The application of a colorant with Trichoderma viride metabolite as effective component in promoting fruit coloring

Abstract

The application discloses application of a colorant with a Trichoderma viride metabolic product as an effective component to promote fruit coloring. The Trichoderma viride metabolic product is prepared by fermentation of a Trichoderma viride strain (CGMCC 3.11450), and the obtained Trichoderma viride metabolic product has a significant promoting effect on fruit coloring. When the Trichoderma viride metabolic product is used in combination with an emulsifier, a dispersing agent and a calcium-containing foliar fertilizer / fruit surface fertilizer, the fruit skin hardness can be enhanced, anthocyanin accumulation can be promoted, and the coloring rate can be improved. The Trichoderma viride metabolic product is creatively applied to promote fruit coloring, the fermentation process is mature, the raw material cost is low, the product is fast, large, stable, the preparation is natural and environment-friendly, the coloring effect is significant, the development and utilization of the beneficial bacteria in agricultural use are widened, a green and environment-friendly biological source preparation for fruit coloring is provided, and the application has good development and application prospect and practical significance.

Description

Technical Field

[0001] This invention belongs to the field of beneficial microorganisms applied to improve fruit quality, specifically involving the application of a colorant with Trichoderma viride metabolites as the active ingredient in promoting fruit coloring. Background Technology

[0002] Trichoderma viride ( Trichoderma viride *Trichoderma viride* is a beneficial microorganism widely distributed in nature, and it is commonly used in agricultural production due to its highly efficient biodegradation and biocontrol functions. Existing research shows that *Trichoderma viride* produces a series of hydrolytic enzymes during its metabolism, such as cellulase, chitinase, and lignin peroxidase, which can efficiently degrade organic matter in the environment. At the same time, as a commonly used agricultural biocontrol bacterium, *Trichoderma viride* has functions such as inhibiting pathogens, promoting plant growth, and improving soil fertility.

[0003] Fruit color is a crucial indicator of fruit quality and a significant factor in determining market acceptance, influencing consumer choice. In actual production, various internal and external factors, such as the external environment and cultivation techniques, lead to uneven coloring in fruits like apples and grapes, severely impacting fruit quality and commercial value. Besides strengthening cultivation techniques, water and fertilizer management, and using methods like bagging and reflective film, the use of plant growth regulators is also a common way to promote fruit coloring.

[0004] Through extensive experimental research and application verification, this invention creatively applies a green Trichoderma metabolite to fruit coloring, achieving excellent coloring results. The combined application of this green Trichoderma metabolite with emulsifiers, dispersants, and calcium-containing foliar / fruit fertilizers significantly enhances its effects, increasing fruit skin firmness, promoting anthocyanin accumulation, and improving coloring rate.

[0005] A search revealed that the prior art document "Microbial Staining of Protein Fibers" (Master's Thesis, Suzhou University, Zhai Hongxia, March 2016) discloses a process for dyeing silk and wool fabrics using yellow pigment prepared from *Trichoderma viride*. It discloses the *Trichoderma viride* strain (GIM3.141) and its source (Guangdong Provincial Microbial Culture Collection Center), as well as the influence of the culture medium and fermentation conditions on the product. This invention discloses the application of a colorant using *Trichoderma viride* metabolites as the active ingredient in promoting fruit coloring. The disclosed *Trichoderma viride* strain (CGMCC 3.11450) is not the same strain as the one described in the prior art document, and the preparation method of the *Trichoderma viride* metabolites disclosed in this invention is significantly different from that in the prior art document. Furthermore, the *Trichoderma viride* metabolites described in this invention are used in the preparation of colorants and have been experimentally verified to have good fruit coloring effects.

