Use of a rhodobacter sphaeroides metabolite in improving the storage tolerance of plant fruits

Abstract

The application discloses application of Rhodobacter sphaeroides metabolite in improving storage resistance of plant fruits. The Rhodobacter sphaeroides metabolite is prepared by fermentation of a Rhodobacter sphaeroides (CGMCC 1.16068), and the obtained Rhodobacter sphaeroides metabolite has a remarkable promoting effect on improving the storage resistance of plant fruits, and can significantly reduce the bad fruit rate and mass loss rate in fruit storage and improve the flesh hardness after application. The application creatively applies the Rhodobacter sphaeroides metabolite to improving the storage resistance of plant fruits, and the application has the remarkable distinguishing features from the existing preservation technology, that is, the application is applied to the fruit growth and development period instead of the fruit storage period after harvesting, and is applied to the fruit tree leaves instead of the fruit surface, and has the excellent characteristics of remarkable and durable effect and environmental protection and no residue. The application widens the direction of development and utilization of beneficial bacteria in agricultural use, provides a green and environmental biological source preparation for improving the storage resistance of fruits, and has good development and application prospect and practical significance.

Description

Technical Field

[0001] This invention belongs to the field of microbial metabolites applied to fruit quality improvement technology, specifically involving the application of a metabolite of Rhodotorula globosum in improving the storage resistance of plant fruits. Background Technology

[0002] Rhodobacter sphaeroides is a purple, non-sulfur bacterium that can grow under various conditions, including aerobic, anaerobic, and dark environments. It possesses multiple metabolic pathways, producing various metabolites such as carotenoids, 5-aminolevulinic acid (5-ALA), coenzyme Q10, and superoxide dismutase. In recent years, it has been widely used in food, medicine, environment, and agriculture. In agriculture, its applications primarily focus on: the production of 5-ALA for the development of herbicides, pesticides, and growth promoters; and the production of carotenoids for aquaculture. Currently, there are no reports on the application of Rhodobacter sphaeroides metabolites in improving the storage life of plant fruits, representing a gap in the application of this species.

[0003] This invention involves the directed cultivation of a specific Rhodophyton floccosum strain (CGMCC 1.16068, purchased from the China General Microbiological Culture Collection Center) to obtain a complex of Rhodophyton floccosum metabolites with multiple biological activities. Further, through extensive concentration gradient experiments, multi-factor response experiments, field effect experiments, and post-harvest storage effect evaluation experiments, a technical solution for the application of Rhodophyton floccosum metabolites in improving the storability of plant fruits was developed. Significant effects were observed in evaluating the rate of fruit spoilage, quality loss, and flesh firmness during apple and grape fruit storage, and it is expected to achieve good results in improving fruit quality, reducing the use of chemical agents, lowering production costs, and increasing fruit growers' income.

[0004] A search revealed that document 1, "Advances in the Application of Rhodopseudomonas aeruginosa" (Microbiology Journal, Chen Lin, June 2016, Vol. 36, No. 3, pp. 105-108), discloses carotenoids, coenzyme Q10, superoxide dismutase (SOD), and 5-aminolevulinic acid (5-ALA) in the metabolites of Rhodopseudomonas aeruginosa. It also discloses the function of these metabolites in degrading pesticide residues, organic wastewater, and toxic and harmful substances such as polycyclic aromatic hydrocarbons (PAHs), and their applications in food, medicine, agriculture, environment, and hydrogen production. However, this document does not describe any application of Rhodopseudomonas aeruginosa and its metabolites in improving the storability of plant fruits, nor does it conduct any experiments or research on the application of Rhodopseudomonas aeruginosa metabolites and their carotenoids, coenzyme Q10, SOD, and 5-ALA in improving the storability of plant fruits. Therefore, document 1 does not provide any inspiration or reference for this invention. This invention is relatively independent and possesses the characteristics of novelty, inventiveness, and practicality.

