Application of bacillus velezensis TCS001 in promoting early maturation of blueberries and improving quality

Abstract

The invention relates to application of bacillus velezensis TCS001 as a medicine for improving the quality of blueberries, after the blueberries are treated by the bacillus velezensis TCS001, the anthocyanin content of the blueberries can be increased by 10%, the soluble sugar content of the blueberries can be increased by 4%, the vitamin C content of the blueberries can be increased by 5%, and the total acid content of the blueberries can be reduced by 20%. The bacillus velezensis TCS001 is found to be capable of promoting the blueberries to be mature and appear on the market 5-7 days in advance for the first time.

Description

Technical Field

[0001] The invention relates to the field of plant growth regulation, in particular to use of Bacillus Velez TCS001 in regulating the growth quality of blueberries, and also to a method for promoting early ripening of blueberries. 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 root irrigation treatment agent used in blueberry production. It has been reported in the literature that spraying 6000 times diluted amino acid oligosaccharide solution on the leaves of blueberries during the young fruit stage, fruit color change stage to full maturity stage can reduce the total acid content by 12.7%.

[0005] In the prior art, there is no report on the application of Bacillus Velez TCS001 to blueberries. Summary of the invention

[0006] The main purpose of the present invention is to provide application of Bacillus Velez TCS001 as a drug for improving the quality of blueberries.

[0007] Another main object of the present invention is to provide a method for improving the quality of blueberries using Bacillus Velez TCS001.

[0008] Preferably, the improving the quality of blueberries includes one or more of increasing the sugar content and anthocyanin of blueberries, reducing the acidity of blueberries, and increasing the vitamin C content, color, size, glossiness, etc.

[0009] Another main object of the present invention is to provide a method for promoting early ripening of blueberries, comprising treating blueberries with Bacillus Velez TCS001.

[0010] Optimization promotes the ripening of blueberries 5-7 days earlier.

[0011] Preferably, the Bacillus Velez subtilis TCS001 is a fermentation broth of Bacillus Velez subtilis TCS001.

[0012] Preferably, the Bacillus Velez TCS001 fermentation broth is prepared as a suspension.

[0013] 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.

[0014] Preferably, the application concentration of the suspension is 300-700 times diluted.

[0015] Preferably, the application concentration of the suspension is 500 times diluted.

[0016] Preferably, the Bacillus Velez subtilis TCS001 is applied by root irrigation and / or spraying.

[0017] Preferably, the Bacillus Velez TCS001 is applied during one or more of the flower bud differentiation period, early flowering period, full flowering period, young fruit period, and harvest period of blueberry.

[0018] Preferably, the Bacillus Velez TCS001 is applied once a week from the young fruit stage of blueberries to the harvest stage until the end of harvest.

[0019] Preferably, the Bacillus Velez TCS001 is applied once within 5 days after harvesting.

[0020] Beneficial technical effects of the present invention

[0021] The present invention first discovered that Bacillus Velez TCS001 can promote the early maturity of blueberries. After root irrigation with 500 times the suspension of Bacillus Velez TCS001, the ripening time was advanced by 5-7 days compared with CK.

[0022] During the blueberry flower bud differentiation period, initial flowering period, peak flowering period, young fruit period to harvest period, root irrigation with 500 times Bacillus Velez TCS001 suspension concentrate can increase the anthocyanin content of blueberries by 10%, soluble sugar by 4%, vitamin C by 5%, and reduce the total acidity of blueberries by 20% compared with CK.

[0023] The quality of blueberry fruit can be significantly improved after being treated with Bacillus Velez TCS001. During the experiment, it was observed that Bacillus Velez TCS001 was significantly better than the blank control in terms of color, size, glossiness and other traits of blueberry, and the taste was good. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are blueberry fruits of the control group and those after root irrigation treatment with Bacillus Velez TCS001. Specific embodiments

[0025] Materials and Methods

[0026] Test Materials:

[0027] The test bacterial agent of the present invention is the suspension concentrate of Bacillus Velezii TCS001 (3.9×10 9 CFU / mL), the specific formula is: 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 Kathon, 0.2% of preservative sodium benzoate, and 53.45% of water.

[0028] Test plants:

[0029] Experimental variety: Blueberry (AFP).

[0030] Main instruments

[0031] ME104E electronic balance (Sartorius Scientific Instruments Co., Ltd.)

[0032] Test methods

[0033] Processing

[0034] Table 1 Treatment methods of blueberry test with Bacillus Velez TCS001 suspension concentrate

[0035]

[0036] After the suspension of Bacillus Velez TCS001 was diluted with water to the corresponding concentration, the roots were irrigated with 500 ml per blueberry tree.

[0037] Root irrigation: irrigate the roots once during the flower bud differentiation period, early flowering period, and peak flowering period of blueberries, and irrigate the roots once a week from the young fruit period to the harvest period.

[0038] Determination of blueberry quality indicators

[0039] Soluble sugar determination method: NY / T2742-2015.

[0040] 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.

[0041] 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 2.0mL, add 400mL 3,5-dinitrosalicylic acid reagent, and heat in a boiling water bath for 5min. 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.

[0042] 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 1.00mL (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.

[0043] 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 10min, 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:

[0044]

[0045] Where:

[0046] X: Soluble sugar content in the sample, in %;

[0047] P: the concentration of reducing sugar in the test solution, in mg / mL;

[0048] V1: sample solution volume, in mL;

[0049] V2: sample volume, in mL;

[0050] V3: fixed volume of sample solution, in mL;

[0051] V4: the volume of the measured liquid absorbed, in mL;

[0052] V5: volume of sample solution, in mL;

[0053] A: Dilution factor, 1 for juicy fruits and 2 for fruits with less water content;

[0054] m: sample mass, in g;

[0055] 10: Conversion factor for converting the measurement result into mass percentage.