[0006] Therefore, the innovation of this invention lies in developing an application of *Trichoderma viride* and its metabolites in fruit coloring agents. *Trichoderma viride* has not been reported in existing coloring applications. The preparation process of the *Trichoderma viride* metabolites is significantly innovative, and the fruit coloring agent prepared from the *Trichoderma viride* metabolites is also significantly innovative. This represents an innovation in the development and application of new microbial strains and has the potential to further develop bio-based, widely applicable coloring agents. Clearly, this is of great significance for the development of beneficial microorganisms for agricultural use and the green and healthy development of fruit and vegetable cultivation. Summary of the Invention

[0007] This invention discloses the application of a colorant with Trichoderma viride metabolites as the active ingredient in promoting fruit coloring, in order to enhance the hardness of the fruit peel, promote the accumulation of anthocyanins, and improve the coloring rate.

[0008] The first objective of this invention is to provide a method for preparing a metabolite of *Trichoderma viride*, comprising the following steps: 1) The *Trichoderma viride* strain CGMCC 3.11450 was activated and then inoculated into a modified PDA medium to cultivate seed culture. 2) When the bacterial concentration in the seed culture reaches 106~109 CFU / mL, it is inoculated into modified Czapek's medium for fermentation culture. The fermentation broth is obtained after 4~8 days. 3) The fermentation broth is treated by flocculation, filtration and sterilization to obtain the filtrate, which is the metabolite of Trichoderma viride.

[0009] The second objective of this invention is to provide a fruit coloring agent formulated with the above-mentioned Trichoderma viride metabolites, wherein the formulation is as follows: Trichoderma viride metabolites, emulsifier, and dispersant are mixed in a weight ratio of 1:0.1~0.45:0.5~1 to obtain the coloring agent.

[0010] A third objective of this invention is to provide the application of the above-mentioned colorant, which uses Trichoderma viride metabolites as the active ingredient, in promoting fruit coloring, and the method is as follows: 1) The metabolites of *Trichoderma viride* are compounded with emulsifiers and dispersants to form a fruit coloring agent, which is then diluted with pure water to form a coloring spray, wherein the mass fraction of *Trichoderma viride* metabolites is 40%~50%; 2) Different application rates should be selected according to different fruit tree species and different fruit development stages. The application rate of coloring spray is 5~30 kg / mu. 3) Choose the application site, such as the leaves of fruit trees or the surface of the fruit, according to different coloring requirements; 4) Select 1 to 3 applications of coloring spray according to the fruit coloring condition and needs, with an interval of 5 to 10 days between adjacent applications.

[0011] The advantages of this invention are: the preparation method of the *Trichoderma viride* metabolite is simple, the fermentation process is mature, the product is stable, the yield is high, and the raw material cost is low, enabling rapid, efficient, and continuous production; the application of the *Trichoderma viride* metabolite in promoting fruit coloring is significantly innovative, creative, and practical. When combined with emulsifiers, dispersants, and calcium-containing foliar / fruit fertilizers, it can enhance fruit skin firmness, promote anthocyanin accumulation, and increase coloring rate. This *Trichoderma viride* metabolite can be developed into a bio-based, widely applicable, and environmentally friendly coloring agent, which is of significant practical importance for promoting the application of beneficial microorganisms in agriculture, improving fruit quality, saving agricultural production costs, and increasing agricultural profits. Detailed Implementation

[0012] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. The parts mentioned in the specific embodiments are parts by weight.

[0013] The *Trichoderma viride* used in this embodiment of the invention was purchased from the China General Microbiological Culture Collection Center (CGMCC 3.11450). Example 1

[0014] A green Trichoderma metabolite, the preparation method of which is as follows: (1) The green Trichoderma strain was activated by PDA solid medium, and single strains were selected for streak culture on slant medium and preserved. The green Trichoderma strains preserved on the slant medium were selected and inoculated into modified PDA medium and cultured at constant temperature and shaking for 24 hours to obtain green Trichoderma seed liquid. (2) The strains in the *Trichoderma viride* seed culture reached the logarithmic growth phase (10) 6 ~10 9 After (CFU / mL), it was inoculated into a modified Czapek fermentation medium and fermented in a fermenter under controlled temperature and aerobic conditions. The concentration of metabolites in the fermentation broth was continuously monitored. Fermentation was stopped after the concentration of metabolites stopped increasing (4-8 days) to obtain the Trichoderma viride fermentation broth. (3) The fermentation broth is subjected to flocculation, filtration and sterilization treatment to obtain the filtrate, which is the metabolite of Trichoderma viride. Example 2

