Sterilization and preservation method of freshly squeezed fruit juice

Abstract

The invention discloses a sterilizing and fresh-keeping method of freshly squeezed fruit juice, which comprises the following steps: adding butanedione not exceeding 0.5 mM into the freshly squeezed fruit juice, irradiating with ultraviolet light for 1-5 minutes, and packaging the fruit juice into a clean and sterile container; according to the method, by inhibiting microbial growth and reducing the activity of polyphenol oxidase, the dual goals of fruit juice sterilization and fresh keeping are achieved, the problems of browning, nutrition loss, flavor change and the like caused by too high processing temperature in fruit juice production are solved, the obtained fruit juice is bright in color and fresh and refreshing in taste, and the original flavor of the fruit juice is reserved to the maximum extent.

Description

Technical Field

[0001] The invention belongs to the field of fruit disinfection, and in particular relates to a method for sterilizing and preserving fresh-squeezed juice by using an ultraviolet light / diacetyl process. Background Art

[0002] In terms of disease transmission, food safety and public health, scientific and effective disinfection is an indispensable measure to maintain health and safety, and is crucial to the stability and sustainable development of the global society (Skowron et al., Lett. Appl. Microbiol., 2022, 74, 482-497). Taking fruit disinfection as an example, fruits are easily contaminated by microorganisms during production, processing and packaging. About 53% of foodborne disease outbreaks are caused by bacteria, 42.5% by viruses, and 4.5% by other parasites (Crit. Rev. Food Sci. Nutr., 2020, 60, 940-975). Representative bacteria include Escherichia coli and Staphylococcus aureus. It is estimated that the economic cost associated with foodborne diseases in the United States is more than $5 billion each year, involving more than 480,000 people, 128,000 hospitalizations and 3,000 deaths (Xiang et al., Crit. Rev. Food Sci. Nutr., 2022, 62, 2250-2268). Therefore, microbial safety has become a challenge for consumers. Secondly, even slight processing operations can cause the fruit to deteriorate and change color. The discoloration of fresh-cut fruits is mostly caused by enzymatic browning, and the key enzyme of enzymatic browning is mainly polyphenol oxidase (PPO) (Navina et.al., Trends Food Sci.Tech., 2023, 104128). Browning not only affects visual perception, but also flavor. Therefore, in the processing of fruits and vegetables, not only sterilization issues should be considered, but also preservation issues.

[0003] Existing disinfection technologies include physical methods, chemical methods and combined methods. The investment and operation costs of most equipment in physical methods are high. The chemical method uses a large amount of reagents, is costly and produces disinfection by-products. Compared with a single technology, the combined method has certain advantages, which can reduce the dosage and reduce the cost (Deng et al., Crit. Rev. Food Sci. Nutr., 2020, 60, 2481-2508). In the combined method, the combination of UV / chemical reagents is more commonly used. The ultraviolet light system is relatively affordable, but it also has some defects. For example, the UV dose used in UV / ClO2 is high, and disinfection by-products chlorate and hypochlorite are produced; the H2O2 used in UV / H2O2 has a high concentration, decomposes quickly, and has plant toxins; the UV / peracetic acid method has a poor sterilization effect and is not allowed to be used to wash fresh-cut fruits (Mendoza et al., LWT, 2022, 165, 113714). In general, the existing technology is difficult to meet the needs of fruit preservation, and it is impossible to take into account the requirements of sterilization and anti-browning at the same time. Therefore, the fruit disinfection industry urgently needs a new disinfection method to meet the dual needs of sterilization and anti-browning.

[0004] In nature, diacetyl (BD) is widely present in the essential oils of many plants. In EU countries, BD is a legal spice and can be used as a food additive. Patent CN110777026A discloses a method for preparing a fermented mare's milk beer beverage, which mentions that the fermentation process of sour mare's milk decomposes lactose to produce lactic acid and volatile substances BD, giving yogurt a typical flavor. BD inhibits the growth of decay-causing pathogens during the acidification process. Patent CN111109558A discloses a high antioxidant lactic acid bacteria complexed Cu 2+ Browning inhibition technology uses the combined effects of Lactobacillus brevis metabolites, including acid, ethanol, BD, H2O2 and other metabolites, to complex Cu 2+ ions, rapidly consume oxygen, produce organic acids and CO2, create an acidic environment that is almost anaerobic, inhibit PPO activity, and thus inhibit enzymatic browning. Patent CN115769835A uses 500-1200 μL stock solution / L solution of BD to dip fresh horticultural products for 30 minutes to delay the aging of fruits. At present, there is no report on the method of preserving fresh squeezed fruits by using the combined action of UV / BD. Summary of the invention

[0005] In view of the defects in the prior art, the present invention provides a novel method for fruit juice disinfection, which utilizes UV / BD to preserve freshly squeezed juice, reduces microbial contamination during juice processing, and ensures the quality and taste of the juice.

