Plant-derived bactericidal composition and application thereof in fruit juice sterilization

Abstract

This invention discloses a plant-derived bactericidal composition and its application in fruit juice sterilization. The bactericidal composition consists of two plant-derived compounds with a synergistic effect, including phloretin and dodecylaldehyde, wherein the mass ratio of dodecylaldehyde to phloretin is (1-10):1. At room temperature and pressure, adding the bactericidal composition directly to fruit juice and maintaining it for 2 days can kill the vegetative cells and spores of *Bacillus cyclophosphamide*. This invention is non-toxic, harmless, simple, convenient, green, energy-saving, has high sterilization efficiency, and has no adverse effects on the nutrients and flavor of the fruit juice. This invention is highly effective in solving the problem of spoilage and deterioration of acidic fruit juice beverages caused by incomplete sterilization and spore killing.

Description

Technical Field

[0001] This invention belongs to the field of plant-derived fungicides, specifically relating to a method for killing vegetative cells and spores of Bacillus cyclophosphamide, a plant-derived fungicide combination, and its application in fruit juice sterilization. Background Technology

[0002] Apple juice, with its sweet and sour taste, contains vitamins, minerals, dietary fiber, and other nutrients. It is easily digested and absorbed by the body, offering benefits such as cleansing the intestines, promoting digestion, preventing high blood pressure, lowering cholesterol, and calming the nerves. However, due to differences in geographical location and growing environment, the quality of apple juice varies greatly from region to region, and most apple juices suffer from excessive levels of *Bacillus cyclophosphamide*. *Bacillus cyclophosphamide* is an acidophilic, heat-resistant, spore-producing Gram-positive bacterium. In the fruit juice industry, *Bacillus cyclophosphamide* can survive pasteurization, and when it contaminates fruit juice, it produces metabolites such as guaiacol, leading to a deterioration in the taste and flavor of the juice, thus severely impacting fruit juice processing and export trade. Furthermore, *Bacillus cyclophosphamide* can form endogenous spores under adverse external conditions. These spores are highly resistant, making them difficult to eradicate with ordinary sterilization methods. Therefore, controlling the vegetative cells and spores of *Bacillus cyclophosphamide* is a crucial aspect of the fruit juice industry.

[0003] Currently, common control methods all have certain drawbacks. (1) Conventional physical sterilization methods. For example, pasteurization is not very effective against Bacillus cyclophosphamide. Ultra-high pressure homogenization technology has disadvantages such as high equipment investment and maintenance costs. Electromagnetic wave treatment methods include ultrasonic and microwave methods. Its advantage is that it will not damage the sensory and composition of food, but the sterilization effect is generally poor and often needs to be combined with other technologies to enhance the sterilization effect. Compared with other non-thermal control methods, ultraviolet (UV) sterilization requires very little energy, but UV has low transmittance in food matrix, resulting in a low absorption coefficient, so it is difficult to apply on a large scale in practice. Irradiation can effectively kill the spores of Bacillus cyclophosphamide, but because radioactivity causes fruit juice to produce off-flavors, consumers have a low acceptance of irradiated food. (2) Conventional chemical sterilization methods. For example, chlorophyll and hypochlorous acid are strong oxidants with good sterilization effects, but chlorophyll and other substances have poor stability and are easily decomposed. For example, as ozone concentration increases and treatment time extends, the number of Bacillus cereus spores decreases accordingly, but ozone treatment is costly. Furthermore, adding preservatives (such as sodium benzoate and potassium sorbate) generally provides better sterilization, but consumers may have concerns about their safety.

[0004] In recent years, plant-derived compounds have been increasingly used in food preservation and sterilization to maintain food quality and safety. Plant-derived compounds are considered to have advantages such as being natural and non-toxic, having low environmental pollution, and being friendly to humans and animals; many plant-derived compounds have already been approved for use as food additives in my country. However, when used alone, plant-derived compounds often require high concentrations to control microorganisms. Therefore, this may adversely affect the nutritional value and flavor of food.

