CN118303464A - Bacteriostat for preventing and treating postharvest diseases of grapefruits and preparation method thereof - Google Patents

Abstract

The invention provides a bacteriostat for preventing and treating postharvest diseases of grapefruits and a preparation method thereof, and relates to the technical field of fruit preservation. The antibacterial agent for preventing and treating the postharvest diseases of the grapefruits is prepared from an extract prepared by fermenting chamomile by mixing saccharomycetes and bacillus amyloliquefaciens, chitosan, caryophyllene, menthol, cinnamaldehyde, an antioxidant, corn starch, a thickening agent and the like through the steps of premixing, stirring, pressure changing, standing and the like. The invention overcomes the defects of the prior art, the prepared bacteriostatic agent is green and pollution-free, the grape fruits are preserved while effectively inhibiting the penicillium expansum caused by the grape fruits, and the disease control effect of the grape fruits after being picked is comprehensively improved.

Description

Antibacterial agent for preventing and controlling post-harvest diseases of grapefruit and preparation method thereof

Technical Field

The invention relates to the technical field of fruit preservation, and in particular to an antibacterial agent for preventing and controlling post-harvest diseases of grapefruit and a preparation method thereof.

Background technique

Grapefruit, also known as pomelo, is a hybrid fruit that is rich in vitamin C and pectin. It can not only improve immunity, but also help alleviate cold symptoms, but also help lower cholesterol in the body and prevent cardiovascular disease. It has a high market value, but grapefruit belongs to one of the four major groups of citrus fruits and is easily infected by green mold, penicillium and other diseases during post-harvest storage.

During the storage of grapefruit, penicillium caused by Penicillium expansum is the most serious disease with the highest incidence rate. At present, the treatment method for grapefruit is usually to directly spray chemical fungicides for sterilization and then store at low temperature. However, the existing chemical fungicides are easy to cause pollution, and with people's pursuit of food safety, the use of chemical fungicides has become more and more cautious. Therefore, it is necessary to develop a green, safe and efficient antibacterial agent to prevent and control grapefruit diseases after harvest.

Summary of the invention

In view of the deficiencies in the prior art, the present invention provides an antibacterial agent for preventing and controlling post-harvest diseases of grapefruit and a preparation method thereof. A long-acting, safe and green antibacterial agent can be obtained by compounding chamomile fermentation extract with multiple ingredients, which can effectively preserve grapefruit and prevent and control post-harvest diseases.

To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

A bacteriostatic agent for preventing and controlling post-harvest diseases of grapefruit, the bacteriostatic agent being prepared by diluting a composite bacteriostatic component, wherein the composite bacteriostatic component comprises the following raw materials in parts by weight: 2-6 parts of chitosan, 1-1.4 parts of chamomile fermented extract, 0.2-0.6 parts of caryophyllene, 0.4-0.6 parts of menthol, 0.2-0.4 parts of cinnamaldehyde, 0.2-0.4 parts of antioxidant, 2-6 parts of corn starch, and 2-4 parts of thickener;

Preferably, the chamomile fermentation extract is a product obtained by extracting chamomile by combined fermentation using yeast and Bacillus amyloliquefaciens.

Preferably, the preparation method of the chamomile fermentation extract comprises the following steps:

S1. Take fresh chamomile, clean it, add 60-70% ethanol solution and grind it into pulp to obtain the pulp for later use;

S2, extracting the slurry in a water bath at 40-60°C with stirring for 60-80 min, and then removing the solvent by rotary evaporation to obtain a preliminary extract for later use;

S3, adding deionized water to the preliminary extract, stirring evenly, adding yeast and amylase-producing Bacillus activation solution, fermenting for 2-4 days, and obtaining a preliminary fermentation material for standby use;

S4, subjecting the above preliminary fermentation material to ultrasonic treatment for 20-30 minutes, followed by filter pressing, adding 2 times the volume of clean water to the filter residue and continuing ultrasonic treatment for 20-30 minutes, filter pressing again, combining the two filtrates to obtain a mixed solution for standby use;

S5. The mixed solution is evaporated, concentrated and freeze-dried to obtain chamomile fermentation extract.