[0015] A colorant using *Trichoderma viride* metabolites as the active ingredient is prepared as follows: The green Trichoderma metabolites prepared in Example 1 were mixed with emulsifier and dispersant at a weight ratio of 1:0.35:0.65 to obtain a colorant. Comparative Example 1

[0016] The method for preparing yellow pigment from *Trichoderma viride* by fermentation was used to prepare *Trichoderma viride* metabolites, replacing the *Trichoderma viride* metabolites in Example 2, as disclosed in Comparative Document 1 ("Microbial staining of protein fibers", Zhai Hongxia, Master's Thesis, Suzhou University, March 2016, Chapter 3: Optimization of culture medium composition and culture conditions for yellowing of *Trichoderma viride*). The remaining steps are the same as in Example 2, to obtain the formulation of Comparative Example 1. Comparative Example 2

[0017] Referring to Examples 1 and 2, the *Trichoderma viride* strain (CGMCC 3.11450) was replaced with the *Trichoderma viride* strain (GIM3.141) disclosed in Comparative Document 1 ("Microbial staining of protein fibers", Zhai Hongxia, Master's thesis of Soochow University, March 2016). The remaining technical features are the same as those in Embodiment 1 and Embodiment 2. Comparative Example 3

[0018] Referring to Examples 1 and 2, an additional step is added between step 2) and step 3) in the preparation of *Trichoderma viride* metabolites: the *Trichoderma viride* fermentation broth is sterilized at 121°C for 25 minutes; The remaining technical features are the same as those in Embodiments 1 and 2.

[0019] Experiment 1

[0020] From July to August 2022, Gala apple trees in an orchard in Guanshui Town, Muping District, Yantai City, were used as the experimental subjects. All rootstocks were M26 and the trees were 8 years old. Six trees treated with natural light as a blank control group, six trees treated with natural light plus a reflective film as a positive control group, six trees treated with natural light plus a colorant prepared in Example 2 as Example 2 group, six trees treated with natural light plus a colorant prepared in Comparative Example 1 as Comparative Example 1 group, six trees treated with natural light plus a colorant prepared in Comparative Example 2 as Comparative Example 2 group, and six trees treated with natural light plus a colorant prepared in Comparative Example 3 as Comparative Example 3 group, for a total of 36 apple trees in 6 groups, were used for the coloring experiment.

[0021] In late July, approximately 25 days before the Gala apples were marketed, the fruit bags on 36 experimental trees were removed. The day after the inner bags were removed, six groups of trees underwent experimental treatment. The leaves and fruit surfaces of the fruiting branches in each example group and the comparative group were sprayed with a 1% diluted colorant solution until droplets appeared on the leaves and fruit surface. The positive control group received reflective film and reflective strips on each apple tree. The negative control group received no treatment. Five and ten days after the initial treatment, the trees in each example group and the comparative group were sprayed with the same concentration of colorant once more.

[0022] Five fruits were randomly selected from each of the six treatment groups at 0, 3, 6, 9 and 12 days after the first treatment for anthocyanin content detection and data analysis.

[0023] The data results are shown in Table 1:

[0024] Table 1. Anthocyanin content in the peel of Gala apples 0–12 days after coloring test.

[0025]

[0026] As shown in Table 1, compared with the blank control group, the anthocyanin content in the peel of Gala apples in the positive control group, each example group, and the comparative group was significantly higher 3-12 days after treatment. P＜0.05 The improvement was significant compared to comparative groups 1-3. P＜0.01 The differences demonstrate that the colorant of the present invention, which uses Trichoderma viride metabolites as the active ingredient, has a significant promoting effect on the coloring of apple fruits, and that the Trichoderma viride strain and the preparation method of the Trichoderma viride metabolites described in the present invention have significant inventiveness compared to other Trichoderma viride strains and their metabolite preparation methods.