[0005] Phloretin is mainly distributed in the peel and root bark of juicy fruits such as apples and pears. Phloretin is a pearly white crystalline powder, odorless, and tasteless at low to medium concentrations, but possesses a sweet aroma at high concentrations, making it suitable for flavoring fruit juices. Lauryl aldehyde, also known as dodecyl aldehyde, is a colorless to pale yellow oily liquid with an aroma similar to pine needle oil and orange oil, and is naturally found in essential oils such as lemon oil and sweet orange oil. Currently, there are few reports in the literature on the use of phloretin, dodecyl aldehyde, or their synergistic combinations in the field of plant-derived fungicides. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention proposes a plant-derived bactericidal composition and its application in fruit juice sterilization.

[0007] The technical solution of the present invention is as follows:

[0008] This invention proposes a plant-derived bactericidal composition that can kill the vegetative cells and spores of Bacillus cereus, wherein the bactericidal composition comprises dodecyl aldehyde and phloretin.

[0009] In some embodiments of the present invention, the mass ratio of dodecylaldehyde to phloretin is (1-10):1.

[0010] In some embodiments of the present invention, the mass ratio of dodecylaldehyde to phloretin is (3-5):1.

[0011] In some embodiments of the present invention, the mass concentration of dodecyl aldehyde in the plant-derived bactericidal combination is 0.1–1.6 mg / mL, and the mass concentration of phloretin is 0.025–0.4 mg / mL.

[0012] In some embodiments of the present invention, the mass concentration of dodecyl aldehyde in the plant-derived bactericidal combination is 0.2–1.2 mg / mL, and the mass concentration of phloretin is 0.05–0.3 mg / mL.

[0013] In some embodiments of the present invention, the mass concentration of dodecyl aldehyde in the plant-derived bactericidal combination is 0.4-0.8 mg / mL, and the mass concentration of phloretin is 0.1-0.2 mg / mL.

[0014] The bactericidal targets of this invention are *Alicyclobacillus acidoterrestris* and its spores.

[0015] The present invention also includes the application of the plant-derived bactericidal composition in the sterilization of fruit juice. Preferably, the plant-derived bactericidal composition is added to the fruit juice for ≥2 days.

[0016] Preferably, the juice is an NFC juice.

[0017] Preferably, the juice is apple juice, kiwi juice, blueberry juice, mulberry juice, orange juice, corn juice, tomato juice, grape juice, mango juice, strawberry juice, etc.

[0018] The principle of this invention: The plant-derived bactericidal combination of this invention, through the synergistic effect of phlorizin and dodecylaldehyde, can cause the cell membrane surface of vegetative cells of *Bacillus cereus* to shrink and break down, leading to the leakage of intracellular nucleic acids and proteins, ultimately killing the bacteria. The normal physiological metabolism and life activities of the bacteria are also affected, such as a significant decrease in biofilm formation capacity. Simultaneously, the plant-derived bactericidal combination can promote the release of DPA from spores, greatly reducing the spores' resistance; it can also cause the spore membrane to collapse and break down, allowing the contents of the spore cells to flow out, ultimately killing the spores. Using a combination of 0.2 mg / mL phlorizin and 0.8 mg / mL dodecylaldehyde for 2 days can kill 5 log-numbers of vegetative cells and 4 log-numbers of spores in fruit juice, achieving a very good effect in inhibiting fruit juice spoilage.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) Non-toxic, harmless, simple, convenient, green, and energy-saving. This invention kills Bacillus cereus and its spores by directly adding a plant-derived bactericidal compound permitted for use as a food additive according to national standards to fruit juice and maintaining it for 2 days. It is simple, convenient, and does not require high equipment costs, avoiding the large energy consumption required by many current sterilization processes. The synergistic effect of the plant-derived bactericidal composition can reduce the concentration of the compound used, which also reduces the cost of the bactericidal composition.

[0021] (2) High sterilization efficiency. The synergistic effect of phlorizin and dodecyl aldehyde in this invention makes it difficult for Bacillus cereus to develop resistance. Under normal temperature and pressure, treatment with the plant-derived bactericidal combination for 2 days can kill 5 log-value vegetative cells and 4 log-value spores in the juice, achieving a very good effect in inhibiting juice spoilage.