Preferably, in step S3, the addition amount of the yeast and Bacillus amyloliquefaciens activation solution is 10%-20% of the total mass of chamomile, and the number of live yeast and Bacillus amyloliquefaciens in the activation solution is 1×10 ⁶ -1×10 ⁹ /mL.

Preferably, the antioxidant is any one or more of sorbic acid, citric acid, and ascorbic acid.

Preferably, the thickener is hydroxypropyl methylcellulose.

The method for preparing the antibacterial agent comprises the following steps:

(1) adding caryophyllene, menthol and cinnamaldehyde to ethanol and stirring and mixing, then adding a thickener, continuing to stir and mix, and then drying and crushing to obtain a premix for use;

(2) Mix the chamomile fermented extract and antioxidant, add deionized water, disperse by ultrasonication, add chitosan, continue to stir evenly, and obtain a premixed solution for use;

(3) Add half of the corn starch to deionized water and gelatinize at 75-80°C in a water bath for 30-40 minutes to obtain a completely gelatinized starch slurry;

(5) Add the remaining half of the corn starch to deionized water and gelatinize it in a water bath at 65-70°C for 20-30 minutes to obtain a preliminary gelatinized starch slurry;

(6) Add the completely gelatinized starch slurry to the premixed liquid, stirring while adding, and after the addition is complete, add the premixed liquid again and stir at a pressure of 0.3-0.6 MPa to obtain a preliminary composite material;

(7) adding the preliminary composite material to the preliminary gelatinized starch slurry, adding dropwise while stirring, and then standing at pressures of 0.2-0.4 MPa, 1-2 MPa and 4-6 MPa, and then concentrating and freeze-drying to obtain a composite sterilized component;

(8) Dilute the above composite sterilization component 100-200 times with deionized water to obtain an antibacterial agent.

Preferably, the temperature of drying and crushing in step (1) is 60-70°C, and the crushed product passes through an 80-mesh sieve.

Preferably, the power of ultrasonic dispersion in step (2) is 400-600 W, and the time of ultrasonic dispersion is 10-15 min.

Preferably, in step (6), the stirring speed at 0.3-0.6 Mpa is 200-400 r/min, and the stirring time is 20-30 min.

Preferably, in step (7), the time for static treatment at a pressure of 0.2-0.4 MPa is 80-100 min, the time for static treatment at a pressure of 1-2 MPa is 40-60 min, and the time for static treatment at a pressure of 4-6 MPa is 15-20 min.

The present invention provides an antibacterial agent for preventing and controlling post-harvest diseases of grapefruit, which has the following advantages over the prior art:

The invention uses chamomile fermentation extract as the main antibacterial component, and chamomile is extracted after being subjected to composite fermentation by yeast and amylolytic bacillus. The component is mixed with caryophyllene, menthol and cinnamaldehyde, which can effectively prevent and treat penicillium in grapefruit during storage after picking. All the components are combined with chitosan, a thickener and corn starch to achieve the purpose of long-term prevention and treatment. The prepared antibacterial agent can form a film on the surface of grapefruit after soaking or spraying the grapefruit, so as to achieve a good antibacterial effect and also achieve the purpose of preservation. Moreover, each component is safe and environmentally friendly, so as to ensure the safety of grapefruit consumption.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram showing the comparison of diseased holes in grapefruits stored for 1 day in Group 9 and Group 2 in the detection of the resistance of each group of antibacterial agents to Penicillium expansum caused by Grapefruit Penicillium expansum in the embodiment of the present invention, wherein no diseased holes appeared in the grapefruits in Group 2;

FIG2 is a schematic diagram showing the comparison of diseased pores in grapefruits stored for 3 days in Group 9 and Group 2 in the test of the resistance of each group of antibacterial agents to Penicillium expansum caused by Grapefruit Penicillium expansum in the embodiment of the present invention, wherein no diseased pores appeared in the grapefruits in Group 2;

