Block polyamino acid, preparation method thereof and application thereof in banana preservation

Abstract

The present invention discloses a block polyamino acid and a preparation method thereof and application thereof in banana preservation. The main chain structure of the block polyamino acid is L -Configuration Polymerization α -amino acid, the side chain is hydroxyethyl and mercapto methylene. L ‑ O ‑tert-Butylthreonine and L‑S‑ (4‑Methoxybenzyl)‑ L The corresponding nitrogen carboxyl lactic anhydride NCA is synthesized by using cysteine ​​as the starting material, and the polymerization reaction is initiated by the corresponding initiator and then the protecting group is removed to obtain the block polyamino acid of the present invention. The main chain of the block polyamino acid prepared by the present invention is L -Configuration Polymerization α -amino acid, the side chain is rich in hydroxyl and thiol, and can be used for banana preservation. The block polyamino acid of the present invention has the characteristics of low cost, safety and non-toxicity, and the banana preserved by the block polyamino acid is stored at room temperature for 5 days without obvious black spots on the appearance.

Description

Technical Field

[0001] The invention belongs to the field of food preservative preparation, and in particular relates to a preparation method of a novel block polyamino acid and application thereof in the field of banana preservation. Background Art

[0002] Fruits and vegetables are rich in key nutrients, including vitamins, minerals, dietary fiber, and phytochemicals with antioxidant properties. In recent years, with the growing concern about the health benefits of a balanced diet, modern dietary trends have shown a significant increase in the consumption of plant-based foods and a corresponding decrease in the intake of animal-based foods. However, the agricultural production of fruits and vegetables is often threatened by pathogenic fungi, resulting in reduced crop yields. In addition, these perishable agricultural products may reduce nutrition and flavor after harvest due to physical damage, microbial spoilage, and their own metabolic activities. These adverse factors lead to significant postharvest losses, which are estimated to account for 35% to 55% of the total production, depending on regional differences. Therefore, there is an urgent need to develop and implement effective methods to maintain the integrity and freshness of fruits and vegetables.

[0003] At present, a variety of preservation strategies are widely used in the industry, including temperature control, atmosphere modification, irradiation treatment, and the use of synthetic or natural preservatives. However, each of these methods has limitations and negative effects. For example, refrigeration consumes a lot of energy and money, and is accompanied by the risk of freezing and mechanical damage. Chemical preservatives may remain in food, potentially threatening the health and safety of consumers. In view of these problems, the research and application of natural extracts as preservatives are gradually gaining attention. Empirical studies have shown that natural extracts such as chitosan, propolis, essential oils, and various oligosaccharides have shown good effects in inhibiting the reproduction of pathogenic microorganisms, thereby effectively extending the shelf life of horticultural products. However, some natural extracts (such as chitosan, propolis, and essential oils) have low solubility in water due to their high molecular weight and polarity, which limits their widespread application in the industrial field. Therefore, it is of great economic value to explore green and environmentally friendly preservatives with excellent properties. Summary of the invention

[0004] In view of the above problems existing in the prior art, the object of the present invention is to provide a method for preparing a novel block polyamino acid for banana preservation and its application in the field of banana preservation.

[0005] The technical solution adopted by the present invention is as follows:

[0006] The block polyamino acid used for banana preservation has a main structural chain of L -Configuration Polymerization α -amino acid, the side chain is hydroxyethyl and mercapto methylene, and its structural formula is shown in Formula I:

[0007] In formula I, x and y are both integers, x is in the range of 6-12, and the molar ratio of x / y is in the range of 1:1 to 1:3.

[0008] Furthermore, the block polyamino acid of the present invention has a molecular weight of 2.0×10 3 -4.0×10 3 g / mol, and its chiral configuration is optically pure L -Configuration.

[0009] The method for preparing a block polyamino acid for preserving bananas comprises the following steps:

[0010] 1) Using THF or DMF as solvent, in the presence of solid phosgene, L - O -tert-Butylthreonine or LS- (4-methoxybenzyl)- L -cysteine ​​as the starting material, stirred at room temperature overnight, and the corresponding nitrogen carboxyl internal anhydride target products were prepared, and the target products were marked as threonine NCA and cysteine ​​NCA respectively; the reaction formulas are as follows:

[0011] ;

[0012] ;

[0013] 2) Using THF or DMF as the reaction solvent, threonine NCA and the initiator are first stirred at room temperature for 20-30 hours in the reaction solvent, and then cysteine ​​NCA is added and stirred at room temperature for 20-30 hours. After the reaction is completed, a side chain protected block polyamino acid intermediate is obtained by post-treatment. The structural formula of the intermediate is as follows:

[0014] ;

[0015] 3) The intermediate obtained in step 2) is added into a trifluoroacetic acid solution containing triisopropylsilane and stirred at room temperature for 1-4 hours. After the reaction is completed, the target product shown in formula I is obtained by post-treatment.

