Application of bacillus velezensis TCS001 to improvement of cherry quality

Abstract

The invention relates to application of bacillus velezensis TCS001 as a medicine for improving cherry quality, after cherries are treated by the bacillus velezensis TCS001, beta-carotene of the cherries can be improved by 13.6%, anthocyanin of the cherries can be improved by 9.8%, soluble sugar of the cherries can be improved by 7.3%, vitamin C of the cherries can be reduced by 19.4%, total acid of the cherries can be reduced by 7%, and the quality of cherry fruits can be remarkably improved.

Description

Technical Field

[0001] The invention relates to the field of plant growth regulation, and in particular to application of Bacillus velez TCS001 in regulating cherry quality. Background Art

[0002] Bacillus velezensis is a new type of biocontrol bacteria. In 2005, Spanish scholars Ruiz-García et al. isolated two strains, CR-14b and CR-502T, which can synthesize a large amount of lipopeptides and have strong antibacterial activity. Bacillus velezensis is widely distributed and can survive in adverse conditions such as drought and salinity. There are few related products, and the development prospects are broad.

[0003] Bacillus velezensis TCS001 was isolated from Bohai silt. The original strain was marine Bacillus CT2628. After mutagenesis and stabilization, it was identified and named Bacillus velezensis TCS001. It has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration (CGMCC) with the deposit number CGMCC No.8921, and was first published in patent ZL201410168402.2. After growing on NA medium, the single colony morphology of TCS001 is nearly round, light yellow, and opaque; the surface of the colony is smooth and the edges are neat in the early stage of culture, and the surface is wrinkled in the later stage, the edges are slightly irregular, there is a bulge in the middle, and it spreads in a cloud-like manner around. Gram staining showed that the Velez Bacillus TCS001 strain was Gram-positive and rod-shaped; it had a certain inhibitory effect on cucumber gray mold, cucumber vine blight, cucumber sclerotinia, cucumber brown spot, cotton wilt and banana leaf spot, among which the inhibition rate against cucumber gray mold was the highest, reaching 87.66%.

[0004] At present, there is no report on applying Bacillus Velez subtilis to cherry in the prior art. Summary of the invention

[0005] The main purpose of the present invention is to provide application of Bacillus Velez TCS001 as a drug for improving cherry quality.

[0006] Another main object of the present invention is to provide a method for improving cherry quality using Bacillus Velez TCS001.

[0007] Preferably, the improving cherry quality comprises one or more of increasing cherry soluble sugar, increasing cherry soluble solids, reducing cherry vitamin C, increasing cherry anthocyanin, increasing cherry β-carotene, reducing cherry acidity, etc.

[0008] Preferably, the Bacillus Velez subtilis TCS001 is a fermentation broth of Bacillus Velez subtilis TCS001.

[0009] Preferably, the Bacillus Velez TCS001 fermentation broth is prepared as a suspension.

[0010] Preferably, the formula of the suspension concentrate includes 40% of Bacillus Velez TCS001 fermentation broth, 3% of dispersant SXC, 3% of dispersant UNA, 0.15% of thickener xanthan gum, 0.2% of preservative kason, 0.2% of preservative sodium benzoate, and 53.45% of water.

[0011] Preferably, the application concentration of the suspension is 300-700 times diluted.

[0012] Preferably, the application concentration of the suspension is 400 times diluted.

[0013] Preferably, the Bacillus Velez subtilis TCS001 is applied by spraying.

[0014] Preferably, the application time of the Bacillus Velez TCS001 is one or more of the cherry flower bud differentiation period, early flowering period, full flowering period, young fruit period, and harvest period;

[0015] Preferably, the Bacillus Velez TCS001 is applied once a week from the young fruit stage to the harvest stage until the end of harvest.

[0016] Preferably, the Bacillus Velez TCS001 is applied once within 5 days after harvesting.

[0017] Beneficial technical effects of the present invention

[0018] During the cherry flower bud differentiation period, initial flowering period, peak flowering period, young fruit period to harvest period, after spraying with 400 times of Bacillus Velez TCS001 suspension, compared with CK, the cherry β-carotene increased by 13.6%, anthocyanin increased by 9.8%, soluble sugar increased by 7.3%, vitamin C decreased by 19.4%, and the total acid of cherry decreased by 7%.

