Phenolic acid inhibitor as well as use method and application thereof

Abstract

The invention discloses a phenolic acid inhibitor and a use method and application thereof.The phenolic acid inhibitor comprises a component A. The component A comprises phenolic acid, polyethylene glycol and a phosphate buffer solution, and the phenolic acid is one or more of cumaric acid, kojic acid, chlorogenic acid, gallic acid and ferulic acid; the phenolic acid inhibitor is prepared from the following raw materials in parts by weight: 1 to 3 parts of phenolic acid, 0.1 to 0.3 part of polyethylene glycol and 8 to 12 parts of phosphate buffer solution, and the phenolic acid is two or three of coumaric acid, chlorogenic acid and ferulic acid. The PPO inhibition activity of the phenolic acid inhibitor can reach 70%-80%, the quality of fruits is well maintained, and browning caused by fruit processing is delayed.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and more specifically, to a phenolic acid inhibitor and a method and application thereof. Background Art

[0002] Fruit processing is an important part of the food industry, aiming to extend the shelf life of fruits, improve flavor and enhance nutritional value. Since fruit raw materials are rich in phenolic compounds and highly active polyphenol oxidase (PPO), it catalyzes the oxidation of phenolic substances into quinones, which then polymerize into colored substances, causing enzymatic browning, which can not only affect the appearance and color of fruits, causing changes in fruit flavor and nutrition, but also may reduce their market value and consumer acceptance. It is common in the processing of fruits such as apples, pears, and bananas.

[0003] At present, there is a hypothesis about the mechanism of PPO enzymatic browning: the regional distribution hypothesis of phenolase points out that there are two forms of PPO in tissue cells, namely, free polyphenol oxidase (sPPO) in the cytoplasm and membrane-bound polyphenol oxidase (mPPO) bound to the cell membrane. When the cell membrane structure is damaged, mPPO is free and converted to sPPO, resulting in enhanced sPPO activity and regional catalytic activity of PPO and phenolic substances. Usually, only when this spatial isolation is broken, PPO will show enzymatic activity. External conditions such as cutting and crushing during fruit processing and storage will lead to the destruction of the membrane system, breaking the regional distribution, allowing the enzyme and substrate to come into contact and causing enzymatic browning. Phenolic acids are a class of organic acids containing phenol rings. The research and application of phenolic acids in food systems are relatively small, so the present application provides an inhibitor containing phenolic acids to inhibit the activity of polyphenol oxidase in fruits. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a phenolic acid inhibitor solution. The phenolic acid inhibitor of the present invention has an inhibitory activity of 70% to 80% on PPO, which better maintains the quality of the fruit and delays the browning caused by fruit processing.

[0006] In order to achieve these purposes and other advantages according to the present invention, a phenolic acid inhibitor is provided, including component A, wherein component A includes the following raw materials: phenolic acid, polyethylene glycol, and phosphate buffer, wherein the pH of the phosphate buffer is 6.8, and the phenolic acid is one or more of coumaric acid, kojic acid, chlorogenic acid, gallic acid, and ferulic acid.

[0007] Preferably, component A comprises the following raw materials in parts by weight: 1 to 3 parts by weight of phenolic acid, 0.1 to 0.3 parts by weight of polyethylene glycol, and 8 to 12 parts by weight of phosphate buffer.

[0008] Preferably, the phenolic acid is one or more of coumaric acid, chlorogenic acid and ferulic acid.

[0009] Preferably, component A includes 0.1-0.3 parts by weight of polyethylene glycol.

[0010] Preferably, the pH of the phosphate buffer is 4-6.8.

[0011] Preferably, the method further comprises component B and component C, wherein component B comprises a 1% potassium citrate aqueous solution, and component C comprises 0.1 parts by weight of purslane extract, 0.2 parts by weight of soybean sprout extract, and 10 parts by weight of phosphate buffer;

[0012] The preparation method of soybean sprout extract is as follows: remove the head and root of soybean sprout, take the white part and chop the white part to obtain pretreated bean sprout, add water 3 times the total mass of the pretreated bean sprout to the pretreated bean sprout, heat at 95-100° C. for 5 hours, filter and concentrate to obtain soybean sprout extract;

[0013] The preparation method of the purslane extract is as follows: add water 4 times the total mass of the purslane to the purslane and heat it at 95-100°C for 3 hours, filter and concentrate the filtrate to obtain an extract, add water to the extract to dissolve it and add petroleum ether to extract it, extract it three times, combine the aqueous phases, concentrate and freeze-dry it to obtain the purslane extract.

