Rosmarinic acid modified chitooligosaccharide, and preparation method and application thereof

Abstract

The application discloses a rosemary acid modified chitooligosaccharide and a preparation method and application thereof. The rosemary acid is used for modifying natural chitooligosaccharide to prepare a rosemary modified chitooligosaccharide (COS-RA), and the compound has an antioxidation activity much higher than that of the natural chitooligosaccharide, has a significant improvement effect on anthocyanin in fruits, and further promotes fruit coloring. After the compound is prepared into an aqueous preparation and is sprayed on the fruits, the total anthocyanin content of the fruits is increased by 40.2% to 54.5%, and the content of various monomer anthocyanins is also significantly increased. Compared with the natural chitooligosaccharide, the compound has a better effect in the application of promoting fruit coloring, has a smaller dosage, has more uniform coloring, has a higher anthocyanin content during the maturing period, and also has the characteristics of the chitooligosaccharide, such as non-toxicity and easy biodegradation, and thus has a better field application effect, and can be widely applied in the field of agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to the preparation of a chitosan oligosaccharide modified derivative and its application in promoting fruit coloring. Background Technology

[0002] Many fruits display their unique colors when ripe, making fruit color a crucial indicator of ripeness in agricultural production. Unripe fruits contain abundant chlorophyll in their peels. As the fruit matures, the chlorophyll gradually breaks down, and anthocyanins accumulate, resulting in vibrant colors. For example, apples, pears, grapes, and peaches turn yellow or red when ripe, while citrus fruits turn orange-yellow. Therefore, the use of coloring agents is particularly important in fruit cultivation. These agents not only increase the anthocyanin content in the peel, accelerating color change and improving fruit quality and sugar content, but also shorten the cultivation time, allowing for earlier market entry and greater economic value.

[0003] Grapes (Vitis vinifera L.) are one of the world's most important economic fruits, and China is the world's largest grape producer, with a total output of 14.8431 million tons in 2020. As grapes mature, the chlorophyll and other pigments in the grape skin gradually diminish and are replaced by anthocyanins and other compounds. Therefore, the appearance and color of grapes are important indicators of grape quality, and anthocyanins are the intrinsic factors that measure grape coloring and are also a crucial indicator of fruit quality.

[0004] Current research on promoting grape coloring mainly focuses on plant hormones, biostimulants, and elemental fertilizers. For example, Chinese patent CN108575997A discloses a benzisoabscisic acid preparation for grape color change, which includes benzisoabscisic acid, an isomer of the hormone abscisic acid, which can promote grape color change. Chinese patent CN104030809 discloses a multifunctional bio-fertilizer that promotes grape coloring and sugar increase, including fermented grape pomace, wood ash, urea, potassium sulfate, diammonium phosphate, calcium magnesium phosphate, borax, and other macro-elements and complex microbial strains. Chinese patent CN102057953A discloses a fruit color enhancer containing biostimulants such as chitin and chitosan, which can promote the coloring of apples, grapes, and cherries. In addition, some studies have shown that biostimulants such as seaweed oligosaccharides and chitosan oligosaccharides can also promote the coloring of citrus fruits and grapes.

[0005] Chitosan oligosaccharides, as a natural biostimulant, are generally believed to primarily function by enhancing the activity of plant antioxidant enzymes, thereby stimulating the synthesis of secondary metabolites such as anthocyanins, and are closely related to their own antioxidant properties. However, current technologies, while using biostimulants such as chitin, chitosan, chitosan oligosaccharides, and seaweed oligosaccharides can promote fruit coloring in a harmless manner, still have drawbacks such as relatively weak effects. Overcoming these drawbacks and providing a non-toxic, harmless fruit coloring agent with a strong promoting effect would have enormous application potential. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a rosmarinic acid-modified chitosan oligosaccharide and its preparation method.

[0007] The second objective of this invention is to provide a water-soluble preparation of chitosan oligosaccharide modified with rosmarinic acid.