[0006] Specifically, the above public objectives are achieved through the following technical solutions:

[0007] (A) Fruit cleaning: Before processing, fruits need to be cleaned by a cleaning machine or manually to fully remove mud impurities and residual pesticides on the surface of the fruits.

[0008] (B) Juicing and clarification: After peeling and removing the core of the fruit, the pulp is homogenized using a homogenizer, then coarsely filtered using gauze, and then centrifuged at 10,000 r / min at 4°C for 5 min. After centrifugation, the precipitated pulp is discarded and the supernatant obtained is the freshly squeezed juice.

[0009] (C) Sterilization: (a) Step: Add BD to freshly squeezed juice and stir evenly. (b) Step: Expose the liquid obtained in step (a) under ultraviolet light. The chemical formula of BD in step (a) is CH3COCOCH3, and the concentration added to the juice does not exceed 0.5mM. In step (b), the juice is exposed to ultraviolet light (such as a mercury lamp) for 5 minutes at an intensity of 0.5mW / m 2 That is, the sterilization and preservation of freshly squeezed juice can be achieved, the browning of juice can be inhibited, the pH of juice can be maintained, the total phenol content of juice can be reduced, and the activity of PPO can be inhibited.

[0010] Furthermore, the above-mentioned fruit includes at least one of apples, peaches and pears.

[0011] The above-mentioned sterilization refers to killing at least one of Escherichia coli or Staphylococcus aureus.

[0012] In the UV / BD system of the present application, BD undergoes Norrish I type cleavage after being excited by UV light, generating acetyl radicals, which can combine with water molecules to form diol radicals or react with dissolved oxygen to generate peroxide radicals. The free radicals can act on bacteria, causing cell lysis and death. In addition, UV / BD treatment also has the ability to resist browning from three aspects. First, it can quickly consume dissolved oxygen and eliminate the browning environment. Second, it weakens the PPO enzyme activity and reduces the browning potential. Third, it converts the substrate or product in the browning reaction and interferes with the polymerization reaction related to browning.

[0013] The present invention not only achieves a good sterilization effect and avoids the quality impact caused by heating in traditional processes, but also prolongs the shelf life of the juice and retains the nutrition and flavor of the juice. Compared with the prior art, it has the following beneficial effects:

[0014] (1) The combination of ultraviolet light and BD has significant sterilization and preservation effects. Compared with single ultraviolet light treatment and single chemical reagent treatment, this method shortens the treatment time, reduces the required ultraviolet light dose and BD dosage, saves costs, and is a green, clean and green technology.

[0015] (2) Compared with other chemical reagents used in juice preservation, such as H2O2, ClO2, and peracetic acid, BD is low-cost, non-toxic, and does not produce harmful disinfection by-products.

[0016] (3) The UV / BD method can not only kill microorganisms in the juice but also prevent browning. The processed juice tastes sweet and fresh without bitterness, thus maintaining the natural quality of the juice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a flow chart of the method for preparing juice and UV / BD sterilization in the present invention.

[0018] Figure 2 This is the killing effect of UV / BD on apple juice concentrate in Example 1.

[0019] Figure 3 is the browning inhibition effect of UV / BD on juice during storage in Example 2;

[0020] Among them, the browning inhibition effect of (a) apple juice, (b) peach juice and (c) pear juice.

[0021] Figure 4 The effect of UV / BD on the quality of apple juice during storage time in Example 3;

[0022] Where (a) is the change in titratable acid, (b) is the change in turbidity, and (c) is the change in pH.

[0023] Figure 5 This is a schematic diagram of the effect of UV / BD on the total phenolic content of apple juice during storage in Example 4.

[0024] Figure 6 This is a schematic diagram of the effect of UV / BD on the activity of polyphenol oxidase in apple juice during storage in Example 5. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the embodiments.

[0026] (1) Examples of Reagents

[0027] The apples used in the examples are Fuji apples purchased from Nanjing Vegetable Market, Jiangsu Province. Diacetyl (hereinafter referred to as BD) was purchased from Shanghai MacLean Biochemical Co., Ltd., China.