[0014] The present invention also provides an application of a phenolic acid inhibitor, wherein the phenolic acid inhibitor is used for inhibiting the activity of polyphenol oxidase in apples.

[0015] The present invention also provides a method for using phenolic acid inhibitors, comprising the following steps: firstly placing fresh-cut apples into component B and immersing them for 20 seconds, then placing them into component C and immersing them for 3 minutes, and then placing them into component A and immersing them for 2 minutes, and storing them at 3-5°C after immersion.

[0016] The present invention has at least the following beneficial effects:

[0017] First, the phenolic acid inhibitor solution prepared by the present invention has an inhibitory activity of 70% to 80% on PPO, which better maintains the quality of fruits, delays the browning caused by fruit processing, and has certain application potential in food preservation.

[0018] Second, the solvents used in the preparation of phenolic acid inhibitors are more environmentally friendly than other methods. PEG is a high molecular weight polymer with excellent solubility, stability and safety. It is widely used in the food industry as a cosolvent and humectant. It can effectively dissolve a variety of substances, including oil-soluble and water-soluble components, and make them evenly distributed in the food system. In addition, PEG can also improve the texture and taste of food and increase the gloss and stability of the product.

[0019] Third, the phenolic acid inhibitor solution prepared by the present invention has low addition concentration, small dosage and high safety, and is very likely to be further developed into a safe and effective food preservative.

[0020] Fourth, there are different forms of PPO in fruits. The sPPO and mPPO involved in the present invention have been less studied in practical applications. The present invention explores the inhibition mechanism of PPO from the perspective of inhibition kinetics.

[0021] Fifth, this shows that the present application has a good protective effect on apples by using component A, component B, and component C, and the protection effect is best when using component B, component C, and component A in sequence.

[0022] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effects of different concentrations of phenolic acid inhibitors of the present invention on the activity of apple sPPO;

[0024] Figure 2 The effects of different concentrations of phenolic acid inhibitors of the present invention on the activity of apple mPPO;

[0025] Figure 3 The inhibition types of different concentrations of the phenolic acid inhibitor (coumaric acid) on apple sPPO;

[0026] Figure 4 The inhibition types of different concentrations of phenolic acid inhibitors (kojic acid) on apple sPPO in the present invention;

[0027] Figure 5 The inhibition types of different concentrations of phenolic acid inhibitors (chlorogenic acid) on apple sPPO of the present invention;

[0028] Figure 6 The inhibition types of different concentrations of phenolic acid inhibitors (gallic acid) of the present invention on apple sPPO;

[0029] Figure 7The inhibition types of different concentrations of phenolic acid inhibitors (ferulic acid) on apple sPPO;

[0030] Figure 8 The inhibition types of apple mPPO by phenolic acid inhibitors (coumaric acid) of different concentrations in the present invention;

[0031] Fig. 9 The inhibition types of apple mPPO by phenolic acid inhibitors (kojic acid) of different concentrations in the present invention;

[0032] Fig.10 The inhibition types of apple mPPO by phenolic acid inhibitors (chlorogenic acid) of different concentrations of the present invention;

[0033] Fig.11 The inhibition types of apple mPPO by phenolic acid inhibitors (gallic acid) of different concentrations of the present invention;

[0034] Fig.12 The inhibition types of apple mPPO by phenolic acid inhibitors (ferulic acid) of different concentrations of the present invention;

[0035] Fig.13 It is a double reciprocal graph of the effects of different concentrations of the phenolic acid inhibitor (coumaric acid) of the present invention on apple sPPO;

[0036] Fig.14 It is a double reciprocal graph of the effects of different concentrations of the phenolic acid inhibitor (kojic acid) of the present invention on apple sPPO;

[0037] Fig.15 It is a double reciprocal graph of different concentrations of phenolic acid inhibitors (chlorogenic acid) of the present invention on apple sPPO;

[0038] Fig.16 It is a double reciprocal graph of the effects of different concentrations of the phenolic acid inhibitor (gallic acid) of the present invention on apple sPPO;

[0039] Fig.17 It is a double reciprocal graph of different concentrations of the phenolic acid inhibitor (ferulic acid) of the present invention on apple sPPO;

[0040] Fig.18 It is a double reciprocal graph of the effects of different concentrations of the phenolic acid inhibitor (coumaric acid) of the present invention on apple mPPO;

[0041] Fig.19 It is a double reciprocal graph of the effects of different concentrations of the phenolic acid inhibitor (kojic acid) of the present invention on apple mPPO;

[0042] Fig. 20 It is a double reciprocal graph of different concentrations of phenolic acid inhibitors (chlorogenic acid) of the present invention on apple mPPO;