3 is a schematic diagram showing the comparison of diseased pores in grapefruits stored for 7 days in Group 9 and Group 2 in the test of the resistance of each group of antibacterial agents to Penicillium expansum caused by Grapefruit Penicillium expansum in the embodiment of the present invention, wherein no diseased pores appeared in the grapefruits in Group 2;

4 is a schematic diagram of a comparison of diseased wells of grapefruit stored for 10 days in Group 9 and Group 2 in detecting the resistance of each group of antibacterial agents to penicillium caused by Penicillium expansum in grapefruit according to an embodiment of the present invention, wherein the bacterial growth range of the diseased wells in Group 9 is significantly larger than the bacterial growth range of the diseased wells in Group 2.

Detailed ways

In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

The yeast used in the following examples is selected from Saccharomyces boulardii; the amyloliquefaciens Bacillus is Bacillus amyloliquefaciens OR2-30 (purchased from Anhui Academy of Agricultural Sciences) and Bacillus amyloliquefaciens G-7 (purchased from Hebei Agricultural University). The activation solution of the yeast and Bacillus amyloliquefaciens is to add the strains to deionized water and let them stand for activation for 4-8 hours.

Embodiment 1:

Preparation of antibacterial agent:

1. Raw material preparation:

1. Preparation of Extract A:

(1) Take fresh chamomile, wash it, add 2 times the volume of 65% ethanol solution, grind it into slurry, and prepare the slurry for later use;

(2) The slurry was stirred and extracted at a speed of 160 r/min at a water bath temperature of 50° C. for 70 min, and then the solvent was removed by rotary evaporation to obtain a preliminary extract for use;

(3) After adding 5 times the volume of deionized water to the preliminary extract and stirring evenly, yeast activation solution with a live bacterial count of 1×10 ⁶ -1×10 ⁹ cells/mL and Bacillus amyloliquefaciens OR2-30 activation solution were added to the preliminary extract in an amount of 15% of the total mass of chamomile, respectively, and fermented at a constant temperature of 40°C for 3 days to obtain a preliminary fermentation material;

(4) The above preliminary fermentation material is subjected to 400W ultrasonic treatment for 25 minutes, and then filtered. The filter residue is added with 2 times the volume of clean water and further subjected to 400W ultrasonic treatment for 25 minutes, and filtered again. The two filtrates are combined to obtain a mixed solution for use;

(5) The mixed solution is evaporated, concentrated and freeze-dried to obtain extract A.

2. Preparation of Extract B:

The preparation method of the extract B is the same as the preparation method of the extract A, except that the Bacillus amyloliquefaciens activation liquid is selected from the Bacillus amyloliquefaciens G-7 activation liquid.

3. Preparation of Extract C:

The yeast activation solution with a viable cell count of 1×10 ⁶ -1×10 ⁹ /mL and the Bacillus amyloliquefaciens OR2-30 activation solution were mixed and freeze-dried to obtain extract C.

4. Preparation of Extract D:

(1) Take fresh chamomile, wash it, add 2 times the volume of 65% ethanol solution, grind it into slurry, and prepare the slurry for later use;

(2) The slurry was stirred and extracted at a speed of 160 r/min at a water bath temperature of 50° C. for 70 min, and then the solvent was removed by rotary evaporation to obtain a preliminary extract for use;

(3) After adding 5 times the volume of deionized water to the preliminary extract and stirring evenly, the mixture was treated with ultrasound at 400W for 25 minutes, and then filtered. The residue was added with 2 times the volume of clean water and treated with ultrasound at 400W for 25 minutes, and then filtered again. The two filtrates were combined to obtain a mixed solution for use;

(4) The mixed solution is evaporated, concentrated and freeze-dried to obtain extract D.

5. Preparation of completely gelatinized starch slurry:

20 g corn starch was added into 6 times volume of deionized water, and gelatinized for 35 min at 78° C. water bath temperature to obtain completely gelatinized starch slurry.

