Method for sterilizing juice by naringenin combined with ultrasound

Abstract

The application discloses a method for sterilizing fruit juice based on naringenin combined with ultrasonic treatment, and takes Bacillus acidiproducens as the research object, and takes naringenin as a sterilization synergist, and after 0.15 mg / mL naringenin is added into the fruit juice, ultrasonic treatment is carried out, and synergistic sterilization effect is realized. The method can ensure the sterilization effect, reduce the problems of browning, nutrient loss and flavor change of the fruit juice, endow the fruit juice with stronger antioxidant capacity, maximize the retention of the original flavor of the fruit juice, has a good industrial application prospect, and can further promote the development and upgrading of the fruit juice processing technology.

Description

Technical Field

[0001] This invention belongs to the field of fruit juice processing technology, specifically relating to a method for sterilizing fruit juice using naringenin combined with ultrasound. Background Technology

[0002] my country is the world's largest fruit producer, with a fruit output reaching 320 million tons in 2023. Relying on its abundant and high-quality raw materials, the fruit juice industry is continuously transforming towards high-end products as consumption upgrades, and the market share of high-quality fruit juices is constantly increasing. To better adapt to the global distribution network and effectively extend the shelf life of fruit juice, it is of significant strategic importance to the industry's development. However, fruit juice is often easily contaminated by *Bacillus cyclophosphamide*, whose spores can withstand pasteurization and grow and multiply during storage and transportation, producing unpleasant odors and causing spoilage. Heat processing, as a key step in fruit juice processing, is commonly used in the food industry to inactivate microorganisms and extend shelf life. However, heat processing easily causes browning of fruit juice, decline in product flavor, and loss of nutrients. Therefore, developing technologies that can reduce nutrient loss, maintain the sensory quality of food, and are suitable for commercial promotion is of significant practical importance.

[0003] Naringenin possesses abundant pharmacological activities, including antibacterial, anti-inflammatory, antioxidant, immunomodulatory, and cardioprotective effects. It is commonly used in fruit juices, candies, and beverages to reduce sweetness and enhance flavor complexity; its significant antioxidant capacity helps delay oxidative spoilage and extend shelf life. The antibacterial effect of naringenin is primarily manifested in its disruption of bacterial cell walls and membranes, as well as its interference with bacterial metabolic pathways. Furthermore, naringenin can affect fundamental physiological processes such as bacterial DNA and protein synthesis, inhibiting bacterial proliferation and the expression of bacterial virulence factors. In summary, naringenin exerts its effects through multiple pathways, demonstrating antibacterial effects against both Gram-positive and Gram-negative bacteria.

[0004] Ultrasound is a sound wave with a frequency exceeding 20 Hz-20 kHz. Ultrasonic sterilization technology utilizes the cavitation effect generated by the mechanical vibration of ultrasound waves in the transmission medium. This cavitation effect, through mechanical and chemical action, kills or inhibits microorganisms in a very short time. Ultrasonic parameters, bacterial state, and food properties all affect the sterilization effect of ultrasound in food processing and the nutritional and sensory evaluation of the product. Some of these effects are positive, while others are negative. For example, high-frequency ultrasound can damage fruit and vegetable tissues, causing nutrient loss and reducing firmness. Furthermore, the high energy requirements and expensive equipment also limit the commercial application of ultrasound. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a novel method for sterilizing fruit juice by organically combining naringenin with ultrasound for fruit juice sterilization.

[0006] The technical solution of the present invention is as follows:

[0007] A method for sterilizing fruit juice using naringin combined with ultrasound, characterized in that: naringin is added to the fruit juice, and then ultrasonic treatment is performed to obtain sterilized fruit juice.

[0008] In some embodiments of the present invention, the concentration of naringenin is 0.15 mg / mL.

[0009] In some embodiments of the present invention, the ultrasonic power is 450-600 W, the ultrasonic time is 30 min, the probe is inserted 1-1.5 cm into the liquid surface, and the working time and the interval time are both set to 2 s.