[0028] Sodium bicarbonate, sodium carbonate, acetic acid, sodium acetate, sodium hydroxide, anhydrous ethanol, catechol, and polyvinyl pyrrolidone were purchased from Shanghai Sinopharm Holding Co., Ltd., China.

[0029] (2) Photochemical reaction device

[0030] The UV reaction device was a Perfek multi-channel photoreactor (PCX-50C), provided by Beijing Perfek Technology Co., Ltd., and the light intensity was 0.5 mW / cm 2 , wavelength 254nm, the light source rotates every 5s.

[0031] (3) Detection method

[0032] The total colony count of apple juice is determined according to the method specified in GB 4789.2-2010. Use a sterile pipette to draw 0.5 mL of sample into a sterile centrifuge tube containing 4.5 mL of physiological saline, mix thoroughly, and make a 1:10 sample homogenate. Prepare 10-fold serial dilution sample homogenates according to the above steps. According to the estimation of the sample contamination status, select 2 to 3 sample homogenates or stock solutions with appropriate dilutions. Pour 15 mL to 20 mL of LB agar culture medium cooled to 46°C into the plate in time, and rotate the plate to mix it evenly. After the agar solidifies, turn the plate over and incubate it at 37°C for 12 hours. Select the plate with a colony count between 30 CFU and 300 CFU and no spreading colony growth to count the total colony count. The colony count of each dilution should be the average of two plates.

[0033] Determination of browning during storage of apple juice: After the apple juice is sealed and stored at 4°C, samples are taken regularly. The absorbance at 420nm is measured using a spectrophotometer. The increase in absorbance is used to indicate the degree of browning of the juice. The determination of the browning degree of peach juice and pear juice is the same as that of apple juice.

[0034] The titratable acidity of apple juice is determined according to the acid-base neutralization principle. Weigh 5 mL of apple juice sample, dilute to 50 mL with distilled water, and shake well. Take 20 mL of filtrate, add 2 drops of 1% phenolphthalein indicator solution, and titrate with 0.01 mol / L sodium hydroxide solution. The titration is considered complete when pink begins to appear in the filtrate and remains unchanged for 30 seconds (pH 8.1-8.3). The titratable acidity content calculation formula (1) is as follows:

[0035]

[0036] Where V is the total volume of sample extract, mL; c is the NaOH concentration, mol / L; V1 is the volume of NaOH consumed in the filtrate, mL; f is the conversion factor 0.067 (malic acid); V s is the volume of the filtrate taken during titration, mL; V m is the apple juice sample volume, mL.

[0037] The turbidity of apple juice was measured using a turbidity meter (Haco, 2100P). 25 mL of apple juice was taken and shaken, and ultrapure water was used as a blank to measure the turbidity of the apple juice.

[0038] The pH of apple juice was measured using a pH meter. 25 mL of apple juice was taken and shaken, and then the pH of the apple juice was measured.

[0039] The total phenol content of apple juice was determined by the Folin-Ciocalteau method. (1) Drawing of gallic acid standard curve Accurately weigh 0.500 g of gallic acid, add distilled water to 100 mL to obtain a 5000 mg / L gallic acid standard solution. Add 0, 4, 8, 12, 16 and 20 mL of gallic acid standard solution to 6 100 mL volumetric flasks, add distilled water to 100 mL, and prepare test solutions with concentrations of 0, 200, 400, 600, 800, and 1000 mg / L respectively. Then take 0.2 mL of 0, 200, 400, 600, 800, and 1000 mg / L gallic acid standard solution, then add 0.5 mL of Folin-Ciocalteau reagent, shake to mix thoroughly, then add 1.5 mL of 20% Na2CO3 solution, mix thoroughly, and dilute to 10 mL. After standing for 1 hour, measure its absorbance at 765nm. Use the concentration of gallic acid standard solution (mg / L) as the horizontal axis and the absorbance value (A) as the vertical axis to obtain the regression equation. (2) Sample determination Accurately pipette 0.2mL of the test solution, and operate according to the steps of the standard curve. Finally, calculate the gallic acid content of total phenols in the test solution based on the standard curve.