[0008] A third objective of this invention is to provide the application of the rosmarinic acid-modified chitosan oligosaccharide water-soluble formulation.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] To overcome the shortcomings of biostimulants and enhance their effect on grape coloring efficiency, this invention creatively modifies chitosan oligosaccharides with rosmarinic acid, synthesizing a novel compound—rosmarinic acid-modified chitosan oligosaccharide. Testing has demonstrated that the rosmarinic acid-modified chitosan oligosaccharide is more effective than natural chitosan oligosaccharide in promoting grape coloring, requiring less dosage and resulting in more uniform coloring. The treated grapes accumulate higher anthocyanin content during ripening, and it retains the non-toxic and harmless advantages of natural chitosan oligosaccharide, exhibiting better field application results than natural chitosan oligosaccharide.

[0011] Therefore, the present invention first provides a rosmarinic acid-modified chitosan oligosaccharide, the chemical structural formula of which is shown in formula (I):

[0012]

[0013] Formula (I)

[0014] Furthermore, in equation (I), the value of n ranges from 1 to 20, and the value of y ranges from 0 to 2.

[0015] Furthermore, the degree of substitution of the rosmarinic acid is 0.113 to 0.287.

[0016] The preparation method of the rosmarinic acid-modified chitosan oligosaccharide includes the following steps: dissolving chitosan oligosaccharide and rosmarinic acid in a citrate buffer solution, heating and stirring while slowly adding laccase, and conducting an oxygen-bubbling reaction. After the reaction, centrifuging is performed to remove the precipitate from the solution, and then the solution is concentrated, dialyzed, and freeze-dried to obtain rosmarinic acid-modified chitosan oligosaccharide powder.

[0017] Furthermore, the chitosan oligosaccharide has a molecular weight ≤2000 Da and a degree of deacetylation ≥95%.

[0018] Furthermore, the purity of the rosmarinic acid is ≥97%.

[0019] Furthermore, the mass ratio of chitosan oligosaccharide to rosmarinic acid is 4 to 10:1.

[0020] Furthermore, the mass ratio of chitosan oligosaccharide to rosmarinic acid is 7:1.

[0021] Furthermore, the enzyme activity of the laccase is 120 U / g.

[0022] Furthermore, the pH value of the citrate buffer solution is 5.5 to 6.5.

[0023] Furthermore, the citrate buffer solution has a pH of 6.0.

[0024] Furthermore, the concentration of the citrate buffer solution is 0.1 mol / L.

[0025] Furthermore, the heating temperature is 20–50°C.

[0026] Furthermore, the temperature is 30°C.

[0027] Furthermore, the stirring reaction time is 18–24 h.

[0028] Furthermore, the stirring reaction time is 24 hours.

[0029] This invention also provides a rosmarinic acid-modified chitosan oligosaccharide water-soluble formulation, comprising rosmarinic acid-modified chitosan oligosaccharide and a surfactant. The surfactant added to the formulation can reduce the surface tension of leaves, thereby enabling the rosmarinic acid-modified chitosan oligosaccharide water-soluble formulation to adhere to the surface of grape leaves and fruits;

[0030] Furthermore, the rosmarinic acid-modified chitosan oligosaccharide content in the water-soluble preparation is 200–300 ppm.

[0031] Furthermore, the concentration of the surfactant is 10–50 ppm.

[0032] Furthermore, the concentration of the surfactant is 30–50 ppm.

[0033] Furthermore, the surfactant is any one or more of sucrose fatty acid esters, tea saponins, and diethyl octanoate.

[0034] Preferably, the rosmarinic acid-modified chitosan oligosaccharide water-soluble preparation also contains a synergist, which can effectively promote the absorption of nutrients by grapes and has an excellent synergistic effect on grape color change and anthocyanin accumulation.

[0035] Furthermore, the concentration of the synergist is 0.2–2.0 g / L.

[0036] Furthermore, the concentration of the synergist is 0.5–1.0 g / L.

[0037] Furthermore, the synergist is any one or more of L-amino acids, potassium nitrate, dipotassium hydrogen phosphate, and sucrose.

[0038] The present invention also provides the application of the rosmarinic acid-modified chitosan oligosaccharide or the water-soluble formulation of the rosmarinic acid-modified chitosan oligosaccharide in promoting fruit coloring.

[0039] The present invention also provides a method for promoting fruit coloring. The specific steps are as follows: starting from 45 to 55 days after flowering, during the fruit coloring period, spray the fruit and leaves with a rosmarinic acid-modified chitosan oligosaccharide water-soluble preparation 6 to 8 times, with an interval of 7 days between each application.

