying oven, and calculating the solid content. And judging the influence of the enzyme treatment on the permeability of the cherry tomato peel according to the calculated solid content of the cherry tomato. As shown in Table 6, the effect of the bio-enzymatic treatment to enhance the permeability of the pericarp of cherry tomato was enhanced with the time of immersion in enzyme. When the enzyme leaching time is 24h, the solid content (peel permeability) is increased nearly twice compared with the blank.
TABLE 6 Effect of Bio-enzymatic treatment on cherry tomato pericarp Permeability under different enzyme treatment time conditions
Example 7: effect of bioenzyme method treatment of cherry tomato pericarp on the delivery effects of different types of sugars
One cherry tomato was soaked in a solution containing 0.1g/LHumicola Insolens-derived cutinase (PDB accession No.: 4 OYY), 0.1% (v/v) cellulase, and 0.01% (m/v) pectinase, and the volume of the solution was 50mL. The control conditions are as follows: temperature 30 ℃, pH =6.5, treatment 24h. Then soaking the mixture in 20% sugar solution of different types at 30 deg.C for 24 hr, and weighing. And finally, drying the cherry tomatoes to constant weight by using an air-blast drying oven, and calculating the solid content. And judging the influence of the enzyme treatment on the permeability of the cherry tomato peel according to the calculated solid content of the cherry tomato. The results are shown in table 7, where the bio-enzymatic treatment enhances the permeability of the cherry tomato pericarp, while the higher the molecular weight of the sugar, the less easily the sugar is transported into the pericarp and the less the solid content is increased.
TABLE 7 solid content variation after enzyme treatment by soaking different types of functional sugars
Example 8: enzyme sugar combined soaking
One cherry tomato was soaked in a solution containing 0.1 or 1g/LHumicola Insolens-derived cutinase (PDB accession No. 4 OYY), 0.1% (v/v) cellulase, 0.01% (m/v) pectinase, and sucrose was added simultaneously to achieve enzyme-sugar co-soaking, ensuring that the sucrose concentration in the solution was 20% and the volume of the solution was 50mL. The control conditions are as follows: the temperature is 30 ℃, the pH =6.5, and the treatment is carried out for 24-48 h. And finally, drying the cherry tomatoes to a constant weight by using an air-blast drying oven, and calculating the solid content. And judging the influence of the enzyme treatment on the permeability of the cherry tomato peel according to the calculated solid content of the cherry tomato. The results are shown in Table 8, in the enzyme-sugar combined soaking process, when the concentration of the cutinase is 1g/L and the soaking time is 24h, the solid content is increased by about 0.7 times compared with the blank; when the cutinase concentration is 0.1g/L and the soaking time is 48h, the solid content can be increased by about 0.9 times than that of the blank. The enzyme and sugar combined soaking can increase the permeability of the peel and reduce the complicated process steps, and is a good choice in the processing.
TABLE 8 solid content change of enzyme sugar in combination with soaking
Example 9: 20% -30% -40% gradient sugar soaking after enzyme treatment
One cherry tomato was soaked in a solution containing 0 to 3g/L Humicola Insolens-derived cutinase (PDB accession No.: 4 OYY), 0.1% (v/v) cellulase, and 0.01% (m/v) pectinase, and the volume of the solution was 50mL. Controlling conditions: temperature 37 ℃, pH =6.5, treatment 24h. And then sequentially soaking in sucrose solutions with mass concentrations of 20%, 30% and 40% for 24h, 24h and 96h respectively, and weighing. And finally, drying the cherry tomatoes to a constant weight by using an air-blast drying oven, and calculating the solid content. And judging the influence of the enzyme treatment on the permeability of the cherry tomato peel according to the calculated solid content of the cherry tomato. As a result, as shown in Table 9, the solid content reached 31.48% when the amount of cutinase was 3 g/L.
TABLE 9 solid content change of 20% -30% -40% gradient infusion after treatment
Example 10: recycling of enzymes
One cherry tomato was soaked in a solution containing 1g/L Humicola Insolens-derived cutinase (PDB accession No.: 4 OYY), 0.1% (v/v) cellulase, and 0.01% (m/v) pectinase, and the volume of the solution was 50mL. Controlling conditions: temperature 30 ℃, pH =6.5, treatment 24h. Then soaked in 20% sucrose solution for 24h and weighed. And finally, drying the cherry tomatoes to constant weight by using an air-blast drying oven, and calculating the solid content. The mixed enzyme solution was used repeatedly six times. And judging the influence of the enzyme treatment on the permeability of the cherry tomato peel according to the calculated solid content of the cherry tomato. As shown in Table 10, the treated cherry tomatoes showed little change in their solid content after 6 repeated uses of the mixed enzyme solution, and still had a similar effect of increasing the peel permeability as the first use. The enzyme liquid is repeatedly used in the processing, which is beneficial to saving economic cost.
TABLE 10 solid content variation for different times of enzyme liquid recycling
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for enhancing the permeability of fruit peel by a biological enzyme method treatment is characterized in that the fruit is soaked in an enzyme solution capable of degrading the components of the cuticle layer of the fruit peel; the enzyme solution contains one or more of cutinase, pectinase and cellulase.
2. The method of claim 1, wherein the fruit is a fruit having a pericarp cuticle comprising cherry tomato, grape, prune, plum.
3. The method according to claim 1 or 2, wherein the peel stratum corneum component is any one or a combination of two or more of cutin, cellulose, pectin, triglycerides, wax esters.
4. The method according to claim 1, wherein the cutinase is any one of Humicola insolens-derived cutinase, thermobifidafa-derived cutinase mutant D204C/E253C and Thermobifidafa-derived cutinase mutant Q92G/H184S/D204C/F209I/I213K/E253C.
5. The method of claim 1, wherein the enzyme solution is further added with natural or synthetic substances to achieve combined soaking.
6. The method of claim 5, wherein the natural substances comprise sucrose, vitamins, sodium chloride, organic acids.
7. The method of claim 5, wherein the synthetic substances include sweeteners, preservatives, color fixatives, antioxidants.
8. The method according to claim 1, wherein the volume of the enzyme solution is 1.5 to 10mL/g of the starting material, and when cutinase is contained in the enzyme solution, the concentration of the cutinase is not less than 0.01g/L.
9. The method according to claim 1, wherein the soaking time is 1-48 h, the temperature is controlled at 25-37 ℃, and the pH value is 5.0-8.0.
10. Use of the method according to any one of claims 1-9 for processing dehydrated fruit, preserved fruit.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5037662A (en) * | 1989-06-23 | 1991-08-06 | Genencor International Inc. | Enzyme assisted degradation of surface membranes of harvested fruits and vegetables |
| CN103710327A (en) * | 2013-12-30 | 2014-04-09 | 福州大世界橄榄有限公司 | Compound enzyme preparation for deep processing of olive and preparation method and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5037662A (en) * | 1989-06-23 | 1991-08-06 | Genencor International Inc. | Enzyme assisted degradation of surface membranes of harvested fruits and vegetables |
| CN103710327A (en) * | 2013-12-30 | 2014-04-09 | 福州大世界橄榄有限公司 | Compound enzyme preparation for deep processing of olive and preparation method and application thereof |
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