[0019] The quality of cherry fruit can be significantly improved after treatment with Bacillus Velez TCS001.

[0020] During the experiment, it was observed that Bacillus Velez TCS001 had significantly better effects on cherry size, glossiness and other traits than the blank control, and the taste was good when eaten. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Cherry fruits after control and Bacillus Velez TCS001 shed treatment;

[0022] Figure 2 Cherry fruits after control and Bacillus Velez TCS001 shed treatment;

[0023] Figure 3 Fruits on cherry branches after treatment with Bacillus Velez TCS001 in the shed;

[0024] Figure 4 Fruits on cherry branches for control;

[0025] Figure 5 Fruits on cherry branches after treatment with Bacillus Velez TCS001 in the shed;

[0026] Figure 6 Fruits on cherry branches for control; Specific embodiments

[0027] Materials and Methods

[0028] Test Materials:

[0029] The test bacterial agent of the present invention is a suspension of Bacillus Velez TCS001 (3.9×109 CFU / mL), and the specific formula is: 40% of Bacillus Velez TCS001 fermentation liquid, 3% of dispersant SXC, 3% of dispersant UNA, 0.15% of thickener xanthan gum, 0.2% of preservative kason, 0.2% of preservative sodium benzoate, and 53.45% of water.

[0030] Test plants:

[0031] Experimental variety: Cherry (Agate Red).

[0032] Main instruments:

[0033] ME104E electronic balance (Sartorius Scientific Instruments Co., Ltd.)

[0034] Test method:

[0035] See Table 1 for treatment methods

[0036] Table 1 Treatment methods of Bacillus Velez TCS001 suspension concentrate cherry test

[0037]

[0038] The suspension concentrate of Bacillus Velez TCS001 was diluted with water to the corresponding concentration and then sprayed.

[0039] Spray treatment: Spray once during the cherry flower bud differentiation period, early flowering period, and peak flowering period, and spray continuously once a week from the young fruit period to the harvest period.

[0040] Determination of cherry quality indexes

[0041] Determination of sugar content: NY / T2742-2015

[0042] 1. Prepare the raw materials: Wash and dry the fruit, take the edible part, chop and mix, sample by quartering, and make a homogenate with a tissue masher (juicy fruits are directly homogenized, and fruits with low water content are added with water in a 1+1 ratio before homogenization). Weigh 10.00g (m) of the sample, wash it with water into a volumetric flask, add 3mL each of potassium ferrocyanide solution (4.5) and zinc acetate solution (4.6), shake well, make up to 250mL (V), let it stand for a while, filter, and use the filtrate for later use.

[0043] 2. Drawing of standard curve: Use a pipette to accurately draw 0mL, 0.2mL, 0.4mL, 0.8mL, 1.0mL, and 1.2mL of glucose standard solution into 6 10mL stoppered test tubes, add water to make the solution volume up to 2.0mL, add 400mL 3,5-dinitrosalicylic acid reagent, and heat in a boiling water bath for 5 minutes. Take it out, immediately put it in cold water, cool to room temperature, make up to volume, and shake well. The concentrations of the obtained series of glucose standard solutions are 0mg / mL, 0.02mg / mL, 0.04mg / mL, 0.08mg / mL, 0.10mg / mL, and 0.12mg / mL, respectively. Use a spectrophotometer to measure the absorbance value at 540nm. Use the glucose concentration (mg / mL) as the ordinate (y) and the absorbance value as the abscissa (x) to draw a standard curve.

[0044] 3. Determination of reducing sugar: According to the sugar content in different samples, use a pipette to draw 5mL~20mL (V2) of the filtrate into a volumetric flask, and dilute to 100mL (V) with water. Draw 1.0mL (V) of sample solution from the volumetric flask into a 10mL (V) volumetric flask or a stoppered graduated test tube, and add water to 2.0mL. Follow the steps for drawing the standard curve above. Record the absorbance value measured, and obtain the concentration of reducing sugar in the test solution from the standard curve.