[0040] Preferably, the fruit is a grape.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] This invention synthesizes a rosmarinic acid-modified chitosan oligosaccharide by modifying chitosan oligosaccharide with rosmarinic acid. Characterization and experiments revealed that this compound exhibits significantly higher antioxidant activity than natural chitosan oligosaccharide, and it significantly increases the anthocyanin content of grape varieties from multiple regions, particularly the content of monomeric anthocyanins such as petunia, delphinidin, and malvidin, thus promoting fruit coloring. When prepared as a water-soluble formulation and sprayed onto the fruit, the total anthocyanin content increased by 40.2%–54.5%, with petunia content increasing by 112.3%–130.9%, delphinidin content by 88.9%–96.3%, and malvidin content by 67.8%–75.3%. Compared with natural chitosan oligosaccharides, this compound is more effective in promoting fruit coloring, requires less dosage, produces more uniform coloring, and accumulates higher anthocyanin content during ripening. It also possesses the same natural non-toxic and biodegradable properties as natural chitosan oligosaccharides, thus exhibiting better field application results and can be widely used in agricultural production. Attached Figure Description

[0043] Figure 1This is a diagram illustrating the synthesis process of rosmarinic acid-modified chitosan oligosaccharide (COS-RA).

[0044] Figure 2 This is a scanning electron microscope image of COS-RA.

[0045] Figure 3 This is the UV-Vis spectrum of COS-RA.

[0046] Figure 4 This is the Fourier transform infrared spectrum of COS-RA.

[0047] Figure 5 This is the COS-RA proton NMR spectrum.

[0048] Figure 6 The COS-RA clearance rate of DPPH. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0050] This invention relates to chitosan oligosaccharide ((C) 12 H 24 N2O9)n, CAS No.: 148411-57, Product No.: C875644, Molecular Weight ≤2000), Rosmarinic Acid (Molecular Formula: C 18 H 16 O8 (CAS No.: 20283-92-5, Catalog No.: R817282, 97%) was purchased from Shanghai Maclean's Co., Ltd., and laccase, sucrose fatty acid esters, L-amino acids, and tea saponins were purchased from Guangzhou Yuanye Co., Ltd. Unless otherwise specified, other reagents and materials used in the following examples were commercially available.

[0051] Example 1: Preparation of Rosmarinic Acid-Modified Chitosan Oligosaccharide (COS-RA) at Different Mass Ratios and pH Levels

[0052] Different mass ratios of chitosan oligosaccharide (COS) and rosmarinic acid (RA) were dissolved in 100 mL of citrate buffer solutions with different pH values. Laccase (9.6 U) was added under constant stirring, and the reaction was carried out at 30 °C with oxygen for 24 h. After the reaction, the precipitate was removed by centrifugation at 5000 rpm for 5 min, concentrated by rotary evaporation, dialyzed against deionized water for 24 h, and then freeze-dried to obtain rosmarinic acid-modified chitosan oligosaccharide (COS-RA) powder. The specific synthesis mechanism is as follows: Figure 1 As shown.

[0053] To screen for the optimal formulation, six schemes with different mass ratios and pH conditions were designed for comparison. The specific schemes are as follows:

[0054] (1) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 10:1, the citrate buffer pH was 5.5, the temperature was 30℃, and the mixture was stirred for 24 hours.

[0055] (2) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 7:1, the citrate buffer pH was 5.5, the temperature was 30℃, and the mixture was stirred for 24 hours.

[0056] (3) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 4:1, the citrate buffer pH was 5.5, the temperature was 30℃, and the mixture was stirred for 24 hours.

[0057] (4) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 10:1, the citrate buffer pH was 6.0, the temperature was 30℃, and the mixture was stirred for 24 hours.

[0058] (5) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 7:1, the citrate buffer pH was 6.0, the temperature was 30℃, and the mixture was stirred for 24 hours.

[0059] (6) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 4:1, the citrate buffer pH was 6.0, the temperature was 30℃, and the mixture was stirred for 24 hours.

[0060] Elemental analysis experiments were conducted to screen for the optimal conditions of different material ratios and pH values.