[0005] Document 2, "Extraction and Stability Study of Coenzyme Q10 and SOD Produced by Rhodotorula globulus 3757" (Food Science and Technology, Li Zuming, Vol. 38, No. 04, 2013, pp. 2-6), discloses that "Coenzyme Q10 and SOD are intracellular products obtained from the fermentation of Rhodotorula globulus" (Page 2, Paragraph 1 of Document 2). It discloses a Rhodotorula globulus strain (CGMCC No. 3757) that produces Coenzyme Q10 and SOD, and a method for extracting Coenzyme Q10 and SOD from Rhodotorula globulus using ultrasonic disruption and freeze-thaw assisted ultrasonic disruption. The Rhodotorula globulus strain (CGMCC 1.16068) described in this invention is not the same strain as the strain described in Document 2, and its culture method and metabolites are significantly different. The Rhodotorula globulus metabolites described in this invention are extracellular secretions, and the extraction process does not require cell disruption. This proves that the Rhodotorula globulus metabolites described in this invention do not contain the "intracellular products" Coenzyme Q10 and SOD. Therefore, the implementation of the application described in this invention does not depend on the presence of coenzyme Q10 or superoxide dismutase as described in documents 1 and 2. The Rhodotorula globulus and its metabolites are significantly different, and this invention has significant inventiveness.

[0006] Document 3, "Optimization of Conditions for Fermentation Production of Carotenoids by Rhodotorula Gluconoidis" (Industrial Microbiology, Kong Lina, February 2015, Vol. 45, No. 1, pp. 25-31), discloses a method for extracting carotenoids from the fermentation broth of Rhodotorula Gluconoidis 3757 (Document 3, page 2, section 1.7, Carotenoid Extraction). This method involves centrifuging the fermentation broth once, retaining the bacterial cells, and discarding the supernatant, demonstrating that the carotenoids in the Rhodotorula Gluconoidis fermentation products described in that document are present intracellularly, not secreted extracellularly. However, the extraction method for the Rhodotorula Gluconoidis metabolites described in this invention involves removing the bacterial cells from the fermentation broth. The two methods use different strains, employ different preparation methods, and yield different portions. Therefore, it can be concluded that the Rhodotorula Gluconoidis metabolites described in this invention do not contain the carotenoids from the Rhodotorula Gluconoidis fermentation products described in Document 3, and the implementation of the application described in this invention does not depend on the presence of the carotenoids in the Rhodotorula Gluconoidis products described in Documents 1 and 3.

[0007] Document 4, "The Effects of 5-Aminolevulinic Acid on Coloring and Postharvest Storage of 'Feizixiao' Lychee" (Master's Thesis, Hainan University, Feng Shun, May 2016), discloses that "pre-harvest 5-ALA treatment has no significant effect on the water loss rate and disease index of 'Feizixiao' lychee at 0, 3, and 6 days postharvest, and pre-harvest 5-ALA treatment has no adverse effect on the postharvest storage of 'Feizixiao' lychee, but to a certain extent helps to alleviate browning and senescence of lychee" (paragraphs 4 and 5 of the abstract of document 4). This indicates that the application of 5-ALA to 'Feizixiao' lychee fruit before harvest cannot effectively improve the postharvest storage properties of the fruit. It does not provide constructive suggestions for the application described in this invention. This invention is relatively independent, novel, and inventive.

[0008] In summary, the innovation of this invention lies in disclosing the application of Rhodotorula globosum metabolites in improving the storability of plant fruits. The Rhodotorula globosum strain is significantly different from existing strains reported for agricultural applications. The Rhodotorula globosum metabolites are significantly different from metabolites extracted from cells. The application effect does not depend on coenzyme Q10, superoxide dismutase (SOD), or carotenoids described in documents 1, 2, and 3. The application is significantly different from post-harvest preservatives applied to the fruit surface. Instead, it is a new technology and application applied during the fruit's growth and development period that significantly improves the fruit's storability, possessing the novelty, inventiveness, and practical applicability required for patent grant. Summary of the Invention

[0009] This invention discloses the application of a metabolite of Rhodotorula globosum in improving the storage resistance of plant fruits, thereby reducing the rate of spoiled fruit and quality loss, increasing fruit firmness, and enhancing the storage resistance of plant fruits.

[0010] The first objective of this invention is to provide a method for preparing metabolites of Rhodopseudomonas spp., comprising the following steps:

[0011] 1) Inoculate the Rhodophyton floccosum strain CGMCC 1.16068 into PDA medium and incubate at a constant temperature in the dark for 16-24 hours;

[0012] 2) Select a single rejuvenated Rhodotorula glutinis and inoculate it into a modified NB medium (nutrient broth medium) and culture it at a constant temperature in the dark for 18-30 hours with shaking until the OD600 value of the bacterial solution reaches 2.0-3.0 to obtain the Rhodotorula glutinis fermentation seed liquid.