[0016] Further, in step 1), the mass of solid phosgene is L - O -tert-Butylthreonine or LS- (4-methoxybenzyl)- L -0.8-1.25 times the mass of the cysteine ​​starting material; Step 1) After the reaction is completed, the following post-treatment steps are also included: After the reaction is completed, the reaction solution is evaporated and concentrated, the concentrated solution is dropped into 3-5°C n-hexane to precipitate, filtered, and the product is frozen for storage.

[0017] Furthermore, in step 2), the initiator is benzylamine, tert-butylamine or n-butylamine, the mass of the initiator is 0.001%-0.004% of the mass of threonine NCA, and the molar ratio of threonine NCA to cysteine ​​NCA is 1:1-3.

[0018] Furthermore, the post-treatment step in step 2) is: after the reaction is completed, the reaction solution is evaporated and concentrated, the concentrated solution is dropped into 3-5°C n-hexane to precipitate, filtered, and the precipitate is collected to obtain the side chain protected block polyamino acid.

[0019] Furthermore, in step 3), the volume concentration of triisopropylsilane in trifluoroacetic acid is 2%-6%, and the solid-liquid ratio of the intermediate to the trifluoroacetic acid solution containing triisopropylsilane is 1g:5-10mL; the post-treatment step after the reaction in step 3) is: the excess trifluoroacetic acid is purged by air flow, the residue is dissolved in anhydrous THF, and then the THF solution containing the polymer is dripped into 3-5 o C in n-hexane to precipitate, filter, and collect the precipitate to obtain the block polyamino acid.

[0020] The blocked polyamino acid of the present invention is used as a preservative in banana preservation and has a good application effect. The application method comprises the following steps:

[0021] S1: The block polyamino acid is dispersed by stirring with deionized water or ethanol to prepare a solution of 10 mg / mL-400 mg / mL;

[0022] S2: The solution prepared in step S1 is evenly sprayed onto the surface of fresh bananas, and then the sprayed bananas are placed in a clean bench and dried with sterile air for 0.5-1 h and then stored at room temperature.

[0023] By adopting the above technology, compared with the prior art, the beneficial effects of the present invention are as follows: the present invention adopts non-toxic natural amino acid derivatives: L - O -tert-Butylthreonine and LS- (4-methoxybenzyl)- L -cysteine ​​as the starting material to prepare the corresponding block polyamino acid. L -Threonine-block- L -cysteine) fresh bananas stored at room temperature for 5 days have basically no obvious black spots (such as Figure 1 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figures are the comparison results of the appearance changes of bananas in the experimental group and the blank control group in Example 3 of the present invention stored at room temperature for 1 to 6 days. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0026] 1. Preparation of polymer monomers

[0027] 1) L - O - Preparation of tert-butylthreonine NCA

[0028] The preparation chemical reaction equation is as follows:

[0029] ;

[0030] 1.75 g L - O -tert-Butylthreonine was added to a pressure-resistant screw-mouth round-bottom flask, followed by 30 mL of anhydrous tetrahydrofuran (or 10 mL of anhydrous DMF) and 7 mL of propylene oxide, and stirred at room temperature for 30 minutes. Next, 1.48 g of solid phosgene was added to the reaction solution, and stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated to 2 mL. Then the concentrated reaction solution was dropped into 4 o C in n-hexane to precipitate. Filter, collect the precipitate, and dry to obtain L - O -tert-butylthreonine corresponding nitrogen carboxyl anhydride target product 1.47 g, yield 73%. The target product is recorded as threonine NCA and stored at -18 o C refrigerator. The structure of the obtained product was characterized by hydrogen nuclear magnetic resonance spectroscopy.

[0031] 1 H-NMR (400 MHz, DMSO- d 6), δ 9.08 (br, 1H), 4.37-4.36 (m, 1 H), 4.07-4.04 (m, 1H), 1.18-1.16 (d, 3H), 1.09 (s, 9 H) ppm.