[0022] (3) It has no adverse effect on the nutrients of the fruit juice. Heat sterilization processes will more or less affect the nutrients of the fruit juice, but the present invention is a non-heat sterilization method, so there will be no such situation. The added plant-derived compounds have no adverse effect on the original nutrients of the fruit juice.

[0023] (4) It has no adverse effect on the flavor of the juice. Phloretin is odorless at low and medium concentrations, but develops a sweet aroma at higher concentrations, which is suitable for the flavor of the juice; dodecyl aldehyde has a natural aroma similar to pine needle oil and orange oil. The synergistic effect of phloretin and dodecyl aldehyde also reduces the concentration of plant-derived compounds used. Therefore, the added plant-derived compounds will not adversely affect the original flavor of the juice. Attached Figure Description

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0025] Figure 1 shows the absorbance curves (A) and the curves (B) showing the relative decrease in absorbance of spores in the example and blank control groups.

[0026] Figure 2 shows the killing of vegetative cells (A) and spores (B) in apple juice in the example and blank control group during the 0-2 day period.

[0027] Figure 3 shows scanning electron microscope images of vegetative cells (A) and spores (B) from the example and blank control groups.

[0028] Figure 4 shows the leakage of proteins (right) and nucleic acids (left) in vegetative cells (A) and spores (B) of the example and blank control group.

[0029] Figure 5 This shows the changes in biofilm formation in the nutrient cells of the example and the blank control group.

[0030] Figure 6 This describes the release of dipyridine carboxylic acid (DPA) within spore cells in the examples and the blank control group.

[0031] Figure 7 The changes in the conventional nutritional indicators of NFC apple juice, namely total acid (A), total sugar (B), total phenol (C), and ascorbic acid (D), are shown in the examples and the blank control group. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to.

[0033] The aforementioned *Alicyclobacillus acidoterrestris* has the accession number ATCC 49025.

[0034] 1. Preparation of Bacillus acidophilus suspension

[0035] Bacillus acidophilus was inoculated into BAM medium and cultured at 37°C and 180 rpm for 20–24 h. The culture was then centrifuged (4000 g, 10 min), the supernatant was discarded, and the precipitate was resuspended in physiological saline. This process was repeated three times. Finally, the OD of the bacterial suspension was adjusted with physiological saline. 600 =0.8.

[0036] 2. Preparation of Bacillus acidophilus spore suspension

[0037] (1) Inoculate Bacillus acidophilus into BAM broth and incubate at 37°C for 18–24 h. Then, take 0.2 mL of the culture medium and place it on BAM agar and incubate at 37°C for 7 days. If the spore formation rate reaches 95% or higher, it can be used for subsequent experiments after staining with malachite green and safranin.

[0038] (2) Take an appropriate amount of sterile saline on BAM medium and gently scrape the spores into the saline with a glass spreader. Then transfer the saline into a sterile test tube.

[0039] (3) Filter the spore suspension with sterile gauze to remove the agar clots.

[0040] (4) Then centrifuge the spore suspension (5000g, 15min), discard the supernatant, and resuspend the precipitate in 50% (v / v) ethanol aqueous solution for 30-60min to destroy the vegetative cells. Then centrifuge the spore suspension again, discard the supernatant, and resuspend the precipitate in physiological saline. Repeat three times.

[0041] (5) Finally, adjust the OD of the spore suspension with physiological saline. 600 =0.2.

[0042] 3. Determination of Minimum Inhibitory Concentration (MIC)

[0043] The MICs of phloretin and dodecylaldehyde used in this invention were determined according to the Clinical and Laboratory Standards Institute (CLSI) standards using the 2-fold broth dilution method, and the results are shown in Table 1.

[0044] Table 1

[0045]

[0046] 4. Evaluation methods for synergistic effects

[0047] The efficacy of combined drug therapy was determined using the fractional concentration inhibition coefficient (FICI) method, and the results are shown in Table 2. The comprehensive formula for FICI is as follows: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, irrelevant effect; FICI > 2, antagonistic effect.

[0048] Table 2

[0049]

[0050] Example 1

[0051] A plant-derived bactericidal composition comprising 0.025 mg / mL phlorizin and 0.1 mg / mL dodecyl aldehyde (hereinafter referred to as LP).