[0013] 3) The Rhodotorula glutinis fermentation seed liquid was inoculated into a fermenter containing modified NA medium fermentation broth at a volume ratio of 10%. The fermentation parameters were set as follows: temperature 25℃~30℃, pH 6.5~8.0, dissolved oxygen 70%~90%. After fermentation for 3~6 days, the Rhodotorula glutinis fermentation broth was obtained.

[0014] 4) The fermentation broth of Rhodotorula globulus is filtered through macroporous resin to remove bacteria, resulting in a clear brown liquid, which is the metabolic product of Rhodotorula globulus.

[0015] The second objective of this invention is to provide an application of the above-mentioned Rhodophyton floccosum metabolites in improving the storability of plant fruits, the method of which is as follows:

[0016] 1) The metabolites of Rhodotorula globulus are diluted with pure water to form an aqueous solution of Rhodotorula globulus metabolites with a mass fraction of 0.5% to 2%, which is the spray solution;

[0017] 2) Different application rates should be selected according to different fruit tree species and different fruit development stages. The application rate of the spray solution is 15-30 kg / mu.

[0018] 3) Select the leaves of the fruit tree or the entire plant near the fruit for spraying;

[0019] 4) Spray 2-3 times during the fruit growth and development period, with an interval of 5-10 days between adjacent applications.

[0020] The advantages of this invention are: it creatively applies the metabolites of *Rhodotorula glutinis* to improve the storability of plant fruits. After application, it significantly reduces the rate of spoiled fruit and quality loss during storage, while increasing the firmness of the fruit flesh. A significant difference between this application and existing preservation technologies is that it is applied during the fruit's growth and development period rather than after harvest, and it is applied to the leaves of fruit trees rather than the fruit surface. This results in a significant, long-lasting effect and is environmentally friendly with no residue. It broadens the scope for the development and utilization of beneficial bacteria in agriculture and provides a green and environmentally friendly biological agent for improving fruit storability, demonstrating promising development and application prospects and practical significance. Detailed Implementation

[0021] 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.

[0022] The Rhodopseudomonas spp. used in this embodiment of the invention was purchased from the China General Microbiological Culture Collection Center (CGMCC 1.16068).

[0023] Example 1

[0024] A metabolite of a type of Rhodoglobinobacter, prepared by the following method:

[0025] 1) Rhodophyton floccosum strain (CGMCC 1.16068) was inoculated into PDA medium and cultured statically in the dark at a constant temperature for 18 hours;

[0026] 2) Select a single rejuvenated Rhodotorula glutinis and inoculate it into a modified NB medium (nutrient broth medium) and culture it at a constant temperature in the dark for 24 hours with shaking until the OD600 value of the bacterial solution reaches 2.0 to obtain the Rhodotorula glutinis fermentation seed liquid.

[0027] 3) The Rhodotorula glutinis fermentation seed liquid was inoculated into a fermenter containing modified NA medium fermentation broth at a volume ratio of 10%. The fermentation parameters were set as follows: temperature 26℃, pH 6.5, dissolved oxygen 90%. After 4 days of fermentation, the Rhodotorula glutinis fermentation broth was obtained.

[0028] 4) The fermentation broth of Rhodotorula globulus is filtered through macroporous resin to remove bacteria, resulting in a clear brown liquid, which is the metabolic product of Rhodotorula globulus.

[0029] Comparative Example 1

[0030] The Rhodopseudomonas spp. strain (CGMCC No. 3757) published in Comparative Document 2 (“Extraction and Stability Study of Coenzyme Q10 and SOD Produced by Rhodopseudomonas spp. 3757”, Li Zuming, Food Science and Technology, Vol. 38, No. 04, 2013, pp. 2-6) was used to replace the Rhodopseudomonas spp. strain (CGMCC 1.16068) in Example 1.

[0031] The remaining steps are the same as in Example 1, to obtain the formulation of Comparative Example 1.

[0032] Comparative Example 2

[0033] The intracellular metabolite extract of Rhodopseudomonas spp. was obtained by replacing step 4 of Example 1 with the cell disruption and extraction method of Rhodopseudomonas spp. 3757 ("Extraction and Stability Study of Coenzyme Q10 and SOD Produced by Rhodopseudomonas spp. 3757", Li Zuming, Food Science and Technology, Vol. 38, No. 04, 2013, pp. 2-6) published in comparative document 2 ("Optimization of Fermentation Conditions for Carotenoid Production by Rhodopseudomonas spp. 3757", Kong Lina, Industrial Microbiology, Vol. 45, No. 1, February 2015, pp. 25-31).