[0061] Specific analysis results:

[0062] Elemental analysis was performed on the rosmarinic acid-modified chitosan oligosaccharides prepared by the above six methods. The results are shown in Table 1. When the mass ratio of chitosan oligosaccharide to rosmarinic acid was 7:1 and the pH was 6.5, the rosmarinic acid-modified chitosan oligosaccharide (COS-RA) had the highest degree of substitution, which was 0.287. Therefore, this mass ratio and pH were selected for further testing.

[0063] Table 1. Elemental analysis results of COS-RA under different material ratios and pH conditions.

[0064]

[0065]

[0066] Example 2: Preparation of Rosmarinic Acid-Modified Chitosan Oligosaccharide (COS-RA) at Different Temperatures and Reaction Times

[0067] Chitosan oligosaccharide (COS) and rosmarinic acid (RA) were dissolved in 100 mL of citrate buffer (0.1 mol / L, pH 6.5). Laccase (9.6 U) was added with constant stirring, and the reaction was carried out under oxygen permeation at 20–50 °C for 18–24 h. After the reaction, the precipitate was removed by centrifugation at 5000 rpm for 5 minutes. The solution was concentrated using a rotary evaporator, then dialyzed against deionized water for 24 h, and finally freeze-dried to obtain rosmarinic acid-modified chitosan oligosaccharide (COS-RA) powder. The specific synthesis mechanism is as follows: Figure 1 As shown.

[0068] To screen for the optimal formulation, six schemes with different temperatures and reaction times were designed for comparison. The specific schemes are as follows:

[0069] (1) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 7:1, the citrate buffer pH was 6.0, the temperature was 20℃, and the mixture was stirred for 18h.

[0070] (2) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 7:1, the citrate buffer pH was 6.0, the temperature was 20℃, and the mixture was stirred for 24 hours.

[0071] (3) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 7:1, the citrate buffer pH was 6.0, the temperature was 30℃, and the mixture was stirred for 18h.

[0072] (4) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 7:1, the citrate buffer pH was 6.0, the temperature was 30℃, and the mixture was stirred for 24 hours.

[0073] (5) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 7:1, the citrate buffer pH was 6.0, the temperature was 50℃, and the mixture was stirred for 18 hours.

[0074] (6) The mass ratio of chitosan oligosaccharide to rosmarinic acid was 7:1, the citrate buffer pH was 6.0, the temperature was 50℃, and the mixture was stirred for 24 hours.

[0075] The optimal conditions were selected by comparing different temperatures and reaction times, and elemental analysis experiments were conducted.

[0076] The results showed that the elemental analysis of the rosmarinic acid-modified chitosan oligosaccharides prepared by the above six methods yielded the highest degree of substitution (0.287) when the reaction temperature of chitosan oligosaccharides and rosmarinic acid was 30℃ and the stirring time was 24h. Therefore, this temperature and reaction time were selected for further characterization.

[0077] Table 2. Elemental analysis results of COS-RA under different temperatures and reaction times.

[0078]

[0079] Example 3 Characterization of Rosmarinic Acid-Modified Chitosan Oligosaccharide (COS-RA)

[0080] Chitosan oligosaccharide (COS) and rosmarinic acid (RA) were dissolved in 100 mL of citrate buffer (0.1 mol / L, pH 6.0) with a 7:1 ratio. Laccase (9.6 U) was added under constant stirring, and the reaction was carried out at 30 °C with oxygen for 24 h. After the reaction, the precipitate was removed by centrifugation at 5000 rpm for 5 min. The solution was concentrated using a rotary evaporator, then dialyzed against deionized water for 24 h, and finally freeze-dried to obtain rosmarinic acid-modified chitosan oligosaccharide (COS-RA) powder produced under the optimal conditions. The specific synthesis mechanism is as follows: Figure 1 As shown.

[0081] To further demonstrate the successful preparation of rosmarinic acid-modified chitosan oligosaccharide, it was characterized, and the specific results are as follows:

[0082] (1) Chitosan oligosaccharide (COS) and rosmarinic acid-modified chitosan oligosaccharide (COS-RA) were observed using a scanning electron microscope (SE). The images are as follows: Figure 2 As shown, the COS surface has more cracks, while the COS-RA surface is relatively smoother, indicating that the addition of rosmarinic acid changed the original morphology of chitosan oligosaccharides.