[0027] Experiment 2

[0028] From August to September 2022, a 6-year-old 'Crimson Seedless' grapevine in a vineyard in Daxindian, Penglai, Yantai, was used as the experimental subject. The rootstock was 110R. Five groups of 15 grapevines were used for the coloring experiment: 3 vines treated with the colorant prepared in Example 2 (Group 2), 3 vines treated with the colorant prepared in Comparative Example 1 (Group 1), 3 vines treated with the colorant prepared in Comparative Example 2 (Group 2), 3 vines treated with the colorant prepared in Comparative Example 3 (Group 3), and 3 vines treated with an equal amount of clearing agent (Group 3).

[0029] In late August, approximately 20 days before the harvest of "Crimson Seedless" grapes, 15 experimental fruit trees underwent treatment. The fruit bunches in each example group and the comparative group were sprayed with a 1% concentration of the corresponding colorant aqueous solution until the bunches were dripping wet; the fruit bunches in the control group were sprayed with plain water until they were dripping wet. Three and seven days after the initial treatment, the example groups and the comparative group were re-sprayed with the same concentration of colorant.

[0030] Fifteen days after the initial treatment, three bunches of grapes were randomly selected from each of the five treatment groups, and ten grapes were picked from each bunch, for a total of 450 grapes. The anthocyanin content in the grape skin was then tested and the data were analyzed.

[0031] The data results are shown in Table 2:

[0032] Table 2. Anthocyanin content in grape skin 15 days after coloring test of "Crimson Seedless" grapes.

[0033]

[0034] As shown in Table 2, after spraying with the colorant aqueous solutions of Examples 2, 1, and 2, the anthocyanin content in the skin of "Crimson Seedless" grapes was significantly higher than that in the control group. P＜0.01 The differences were statistically significant, and both Example 2 and Comparative Examples 1-3 showed extremely significant differences. P＜0.01 The differences are evident. This invention demonstrates that the colorant using *Trichoderma viride* metabolites as the active ingredient significantly promotes grape fruit coloring, and that the *Trichoderma viride* strain and its metabolite preparation method described in this invention are significantly different from other *Trichoderma viride* strains and their metabolite preparation methods.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a colorant using *Trichoderma viride* metabolites as the active ingredient in promoting the coloring of apples or grapes, characterized in that: The *Trichoderma viride* metabolite is prepared by fermentation of *Trichoderma viride* strain with accession number CGMCC 3.11450. The colorant is a compound of *Trichoderma viride* metabolite, emulsifier, and dispersant in a weight ratio of 1:0.1~0.45:0.5~1. The preparation method of the *Trichoderma viride* metabolite includes the following steps: 1) After activating the *Trichoderma viride* strain CGMCC 3.11450, inoculate it into modified PDA medium and culture for 8-24 hours to obtain seed culture; 2) When the bacterial cell concentration in the seed culture reaches 10 6 ~10 9 When the concentration of CFU / mL is 1, it is inoculated into a modified Czapek medium and fermented for 4-8 days to obtain the fermentation broth. 3) The fermentation broth is subjected to flocculation, filtration and sterilization treatment to obtain filtrate, which is the metabolite of Trichoderma viride; wherein, the modified PDA medium is PDA medium with agar removed and pH adjusted to 4.5~6.0, and the modified Czapek's medium is Czapek's medium with agar removed and pH adjusted to 4.0~5.

0.

2. The application according to claim 1, characterized in that: The colorant is diluted with pure water to form a coloring spray, wherein the mass fraction of Trichoderma viride metabolites is 40%~50%, the application rate is 5~30 kg / mu, and it is applied 1~3 times according to the fruit coloring situation, with an interval of 5~10 days between adjacent applications.

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