A method for improving wine grape tannin using bacillus megaterium

Abstract

The application discloses a method for improving tannin of wine grape by using bacillus megaterium, and belongs to the technical field of plant cultivation. The method comprises the following steps: (1) inoculating bacillus megaterium strains into a culture solution, carrying out fermentation culture, and obtaining a bacterial liquid; (2) mixing the bacterial liquid with a nutrient solution, and obtaining a bacterial agent; (3) respectively applying the bacterial agent to root irrigation in a germination period and an initial fruiting period of the wine grape, and performing foliar spraying in a color changing period; and the bacillus megaterium strains are composed of bacillus megaterium CGMCC 1.8788 and bacillus megaterium CGMCC 1.8569. The bacterial agent prepared by using the bacillus megaterium is applied in different periods of wine grape growth, can promote the accumulation of tannin in the wine grape, improves the quality of the wine grape, provides a reference for a new application of the bacillus megaterium, and provides an experience method for the cultivation of the wine grape.

Description

Technical Field

[0001] This invention relates to the field of plant cultivation technology, and in particular to a method for increasing tannins in wine grapes using Bacillus megaterium. Background Technology

[0002] Tannins are complex phenolic compounds with strong antioxidant and astringent properties, significantly influencing the color, taste, and finish of wine. Tannins can bind with pigments in grape skins, giving wine its beautiful color; appropriate amounts of tannins can increase the complexity and depth of a wine's flavor, and prolong its finish. Therefore, tannins are one of the key factors determining wine quality.

[0003] Tannins are natural tannins, primarily derived from grape skins, seeds, and stems. They are formed during wine fermentation and aging by combining with other compounds. Therefore, the tannin content determines the quality of wine. Wine grapes refer to grape varieties primarily used for winemaking, with suitable sugar and acidity levels. However, with the diversification of wine types and increased demands for quality, further increasing the tannin content of wine grapes is crucial for developing more diverse wine varieties and for the rapid development of the wine industry. Summary of the Invention

[0004] The purpose of this invention is to provide a method for increasing tannins in wine grapes using Bacillus megaterium, thereby addressing the problems existing in the prior art. This invention utilizes a Bacillus megaterium-based inoculant, applied at different stages of wine grape growth, to promote tannin accumulation and improve grape quality. This provides a reference for new applications of Bacillus megaterium and offers an empirical method for wine grape cultivation.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for increasing tannins in wine grapes using Bacillus megaterium, comprising the following steps:

[0007] (1) Inoculate the Bacillus megaterium strain into the culture medium and ferment it to obtain the bacterial solution;

[0008] (2) The bacterial solution is mixed with the nutrient solution to obtain the bacterial agent;

[0009] (3) Apply the microbial agent to the roots during the budding and early fruiting stages of wine grapes, and spray it on the leaves during the color-changing stage.

[0010] The Bacillus megaterium strain consists of Bacillus megaterium CGMCC 1.8788 and Bacillus megaterium CGMCC 1.8569.

[0011] Preferably, the ratio of viable Bacillus megaterium CGMCC 1.8788 to Bacillus megaterium CGMCC 1.8569 is (2.5-3):1.

[0012] Preferably, the inoculation amount is 2-4%.

[0013] Preferably, the fermentation temperature is 33–35°C; the bacterial count in the bacterial solution is 5 × 10⁻⁶. 8～12 CFU / mL.

[0014] Preferably, the culture medium is formulated as follows: peptone 4.0–8.0 g / L, glucose 10–25 g / L, sodium chloride 0.1–0.5 g / L, calcium carbonate 0.1–0.3 g / L, dipotassium hydrogen phosphate 0.4–1.0 g / L, vitamin B1 0.5–0.8 g / L, vitamin B2… 12 0.5~0.8g / L, pH 7.2~7.4.

[0015] Preferably, the nutrient solution is formulated as follows: nitrogen 5-15 g / L, potassium nitrate 150-200 mg / L, magnesium sulfate 250-300 mg / L, ammonium dihydrogen phosphate 20-40 mg / L, boric acid 1-2 mg / L, manganese chloride 0.5-0.8 mg / L, copper sulfate 0.1-0.2 mg / L, and zinc sulfate 0.05-0.1 mg / L.