[0034] The remaining technical features are the same as in Example 1.

[0035] Comparative Example 3

[0036] Referring to comparative document 1 (“Advances in the application of Rhodopseudomonas aeruginosa”, Chen Lin, Journal of Microbiology, June 2016, Vol. 36, No. 3, pp. 105-108), a mixed preparation of carotenoids, coenzyme Q10, superoxide dismutase and 5-aminolevulinic acid was prepared to replace the Rhodopseudomonas aeruginosa metabolites prepared in Example 1.

[0037] Experiment 1

[0038] From August to November 2022, the "Yanfu 3" apple tree in an experimental park of Yantai Kaido Marine Biology Research Institute was used as the experimental subject. The rootstock was M26 and the tree age was 6 years. Three fruit trees sprayed with water served as the control group, and three trees each of Example 1 and Comparative Examples 1-3 were sprayed as the experimental group. An apple fruit storage resistance test was designed, with a total of 15 apple trees in 5 groups.

[0039] In late August, before removing the bags from the "Yanfu 3" apple trees, the leaves of three control group trees were sprayed with clean water. The leaves of each plant in Example 1 and Comparative Examples 1-3 were sprayed with a 1000-fold dilution of the corresponding formulation, spraying until droplets of liquid dripped from the leaves. The same treatment was repeated once each at 10 and 20 days after the initial spraying.

[0040] During the mid-September harvest season, 10 fruits were randomly picked from each group of apple trees, totaling 150 "Yanfu 3" apples from 15 trees in 5 groups. These were stored at room temperature. Fruit firmness was measured, individual fruit weight was measured, and damaged fruit was counted at 0 days and 60 days. The fruit firmness, weight loss rate, and damaged fruit rate of "Yanfu 3" apples after 60 days of storage at room temperature were calculated, and the data are shown in Table 1.

[0041] Table 1. Fruit firmness, weight loss rate, and spoilage rate of "Yanfu 3" apples 60 days after treatment.

[0042]

[0043]

[0044] As shown in Table 1, compared with the water control group, Comparative Example 2 had no significant effect on the firmness of "Yanfu 3" apples (P < 0.05). Example 1, Comparative Example 1, and Comparative Example 3 all significantly improved the firmness of apples (P < 0.05), and Example 1 was highly significant compared with all the comparative examples (P < 0.01). Example 1 and all the comparative examples significantly reduced the weight loss rate of "Yanfu 3" apples (P < 0.05), while Example 1 was more effective than all the comparative examples and was highly significant (P < 0.01). Example 1 and all the comparative examples significantly reduced the spoilage rate of "Yanfu 3" apples, while Example 1 was more effective than all the comparative examples and was highly significant. This invention demonstrates that the Rhodotorula globosum metabolite described in this invention has a significant effect on improving the storability of plant fruits. Furthermore, the Rhodotorula globosum strain and its metabolite described in this invention are significantly different from other Rhodotorula globosum strains and their metabolite preparation methods in improving the storability of apple fruits.

[0045] Experiment 2

[0046] From August to September 2022, the "Crimson Seedless" grapevines in a grape planting and promotion demonstration garden located in Daxindian, Penglai, Yantai, by the Yantai Kaido Marine Biological Research Institute were used as experimental subjects. The rootstock was 110R, and the vines were 6 years old. Three grapevines treated with the metabolites of Rhodophyton floccosum prepared in Example 1 were designated as Example 1 group; three grapevines treated with the formulation prepared in Comparative Example 1 were designated as Comparative Example 1 group; three grapevines treated with the formulation prepared in Comparative Example 2 were designated as Comparative Example 2 group; three grapevines treated with the formulation prepared in Comparative Example 3 were designated as Comparative Example 3 group; and three grapevines treated with an equal amount of Qingguo fruit were designated as the control group. A total of 15 grapevines from 5 groups were used in the fruit storage tolerance spraying experiment.