[0045] 4. Determination of soluble sugar: According to the sugar content of the sample, use a pipette to draw 5mL~10mL (V) of the sample solution into a volumetric flask, add 1mL of 6mol / L hydrochloric acid solution, place in a constant temperature water bath (80±2)℃ and heat for 10 minutes, take out, place in a cold water tank and cool to room temperature, add 3 drops of methyl red indicator and neutralize with 6mol / L sodium hydroxide solution to light orange, dilute to 100mL (V) with water and mix well. The following steps are followed for the determination of reducing sugar. The soluble sugar content of the sample is calculated by mass fraction according to the following formula:

[0046]

[0047] Where:

[0048] X: Soluble sugar content in the sample, in %;

[0049] P: the concentration of reducing sugar in the test solution, in mg / mL;

[0050] V1: sample solution volume, in mL;

[0051] V2: sample volume, in mL;

[0052] V3: fixed volume of sample solution, in mL;

[0053] V4: the volume of the measured liquid absorbed, in mL;

[0054] V5: volume of sample solution, in mL;

[0055] A: Dilution factor, 1 for juicy fruits and 2 for fruits with less water content;

[0056] m: sample mass, in g;

[0057] 10: Conversion factor for converting the measurement result into mass percentage.

[0058] Total acid determination method: GB12456-2021

[0059] The determination of titratable acidity is as follows:

[0060] 1. Prepare the raw materials: Use the quartering method to divide the edible part, chop and mix evenly, weigh 250g, accurate to 0.1g, put it into a high-speed tissue pounder, add an equal amount of water, and pound for 1 to 2 minutes. Every 2g of homogenate is converted into 1g of sample, weigh 50g of homogenate, accurate to 0.1g, wash it into a 250mL container bottle with 100mL of water, heat it in a water bath at 75℃ to 80℃ for 30 minutes, shake it several times, take it out to cool, add water to the scale, shake well and filter.

[0061] 2. Indicator titration method: According to the predicted acidity, take 50 mL of sample solution with a pipette, add 5 drops of phenolphthalein indicator, and titrate with standard sodium hydroxide solution until a slightly reddish color appears that does not fade within 30 seconds. Record the consumed volume.

[0062] The measurement results are calculated according to the following formula:

[0063]

[0064] Where:

[0065] V1: Volume of sodium hydroxide standard solution consumed during titration, mL

[0066] C: molar concentration of sodium hydroxide standard solution, mol / L;

[0067] m: sample mass, g;

[0068] 250: Fixed volume of the sample after extraction, mL.

[0069] Determination of soluble solids: NY / T 2637-2014

[0070] The refractometer method is as follows:

[0071] 1. Sample solution preparation: Take cherries and chop them and mix them evenly. Weigh an appropriate amount of sample (generally weigh 250g for samples with high water content and 125g for samples with low water content), add appropriate amount of water, put them in a high-speed tissue grinder and grind them. Use two layers of mirror paper or four layers of gauze to squeeze out the homogenized juice for determination.

[0072] 2. Instrument calibration: Calibrate the refractometer with distilled water at 20°C and adjust the soluble solids content reading to 0.

[0073] 3. Sample liquid measurement: Keep the measurement temperature stable, and the fluctuation range should not exceed ±0.5℃. Wipe the surface of the prism with a soft flannel, add 2-3 drops of the sample liquid to be tested, and evenly distribute the sample liquid on the entire prism surface. Aim at the light source (for non-digital refractometers, turn the achromatic adjustment knob to divide the field of view into light and dark parts, and then turn the prism knob to make the light and dark dividing line fit at the cross point of the objective lens), and record the refractometer reading. For refractometers without automatic temperature compensation function, record the measurement temperature. Wipe the prism surface with distilled water and a soft flannel.

[0074] 4. Calculation of results: For undiluted samples, the refractometer reading is the soluble solid content of the sample. For samples diluted with distilled water, the soluble solid content is calculated according to the formula.

[0075] The measurement results are calculated according to the following formula:

[0076]

[0077] Where:

[0078] X: Soluble solid content of the sample, in percentage (%)

[0079] P: Soluble solid content of sample solution, in percentage (%)

[0080] m0: sample mass, in grams (g);

[0081] m1: The mass of distilled water added to the sample, in grams (g).

[0082] Vitamin C (ascorbic acid) determination method: GB5009.86-2016

[0083] Determination of Vitamin C (Ascorbic Acid)

[0084] 1. Sample solution preparation: weigh 10g of slurry sample, transfer the sample into a 100mL volumetric flask with an extractant, dilute to scale, shake well and filter. If the filtrate is colored, add 0.4g of white clay per gram of sample to decolorize it before filtering.