[0043] Fig.21 It is a double reciprocal graph of the effects of different concentrations of the phenolic acid inhibitor (gallic acid) of the present invention on apple mPPO;

[0044] Fig. 22 It is a double reciprocal graph of the effects of different concentrations of the phenolic acid inhibitor (ferulic acid) of the present invention on apple mPPO;

[0045] Fig.23 The pictures are the color phenotype characteristics of apples affected by the coatings of different phenolic acid inhibitors of the present invention;

[0046] Figure 3-22 In the figure, the black square indicates that the concentration of the phenolic acid inhibitor phenolic acid is 1 mM, the red circle indicates that the concentration of the phenolic acid inhibitor phenolic acid is 2.5 mM, the blue triangle indicates that the concentration of the phenolic acid inhibitor phenolic acid is 5 mM, and the purple triangle indicates that the concentration of the phenolic acid inhibitor phenolic acid is 10 mM. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0048] <Example 1>

[0049] A phenolic acid inhibitor, comprising component A, wherein component A comprises 1 part by weight of coumaric acid, 0.1 part by weight of polyethylene glycol, and 8 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8;

[0050] The preparation method of the phenolic acid inhibitor is as follows: coumaric acid is firstly dissolved in polyethylene glycol, ultrasonicated at room temperature for 10 minutes, and then a phosphate buffer is added to the system.

[0051] <Example 2>

[0052] A phenolic acid inhibitor, comprising component A, wherein component A comprises 1 part by weight of kojic acid and 8 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8;

[0053] Dissolve kojic acid in phosphate buffered saline and dilute.

[0054] <Example 3>

[0055] A phenolic acid inhibitor, comprising component A, wherein component A comprises 1 part by weight of chlorogenic acid, 0.1 part by weight of 8 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8;

[0056] The preparation method of the phenolic acid inhibitor is as follows: chlorogenic acid is first dissolved in polyethylene glycol, ultrasonicated at room temperature for 10 minutes, and then phosphate buffer is added to the system.

[0057] <Example 4>

[0058] A phenolic acid inhibitor, comprising component A, wherein component A comprises 1 part by weight of gallic acid, 0.1 part by weight of polyethylene glycol, and 8 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8;

[0059] The preparation method of the phenolic acid inhibitor is as follows: gallic acid is first dissolved in polyethylene glycol, ultrasonicated at room temperature for 10 minutes, and then 0.05 mM phosphate buffer is added to the system.

[0060] <Example 5>

[0061] A phenolic acid inhibitor, comprising component A, wherein component A comprises 1 part by weight of ferulic acid, 0.1 part by weight of polyethylene glycol, and 8 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8;

[0062] The preparation method of the phenolic acid inhibitor is as follows: ferulic acid is first dissolved in polyethylene glycol, ultrasonicated at room temperature for 10 minutes, and then 0.05 mM phosphate buffer (0.05 mM phosphate buffer has a pH of 6.8) is added.

[0063] <Example 6>

[0064] A phenolic acid inhibitor comprises a component A, wherein the component A comprises the following raw materials in parts by weight: 0.5 parts by weight of coumaric acid, 0.5 parts by weight of chlorogenic acid, 0.5 parts by weight of ferulic acid, 0.1 parts by weight of polyethylene glycol, and 8 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8.

[0065] The preparation method is as follows: firstly, coumaric acid, chlorogenic acid and ferulic acid are dissolved in polyethylene glycol, and then a phosphate buffer solution is added.

[0066] <Example 7>

[0067] A phenolic acid inhibitor comprises a component A, wherein the component A comprises the following raw materials in parts by weight: 1 part by weight of coumaric acid, 1 part by weight of chlorogenic acid, 1 part by weight of ferulic acid, 0.3 part by weight of polyethylene glycol, and 12 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8.

[0068] The preparation method is as follows: firstly, coumaric acid, chlorogenic acid and ferulic acid are dissolved in polyethylene glycol, and then a phosphate buffer solution is added.

[0069] <Example 8>

[0070] A phenolic acid inhibitor comprises a component A, wherein the component A comprises the following raw materials in parts by weight: 0.67 parts by weight of coumaric acid, 0.67 parts by weight of chlorogenic acid, 0.67 parts by weight of ferulic acid, 0.2 parts by weight of polyethylene glycol, and 10 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8.

[0071] The preparation method is as follows: firstly, coumaric acid, chlorogenic acid and ferulic acid are dissolved in polyethylene glycol, and then a phosphate buffer solution is added.