[0040] Apple juice PPO enzyme activity assay. (1) Crude enzyme extract: Wash, peel, core and juice the apples. Add 100g of apples to 100mL of 0.05M phosphate buffer solution (containing 3% (w / v) PVP) precooled at 4°C, mix well, pass through gauze, centrifuge at 10000rpm and 4°C for 5min to obtain the supernatant. (2) PPO activity assay: Use 0.1M catechol as the reaction substrate and prepare 0.1M catechol buffer solution with 0.05M phosphate buffer solution with a pH of 6.0. Pipette 0.5mL of enzyme solution into a cuvette, add 2.5mL of the above-prepared catechol buffer, shake well, put into a spectrophotometer and measure the absorbance at 390nm. Define the absorbance change of 0.01 per minute as 1 enzyme activity unit. The calculation formula (2) for apple juice PPO activity is as follows:

[0041]

[0042] Where V is the total volume of crude enzyme extract, mL; V1 is the volume of enzyme extract during measurement, mL; and m is the mass of apple, g.

[0043] The following examples of juice preparation methods:

[0044] The fruits are picked manually, and those that are rotten, severely mechanically damaged, or have diseases and insect pests are removed. They are manually cleaned before processing to remove mud impurities and residual pesticides on the surface of the fruits. After the fruits are peeled and cored, the pulp is homogenized using a homogenizer, then coarsely filtered using gauze, and then centrifuged at 10,000 r / min at 4°C for 5 min. The precipitated pulp is discarded, and the supernatant is the juice to be tested.

[0045] Example 1

[0046] The inhibitory effect of UV / BD on the total bacterial count in apple juice

[0047] 8 mL of apple juice was added to the Perfil reaction bottle, and then 2 mL of 2.5 mM BD diluent was added to make the final concentration of BD in the apple juice 0.5 mM. A blank control group (CK) without light and without BD was set up; a control group (UV) with only light and no BD added, and a control group (BD) with only BD added but no light. The quartz reaction bottle was sealed with a fully transparent film and placed in a photoreactor (0.5 mW / cm 2 ), after 300 seconds of reaction, take 0.5 mL of sample and place in a sterile centrifuge tube, dilute it in gradient, and take 100 μL of apple juice to spread. Then turn the plate over and place it in an incubator for incubation. After 12 hours, count the bacteria and record them. The remaining apple juice was sealed and stored in a refrigerator at 4°C, and samples were taken after 1, 3, and 5 days.

[0048] Figure 2 The figure shows the changes in the total plate count of the above-mentioned apple juice treatment groups after 300s of illumination. It can be seen from the figure that the initial colony count of freshly squeezed apple juice is 2.06 logarithms. After UV, BD and UV / BD treatment, the colony count of UV / BD is significantly different from that of the other three groups, and the bactericidal effect is the best, which is 0.43 logarithms. From the third day during storage, the colony count of apple juice treated with UV / BD dropped to 0, and there was no further growth until the fifth day. This is because once BD is excited by UV, it undergoes Norrish I type cleavage to produce acetyl groups. Acetyl groups can react with DO to generate peroxyacetyl radicals, which have strong oxidizing properties and attack bacteria, causing cell lysis and death. There are signs of a decrease and then an increase in the BD group. The possible reason is that BD has antibacterial activity. After BD treatment, bacteria will be damaged by oxidation, and then the bacteria will mobilize antioxidant mechanisms to cope with oxidative stress to protect bacteria from the destructive effects of ROS.

[0049] Example 2

[0050] The processing flow of this embodiment is as follows Figure 1 shown.

[0051] 8 mL of apple juice was added to the Perfil reaction bottle, and then 2 mL of 2.5 mM BD diluent was added to make the final concentration of BD in the apple juice 0.5 mM. A blank control group (CK) without light and without BD was set up; a control group (UV) with only light and no BD added, and a control group (BD) with only BD added but no light. The quartz reaction bottle was sealed with a fully transparent film and placed in a photoreactor (0.5 mW / cm 2 ) and react for 5 min. After the reaction, take 1 mL of apple juice and measure the absorbance at a wavelength of 420 nm. The remaining apple juice was sealed and stored in a refrigerator at 4°C, and samples were taken and the absorbance was measured after 1, 3, and 5 days.

[0052] Figure 3 (a)-(c) are the browning inhibition rate changes of the apple juice, peach juice and pear juice treatment groups after 300s of light exposure. Figure 3 It can be seen that after UV / BD treatment, the browning degree of apple juice was significantly inhibited compared with UV (P < 0.05), and the inhibition rate was 20.85%. During storage, the anti-browning effect of apple juice treated with UV / BD was always higher than that of the BD group, and the highest inhibition rate was 37.76% on the second day, while the anti-browning effect of UV was poor, and the browning inhibition rate remained at around 2%. For peach juice, the browning inhibition rate of UV / BD remained the highest during storage compared with the UV group and BD group (P < 0.05). Similarly, the preservation ability of UV / BD on pear juice showed an upward trend during storage, and the inhibition rate could reach up to 25.2%. In summary, UV / BD has a significant inhibitory effect on the browning of juice during storage and has a protective effect on the color of juice.