[0085] 2. Titration: Pipette an appropriate amount of filtrate into a 50mL conical flask and titrate with a calibrated 2,6-dichloroindophenol solution until the solution turns pink and does not fade for 15 seconds. Perform a blank test at the same time. Vitamin C is calculated as follows:

[0086]

[0087] Where:

[0088] V1: volume of dye solution consumed when titrating the sample (mL);

[0089] V0: volume of dye solution consumed during blank titration (mL);

[0090] T: 2,6-dichloroindophenol dye titer (mg / mL);

[0091] F: dilution factor;

[0092] M: sample weight (g).

[0093] Anthocyanin determination method: T / QAS075-2022

[0094] Determination of anthocyanins:

[0095] 1. Prepare the raw materials: Take an appropriate amount or all of them by quartering, remove the branches, leaves, broken fruits and other impurities visible to the naked eye except the fruit and stalk, and use a homogenizer to homogenize and disperse at 15000r / min for 20 seconds to make a homogenate, which should be used for testing immediately. The entire testing process should be carried out under light-proof conditions.

[0096] 2. Weigh 5.0g of fresh fruit homogenate, accurate to ±1mg; 0.1g of extract, accurate to ±0.1mg, and place in a 150mL stoppered conical flask, accurately add 50mL of extract (concentrated HCl: 80% ethanol solution = 3:97, v / v), seal, and weigh. Place in an ultrasonic cleaner and extract at 50℃ for 30 minutes, shake once every 10 minutes to keep the solid phase completely dispersed. After the extraction is completed, cool to room temperature, weigh again, make up the lost weight with the extract, and mix well. Take part of the extract and centrifuge at 8000r / min for 3 minutes, and take the supernatant for later use.

[0097] 3. The prepared test solution was first diluted 2 to 10 times (containing 5 mg to 60 mg anthocyanins) by adding the extract solution (concentrated hydrochloric acid: 80% ethanol solution = 3:97, v / v), and then diluted 5 times with the buffer solution to prepare two test solutions. One was diluted with potassium chloride buffer solution (0.025 mol / L, pH 1.0) (1 mL test solution + 4 mL potassium chloride buffer solution), and the other was diluted with sodium acetate buffer solution (0.4 mol / L, pH 4.5) (1 mL test solution + 4 mL sodium acetate buffer solution).

[0098] 4. After the test solution is allowed to stand for 10 minutes, scan the visible band between 400nm and 600nm to determine the maximum absorption wavelength. The absorbance values ​​of the test solution diluted with pH1.0 buffer solution and the test solution diluted with pH4.5 buffer solution are measured at the maximum absorption wavelength and 700nm wavelength, respectively.

[0099] The content of anthocyanidins (anthocyanins) X (calculated as petunia components) is calculated according to the following formula:

[0100]

[0101] Where:

[0102] X: the content of anthocyanins in cherries as petunidin components, in grams per 100 grams (g / 100g);

[0103] A: The absorbance difference between the test solution at pH 1.0 and pH 4.5, A = (Amax nm - A700 nm) pH 1.0 - (Amax nm - A700 nm) pH 4.5;

[0104] MW: 912.7, the average molar mass of petunidin components, in grams per mole (g / mol);

[0105] DF: dilution factor;

[0106] V: total volume of the extract, in milliliters (mL);

[0107] ε: 29591, the average molar extinction coefficient of petunidin components, in liters per mole centimeter (L / (mol·cm));

[0108] l: cuvette thickness, in centimeters (cm);

[0109] m: the mass of the sample weighed, in grams (g);

[0110] 10: Conversion factor from g / kg to g / 100g.

[0111] β-carotene determination method: GB 5009.83-2016

[0112] Reverse phase chromatography determination, the steps are as follows:

[0113] 1. Prepare the raw materials: accurately weigh 1-5 g (accurate to 0.001 g) of the mixed sample, transfer it to a 250 mL conical flask, add 1 g ascorbic acid and 75 mL anhydrous ethanol, and shake in a water bath at 60°C ± 1°C for 30 minutes.

[0114] 2. Saponification: Add 25mL potassium hydroxide solution and cover the bottle with a stopper. Place in a constant temperature vibration water bath preheated to 53℃±2℃ and saponify for 30 minutes. Take out, let stand, and cool to room temperature.