[0052] Example 2

[0053] A plant-derived bactericidal composition comprising 0.05 mg / mL phlorizin and 0.2 mg / mL dodecyl aldehyde (hereinafter referred to as 2LP).

[0054] Example 3

[0055] A plant-derived bactericidal composition comprising 0.1 mg / mL phlorizin and 0.4 mg / mL dodecyl aldehyde (hereinafter referred to as 4LP).

[0056] Example 4

[0057] A plant-derived bactericidal composition comprising 0.2 mg / mL phlorizin and 0.8 mg / mL dodecyl aldehyde (hereinafter referred to as 8LP).

[0058] Blank control group

[0059] A blank control group was set up, and the plant-derived compound bactericidal combination was replaced with a background solution (hereinafter referred to as CK).

[0060] Test case

[0061] Examples 1-4 and the control group were added to sterilized BAM liquid culture medium, and Bacillus acidophilus spores were inoculated into the medium. The cultures were incubated at 37±2℃ and 180 r / min. Samples were taken at different time points, and the absorbance at 600 nm was measured. The results are shown in Figure 1(A). The absorbance at 600 nm is often used to estimate the release of spore contents. The release of spore contents is closely related to the permeability of the spore inner membrane. The greater the release of spore contents, the lower the absorbance, indicating greater damage to the spore inner membrane caused by the plant-derived bactericide combination. Figure 1(A) shows that the absorbance at 600 nm in the blank control group continuously increased, indicating that a large number of spores germinated into vegetative cells. Figure 1(B) shows the relative decrease in absorbance at 600 nm for Examples 1-4. As shown in Figure 1(B), the relative decrease in absorbance in Examples 1-4 initially increased. Specifically, the relative decrease in absorbance in Example 4 consistently increased, indicating that the damage to the spores in Example 4 was time-dependent; the damage to the spore inner membrane increased with treatment time. In contrast, the relative decrease in absorbance in Examples 1-3 showed a trend of first increasing and then decreasing, indicating that Examples 1-3 initially caused greater damage to the spores, but the effect gradually weakened. Furthermore, the relative decrease in absorbance became negative at 22 hours for Example 1 and at 25 hours for Example 2, indicating that the absorbance began to increase relative to the initial absorbance, and the spores began to germinate into vegetative cells.

[0062] Figure 2 shows the number of vegetative cells (A) and spores (B) of *Bacillus cyclophosphamide* in apple juice after treatment on days 0, 1, and 2, in both the examples and the blank control group. As can be seen from Figures 2(A) and (B), the number of vegetative cells and spores decreased in Examples 1-4 compared to the blank control group, and this decrease was concentration-dependent; the higher the concentration of the bactericidal composition, the better the bactericidal and spore-killing effects. On day 2, in Example 4, the number of vegetative cells decreased by 5 logs and the number of spores decreased by 4 logs compared to the blank control group.

[0063] Figure 3 shows scanning electron microscope (SEM) images of *Bacillus cyclophosphamide* vegetative cells (A) and spores (B) after treatment in the examples and the blank control group. Figure 3(A) shows that, compared to the blank control group, the vegetative cell surfaces in Examples 1-4 were shrunken and damaged, and the number of shrunken and damaged bacteria increased with increasing concentration of the bactericidal composition, with Example 4 showing the highest number. Figure 3(B) shows that, compared to the blank control group, with increasing concentration of the bactericidal composition in Examples 1-4, the irregular wrinkles on the spore surface disappeared, the spore surface became smooth, the degree of spore depression and breakage increased, and the number of ruptured and disintegrated spores also increased, with Example 4 showing the highest number. These phenomena indicate that with increasing concentration of the bactericidal composition, the damage and depression to the cell membranes of vegetative cells and spores become more severe, and the leakage of contents increases, thereby achieving the effects of bactericidal and spore-killing.

[0064] Figure 4(A) shows the absorbance of the supernatant at 260 nm and 280 nm after treatment of vegetative cells of *Bacillus cereus* with the plant-derived bactericidal composition, corresponding to the leakage of nucleic acids and proteins, respectively. Similarly, Figure 4(B) also corresponds to the leakage of nucleic acids and proteins from the spores. Figures 4(A) and (B) show that, compared to the blank control group, the leakage of nucleic acids and proteins in Examples 1-4 increased; and the leakage of nucleic acids and proteins also increased with the increase of the concentration of the bactericidal composition. Since proteins and nucleic acids play an important role in the life activities of vegetative cells and spores, their leakage will accelerate the death of vegetative cells and spores.