[0072] <Example 9>

[0073] A phenolic acid inhibitor comprises a component A, wherein the component A comprises the following raw materials in parts by weight: 1 part by weight of coumaric acid, 1 part by weight of chlorogenic acid, 0.2 parts by weight of polyethylene glycol, and 10 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8.

[0074] The preparation method is as follows: firstly, coumaric acid and chlorogenic acid are dissolved in polyethylene glycol, and then a phosphate buffer solution is added.

[0075] <Example 10>

[0076] A phenolic acid inhibitor comprises a component A, wherein the component A comprises the following raw materials in parts by weight: 1 part by weight of ferulic acid, 1 part by weight of chlorogenic acid, 0.2 part by weight of polyethylene glycol, and 10 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8.

[0077] The preparation method is as follows: firstly, ferulic acid and chlorogenic acid are dissolved in polyethylene glycol, and then a phosphate buffer solution is added.

[0078] <Example 11>

[0079] A phenolic acid inhibitor comprises a component A, wherein the component A comprises the following raw materials in parts by weight: 1 part by weight of ferulic acid, 1 part by weight of coumaric acid, 0.2 part by weight of polyethylene glycol, and 10 parts by weight of phosphate buffer, wherein the pH value of the phosphate buffer is 6.8.

[0080] The preparation method is as follows: firstly, ferulic acid and coumaric acid are dissolved in polyethylene glycol, and then a phosphate buffer solution is added.

[0081] <Example 12>

[0082] A phenolic acid inhibitor comprises a component A, wherein the component A comprises the following raw materials in parts by weight: 0.67 parts by weight of coumaric acid, 0.67 parts by weight of chlorogenic acid, 0.67 parts by weight of ferulic acid, 0.2 parts by weight of polyethylene glycol, and 10 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 6.8.

[0083] The preparation method of component A is as follows: firstly, coumaric acid, chlorogenic acid and ferulic acid are dissolved in polyethylene glycol, and then a phosphate buffer solution is added.

[0084] It also includes a component B and a component C, wherein the component B includes a potassium citrate aqueous solution with a mass fraction of 1%, and the component C includes 0.1 parts by weight of a purslane extract, 0.2 parts by weight of a soybean sprout extract, and 10 parts by weight of a phosphate buffer;

[0085] The preparation method of soybean sprout extract is as follows: remove the head and root of soybean sprout, take the white part and chop the white part to obtain pretreated bean sprout, add 3 times the total mass of water to the pretreated bean sprout, heat at 95-100°C for 5h, filter and concentrate to obtain soybean sprout extract.

[0086] The preparation method of the purslane extract is as follows: add water 4 times the total mass of the purslane to the purslane and heat it at 95-100°C for 3 hours, filter and concentrate the filtrate to obtain an extract, add water to the extract to dissolve it and add petroleum ether to extract it, extract it three times, combine the aqueous phases, concentrate and freeze-dry it to obtain the purslane extract.

[0087] A method for using a phenolic acid inhibitor comprises the following steps: firstly placing fresh-cut apples into a component B and dipping them for 20 seconds, then placing them into a component C and dipping them for 3 minutes, and then placing them into a component A and dipping them for 2 minutes, and storing them at 3-5°C after the dipping is completed.

[0088] The method of using the phenolic acid inhibitor in Examples 6-11 is to immerse fresh-cut apples in component A for 2 minutes, and then store them at 3-5°C after immersion.

[0089] <Example 13>

[0090] A phenolic acid inhibitor, comprising component A, wherein component A comprises 1 part by weight of coumaric acid, 0.1 part by weight of polyethylene glycol, and 8 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 4;

[0091] The preparation method of the phenolic acid inhibitor is as follows: coumaric acid is firstly dissolved in polyethylene glycol, ultrasonicated at room temperature for 10 minutes, and then a phosphate buffer is added to the system.

[0092] <Example 14>

[0093] A phenolic acid inhibitor, comprising component A, wherein component A comprises 1 part by weight of kojic acid and 8 parts by weight of phosphate buffer, wherein the pH of the phosphate buffer is 5.5;

[0094] Dissolve kojic acid in phosphate buffered saline and dilute.

[0095] <Comparative Example 1>

[0096] The method of Example 12 was adopted to prepare phenolic acid inhibitors, wherein the difference was that the phenolic acid inhibitors included the following raw materials in parts by weight: 0.67 parts by weight of coumaric acid, 0.67 parts by weight of chlorogenic acid, 0.67 parts by weight of ferulic acid, 0.2 parts by weight of polyethylene glycol, 10 parts by weight of phosphate buffer, 0.1 parts by weight of Portulaca oleracea extract, 0.2 parts by weight of soybean sprout extract, and potassium citrate, wherein the mass fraction of potassium citrate in the system was 1%.