[0115] 3. Sample extraction: Transfer the saponified liquid into a 500mL separatory funnel, add 100mL petroleum ether, shake gently to exhaust, cover the bottle stopper, shake at room temperature for 10 minutes, let stand and separate, transfer the aqueous phase into another separatory funnel for a second extraction according to the above method. Combine the organic phases and wash with water until nearly neutral. Discard the aqueous phase, and filter and dehydrate the organic phase through anhydrous sodium sulfate. Collect the filtrate into a 500mL evaporating flask, and concentrate it under reduced pressure at 40℃±2℃ on a rotary evaporator to near dryness. Blow dry with nitrogen, accurately add 5.0mL of dichloromethane with a pipette, cover the bottle stopper, and fully dissolve the extract. After filtering through a 0.45μm membrane, discard the initial 1mL of filtrate and collect it in a sample injection bottle for use.

[0116] 4. Sample determination: Under the same chromatographic conditions, the sample solutions to be tested were injected into the liquid chromatograph for HPLC analysis. The retention time was used for qualitative analysis, the peak area was used for quantitative analysis, and the concentration of β-carotene in the test solution was calculated according to the standard curve regression equation.

[0117] The measurement results are calculated according to the following formula:

[0118]

[0119] Where:

[0120] ρβ: β-carotene concentration in the test solution obtained from the standard curve2, in μg / mL;

[0121] V: fixed volume of sample solution, in mL;

[0122] 100: coefficient to express the result as μg / 10g2;

[0123] m: sample mass, in g.

[0124] As shown in Table 2, the Bacillus Velez TCS001 suspension was diluted 400 times and sprayed once during the cherry flower bud differentiation period, the initial flowering period and the full flowering period, and once a week from the young fruit period to the harvest period; it can increase the β-carotene content of cherry by 13.6%, anthocyanin by 9.8%, soluble sugar by 7.3%, reduce vitamin C by 19.4%, and reduce the total acid of cherry by 7%.

[0125] Table 2 Cherry quality test results

[0126]

[0127] During the experiment, it was observed that Bacillus Velez TCS001 had a positive effect on the size, glossiness and other traits of cherries. The intrinsic quality can directly affect the taste of the fruit, so the taste was good when tasted.

[0128] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of Bacillus Velez TCS001 as a drug for improving cherry quality.

2. The use according to claim 1, characterized in that: The improvement of cherry quality includes one or more of increasing cherry soluble sugar, increasing cherry soluble solids, reducing cherry vitamin C, increasing cherry anthocyanin, increasing cherry beta-carotene, reducing cherry acidity, etc.

3. A method for improving cherry quality using Bacillus Velez TCS001, characterized in that: The method of improving cherry quality includes one or more of increasing cherry soluble sugar, increasing cherry anthocyanin, increasing cherry soluble solids, reducing cherry vitamin C, increasing cherry anthocyanin, increasing cherry beta-carotene, reducing cherry acidity, etc.

4. The use according to any one of claims 1-2 and the method according to claim 3, characterized in that: The Bacillus Velez TCS001 is the fermentation liquid of Bacillus Velez TCS001.

5. The use according to claim 4 or the method according to claim 4, characterized in that: The Bacillus Velez TCS001 fermentation liquid is prepared into a suspension.

6. The use according to claim 5 or the method according to claim 5, characterized in that: The formula of the suspension comprises 40% of Bacillus Velez TCS001 fermentation liquid, 3% of dispersant SXC, 3% of dispersant UNA, 0.15% of thickener xanthan gum, 0.2% of preservative kason, 0.2% of preservative sodium benzoate and 53.45% of water.

7. The use according to claim 5 or the method according to claim 5, characterized in that: The application concentration of the suspension is 300-700 times diluted; preferably, the application concentration is 400 times diluted.

8. The use according to any one of claims 1-2 and the method according to claim 3, characterized in that: The application time of the Bacillus Velez TCS001 can be one or more of the flower bud differentiation period, the initial flowering period, the full flowering period, the young fruit period, and the harvest period.

9. The use according to any one of claims 1-2 and the method according to claim 3, characterized in that: The application mode of the Bacillus Velez TCS001 is spraying treatment.

10. A method for increasing the size and / or glossiness and / or taste of cherries, characterized in that: Including the use of Bacillus Velez TCS001 to treat cherries.

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