[0010] In some embodiments of the present invention, the temperature is controlled below 30°C during the ultrasonic treatment process.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] (1) Safe, environmentally friendly, energy-saving and cost-reducing. This invention adds a plant-derived bactericide—naringenin—and combines it with ultrasonic treatment. While killing Bacillus cereus and its spores, it significantly reduces the concentration of naringenin and the ultrasonic power, saving production costs and meeting the requirements of green manufacturing.

[0013] (2) Significant sterilization effect. The synergistic effect of naringenin and ultrasound in this invention can inactivate the vegetative cells of Bacillus cyclophosphamide in a short time and inhibit the growth of residual spores, thus significantly delaying the spoilage of the juice and extending its shelf life.

[0014] (3) Preserving nutrients and improving quality. Compared with traditional heat sterilization processes, this invention is a non-heat sterilization technology, which can significantly reduce the damage to the nutrients in the juice. The synergistic treatment of this invention not only does not significantly affect the quality of the juice, but the lower concentration of naringenin can also reduce the bitterness of the juice and improve its flavor. In addition, due to the antioxidant properties of naringenin itself, it can also enhance the overall antioxidant capacity of the juice. Attached Figure Description

[0015] Figure 1 This is a diagram showing the inhibition zone of naringenin against Bacillus cyclophosphamide.

[0016] Figure 2 The study aimed to investigate the inactivation effects of different concentrations of naringenin combined with ultrasound on the vegetative cells (a) and spores (b) of Bacillus cereus.

[0017] Figure 3To investigate the synergistic effect of naringenin with different ultrasonic powers on the inactivation of vegetative bodies (a) and spores (b) of Bacillus cereus.

[0018] Figure 4 The effect of initial bacterial concentration on the synergistic effect of naringenin on ultrasonic inactivation of Bacillus cereus vegetative cells (a) and spores (b).

[0019] Figure 5 Changes in Bacillus cereus vegetative cells in apple juice treated with ultrasound, combining naringenin and anaerobic acid, during storage at 4°C (a) and 25°C (b).

[0020] Figure 6 Changes in Bacillus cereus spores in apple juice treated with ultrasound, including those containing naringenin, during storage at 4°C (a) and 25°C (b).

[0021] Figure 7 The electronic nose was used to determine the flavor of apple juice under different treatments at storage conditions of 4℃ (a) and 25℃ (b). Detailed Implementation

[0022] Example 1: Study on the antibacterial ability of naringenin

[0023] (1) Preparation of vegetative suspension and spore suspension of Bacillus acidophilus

[0024] The activated bacterial strain was inoculated into AAM liquid medium and cultured for 6 h. The culture was then washed with sterile physiological saline, resuspended, and the bacterial suspension concentration was adjusted to 10. 7 CFU / mL refers to the vegetative cell suspension. The activated strain is inoculated into liquid culture medium and cultured at 45°C for approximately 7 days. After heat treatment at 80°C for 10 minutes to kill any remaining vegetative cells, spores are collected by centrifugation. Then, residual substances and cell debris in the culture medium are removed using sterile physiological saline. Finally, the concentration is adjusted to approximately 10. 7 CFU / mL.

[0025] (2) Determination of inhibition zone

[0026] The inhibitory effect of naringenin on Bacillus cyclophosphamide was determined using the perforation method, and the results are as follows: Figure 1 As shown, the inhibition zone was measured to be 26.10 ± 0.27 mm, indicating that naringenin has a strong inhibitory effect on Bacillus cereus.

[0027] (3) Measurement of MIC and MBC

[0028] Bacillus acidophilus was inoculated into AAM liquid medium and fruit juice containing different concentrations of naringenin. OD was measured after 24 h of culture. 600 Value. MIC is the OD value of the culture medium. 600The minimum concentration of antibacterial substances with a variation value less than 0.05. The OD of the culture medium... 600 Culture media with a variation value less than 0.05 were spread onto AAM solid medium. The minimum concentration of the inhibitory substance that resulted in no microbial growth was considered the MBC, and the results are shown in Table 1. In AAM medium, the MIC of naringenin against both vegetative cells and spores of *Bacillus cereus* was 0.15 mg / mL, indicating that 0.15 mg / mL of naringenin could completely inhibit the growth of *Bacillus cereus*. While the MBC of naringenin against vegetative cells was 0.15 mg / mL, naringenin could not kill *Bacillus cereus* spores. In fruit juice, the MIC of naringenin against *Bacillus cereus* decreased to 0.05 mg / mL, but even at concentrations as high as 5 mg / mL, naringenin still could not completely kill the spores, possibly due to the strong resistance of the spores.