[0097] The preparation method is as follows: firstly, coumaric acid, chlorogenic acid and ferulic acid are dissolved in polyethylene glycol, then a phosphate buffer solution is added, and then a purslane extract, a soybean sprout extract and potassium citrate are added.

[0098] The method of use is to immerse fresh-cut apples in phenolic acid inhibitors for 2 minutes, and then store them at 3-5℃ after immersion.

[0099] <Comparative Example 2>

[0100] The method of Example 12 is used to prepare the phenolic acid inhibitor, wherein the difference is that: it includes component A and component C;

[0101] The method of use is to immerse fresh-cut apples in component C for 3 minutes, and then immerse them in component A for 2 minutes. After immersion, store them at 3-5°C.

[0102] <Comparative Example 3>

[0103] The method of Example 12 is used to prepare the phenolic acid inhibitor, wherein the difference is that: it includes component A and component B;

[0104] The method of use is to immerse the fresh-cut apples in component B for 20 seconds, and then immerse them in component A for 2 minutes. After immersion, store them at 3-5°C.

[0105] <Experimental Data>

[0106] 1. Inhibition of apple sPPO and mPPO

[0107] 1.1 Extraction of apple sPPO and mPPO crude enzyme solutions

[0108] Take 25 parts by weight of diced apples, add 25 parts by weight of 0.05M disodium hydrogen phosphate-citric acid buffer (pH 6.8) containing 0.11 parts by weight of EDTA (ethylenediaminetetraacetic acid), 0.25 parts by weight of PMSF (phenylmethylsulfonyl fluoride) and 5 parts by weight of PVPP (cross-linked polyvinyl pyrrolidone), and stir and homogenize for 90-100 seconds. After homogenization, filter the residue through four layers of gauze, and centrifuge the filtrate at 4°C and 10000 rpm for 20 minutes to obtain a precipitate and a supernatant, which is the sPPO crude enzyme solution;

[0109] Take the sPPO crude enzyme liquid extraction method to collect the precipitate after centrifugation, rinse it with 20 parts by weight of deionized water, add 20 parts by weight of 0.05M Tris-HCl buffer solution (pH 6.8) containing 0.2 parts by weight of Triton X-114, dissolve it, and stir it immediately to dissolve it completely. The resulting solution needs to be extracted by ultrasonic for 10 minutes, and then stand in a 4°C refrigerator for 30 minutes. Then centrifuge at 4°C, 10000rpm for 15 minutes, and the resulting supernatant needs to stand in a 4°C environment for 30 minutes, and then bathe at 37.5°C for 15 minutes. Immediately after the temperature induction is completed, centrifuge at 25°C, 11000rpm for 15 minutes. After the centrifugation is completed, slowly pour out the upper aqueous phase and retain the aqueous phase, which is the mPPO crude extract.

[0110] 1.2 Preparation of phenolic acid inhibitors

[0111] Phenolic acid inhibitors were prepared according to the components and methods of Examples 1-5, and the concentrations of phenolic acid inhibitors in each group of Examples 1-5 were prepared as 0, 1.0, 2.5, 5 and 10 mM. That is, Example 1 corresponds to coumaric acid, Example 2 corresponds to kojic acid, Example 3 corresponds to chlorogenic acid, Example 4 corresponds to gallic acid, and Example 5 corresponds to ferulic acid.

[0112] 1.3 Determination of inhibitory activity of PPO

[0113] The activity of PPO after treatment with each concentration of inhibitor in Examples 1-5 was measured using an ultraviolet spectrophotometer. The treatment method for each concentration of inhibitor was as follows: 0.2 mL of inhibitor and 0.2 mL of PPO solution were added to a test tube in sequence, mixed, and incubated at 37.5°C for 15 min. Then 2 mL of 0.2 M catechol solution (prepared using 0.05 M pH 6.8 phosphate buffer) was added, and the absorbance value was immediately measured at room temperature (25 ± 1°C) and 420 nm wavelength for 1 min. Each group of samples was measured three times, and the average value was taken. Figure 1 and Figure 2 As shown;

[0114] Relative activity = enzyme activity of PPO after inhibition by inhibitor / enzyme activity of PPO without addition of inhibitor.

[0115] 1.4 Determination of the inhibitory type of PPO

[0116] The enzymatic reaction rate of PPO treated with different concentrations of inhibitors and 0.2 mM catechol was measured, and the changes in its absorbance with the increase of PPO concentration were recorded. The curves were drawn with PPO concentration and reaction rate as the horizontal and vertical coordinates, and the inhibition type of the inhibitor on sPPO and mPPO was determined based on the correlation of the curves, as shown in Table 1.