[0056] Total acid determination method: GB12456-2021 titratable acidity determination, the steps are as follows:

[0057] 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 2min. 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 30min, shake it several times, take it out to cool, add water to the scale, shake well and filter.

[0058] 2. Indicator titration

[0059] 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.

[0060] The measurement results are calculated according to the following formula:

[0061]

[0062] Where:

[0063] V1: Volume of sodium hydroxide standard solution consumed during titration, mL

[0064] C: molar concentration of sodium hydroxide standard solution, mol / L;

[0065] m: sample mass, g;

[0066] 250: Fixed volume of the sample after extraction, mL.

[0067] Soluble solids determination method: NY / T2637-2014

[0068] Determination of soluble solids:

[0069] 1. Prepare the raw materials: Take the edible part of the sample, chop it and mix it evenly (frozen products must be thawed in advance), weigh 250g, accurate to 0.1g, put it into a high-speed tissue grinder and grind it, and use two layers of lens paper or gauze to squeeze out the homogenized juice for determination.

[0070] 2. Determination:

[0071] 1) Adjust the temperature of the circulating water in the constant temperature water bath to 20±0.5℃, and let the water flow through the thermostat of the refractometer. The circulating water can also be adjusted in the range of 15~25℃, and the temperature should not exceed ±0.5℃.

[0072] 2) Calibrate the refractometer reading with distilled water and adjust the soluble solids to 0% at 20°C; when the temperature is not 20°C, calibrate according to the correction values ​​in the table below.

[0073] 3) After wiping the surface of the prism dry, add 2 to 3 drops of the sample solution to be tested to the center of the prism, immediately close the upper and lower prisms, aim at the light source, turn the achromatization adjustment knob to divide the field of view into light and dark parts, then turn the prism knob to make the light and dark dividing line fit at the cross point of the objective lens, read the percentage shown on the scale, and record the temperature during the measurement.

[0074] Vitamin C (ascorbic acid) determination method: GB5009.86-2016

[0075] 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.

[0076] 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:

[0077]

[0078] Where:

[0079] V1: volume of dye solution consumed when titrating the sample (mL);

[0080] V0: volume of dye solution consumed during blank titration (mL);

[0081] T: 2,6-dichloroindophenol dye titer (mg / mL);

[0082] F: dilution factor;

[0083] M: sample weight (g).

[0084] Anthocyanin determination method: T / QAS075-2022;

[0085] Anthocyanin determination method:

[0086] 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 20s to make a homogenate, which should be used for testing immediately. The entire testing process should be carried out under light-proof conditions.

[0087] 2. Weigh 5.0g of fresh fruit homogenate, accurate to ±1mg; 0.1g of extract, accurate to ±0.1mg), 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 30min, shake once every 10min 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 3min, and take the supernatant for later use.

[0088] 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).

[0089] 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 respectively.

[0090] The content of anthocyanidins (anthocyanins) X (calculated as petunia components) is calculated according to the following formula:

[0091]

[0092] Where:

[0093] X: the content of anthocyanins in blueberries calculated as petunidin components, in grams per 100 grams (g / 100g);

[0094] 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;

[0095] MW: 912.7, the average molar mass of petunidin components, in grams per mole (g / mol);

[0096] DF: dilution factor;

[0097] V: total volume of the extract, in milliliters (mL);

[0098] ε: 29591, the average molar extinction coefficient of petunidin components, in liters per mole centimeter (L / (mol·cm));

[0099] l: cuvette thickness, in centimeters (cm);

[0100] m: the mass of the sample weighed, in grams (g);

[0101] 10: Conversion factor from g / kg to g / 100g.

[0102] Test results:

[0103] As shown in Table 2, the root irrigation treatment with Bacillus Velez TCS001 suspension diluted 500 times, once during the blueberry flower bud differentiation period, initial flowering period and full flowering period, and once a week from the young fruit stage to the harvest period can increase the anthocyanin content of blueberries by 10%, the soluble sugar content by 4%, the vitamin C content by 5%, and reduce the total acidity of blueberries by 20%.

[0104] Table 2 Blueberry quality test results

[0105]

[0106] During the test, it was observed that Bacillus Velez TCS001 had a positive effect on the color, size, glossiness and other characteristics of blueberries, and the taste of the five groups of people was good, as shown in Table 3 below.

[0107] Table 3 Blueberry tasting results

[0108]

[0109] 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 the quality of blueberries.

2. The use according to claim 1, characterized in that: The improving of blueberry quality comprises increasing the sugar content and anthocyanin of blueberries, reducing the acidity of blueberries, and increasing one or more of the following: vitamin C content, color, size, glossiness, etc.

3. A method for improving the quality of blueberries using Bacillus Velez TCS001, characterized in that: The improving of blueberry quality comprises increasing the sugar content and anthocyanin of blueberries, reducing the acidity of blueberries, and increasing one or more of the following: vitamin C content, color, size, glossiness, etc.

4. A method for promoting early ripening of blueberries, characterized in that: The method comprises treating blueberries with Bacillus Velez TCS001; preferably, the blueberries are promoted to mature 5-7 days earlier.

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

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

7. The use according to claim 6 or the method according to claim 6, 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.

8. The use according to claim 7 or the method according to claim 7, characterized in that: The application concentration of the suspension is 300-700 times diluted; preferably, the application concentration is 500 times diluted.

9. The use according to any one of claims 1-2 or the method according to any one of claims 3-4, 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.

10. The use according to any one of claims 1-2 or the method according to any one of claims 3-4, characterized in that: The application method of the Bacillus Velez TCS001 is root irrigation and / or spraying treatment.

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