[0029] Table 1. MIC and MBC of naringenin against Bacillus cyclophosphamide.

[0030]

[0031] Example 2: The killing effect of naringenin combined with ultrasound on Bacillus cyclophosphamide in fruit juice

[0032] (1) Study on the inactivation effect of different concentrations of naringenin combined with ultrasound on Bacillus cyclophosphamide.

[0033] Adjust the nutrient suspension with apple juice (10) 5 CFU / mL) and spore suspension (10 4 Naringenin was added at concentrations of 0, 1 / 2 MIC, 1 MIC, and 2 MIC. The cells were sonicated using an intermittent ultrasonic cell disruptor equipped with a 6 mm diameter amplitude transformer, which was inserted 1-1.5 cm into the suspension. The working and intermittent times were both set to 2 s. The temperature was maintained below 30℃ using a constant-temperature water bath. Alternatively, sonication was performed at 300 W. Samples were taken at different time points, diluted 10-fold to the appropriate concentration, plated on AAM plates, and incubated at 45℃ for 24 h for cell counting. Results are as follows: Figure 2 As shown, 1 MIC of naringenin, in synergy with ultrasound, reduced the number of vegetative cells of *Bacillus cereus* by 4.04 log, an increase of 0.99 log compared to the control group, and reduced the number of spores by 1.05 log. In contrast, ultrasound alone had almost no bactericidal effect on spores. The results indicate that the addition of naringenin significantly enhanced the inactivation effect of ultrasound on both vegetative cells and spores of *Bacillus cereus*.

[0034] (2) Study on the synergistic effect of different ultrasonic powers and naringenin on the inactivation of Bacillus cyclophosphamide

[0035] Adjust the concentration of the nutrient and spore suspension to 10 using apple juice. 5 CFU / mL, 1MIC concentration of naringenin was added to the prepared bacterial suspension, and the power of the intermittent ultrasonic cell disruptor was adjusted to 150 W, 300 W, 450 W, and 600 W for ultrasonic treatment. Samples were then taken and counted after treatment. Results are as follows: Figure 3 As shown, the inactivation effect of naringenin synergistic with ultrasound on Bacillus cyclophosphamide gradually increased with increasing ultrasound power; 600 W ultrasound reduced vegetative somatic cells by 5.18 log and spores by 1.33 log. Power mainly affects the bactericidal effect by influencing cavitation; higher power often induces stronger cavitation and produces a stronger bactericidal effect.

[0036] (3) Study on the effect of different initial bacterial concentrations on the synergistic effect of ultrasound and naringenin on the inactivation of Bacillus cyclophosphamide.

[0037] Adjust the nutrient suspension with apple juice (10) 5 10 6 10 7 10 8 CFU / mL) and spore suspension (10 3 10 4 10 5 10 6 A concentration of naringenin (CFU / mL) was added to the prepared bacterial suspension, and the suspension was subjected to ultrasonic treatment at 300 W before sampling and counting. The results are as follows: Figure 4 As shown, the synergistic bactericidal ability gradually weakens with increasing initial bacterial concentration. When the initial trophoblast cell concentration is 10... 5 At a concentration of log CFU / mL, naringenin combined with sonication can kill all bacteria. When the trophoblast concentration is 10... 6 At a concentration of log CFU / mL, treatment for 30 min killed all bacteria. Studies have shown that the lower the concentration of vegetative somatic cells, the stronger the synergistic bactericidal ability. However, for spore suspensions, when the initial bacterial concentration was 10... 3 ~10 6 At that time, the reduction in spores under synergistic treatment was in the range of 0.96 to 0.99 log, indicating that the inactivation effect of synergistic treatment on spores did not vary significantly with changes in the initial bacterial concentration.