[0032] 2) LS- (4-methoxybenzyl)- L -Cysteine ​​NCA Preparation

[0033] The preparation chemical reaction equation is as follows:

[0034] ;

[0035] 1.2 g LS- (4-methoxybenzyl)- L-cysteine ​​was added to a pressure-resistant screw-capped round-bottom flask, followed by 30 mL of anhydrous tetrahydrofuran (or 10 mL of anhydrous DMF) and 7 mL of propylene oxide, and stirred at room temperature for 30 minutes. Next, 1.48 g of solid phosgene was added to the reaction solution, and stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated to 2 mL. The concentrated reaction solution was then dropped into 4 o C in n-hexane to precipitate. Filter, collect the precipitate, and dry to obtain LS- (4-methoxybenzyl)- L -cysteine ​​corresponding nitrogen carboxyl internal anhydride target product 1.02 g product, yield 78%. The target product is recorded as cysteine ​​NCA and stored at -18 o C refrigerator. The structure of the obtained product was characterized by hydrogen nuclear magnetic resonance spectroscopy.

[0036] 1 H-NMR (400 MHz, DMSO- d 6), δ 9.08 (br, 1H), 7.02-6.98 (m, 4 H), 4.35-4.33 (m, 1 H), 3.80 (s, 3H), 3.75-3.66 (m, 2 H), 2.98-2.93 (m, 2H) ppm.

[0037] 2. Preparation method of block polyamino acid

[0038] The preparation chemical reaction equation is as follows:

[0039] . Example 1

[0040] Preparation of block polyamino acids with a 1:1 ratio of threonine to cysteine

[0041] 1.0 g L - O -tert-Butylthreonine NCA was dissolved in 20 mL of anhydrous THF, and then 0.01 mg of tert-butylamine was added and stirred at room temperature for 24 hours under nitrogen protection. 1.34 g LS- (4-methoxybenzyl)- L -cysteine ​​NCA was added and stirred for 24 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated to 2 mL. Then the concentrated reaction solution was added dropwise to 4 o The precipitate was precipitated in n-hexane at 400 °C. The precipitate was collected by filtration and dried to obtain 1.54 g of side chain protected block polyamino acid with a yield of 65%.

[0042] Take 0.6 g of the side chain protected amino acid obtained in the above step, add 5 mL of trifluoroacetic acid and 0.1 mL of triisopropylsilane, and stir at room temperature for 2 hours. After the reaction is completed, the excess trifluoroacetic acid is purged out by air flow. The residue is dissolved in 2 mL of anhydrous THF, and then the THF solution containing the polymer is dropped into 4 o C in n-hexane to precipitate. Filter, collect and dry the precipitate to obtain 0.32 g of block polyamino acid with a yield of 92%. The molecular weight of the obtained target polymer was characterized by gel permeation chromatography with THF as the mobile phase. The test results showed that the molecular weight of the obtained polymer was: 2.1×10 3 g / mol. Example 2

[0043] Preparation of block polyamino acids with a ratio of threonine to cysteine ​​of 1:3

[0044] 1.0 g L - O -tert-Butylthreonine NCA was dissolved in 10 mL of anhydrous DMF, and then 0.04 mg of n-butylamine was added and stirred at room temperature for 36 hours under nitrogen protection. 4.0 g LS- (4-methoxybenzyl)- L -cysteine ​​NCA was added and stirred for 36 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated to 2 mL. Then the concentrated reaction solution was added dropwise to 4 o The precipitate was precipitated in n-hexane at 400 °C. The precipitate was collected by filtration and dried to obtain 3.65 g of side chain protected block polyamino acid with a yield of 73%.

[0045] Take 0.8 g of the side chain protected amino acid obtained in the above step, add 5 mL of trifluoroacetic acid and 0.3 mL of triisopropylsilane, and stir at room temperature for 4 hours. After the reaction is completed, the excess trifluoroacetic acid is purged out by air flow. The residue is dissolved in 2 mL of anhydrous THF, and then the THF solution containing the polymer is dropped into 4 o C in n-hexane to precipitate. Filter, collect and dry the precipitate to obtain 0.41 g of block polyamino acid with a yield of 90%. The molecular weight of the obtained target polymer was characterized by gel permeation chromatography with THF as the mobile phase. The test results showed that the molecular weight of the obtained polymer was: 3.98×10 3 g / mol. Example 3

[0046] An experimental group was set up: the block polyamino acid prepared in Example 2 was dissolved and dispersed in anhydrous ethanol to prepare a preservative solution with a concentration of 300 mg / mL. The preservative solution was evenly sprayed on the surface of fresh bananas, and then the bananas sprayed with the preservative were placed in a clean bench and dried with sterile air for half an hour and then stored at room temperature.