[0016] Preferably, the volume ratio of the bacterial solution to the nutrient solution is 1:(60-100).

[0017] Preferably, the dosage for root irrigation is 40-70 L / mu; the dosage for foliar spraying is 15-30 L / mu.

[0018] The present invention discloses the following technical effects:

[0019] This invention screens out two Bacillus megaterium species that promote the growth of wine grapes, prepares bacterial solutions in a specific ratio, and then mixes them with nutrient solutions. By applying these solutions during specific growth stages of wine grapes, the accumulation of tannins and other phenolic compounds is promoted, thereby improving the quality and yield of wine grapes and promoting the development of the wine industry. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] The strains involved in the embodiments of this invention are sourced from:

[0026] Bacillus megaterium CGMCC 1.8788, Bacillus megaterium CGMCC 1.8569, Bacillus subtilis CGMCC 1.15792 and Bacillus subtilis CGMCC 1.12938 were all derived from the China General Microbiological Culture Collection Center.

[0027] All of the above strains can be purchased from the aforementioned preservation institutions.

[0028] Example 1

[0029] This embodiment of the method for increasing tannins in wine grapes using Bacillus megaterium includes the following steps:

[0030] (1) Bacillus megaterium CGMCC 1.8788 and Bacillus megaterium CGMCC 1.8569 were activated in brown sugar water for 3 hours, and then the two were mixed at a live cell ratio of 2.8:1 to prepare a bacterial inoculum. The obtained bacterial inoculum was inoculated into the culture medium at an inoculation rate of 3% and fermented at 34℃ until the concentration of the culture medium reached 5×10⁻⁶. 10 CFU / mL was used to obtain bacterial culture.

[0031] The culture medium formula is as follows: peptone 5.0 g / L, glucose 20 g / L, sodium chloride 0.4 g / L, calcium carbonate 0.2 g / L, dipotassium hydrogen phosphate 0.6 g / L, vitamin B1 0.6 g / L, vitamin B2... 12 0.6 g / L, pH 7.2.

[0032] (2) Prepare the nutrient solution according to the formula: nitrogen 12g / L, potassium nitrate 180mg / L, magnesium sulfate 270mg / L, ammonium dihydrogen phosphate 30mg / L, boric acid 1.5mg / L, manganese chloride 0.7mg / L, copper sulfate 0.14mg / L, and zinc sulfate 0.08mg / L. Mix the bacterial solution and nutrient solution evenly at a volume ratio of 1:80 to obtain the bacterial agent.

[0033] (3) Apply the microbial agent to the roots once during the budding and early fruiting stages of wine grapes at a dosage of 50L / mu; and spray the leaves of the microbial agent once during the color-changing stage of wine grapes at a dosage of 20L / mu.

[0034] Example 2

[0035] This embodiment of the method for increasing tannins in wine grapes using Bacillus megaterium includes the following steps:

[0036] (1) Bacillus megaterium CGMCC 1.8788 and Bacillus megaterium CGMCC 1.8569 were activated in brown sugar water for 3 hours, and then the two were mixed at a live cell ratio of 2.5:1 to prepare a bacterial inoculum. The obtained bacterial inoculum was inoculated into the culture medium at an inoculation rate of 4%, and fermented at 34℃ until the concentration of the culture medium reached 5×10⁻⁶. 10 CFU / mL was used to obtain bacterial culture.

[0037] The culture medium formula is as follows: peptone 4.0 g / L, glucose 25 g / L, sodium chloride 0.1 g / L, calcium carbonate 0.3 g / L, dipotassium hydrogen phosphate 1.0 g / L, vitamin B1 0.8 g / L, vitamin B2... 12 0.8 g / L, pH 7.2.