[0083] (2) Figure 3 The UV-Vis absorption spectra of chitosan oligosaccharide (COS) and rosmarinic acid-modified chitosan oligosaccharide (COS-RA) are shown below. Figure 3 As can be seen, new absorption peaks were observed on COS-RA, along with some characteristic peaks of rosmarinic acid (RA) and chitosan oligosaccharide (COS), indicating that RA was successfully grafted onto COS.

[0084] (3) Figure 4 The Fourier transform infrared absorption spectra of COS and COS-RA are given by... Figure 4 It can be seen that COS-RA's (1410cm) -1 The deformation caused the characteristic absorption band to shrink, indicating that the amino group of COS was replaced by RA, forming a new bond and generating a new derivative.

[0085] (4) Figure 5 The images show the proton NMR spectra of COS and COS-RA. Multiple proton characteristic peaks (6.77–7.50 ppm) were observed in region 2 of the COS-RA spectrum, belonging to the aromatic protons of the conjugated RA.

[0086] (5) Figure 6 To determine the antioxidant activity of COS and COS-RA using the DPPH free radical scavenging rate assay, and to analyze... Figure 6It can be seen that the DPPH scavenging rate of COS-RA is significantly higher than that of COS, and at a concentration of 0.25 mg / mL, the scavenging rate is approximately 12.96 times that of COS.

[0087] In summary, this manufacturing method enables rosmarinic acid to be successfully grafted onto chitosan oligosaccharides, resulting in the formation of rosmarinic acid-modified chitosan oligosaccharides, which possess antioxidant properties far exceeding those of natural chitosan oligosaccharides.

[0088] Example 4: Color-changing experiment of Kyoho grapes in Baiyun District, Guangdong Province

[0089] The rosmarinic acid-modified chitosan oligosaccharide (degree of substitution 0.287) prepared in Example 3, amino acids, and sucrose fatty acid esters were weighed and dissolved in water in sequence to obtain a water-soluble preparation of rosmarinic acid-modified chitosan oligosaccharide.

[0090] To test its effect on promoting grape coloring, five groups of water-soluble formulations were designed and compared. Each 1L of solution contained the following components:

[0091] (1) 250mg rosmarinic acid modified chitosan oligosaccharide, 0.5g L-amino acids, 50mg sucrose fatty acid ester;

[0092] (2) 250mg chitosan oligosaccharide, 0.5g L-amino acids, 50mg sucrose fatty acid esters;

[0093] (3) Water, 0.5g L-amino acids, 50mg sucrose fatty acid esters;

[0094] (4) Water, water, 50mg sucrose fatty acid ester;

[0095] (5) Clean water, clean water, clean water;

[0096] The grape variety was Kyoho. The experimental site was Baiyun District, Guangzhou City, Guangdong Province. Spraying was carried out 50 days after flowering (when the grapes began to change color), once every seven days, for a total of 6 sprays. Samples were collected and measured on the 45th day. Each vine was a plot, and the results were replicated three times. The measured indicators were single fruit weight, a* value, soluble solids, total soluble sugar, titratable acid, and total anthocyanin content.

[0097] Specific measurement method:

[0098] (1) Single fruit weight and soluble solids (SSC)

[0099] Eighteen berries were sampled from the top, middle, and bottom of each embodiment, and their weight was measured using an electronic scale. After washing the berry surface, the a* value was recorded at two points along the equator, and the average value was taken. Soluble solids content (SSC) was measured using a digital refractometer (TD45).

[0100] (2) Determination of total soluble sugars

[0101] Weigh 0.5g of grapes, grind them, add 7ml of 80% ethanol to homogenize, and incubate in an 80℃ water bath for 30min. After cooling, centrifuge at 4000r / min for 10min, and take the supernatant to make up to 100ml. Take 0.2ml of the supernatant, add 5ml of anthrone sulfuric acid and distilled water in sequence, shake well, react in a 100℃ water bath for 10min, remove and cool, and measure the wavelength at 620nm. Calculate the soluble sugar content according to the standard curve.

[0102] (3) Titratable acid determination (TA)

[0103] Weigh 0.5g of grapes, grind them, and bring the volume to 10mL. Homogenize the mixture and incubate it in an 80℃ water bath for 0.5h. After centrifugation, take the supernatant and bring the volume to 100mL. Titrate with 0.05mol / L NaOH solution and calculate the titratable acid content according to the formula.