[0065] Figure 5 This display shows the changes in the amount of biofilm formed by vegetative cells. Biofilms are formed by bacteria to provide a stable growth environment and resist adverse external environments, serving to adhere to solid surfaces and increase their resistance. Figure 5 The results show that Examples 1-4 achieved biofilm inhibition rates of 40.9±3.8%, 85.1±0.016%, 85.2±0.001%, and 85.5±0.016%, respectively. These data indicate that plant-derived bactericidal compositions, especially high-concentration compositions, can significantly affect the formation of biofilms in *Bacillus cyclophosphamide*, thereby impacting the normal life activities of the bacteria.

[0066] Figure 6This displays the release of dipyridine carboxylic acid (DPA). The experiment used a high-temperature, high-pressure sterilization method at 121°C for 30 minutes to determine 100% DPA release from the spores. The release levels and specific amounts of DPA in Examples 1-4 and the blank control group were obtained using a colorimetric method. DPA is an important component of the spore nucleus and is a substance unique to spores. When the external environment damages the spore inner membrane, leading to increased permeability, a large amount of DPA is released, causing the spore nucleus to absorb water and swell. This reduces the spore's resistance to the external environment; therefore, DPA release can be used to characterize changes in the permeability of the spore inner membrane. Figure 6 As shown, compared to the blank control group, the DPA release in Examples 1-4 was increased; moreover, the DPA release increased with the increase of the concentration of the bactericidal composition, with the DPA release rate in Example 4 reaching 95.1±6.9% and the DPA release amount reaching 10.8±0.8 μg / mL. These results indicate that the plant-derived bactericidal composition increases the permeability of the spore inner membrane, leading to a large release of DPA, significantly reducing the spore's resistance, and ultimately killing the spore.

[0067] Figure 7 The display shows the changes in total acid (A), total sugar (B), total phenol (C), and ascorbic acid (D) in NFC apple juice. Figure 7 The results showed that after treatment with Examples 1-4 and the blank control group, there were no significant differences in total acid, total sugar, and ascorbic acid. However, the total phenol content increased with the increase of the concentration of the plant-derived bactericide composition. This is mainly because phloretin contains phenolic hydroxyl groups, and the increase in the concentration of phloretin leads to an increase in the total phenol content. The above changes in nutrients indicate that the addition of the plant-derived bactericide composition has no adverse effect on the nutrients of NFC apple juice.

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A plant-derived bactericidal composition for the preparation of a product that inhibits the growth of Bacillus cyclophosphamide (Bacillus acidophilus). Alicyclobacillus acidoterrestris Its application in bactericides and their spore-killing agents, characterized in that, phloretin and dodecanal, the mass ratio of the dodecanal and the phloretin being (1-10):1; the mass concentration of the dodecanal being 0.1-1.6 mg / mL, and the mass concentration of the phloretin being 0.025-0.4 mg / mL.

2. Use according to claim 1, characterized in that, The mass ratio of the dodecanal and the phloretin is (3-5):

1.

3. Use according to claim 2, characterized in that, The mass concentration of the dodecanal is 0.2-1.2 mg / mL, and the mass concentration of the phloretin is 0.05-0.3 mg / mL.

4. Use according to claim 3, characterized in that, The mass concentration of the dodecanal is 0.4-0.8 mg / mL, and the mass concentration of the phloretin is 0.1-0.2 mg / mL.

5. Use of the plant-derived sterilization composition according to any one of claims 1-4 in sterilization of fruit juice.

6. Use according to claim 5, characterized in that, The plant-derived sterilization composition is kept in the fruit juice for ≥2 d.

7. Use according to claim 6, characterized in that, The fruit juice is NFC fruit juice.

8. Use according to claim 5 or 6 or 7, characterized in that, The fruit juice is apple juice, kiwi juice, blueberry juice, mulberry juice, orange juice, corn juice, tomato juice, grape juice, mango juice, and strawberry juice.

Images