[0117] 1.5 Determination of the inhibitory kinetics of PPO

[0118] The enzymatic reaction rate of PPO and catechol treated with different concentrations of inhibitors was measured, and the changes in its absorbance with the increase of catechol concentration were recorded. The Lineweaver-Burk double reciprocal graph was drawn with the reciprocal of substrate concentration and the reciprocal of reaction rate as the horizontal and vertical coordinates respectively.

[0119] K i and K is Calculated according to the following formula:

[0120] Slope=K m / V max +[I] / V max K i

[0121] Y-intercept=1 / V max (1+[I] / K is )

[0122] In the formula, V, V max , K i , K m and K is [S] and [I] represent the concentration of substrate and inhibitor, respectively. The results are shown in Table 1.

[0123] 1.6 Results Analysis

[0124] Figure 1 The effects of different concentrations of phenolic acid inhibitors on apple sPPO activity are shown. Figure 2 The effect of different concentrations of phenolic acid inhibitors on apple mPPO activity is shown. Figure 1 and Figure 2It can be seen that with the increase of inhibitor concentration, the activities of sPPO and mPPO gradually decreased, but the inhibition rate of the same inhibitor on mPPO was lower than that on sPPO. As shown in Table 1, the IC values ​​of p-coumaric acid (Example 1), kojic acid (Example 2), chlorogenic acid (Example 3), gallic acid (Example 4) and ferulic acid (Example 5) on sPPO were 50 The values ​​were 1.95, 2.33, 2.06, 4.73 and 3.21 mM respectively; while the IC values ​​of p-coumaric acid, kojic acid, chlorogenic acid, gallic acid and ferulic acid for mPPO were 50 The values ​​were 3.02, 3.25, 2.14, 3.75 and 2.39 mM, respectively. It can be seen that p-coumaric acid and chlorogenic acid have strong inhibitory effects on sPPO and mPPO, and the same inhibitor has a stronger inhibitory effect on sPPO than mPPO. In addition, gallic acid and ferulic acid have better inhibitory effects on mPPO than sPPO.

[0125] Table 1 Inhibition types and inhibition kinetics analysis of inhibitors on apple sPPO and mPPO

[0126]

[0127]

[0128] By studying the relationship between the enzyme reaction rate and PPO concentration, the inhibition types of these inhibitors on PPO were analyzed, such as Figure 3-12 As shown, with the increase of the concentration of p-coumaric acid, kojic acid, chlorogenic acid, gallic acid and ferulic acid, the slope of the straight line gradually decreases, and all pass through the origin, indicating that p-coumaric acid, kojic acid, chlorogenic acid, gallic acid and ferulic acid are reversible inhibitors of sPPO and mPPO.

[0129] In order to study the inhibitory mechanism of PPO, the Lineweaver-Burk plot was used to analyze the inhibitory kinetics of sPPO and mPPO. Figure 3-22 As shown in m The value of (horizontal coordinate intercept) gradually increases with the increase of p-coumaric acid and kojic acid concentrations, while 1 / V max The value of (vertical coordinate intercept) is unchanged, indicating that p-coumaric acid and kojic acid show competitive inhibition on sPPO and mPPO. max The value of gradually increases, while -1 / K m The intersection point is in the second quadrant, which is mixed inhibition. As the concentration of gallic acid and ferulic acid increases, 1 / V max The value of gradually increases, while -1 / K mThe value of intersects at one point on the X axis, indicating that gallic acid and ferulic acid exhibit non-competitive inhibition on sPPO and mPPO.

[0130] K i and K is represent the dissociation constants of free enzyme and enzyme-substrate complex binding to inhibitor, respectively, K i The lower the value, the stronger the binding effect between the inhibitor and the enzyme molecule. As shown in Table 1, p-coumaric acid and kojic acid have a significant effect on the K i The values ​​were lower than those of mPPO, indicating that the binding between the inhibitor and sPPO was stronger than that between mPPO, so the inhibitory effects of p-coumaric acid and kojic acid on sPPO were stronger than those on mPPO. i and K is In terms of IC values, chlorogenic acid has a stronger inhibitory effect on sPPO and mPPO than gallic acid and ferulic acid, which is consistent with the 50 The values ​​are consistent.

[0131] 2. Impact on the storage quality of fresh-cut apples

[0132] The methods of Examples 8-12 and Comparative Examples 1-3 were used to prepare phenolic acid inhibitors and treat fresh-cut apples, and fresh-cut apples were soaked in deionized water as a control experiment. Each group of experiments treated 20 fresh-cut apple slices.