[0104] TTA(%)=(H×0.1×K×C) / (W×D)×100

[0105] Where: K: coefficient of tartaric acid (K=0.075); H: amount of NaOH consumed; C: total dilution; W: sample weight 10g; D: sample volume 10ml.

[0106] (4) Determination of total anthocyanin content

[0107] The absorbance was determined using pH differential spectrophotometry. 0.5 g of pericarp was ground in 5 mL of cold extraction buffer (methanol:formic acid:ethanol = 70:2:28, v / v / v) for 30 minutes, then extracted in a shaker at 30°C and 250 rpm for 2 hours. After centrifugation and filtration, the absorbance at 510 nm and 700 nm was recorded using a UV-Vis spectrophotometer (UV-1600) at pH 1.0 and pH 4.5, respectively, using the supernatant. The absorbance was calculated using the following formula.

[0108] Anthocyanin concentration C (mg / L) = A × MW × DF × 1000 / (ε × l)

[0109] A=(A510-A700)pH1-(A510-A700)pH4.5

[0110] Where: MW = 449.2 (molecular weight of cornflower-3-glucoside, mg / mol); DF = dilution factor of sample solution; ε = 26900 (molar extinction coefficient of cornflower-3-glucoside, mol-1); l = optical path diameter of cuvette, 1 cm.

[0111] (5) Determination of anthocyanins by monomers.

[0112] Following the method of Cho et al. (2004), a 250×4.6mm Symmetry was used. Anthocyanins were analyzed by HPLC using a column (Waters Corp., Milford, MA, USA). The mobile phase consisted of a binary gradient of 5% formic acid (A) and 100% methanol (B). The flow rate was 1 mL / min, and the linear gradient was from 2%B to 60%B over 60 min. The anthocyanin peak was quantified at 510 nm using a Waters 996 photodiode array detector (Milford). All anthocyanins were quantified as anthocyanin-3-glucosinolate equivalents (C3Gluc) using an external calibration curve with standards ranging from 5 to 125 μg / mL (Polyphenols Laboratories AS, Sandnes, Norway). Anthocyanin concentrations are expressed as mg malvidin-3-O-glucosinolate equivalents per gram of berry peel.

[0113] The results of the measurements for each indicator are shown in Tables 3 and 4:

[0114] Table 3. Effects of COS-RA and COS on the fruit quality of Kyoho grapes

[0115]

[0116] *Different letters after the data in the same column indicate significant differences (P<0.05), the same applies below.

[0117] Table 4. Effects of COS-RA and COS on anthocyanins in Kyoho grapes

[0118]

[0119]

[0120] The results showed that treatment with the rosmarinic acid-modified chitosan oligosaccharide water-soluble preparation of this embodiment resulted in a certain increase in yield of Kyoho grapes after harvest compared to the water control group. Soluble solids and soluble sugar increased by 35.6% and 26.6%, respectively, and titratable acid was also significantly lower than the control, indicating improved fruit quality. The chitosan oligosaccharide treatment group also showed promotion, but the promotion effect was significantly lower than that of the COS-RA treatment group. Most importantly, compared to the water control group, the a* value (the difference between red and green on the fruit surface, with positive numbers indicating red and negative numbers indicating green, and higher absolute values ​​indicating deeper color) was significantly higher by 32.3%, and the total anthocyanin content increased by 40.2%. Furthermore, the increased content of petunidin (112.3%), delphinidin (88.9%), and malvidin (67.8%) led to greater accumulation of total anthocyanins.

[0121] Example 5: Color-changing experiment of Xiahei grapes in Zengcheng District, Guangdong Province

[0122] Rosmarinic acid-modified chitosan oligosaccharide, potassium sulfate, and tea saponin were weighed and dissolved in water in sequence to prepare a water-soluble preparation of rosmarinic acid-modified chitosan oligosaccharide.

[0123] To test its effect on promoting grape coloring, five groups of water-soluble formulations were designed and compared. Each 1L of solution contained the following components:

[0124] (1) 250mg rosmarinic acid modified chitosan oligosaccharide, 0.5g potassium sulfate, 30mg tea saponin;

[0125] (2) 250mg chitosan oligosaccharide, 0.5g potassium sulfate, 30mg tea saponin;

[0126] (3) Water, 0.5g potassium sulfate, 30mg tea saponin;

[0127] (4) Water, water, 30mg tea saponin

[0128] (5) Clean water, clean water, clean water;

[0129] The grape variety was Xiahei. The test site was Zengcheng District, Guangzhou City, Guangdong Province. Spraying was carried out 50 days after flowering (when the grapes began to change color), once every seven days, for a total of 6 sprays. Samples were collected and measured on the 45th day. Each vine was a plot, and the results were replicated three times. The measured indicators were single fruit weight, a* value, soluble solids, total soluble sugar, titratable acid, and total anthocyanin content.