6. Preparation of preliminary gelatinized starch slurry:

20 g corn starch was added into 6 times volume of deionized water, and gelatinized for 35 min at 68° C. water bath temperature to obtain preliminary gelatinized starch slurry.

7. Starch slurry:

40 g of corn starch was added into 6 times volume of deionized water, and the mixture was stirred and mixed for 35 min to obtain starch slurry.

2. Preparation process:

1. Prepare materials: chitosan 40g, extract 12g, caryophyllene 4g, menthol 5g, cinnamaldehyde 3g, citric acid 3g, hydroxypropyl methylcellulose 30g;

2. Add caryophyllene, menthol and cinnamaldehyde to 500 ml of ethanol and stir to mix, then add hydroxypropyl methylcellulose, continue stirring to mix, and then dry at 65°C to constant weight, remove ethanol, and then crush through an 80-mesh sieve to obtain a premix for use;

3. After mixing the extract and citric acid, add 500 ml of deionized water, then disperse by ultrasonic at 400 W for 12 min, then add chitosan and continue stirring to obtain a premixed solution for use;

4. Add the first slurry to the premixed liquid and stir while adding. After the addition is complete, add the premixed liquid again and stir at a speed of 400 r/min under a pressure of 0.4 Mpa for 25 minutes to obtain a preliminary composite material;

5. Add the above preliminary composite material to the second slurry, add dropwise while stirring, then let it stand for 90 minutes at a pressure of 0.3Mpa, adjust the pressure to 1.5Mpa and let it stand for 50 minutes, then adjust the pressure to 5Mpa and let it stand for 18 minutes, then concentrate and freeze-dry to obtain a composite sterilization component;

6. Dilute the above composite sterilization component 150 times with deionized water to obtain an antibacterial agent.

According to the above preparation method, the following groups of antibacterial agents were prepared, and the selection of extracts, first slurries, and second slurries in each group of antibacterial agents is shown in Table 1 below:

Table 1

Comparative Example 1:

Preparation of antibacterial agent:

(1) Preparation: chitosan 40 g, extract A 12 g, citric acid 3 g, hydroxypropyl methylcellulose 30 g;

(2) After extract A and citric acid are mixed, 500 ml of deionized water is added, and then ultrasonic dispersion is performed at 400 W for 12 min. Then chitosan is added and stirred evenly to obtain a premixed solution for use;

(3) Add the above-mentioned completely gelatinized starch slurry to the premixed liquid, stirring while adding, and then add hydroxypropyl methylcellulose after the addition is complete, and stir at a speed of 400 r/min under a pressure of 0.4 MPa for 25 minutes to obtain a preliminary composite material;

(4) Add the above preliminary composite material to the preliminary gelatinized starch slurry, add dropwise while stirring, then let it stand for 90 minutes at a pressure of 0.3 MPa, adjust the pressure to 1.5 MPa and let it stand for 50 minutes, then adjust the pressure to 5 MPa and let it stand for 18 minutes, then concentrate and freeze-dry to obtain the composite sterilized component.

(5) Dilute the above composite sterilization component 150 times with deionized water to obtain an antibacterial agent.

Comparative Example 2:

Preparation of antibacterial agent:

(1) Preparation: chitosan 40 g, extract A 12 g, caryophyllene 4 g, menthol 5 g, cinnamaldehyde 3 g, citric acid 3 g, hydroxypropyl methylcellulose 30 g;

(2) Add caryophyllene, menthol and cinnamaldehyde to 500 ml of ethanol and stir to mix, then add hydroxypropyl methylcellulose, continue stirring to mix, then dry at 65°C to constant weight, remove ethanol and grind through an 80-mesh sieve to obtain a premix for use;

(3) After extract A and citric acid are mixed, 500 ml of deionized water is added, and then ultrasonic dispersion is performed at 400 W for 12 min. Chitosan is then added and stirred evenly to obtain a premixed solution for use;

(4) Add the completely gelatinized starch slurry to the premixed liquid, stirring while adding, and after the addition is complete, add the premixed liquid again and stir at a speed of 400 r/min for 25 min to obtain a preliminary composite material;

(5) adding the preliminary composite material to the preliminary gelatinized starch slurry, adding dropwise while stirring, and then standing for 150 minutes, and then concentrating and freeze-drying to obtain a composite sterilized component;

(6) Dilute the above composite sterilization component 150 times with deionized water to obtain an antibacterial agent.