[0053] Example 3

[0054] The titratable acidity and pH of juice are important factors affecting the taste. The changes of titratable acidity and pH of apple juice treated with different methods during storage are as follows: Figure 4 As shown in (a) and (c). The UV, BD and UV / BD treatment groups had no significant effect on the titratable acid and pH of apple juice. During the storage period, the pH of apple juice treated with BD increased fastest within the third day, reaching 3.98, and then decreased to 3.88, close to the initial pH of 3.90. The pH of the UV group decreased to 3.77 on the fifth day, which may be caused by the decrease in the pH value of the control group CK. The pH of the UV / BD group remained generally balanced during the storage period.

[0055] Turbidity is a measure of the degree of light scattering by soluble solids and suspended particles in water. The turbidity of the solution system directly affects the effectiveness of UV sterilization. The changes in turbidity of apple juice treated with different methods during storage are shown in Figure 2. Figure 4 As shown in (b), the turbidity change trends of the treatment groups and their respective CKs were consistent, with no significant difference, and they generally showed an upward trend during the storage period.

[0056] Example 4

[0057] Phenolic compounds are one of the main contributors to the antioxidant effect of fruit juice. Polyphenols have a variety of biological properties, such as anti-inflammatory, antioxidant, antiviral, and anti-cancer functions. During processing and storage, phenolic substances degrade, oxidize, and rapidly polymerize, so the total phenol content is an important indicator of juice quality. Figure 5 The results show the changes of total polyphenol contents in apple juice treated with different treatments during storage time. On the 0th and 1st day, the UV, BD and UV / BD treatment groups had no significant effect on the total polyphenol contents of apple juice. From the 3rd day onwards, UV treatment could significantly reduce the total polyphenol contents of apple juice (P < 0.05), while there was still no difference between BD and UV / BD and CK groups. On the 5th day, the total polyphenol contents of apple juice treated with BD and UV / BD were significantly reduced compared with those on the 3rd day, but this phenomenon was not observed in the UV group. However, on the 5th day, the total polyphenol contents of the UV / BD group were higher than those of the CK group. During the entire storage period, the total polyphenol contents of apple juice treated with UV increased, the total polyphenol contents of apple juice treated with BD remained balanced, and the total polyphenol contents of apple juice treated with UV / BD decreased.

[0058] Example 5

[0059] The oxidation reaction that occurs during the processing is related to the catalytic activity of the inherent enzymes in the fruit, and this process is mainly mediated by PPO. Inhibiting the activity of this enzyme is beneficial to reduce the degree of enzymatic browning of the juice, thereby maintaining the color and nutritional content of the juice.

[0060] The changes in PPO activity of apple juice treated with different methods during storage are shown in Figure 2. Figure 6 As shown. The PPO activity of apple juice treated with UV was significantly reduced (P < 0.05), and there was no significant change in other treatment groups compared with their respective CK groups. The PPO activity showed an overall downward trend with the extension of storage time. During the first to third days, the PPO activity of apple juice in the BD and UV / BD groups was significantly reduced compared with that in the CK group (P < 0.05), and the PPO activity of apple juice in the latter was the lowest at 0.592 U / g·min.

[0061] The above results show that after low-concentration BD and low-dose UV treatment, freshly squeezed apple juice has a small amount of phenolic substances under the action of PPO, and the production of quinone compounds is also small, which further reduces the browning degree of apple juice.

Claims

1. A method for sterilizing and preserving freshly squeezed juice, characterized in that: Add diacetyl to freshly squeezed juice to obtain a mixed solution; then place the mixed solution under ultraviolet light to sterilize and preserve the freshly squeezed juice.

2. The method according to claim 1, characterized in that In the mixed solution, the concentration of diacetyl does not exceed 0.5 mM.

3. The method according to claim 1, characterized in that The ultraviolet light intensity does not exceed 0.5 mW / m 2 .

4. The method according to claim 1, characterized in that: The irradiation time under ultraviolet light is 1-5 min.

5. The method according to claim 1, characterized in that The freshly squeezed juice is obtained by the following method: taking the pulp of vegetables and fruits, homogenizing it, filtering it and then centrifuging it, and the obtained supernatant is the freshly squeezed juice.

6. The method according to claim 1, characterized in that The freshly squeezed juice includes at least one of apple juice, peach juice or pear juice.

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