[0130] The detection indicators are referenced in Example 2, and the measurement results are shown in Tables 5 and 6.

[0131] Table 5. Effects of COS-RA on the fruit quality of Xiahei grapes

[0132]

[0133]

[0134] Table 6. Effects of COS-RA and COS on anthocyanins in Kyoho grapes

[0135]

[0136] The results showed that treatment with the rosmarinic acid-modified chitosan oligosaccharide water-soluble preparation of this embodiment did not increase the yield of Xiahei grapes. However, compared with the water control group, the soluble solids and soluble sugar increased by 31.7% and 37.8% respectively, the a* value was significantly higher than 38.5%, the total anthocyanin increased by 54.5%, and the contents of petunidin (130.9%), delphinidin (96.3%), and malvidin (75.3%) were also significantly higher than those of the water control group, and also showed a significant improvement compared with the chitosan oligosaccharide group.

[0137] The above embodiments illustrate that the rosmarinic acid-modified chitosan oligosaccharide water-soluble preparation can enhance the anthocyanin content of Summer Black and Kyoho grapes. Clearly, this invention is not limited to the two grape varieties described, nor is it limited to grapes alone; it has a significant effect on promoting coloring in various fruits. For those skilled in the art, other variations or modifications can be made based on the above description and ideas; it is neither necessary nor possible to exhaustively list all possible implementations here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of the claims of this invention.

Claims

1. An aqueous formulation of rosmarinic acid-modified chitooligosaccharide, characterized by, The rosmarinic acid modified chitosan oligosaccharide is composed of rosmarinic acid modified chitosan oligosaccharide, surfactant and synergist. The chemical structural formula of the rosmarinic acid modified chitosan oligosaccharide is shown as formula (I): Formula (I); in formula (I), the value range of n is 1-20, and the value range of y is 0-2; the concentration of the rosmarinic acid modified chitosan oligosaccharide is 200-500 ppm. The surfactant is sucrose fatty acid ester; the concentration of the surfactant is 10-50 ppm. The synergist is L-amino acid; the concentration of the synergist is 0.2-2.0 g / L.

2. The aqueous formulation of rosemary acid-modified COS according to claim 1, characterized in that, The degree of substitution of the rosmarinic acid modified chitosan oligosaccharide is 0.113-0.

287.

3. The aqueous rosmarinic acid-modified COS preparation according to any one of claims 1 to 2, characterized by, The preparation method of the rosmarinic acid modified chitosan oligosaccharide is that chitosan oligosaccharide and rosmarinic acid are dissolved in a citrate buffer solution, laccase is added, heating and stirring are conducted, oxygen bubbling reaction is conducted, the product is subjected to ultracentrifugation and dialysis, and freeze-drying is conducted to obtain the product.

4. The aqueous formulation of rosmarinic acid-modified COS according to claim 3, wherein, The molecular weight of the chitosan oligosaccharide is ≤2000 Da, and the degree of deacetylation is ≥95%.

5. The aqueous formulation of rosmarinic acid-modified COS according to claim 3, wherein the concentration of rosmarinic acid is 0.01 to 0.1% (w / v). The purity of the rosmarinic acid is ≥97%.

6. The aqueous formulation of rosmarinic acid-modified COS according to claim 3, wherein The mass ratio of the chitosan oligosaccharide to the rosmarinic acid is 4-10:

1.

7. The aqueous formulation of rosmarinic acid-modified COS according to claim 3, wherein The pH value of the citrate buffer solution is 5.5-6.

5.

8. The aqueous formulation of rosmarinic acid-modified COS according to claim 3, wherein The reaction temperature is 20-50 ℃, and the reaction time is 18-24 h.

9. Application of the rosmarinic acid modified chitosan oligosaccharide aqueous preparation according to any one of claims 1-8 in promoting fruit coloring.

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