[0133] Each group of apples was photographed every day (7 days), and the L* value, a* value and b* value of each group were measured using a colorimeter. The ΔE value was calculated according to the formula as a parameter for judging the degree of browning on the apple surface. Each group of samples was measured three times, and the results are shown in 23.

[0134] Color difference determination: In each group of experiments, 10 apple slices were randomly selected, and the L* (whiteness), a* (yellowness), and b* (redness) of the front and back sides of the apple slices were measured using a colorimeter, and the color difference ΔE was calculated, as shown in Table 2;

[0135] Chromatic difference change rate ΔE (%) = (ΔE n -ΔE0) / ΔE0×100%;

[0136] Among them, ΔE n is the color difference of fresh-cut apples on the nth day of storage, and ΔE0 is the color difference of fresh-cut apples on the 0th day of storage.

[0137] Table 2 shows the color difference change rate ΔE (%) of fresh-cut apples in the control group, Example 8-Example 11

[0138] Control group Example 9 Example 10 Embodiment 11 Example 8 1d <![CDATA[0.85±0.13 Cg ]]> <![CDATA[1.69±0.28 Ag ]]> <![CDATA[1.72±0.19 Ag ]]> <![CDATA[1.62±0.29 Ag ]]> <![CDATA[1.08±0.14 Bg ]]> 2d <![CDATA[5.60±0.72 Af ]]> <![CDATA[3.25±0.07 Bf ]]> <![CDATA[2.55±0.10 Bf ]]> <![CDATA[5.49±0.37 Af ]]> <![CDATA[2.04±0.41 Cf ]]> 3d <![CDATA[10.40±0.35 Ae ]]> <![CDATA[6.6±0.30 Ce ]]> <![CDATA[5.6±0.24 De ]]> <![CDATA[7.60±0.02 Be ]]> <![CDATA[3.12±0.19 Ee ]]> 4d <![CDATA[15.17±0.21 Ad ]]> <![CDATA[9.39±0.24 Cd ]]> <![CDATA[7.72±0.39 Dd ]]> <![CDATA[10.36±0.18 Bd ]]> <![CDATA[5.14±0.08 Ed ]]> 5d <![CDATA[16.92±0.4 Ac ]]> <![CDATA[10.63±0.57 Cc ]]> <![CDATA[8.75±0.24 Dc ]]> <![CDATA[11.86±1.05 Bc ]]> <![CDATA[7.29±0.24 Ec ]]> 6d <![CDATA[19.29±1.11 Ab ]]> <![CDATA[13.87±0.78 Bb ]]> <![CDATA[11.29±0.91 Cb ]]> <![CDATA[13.43±0.46 Bb ]]> <![CDATA[9.10±0.11 Db ]]> 7d <![CDATA[23.49±1.16 Aa ]]> <![CDATA[14.37±1.35 BCa ]]> <![CDATA[12.99±0.47 Ca ]]> <![CDATA[15.43±0.41 Ba ]]> <![CDATA[11.22±0.04 Da ]]>

[0139] Table 3 shows the color difference change rate ΔE (%) of fresh-cut apples in Example 12 and Comparative Examples 1-3.

[0140] Example 12 Comparative Example 1 Comparative Example 2 Comparative Example 3 1d <![CDATA[0.74±0.23 Bf ]]> <![CDATA[0.94±0.06 Bf ]]> <![CDATA[1.43±0.16 Ag ]]> <![CDATA[1.02±0.28 Bg ]]> 2d <![CDATA[1.06±0.02 Bf ]]> <![CDATA[1.29±0.04 Bf ]]> <![CDATA[2.72±0.95 Af ]]> <![CDATA[1.96±0.64 ABf ]]> 3d <![CDATA[1.90±0.03 Ce ]]> <![CDATA[2.49±0.48 BCe ]]> <![CDATA[4.96±0.91 Ae ]]> <![CDATA[3.17±0.57 Be ]]> 4d <![CDATA[3.05±0.25 Cd ]]> <![CDATA[4.53±0.07 Bd ]]> <![CDATA[6.34±0.56 Ad ]]> <![CDATA[5.11±0.02 Bd ]]> 5d <![CDATA[4.80±0.42 Dc ]]> <![CDATA[5.77±0.33 Cc ]]> <![CDATA[8.74±0.62 Ac ]]> <![CDATA[7.07±0.34 Bc ]]> 6d <![CDATA[6.95±0.40 Db ]]> <![CDATA[7.95±0.62 Cb ]]> <![CDATA[10.07±0.49 Ab ]]> <![CDATA[9.22±0.09 Bb ]]> 7d <![CDATA[8.48±0.06 Da ]]> <![CDATA[10.43±0.44 Ca ]]> <![CDATA[12.52±0.30 Aa ]]> <![CDATA[11.16±0.29 Ba ]]>