[0038] Example 3: Application of naringenin in synergistic ultrasound in apple juice

[0039] To investigate the effect of naringenin synergistic ultrasound on fruit juice quality, apple juice samples were divided into four groups according to the following procedure: CK: untreated blank apple juice; N: apple juice with naringenin added at a concentration of 1 MIC; U: apple juice ultrasonicated at 300W for 30 min; UN: apple juice with naringenin added at a concentration of 1 MIC and ultrasonicated at the optimal ultrasonic power for 30 min, then stored at 25℃ for 30 days. Changes in bacterial count during storage were measured, and after storage, basic physicochemical indicators, total phenol content, antioxidant capacity, volatile flavor components, and electronic nose analysis were performed. The changes in bacterial count during fruit juice storage are shown in the figure below. Figure 5 , 6 As shown in the figure, bacterial cells in the control group grew normally and reached a stationary phase after 5 days. The ultrasonic treatment group failed to completely inactivate all cells, and *Bacillus cereus* continued to proliferate normally during storage. In the naringin-treated and synergistic treatment groups, the juice inoculated with *Bacillus cereus* vegetative cell suspension showed complete bacterial death on the 2nd day of storage; the bacterial count in the juice inoculated with spore suspension initially decreased to some extent, then stabilized. The synergistic treatment group, due to its stronger inactivation ability, killed more spores, resulting in the lowest residual bacterial count in the juice after 30 days of storage. The synergistic treatment of naringin and ultrasound significantly inhibited and killed *Bacillus cereus* vegetative cells, and its inactivation effect on spores was superior to ultrasound treatment alone, ensuring that the juice maintained a lower bacterial count during the 30-day storage period and preventing spoilage.

[0040] Table 2 shows the changes in basic physicochemical properties and antioxidant capacity of apple juice after storage under different treatments. There was no significant difference in soluble solids between the ultrasonic treatment group and the control group. The soluble solids content increased somewhat in the naringin treatment group and the synergistic treatment group, which is attributed to the increased soluble solids content of the apple juice due to the addition of naringin. The pH of all treatment groups ranged from 3.86 to 3.91, and the total acid content ranged from 2.42 to 2.68 g / L. The color characteristics (L*, a*, b*) and color difference (ΔE) of the juice are shown in Table 3. Under storage conditions of 4°C and 25°C, the synergistic treatment had little effect on the color of the apple juice. Compared with the control group, there were no significant differences in total phenol content, DPPH scavenging rate, and ABTS scavenging rate in the apple juice after ultrasonic treatment. When naringin was added, the total phenol content, DPPH scavenging rate, and ABTS scavenging rate of the apple juice all increased significantly, which is attributed to the enhanced antioxidant capacity of the apple juice due to the dissolution of naringin, corresponding to the results of soluble solids. The electronic nose test results are as follows: Figure 7 Different treatment groups had no significant effect on the aroma of apple juice, and the effect did not change significantly with storage temperature.

[0041] Table 2. Determination of physicochemical properties and antioxidant capacity of apple juice under different treatments.

[0042]

[0043] Table 3. Determination of color values ​​of apple juice under different treatments.

[0044]

Claims

1. A method for sterilizing fruit juice using naringin combined with ultrasound, characterized in that: Naringin is added to the juice, and then it is subjected to ultrasonic treatment to obtain sterilized juice.

2. The method for sterilizing fruit juice using naringin combined with ultrasound according to claim 1, characterized in that: The concentration of naringenin in the juice was controlled at 0.15 mg / mL.

3. The method for sterilizing fruit juice using naringin combined with ultrasound according to claims 1 and 2, characterized in that: The ultrasonic power is 450–600 W, the ultrasonic time is 30 min, the probe is inserted 1–1.5 cm into the liquid surface, and the working time and interval time are both set to 2 s.

4. The method for sterilizing fruit juice using naringin combined with ultrasound according to claims 1, 2, and 3, characterized in that: During ultrasonic treatment, the temperature is controlled below 30℃.

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