[0047] In mid-August, 15 experimental fruit trees were treated. The entire leaves of the plants in each example group and the comparative group were sprayed with a 0.5% aqueous solution of the corresponding formulation until dripping water appeared at the leaf edges. The entire leaves of the control group were sprayed with plain water until dripping water appeared at the leaf edges. Seven and fourteen days after treatment, the example groups and the comparative group were each re-sprayed with the same concentration of the formulation once.

[0048] In early September, during the harvest season, three bunches of grapes were randomly selected from each group of grapevines, totaling 45 bunches of "Crimson Seedless" grapes from the five treatments. The grapes were stored at room temperature, and fruit firmness, individual fruit weight, and spoilage statistics were performed at 0 and 15 days. The fruit firmness, weight loss rate, and spoilage rate of "Crimson Seedless" grapes after 15 days of storage at room temperature were calculated, and the data are shown in Table 2.

[0049] Table 2. Fruit firmness, weight loss rate, and spoilage rate of "Crimson Seedless" grapes 15 days after treatment.

[0050]

[0051] As shown in Table 2, compared with the water control group, the Example 1 group and each comparative group significantly improved the firmness of "Crimson Seedless" grapes (P < 0.05), and the Example 1 group showed a highly significant improvement compared with each of the comparative groups (P < 0.01). The Comparative Group 1 had no significant effect on the grape fruit weight loss rate (P < 0.05), while the Example 1, Comparative Group 2, and Comparative Group 3 all significantly reduced the weight loss rate of "Crimson Seedless" grapes (P < 0.05), and the Example 1 group showed a more significant improvement compared with each of the comparative groups (P < 0.01). The Example 1 group and each comparative group significantly reduced the rate of damaged fruit in "Crimson Seedless" grapes, and the Example 1 group showed a more significant improvement compared with each of the comparative groups (P < 0.01). This invention demonstrates that the Rhodotorula globosum metabolite described in this invention has a significant effect on improving the storage resistance of plant fruits. Furthermore, the Rhodotorula globosum strain and its metabolite described in this invention are significantly different from other Rhodotorula globosum strains and their metabolite preparation methods in improving the storage resistance of grape fruits.

[0052] 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. A type of Rhodococcus-like bacterium Rhodobacter sphaeroides The application of metabolites during fruit growth and development to improve the storability of plant fruits is described below, and the preparation process of the Rhodophyton floccosum metabolites is as follows: 1) Inoculate the Rhodophyton floccosum strain CGMCC 1.16068 into PDA medium and incubate at a constant temperature in the dark for 16-24 hours; 2) Select a single rejuvenated Rhodotorula globulus, inoculate it into a modified NB medium, and culture it at a constant temperature in the dark for 18-30 hours with shaking until the OD600 value of the bacterial solution reaches 2.0-3.0 to obtain the Rhodotorula globulus fermentation seed liquid; 3) The Rhodotorula glutinis fermentation seed culture was inoculated into a fermenter containing modified NA medium fermentation broth at a volume ratio of 10%. The fermentation parameters were set as follows: temperature 25℃~30℃, pH 6.5~8.0, dissolved oxygen 70%~90%. The fermentation broth of Rhodotorula glutinis was obtained after 3~6 days of fermentation. 4) The fermentation broth of Rhodotorula globulus is filtered through macroporous resin to remove bacteria, resulting in a clear brown liquid, which is the metabolic product of Rhodotorula globulus.

2. The application according to claim 1, characterized in that, The modified NB medium used in the preparation process of the metabolites of Rhodotorula globulus is: 15 g / L peptone, 5 g / L beef extract, 5 g / L sodium chloride, 5 g / L glucose, pH 6.5; the modified NA medium is: 5 g / L peptone, 5 g / L beef extract, 3 g / L sodium chloride, 2 g / L glucose, pH 6.8~7.

2.

3. The application according to claim 1, characterized in that, The plant fruit is an apple or a grape.

4. The application according to any one of claims 1 to 3, characterized in that, The application method is as follows: 1) The metabolites of Rhodotorula globulus are diluted with pure water to form an aqueous solution of Rhodotorula globulus metabolites with a mass fraction of 0.5%~2%, which is the spray solution; 2) Different application rates should be selected according to different fruit tree species and different fruit development stages. The application rate of the spray solution is 15~30 kg / mu. 3) Select the leaves of the fruit tree or the entire plant near the fruit for spraying; 4) Spray 2-3 times during the fruit growth and development period, with an interval of 5-10 days between adjacent applications.

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