[0141] Note: In Table 2 and Table 3, different superscript letters (a-g) in the same column of data indicate significant differences (P<0.05), and different superscript letters (A-E) in the same row of data indicate significant differences (P<0.05);

[0142] From Table 2, Table 3 and Fig.23 It can be seen that the browning effect on the surface of apples in the blank control group was the most obvious as the storage time prolonged, and the color difference change rate showed the greatest upward trend with the increase in storage time; the apples treated with a composite coating composed of three phenolic acid inhibitors: 0.67 parts of p-coumaric acid, 0.67 parts of chlorogenic acid, 0.67 parts of ferulic acid, 0.2 parts of polyethylene glycol, and 10 parts of phosphate buffer had a small amount of browning on the surface after 7 days, and the color difference change rate was low within 7 days of storage, which can effectively protect the color of fresh-cut apples; the effect of apples treated with a composite coating of two phenolic acid inhibitors was second, but the color of fresh-cut apples treated with phenolic acid inhibitors was better than that of untreated apples. The three inhibitors had a good inhibitory effect on sPPO and mPPO, so they can also better inhibit the activity of PPO in apples to reduce enzymatic browning, playing a role in the anti-browning and preservation of fruits.

[0143] By comparing and analyzing the color difference change rate ΔE in Table 2 and Table 3, it can be seen that the present application can effectively slow down the browning of fruits and vegetables by using component A, component B and component C for treatment. By comparing and analyzing Example 12 and Comparative Example 1, it can be seen that the anti-browning effect of mixing component A, component B and component C together for treating apples is significantly lower than that of using component A, component B and component C separately for treatment. By comparing and analyzing Example 12 with Comparative Examples 2 and 3, it can be seen that when only component A and component C are used, and only component A and component B are used for treating apples, the anti-browning effect of phenolic acid inhibitors is significantly lower than that of Example 12. This shows that the present application has a better protective effect on apples by using component A, component B and component C, and the protection effect is best when component B, component C and component A are used in sequence.

[0144] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A phenolic acid inhibitor, characterized in that: Including component A, component A includes the following raw materials: Phenolic acid and phosphate buffer, wherein the phenolic acid is one or more of coumaric acid, kojic acid, chlorogenic acid, gallic acid and ferulic acid.

2. The phenolic acid inhibitor according to claim 1, characterized in that Component A comprises the following raw materials in parts by weight: 1 to 3 parts by weight of phenolic acid and 8 to 12 parts by weight of phosphate buffer.

3. The phenolic acid inhibitor according to claim 2, characterized in that The phenolic acid is one or more of coumaric acid, chlorogenic acid and ferulic acid.

4. The phenolic acid inhibitor according to claim 3, characterized in that Component A also includes 0.1-0.3 parts by weight of polyethylene glycol.

5. The phenolic acid inhibitor according to claim 4, characterized in that The pH of phosphate buffer is 4-6.

8.

6. The phenolic acid inhibitor according to claim 5, characterized in that It also includes a component B and a component C, wherein the component B includes a potassium citrate aqueous solution with a mass fraction of 1%, and the component C includes 0.1 parts by weight of a purslane extract, 0.2 parts by weight of a soybean sprout extract, and 10 parts by weight of a phosphate buffer; The preparation method of soybean sprout extract is as follows: remove the head and root of soybean sprout, take the white part and chop the white part to obtain pretreated bean sprout, add water 3 times the total mass of the pretreated bean sprout to the pretreated bean sprout, heat at 95-100° C. for 5 hours, filter and concentrate to obtain soybean sprout extract; The preparation method of the purslane extract is as follows: add water 4 times the total mass of the purslane to the purslane and heat it at 95-100°C for 3 hours, filter and concentrate the filtrate to obtain an extract, add water to the extract to dissolve it and add petroleum ether to extract it, extract it three times, combine the aqueous phases, concentrate and freeze-dry it to obtain the purslane extract.

7. The use of the phenolic acid inhibitor according to any one of claims 1 to 6, characterized in that: The phenolic acid inhibitor is used for inhibiting the activity of polyphenol oxidase in apples.

8. A method of using the phenolic acid inhibitor according to claim 5, characterized in that: The following steps are involved: First, immerse the fresh-cut apples in component B for 20 seconds, then in component C for 3 minutes, and then in component A for 2 minutes. After immersion, store them at 3-5°C.

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