Detection:

The antibacterial agents prepared in the experimental groups 1-7 and comparative examples 1-2 in the above embodiment 1 were subjected to relevant tests:

Testing materials: "Rio Red" grapefruit, purchased from a grapefruit orchard in Gasa Town, Yuxi City, Yunnan Province; Penicillium expansum (P. expansum), isolated from the surface of diseased grapefruit fruit;

1. Detect the resistance of each group of antibacterial agents to Penicillium expansum caused by grapefruit:

(1) 30 grapefruits of the same size, color, and maturity were selected and randomly divided into 10 groups, with 3 grapefruits in each group. The grapefruits in each group were washed with distilled water, and then the surface of the fruit was wiped with 75% alcohol for disinfection. The grapefruits were placed at room temperature for 6 hours before use.

(2) Activation treatment of Penicillium expansum: inoculate Penicillium expansum onto PDA medium, culture at 28°C, and dilute with sterile water to prepare a 1×10 ⁵ CFU/mL Penicillium expansum spore suspension for later use;

(3) Use a sterilized punch needle to punch holes in four directions at the equatorial part of each group of grapefruit (with a diameter of 5 mm and a depth of about 6 mm). Inoculate each hole with 20 μL of Penicillium expansum spore suspension. Then spray the surface of each group of grapefruit with an antibacterial agent (20 mL/piece), take out the grapefruit and place it in a perforated plastic box. Store it at a constant temperature of 17±1℃ and observe the incidence of grapefruit in each group.

Incidence rate = number of diseased holes/total number of holes × 100%;

The specific results are shown in Table 2 below:

Table 2

It can be seen from the above table that the antibacterial agents prepared using experimental groups 1 and 2 have better antibacterial effects than the antibacterial agents prepared using experimental groups 3-7 and comparative examples 1-2, and the overall antibacterial effect is also longer-lasting.

2. Test the preservation effect of each group of antibacterial agents on grapefruit:

(1) 30 grapefruits of the same size, color, and maturity were selected and randomly divided into 10 groups, with 3 grapefruits in each group. The grapefruits in each group were washed with distilled water, and then the surface of the fruit was wiped with 75% alcohol for disinfection. The grapefruits were placed at room temperature for 6 hours before use.

(2) Soak the grapefruits of each group in the antibacterial agent for 2 minutes, take them out and let them stand for 6 hours before weighing them, which is m0. Then put them in a perforated plastic box and store them at a constant temperature of 17±1℃. Weigh them at 5d, 10d, 15d, 20d, and 25d, which is m1. Calculate the average weight loss rate of the grapefruits of each group at different times.

Weight loss rate = (m0-m1)/m0×100%;

The specific results are shown in Table 3 below:

table 3

It can be seen from the above table that the antibacterial agents prepared using experimental groups 1 and 2 have better grapefruit preservation effects than the antibacterial agents prepared using experimental groups 3-7 and comparative examples 1-2.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. An antibacterial agent for preventing and controlling post-harvest diseases of grapefruit, characterized in that the antibacterial agent is prepared by diluting a composite antibacterial component, and the composite antibacterial component comprises the following raw materials in parts by weight: 2-6 parts of chitosan, 1-1.4 parts of chamomile fermented extract, 0.2-0.6 parts of caryophyllene, 0.4-0.6 parts of menthol, 0.2-0.4 parts of cinnamaldehyde, 0.2-0.4 parts of antioxidant, 2-6 parts of corn starch, and 2-4 parts of thickener; The chamomile fermentation extract is a product obtained by extracting chamomile by combined fermentation with yeast and amylolytic bacillus. 2. The antibacterial agent according to claim 1, characterized in that the preparation method of the chamomile fermentation extract comprises the following steps: S1. Take fresh chamomile, clean it, add 60-70% ethanol solution and grind it into pulp to obtain the pulp for later use; S2, extracting the slurry in a water bath at 40-60°C with stirring for 60-80 min, and then removing the solvent by rotary evaporation to obtain a preliminary extract for later use; S3, adding deionized water to the preliminary extract, stirring evenly, adding yeast and amylase-producing Bacillus activation solution, fermenting for 2-4 days, and obtaining a preliminary fermentation material for standby use; S4, subjecting the above preliminary fermentation material to ultrasonic treatment for 20-30 minutes, followed by filter pressing, adding 2 times the volume of clean water to the filter residue and continuing ultrasonic treatment for 20-30 minutes, filter pressing again, combining the two filtrates to obtain a mixed solution for standby use; S5. The mixed solution is evaporated, concentrated and freeze-dried to obtain chamomile fermentation extract. 3. The antibacterial agent according to claim 2, characterized in that the addition amount of the yeast and amylolytic Bacillus activation solution in step S3 is 10%-20% of the total mass of chamomile, and the number of live yeast and amylolytic Bacillus in the activation solution is 1×10 ⁶ -1×10 ⁹ /mL. 4. The antibacterial agent according to claim 2, characterized in that the antioxidant is any one or more of sorbic acid, citric acid, and ascorbic acid. 5. The antibacterial agent according to claim 2, characterized in that the thickener is hydroxypropyl methylcellulose. 6. A method for preparing the antibacterial agent according to any one of claims 1 to 5, characterized in that the preparation method comprises the following steps: (1) adding caryophyllene, menthol and cinnamaldehyde to ethanol and stirring and mixing, then adding a thickener, continuing to stir and mix, and then drying and crushing to obtain a premix for use; (2) Mix the chamomile fermented extract and antioxidant, add deionized water, disperse by ultrasonication, add chitosan, continue to stir evenly, and obtain a premixed solution for use; (3) Add half of the corn starch to deionized water and gelatinize at 75-80°C in a water bath for 30-40 minutes to obtain a completely gelatinized starch slurry; (5) Add the remaining half of the corn starch to deionized water and gelatinize it in a water bath at 65-70°C for 20-30 minutes to obtain a preliminary gelatinized starch slurry; (6) Add the completely gelatinized starch slurry to the premixed liquid, stirring while adding, and after the addition is complete, add the premixed liquid again and stir at a pressure of 0.3-0.6 MPa to obtain a preliminary composite material; (7) adding the preliminary composite material to the preliminary gelatinized starch slurry, adding dropwise while stirring, and then standing at pressures of 0.2-0.4 MPa, 1-2 MPa and 4-6 MPa, and then concentrating and freeze-drying to obtain a composite sterilized component; (8) Dilute the above composite sterilization component 100-200 times with deionized water to obtain an antibacterial agent. 7. The preparation method according to claim 6, characterized in that the temperature of the drying and crushing in step (1) is 60-70°C, and the crushing passes through an 80-mesh sieve. 8. The preparation method according to claim 6, characterized in that: the power of ultrasonic dispersion in step (2) is 400-600W, and the time of ultrasonic dispersion is 10-15min. 9. The preparation method according to claim 6, characterized in that: in the step (6), the speed of the stirring treatment at 0.3-0.6Mpa is 200-400r/min, and the stirring time is 20-30min. 10. The preparation method according to claim 6, characterized in that: in the step (7), the time for the static treatment at a pressure of 0.2-0.4 MPa is 80-100 min, the time for the static treatment at a pressure of 1-2 MPa is 40-60 min, and the time for the static treatment at a pressure of 4-6 MPa is 15-20 min.