[0047] In addition, a blank control group was set up: anhydrous ethanol solvent was evenly sprayed on the surface of fresh bananas, and then the bananas were placed in a clean bench and dried with sterile air for half an hour and then stored at room temperature.

[0048] The appearance of bananas in the experimental group and the blank control group were measured within one week, and the weight, reducing sugar content, total phenol content and relative activity of polyphenol oxidase (PPO) of banana samples were tested on the first and sixth days. The results are shown in Tables 1 and Figure 1 The test method is as follows:

[0049] Reducing sugar content: The reducing sugar content in banana pulp was determined by DNS method. Banana pulp was homogenized with distilled water at a mass ratio of 1:5. 1 mL of centrifugal supernatant and 1.5 mL of DNS reagent were heated in a boiling water bath for 5 min to develop color. The absorbance was measured at a wavelength of 540 nm, with distilled water as a blank control. The reducing sugar content was calculated using the glucose standard curve, and the results were expressed in mg / g.

[0050] Titratable acid content: Banana pulp was homogenized with distilled water at a mass ratio of 1:5, and the banana pulp was homogenized and heated in a boiling water bath for 30 min. After cooling, it was filtered, 1 mL of the filtrate, 5 mL of water and 3 drops of 1% phenolphthalein indicator were mixed, and titrated with 0.01 mol / L sodium hydroxide until red. The results were expressed as mass fraction (%).

[0051] Total phenolic content: Banana pulp was mixed with distilled water in a mass ratio of 1:5 and homogenized in a water bath at 80°C for 40 min. The centrifugal supernatant, leaf phenol and 10% Na2CO3 (w / v) solution were mixed in a ratio of 1:1:4 (v / v / v). The absorbance of the mixed solution was measured at a wavelength of 760 nm using a UV spectrophotometer. A standard curve was drawn using gallic acid standards, and the total phenolic content was calculated in mg / g.

[0052] Relative activity of PPO: Take 5 g of banana pulp, add 10 mL of phosphate buffer (pH = 7.5), homogenize, and centrifuge to extract the supernatant to obtain a crude enzyme solution rich in PPO. Take 0.1 mL of the crude enzyme solution, add 4 mL of phosphate buffer (pH = 7.5) and 1 mL of catechol solution (10%, mass ratio, solvent is deionized water), mix, and then measure and record its absorbance at 525 nm using a UV-visible spectrophotometer. After standing for ten minutes, continue to measure and record its absorbance at 525 nm using a UV-visible spectrophotometer. Calculate the PPO enzyme activity based on the transformation of the two absorbance values. The calculation formula is:

[0053] ;

[0054] In the formula is the absorbance change value; W is the weight of fresh banana pulp, g; t is the standing time, min; D is the dilution multiple.

[0055] From the data in Table 1, we can see that compared with the blank control group, the mass reduction rate of the bananas preserved by blocked polyamino acids was 16% after six days, while the mass reduction rate of the blank control group was 34%, indicating that the respiration of the bananas preserved by blocked polyamino acids was significantly inhibited and the water volatilization was greatly reduced. In addition, respiration will also consume the reducing sugar content in bananas. The experimental results show that the reducing sugar content of bananas preserved by blocked polyamino acids is much higher than that of the blank control group after six days, which also confirms that the respiration of bananas is inhibited. An important reason for the browning of the banana surface is that polyphenol oxidase catalyzes the enzymatic browning reaction of polyphenols in bananas, thereby producing black spots. From Table 1, it can be seen that the total phenol content in the bananas preserved by blocked polyamino acids is much higher than that of the blank control group, while the activity of polyphenol oxidase is much lower than that of the blank control group. In addition, titratable acid is an important product of enzymatic browning. The content of bananas preserved by blocked polyamino acids is also much lower than that of the blank control group after 6 days of storage. These experimental data show that the enzymatic browning of bananas preserved by blocked polyamino acids is inhibited, thereby delaying the formation of black spots on the banana surface. This experimental result is consistent with Figure 1 The overall observed changes in the banana surface were consistent.