[0038] (2) Prepare the nutrient solution according to the formula: nitrogen 15g / L, potassium nitrate 150mg / L, magnesium sulfate 250mg / L, ammonium dihydrogen phosphate 20mg / L, boric acid 1mg / L, manganese chloride 0.5mg / L, copper sulfate 0.2mg / L, and zinc sulfate 0.05mg / L. Mix the bacterial solution and nutrient solution at a volume ratio of 1:100 to obtain the bacterial agent.

[0039] (3) Apply the microbial agent to the roots once during the budding and early fruiting stages of wine grapes at a dosage of 70L / mu; and spray the leaves of the microbial agent once during the color-changing stage of wine grapes at a dosage of 30L / mu.

[0040] Example 3

[0041] This embodiment of the method for increasing tannins in wine grapes using Bacillus megaterium includes the following steps:

[0042] (1) Bacillus megaterium CGMCC 1.8788 and Bacillus megaterium CGMCC 1.8569 were activated in brown sugar water for 3 hours, and then the two were mixed at a live cell ratio of 3:1 to prepare a bacterial inoculum. The obtained bacterial inoculum was inoculated into the culture medium at an inoculation rate of 3% and fermented at 34°C until the concentration of the culture medium reached 5×10⁻⁶. 10 CFU / mL was used to obtain bacterial culture.

[0043] The culture medium formula is as follows: peptone 8.0 g / L, glucose 20 g / L, sodium chloride 0.5 g / L, calcium carbonate 0.1 g / L, dipotassium hydrogen phosphate 0.4 g / L, vitamin B1 0.5 g / L, vitamin B2... 12 0.5 g / L, pH 7.2.

[0044] (2) Prepare the nutrient solution according to the formula: nitrogen 10g / L, potassium nitrate 200mg / L, magnesium sulfate 300mg / L, ammonium dihydrogen phosphate 40mg / L, boric acid 2mg / L, manganese chloride 0.8mg / L, copper sulfate 0.1mg / L, and zinc sulfate 0.1mg / L. Mix the bacterial solution and nutrient solution evenly at a volume ratio of 1:60 to obtain the bacterial agent.

[0045] (3) Apply the microbial agent to the roots once during the budding and early fruiting stages of wine grapes at a dosage of 40L / mu; and spray the microbial agent on the leaves once during the color-changing stage of wine grapes at a dosage of 15L / mu.

[0046] Comparative Example 1

[0047] The difference between this comparative example and Example 1 is that Bacillus megaterium CGMCC 1.8569 is omitted.

[0048] Comparative Example 2

[0049] The difference between this comparative example and Example 1 is that Bacillus megaterium CGMCC 1.8788 is omitted.

[0050] Comparative Example 3

[0051] The difference between this comparative example and Example 1 is that the ratio of viable bacteria of Bacillus megaterium CGMCC 1.8788 and Bacillus megaterium CGMCC 1.8569 was adjusted to 1:1.

[0052] Comparative Example 4

[0053] The difference between this comparative example and Example 1 is that Bacillus megaterium CGMCC 1.8788 and Bacillus megaterium CGMCC 1.8569 are replaced with Bacillus subtilis CGMCC 1.15792 and Bacillus subtilis CGMCC 1.12938.

[0054] Comparative Example 5

[0055] The difference between this comparative example and Example 1 is that, at a dosage of 50L / acre, the microbial agent was applied once with water to the roots during the budding, flowering, and early fruiting stages of the wine grapes, and at a dosage of 20L / acre, it was applied once as a foliar spray during the color-changing stage of the wine grapes.

[0056] Comparative Example 6

[0057] The difference between this comparative example and Example 1 is that, at a dosage of 50L / acre, the microbial agent was applied to the roots once during the budding, flowering, and early fruiting stages of the wine grapes; and the foliar spraying during the color-changing stage was omitted.

[0058] Experimental Example 1

[0059] At the Jinshan Guanlan Grape Base, Cabernet Sauvignon wine grapes were used as the experimental variety. Experimental plots were randomly divided into plots of equal size, with a planting density of 60×320cm for all plots. Each plot was cultivated according to the methods described in Examples 1–3 and Comparative Examples 1–6, respectively, with a blank control group also included. Apart from the above treatments, other field management practices were carried out according to local routine procedures.

[0060] At harvest, 10 bunches of grapes were randomly selected from each experimental plot. From each bunch, 5 grapes were randomly selected from the top, middle, and bottom parts. The tannin content was determined using the Folin-Denis method, and the grape yield of each plot was recorded. The results are shown in Table 1.

[0061] Table 1. Results of Tannin Content and Grape Yield in Wine Grapes from Each Experimental Plot

[0062] Group Tannin content (mg / g) Grape yield (kg / mu) Blank control group 15.14 420.5 Example 1 18.26 444.6 Example 2 18.24 439.7 Example 3 18.01 435.0 Comparative Example 1 16.88 416.4 Comparative Example 2 15.97 422.3 Comparative Example 3 17.17 425.1 Comparative Example 4 14.55 472.7 Comparative Example 5 16.12 423.9 Comparative Example 6 15.45 426.5

[0063] As shown in Table 1, compared with the blank control group, the methods in Examples 1-3 significantly improved the tannin content and yield of grapes. However, Comparative Examples 1 and 2 show that the effect of a single *Bacillus megaterium* strain on tannin content and yield is lower than that of the combined application of the two *Bacillus megaterium* strains in this invention. Comparative Example 3 shows that this improvement effect depends on the specific ratio of the two *Bacillus megaterium* strains. Comparative Example 4 shows that different types of Bacillus have different growth-promoting effects on wine grapes; *Bacillus subtilis* has a better effect on improving grape yield than *Bacillus megaterium*, but *Bacillus subtilis* does not improve grape tannin content. Therefore, *Bacillus megaterium* was chosen to improve grape tannin content. Comparative Examples 5 and 6 show that the effect of *Bacillus megaterium* on improving grape tannin content depends on the specific application period and does not show a significant improvement effect with increasing application frequency.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for increasing tannins in wine grapes using Bacillus megaterium, characterized in that, Includes the following steps: (1) Inoculate the Bacillus megaterium strain into the culture medium and ferment it to obtain the bacterial solution; (2) The bacterial solution is mixed with the nutrient solution to obtain the bacterial agent; (3) Apply the microbial agent to the roots during the budding and early fruiting stages of wine grapes, and spray it on the leaves during the color-changing stage; The Bacillus megaterium strain consists of Bacillus megaterium CGMCC 1.8788 and Bacillus megaterium CGMCC 1.8569; The viable cell ratio of Bacillus megaterium CGMCC 1.8788 and Bacillus megaterium CGMCC 1.8569 is (2.5~3):1; The dosage for root irrigation is 40-70 L / mu; the dosage for foliar spraying is 15-30 L / mu.

2. The method according to claim 1, characterized in that, The inoculation amount is 2-4%.

3. The method according to claim 1, characterized in that, The fermentation culture temperature is 33~35℃; the bacterial count of the bacterial solution is 5×10⁻⁶. 8~12 CFU / mL.

4. The method according to claim 1, characterized in that, The culture medium is formulated as follows: peptone 4.0~8.0 g / L, glucose 20~25 g / L, sodium chloride 0.1~0.5 g / L, calcium carbonate 0.1~0.3 g / L, dipotassium hydrogen phosphate 0.4~1.0 g / L, vitamin B1 0.5~0.8 g / L, vitamin B2... 12 0.5~0.8g / L, pH 7.2~7.

4.

5. The method according to claim 1, characterized in that, The nutrient solution formula is as follows: nitrogen 10-15 g / L, potassium nitrate 150-200 mg / L, magnesium sulfate 250-300 mg / L, ammonium dihydrogen phosphate 20-40 mg / L, boric acid 1-2 mg / L, manganese chloride 0.5-0.8 mg / L, copper sulfate 0.1-0.2 mg / L, and zinc sulfate 0.05-0.1 mg / L.

6. The method according to claim 1, characterized in that, The volume ratio of the bacterial solution to the nutrient solution is 1:(60~100).