[0056]

[0057] Figure 1 The comparison results of the appearance changes of bananas in the experimental group and the blank control group in Example 3 of the present invention after being stored at room temperature for 1 to 6 days are shown. Figure 1 From left to right, A shows the appearance of bananas in the experimental group on the 1st, 2nd, 3rd, 4th, 5th, and 6th days. Figure 1 B in the figure shows the appearance photos of bananas in the blank control group on the 1st day, 2nd day, 3rd day, 4th day, 5th day and 6th day from left to right.

[0058] from Figure 1 It can be seen that the use of poly ( L -Threonine-block- L -cysteine) were stored at room temperature for 5 days without obvious black spots on the appearance.

Claims

1. A block polyamino acid for banana preservation, characterized in that Its main structural chain is L -Configuration Polymerization α -amino acid, the side chain is hydroxyethyl and mercapto methylene, and its structural formula is shown in Formula I: ； In formula I, x and y are both integers, x is in the range of 6-12, and the molar ratio of x / y is in the range of 1:1 to 1:

3.

2. The block polyamino acid for banana preservation according to claim 1, characterized in that Its molecular weight is 2.0×10 3 -4.0×10 3 g / mol.

3. The method for preparing a block polyamino acid for banana preservation according to claim 1, characterized in that The steps include: 1) Using THF or DMF as solvent, in the presence of solid phosgene, L - O -tert-Butylthreonine or LS- (4-methoxybenzyl)- L -cysteine ​​as the starting material, stirred at room temperature overnight, and the corresponding nitrogen carboxyl internal anhydride target products were prepared, and the target products were marked as threonine NCA and cysteine ​​NCA respectively; the reaction formulas are as follows: ； ； 2) Using THF or DMF as the reaction solvent, threonine NCA and the initiator are first stirred at room temperature for 20-30 hours in the reaction solvent, and then cysteine ​​NCA is added and stirred at room temperature for 20-30 hours. After the reaction is completed, a side chain protected block polyamino acid intermediate is obtained by post-treatment. The structural formula of the intermediate is as follows: ； 3) The intermediate obtained in step 2) is added into a trifluoroacetic acid solution containing triisopropylsilane and stirred at room temperature for 1-4 hours. After the reaction is completed, the target product shown in formula I is obtained by post-treatment.

4. The method for preparing a block polyamino acid for banana preservation according to claim 3, characterized in that In step 1), the mass of solid phosgene is L - O -tert-Butylthreonine or LS- (4-methoxybenzyl)- L -0.8-1.25 times the mass of the cysteine ​​starting material; Step 1) After the reaction is completed, the following post-treatment steps are also included: After the reaction is completed, the reaction solution is evaporated and concentrated, the concentrated solution is dropped into 3-5°C n-hexane to precipitate, filtered, and the product is frozen for storage.

5. The method for preparing a block polyamino acid for banana preservation according to claim 3, characterized in that In step 2), the initiator is benzylamine, tert-butylamine or n-butylamine, the mass of the initiator is 0.001%-0.004% of the mass of threonine NCA, and the molar ratio of threonine NCA to cysteine ​​NCA is 1:1-3.

6. The method for preparing a block polyamino acid for banana preservation according to claim 3, characterized in that Step 2) The post-treatment step is: after the reaction is completed, the reaction solution is evaporated and concentrated, the concentrated solution is dropped into 3-5°C n-hexane to precipitate, filtered, and the precipitate is collected to obtain the side chain protected block polyamino acid.

7. The method for preparing a block polyamino acid for banana preservation according to claim 3, characterized in that In step 3), the volume concentration of triisopropylsilane in trifluoroacetic acid is 2%-6%, and the solid-liquid ratio of the intermediate to the trifluoroacetic acid solution containing triisopropylsilane is 1g:5-10mL; after the reaction in step 3), the post-treatment step is: the excess trifluoroacetic acid is purged by air flow, the residue is dissolved in anhydrous THF, and then the THF solution containing the polymer is dripped into 3-5 o C in n-hexane to precipitate, filter, and collect the precipitate to obtain the block polyamino acid.

8. Use of the block polyamino acid as claimed in claim 1 as a preservative for preserving bananas.

9. The use according to claim 8, characterized in that The application method comprises the following steps: S1: The block polyamino acid is dispersed by stirring with ethanol to prepare a solution of 10 mg / mL-400 mg / mL; S2: The solution prepared in step S1 is evenly sprayed onto the surface of fresh bananas, and then the sprayed bananas are placed in a clean bench and dried with sterile air for 0.5-1 h and then stored at room temperature.

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