Effect of riboflavin for delaying reduction of antioxidant substances after broccoli harvesting and application of riboflavin

Abstract

The invention provides the effect of riboflavin for delaying the reduction of the content of antioxidant substances in broccoli after harvesting and the application of riboflavin, aiming at the broccoli treated by riboflavin, the content change of ascorbic acid, glutathione, glucosinolate and vitamin B2 in the storage period of the broccoli is measured, and the content of the antioxidant substances in the broccoli is determined. And a real-time fluorescent quantitative PCR method is utilized to research the expression difference of the metabolism genes related to the antioxidant substances between a riboflavin treatment group and a control group, so that a theoretical basis is provided for delaying the reduction of the content of the antioxidant substances after the broccoli is picked by riboflavin treatment.

Description

Technical Field

[0001] This invention belongs to the field of vegetable storage technology, specifically relating to the efficacy and application of riboflavin in delaying the decline of antioxidant substances in broccoli after harvest. Background Technology

[0002] Broccoli, also known as Brussels sprouts or young cauliflower, belongs to the Brassicaceae family. Rich in glucosinolates, sulforaphane, and other active substances, it possesses anti-cancer properties. Broccoli enjoys a high reputation in Ningbo, Zhejiang Province, especially during its optimal growing season in autumn, when its abundant yield effectively supplements the autumn vegetable market and meets consumers' demand for healthy vegetables. Broccoli's rich dietary fiber, vitamin C, and various minerals have won it widespread consumer favor. However, broccoli is highly susceptible to mechanical damage during harvesting and transportation. Its high water content and large surface area pose a significant challenge to transpiration after harvest, leading to rapid water loss and wilting, severely impacting its commercial value and edible quality. Typically, packaged broccoli can only maintain its freshness for about a week at room temperature, after which its quality declines considerably. Given these challenges, exploring environmentally friendly and efficient preservation technologies to extend the shelf life of broccoli and maintain its excellent quality has become an important direction in post-harvest physiology and preservation technology research for vegetables both domestically and internationally.

[0003] Riboflavin (VB2), a natural antioxidant widely distributed in various fresh agricultural products, is a valuable source of vitamins, minerals, and natural antioxidants such as carotenoids and flavonoids. In recent years, riboflavin, as an innovative and environmentally friendly preservation technology, has shown great application potential in the field of post-harvest preservation of fruits and vegetables due to its unique advantages of being non-toxic, harmless, and eco-friendly.

[0004] Patent CN120770430A has explored the mechanism by which riboflavin inhibits broccoli yellowing, finding that riboflavin can inhibit the expression of chlorophyll degradation genes and chlorophyll senescence genes in broccoli, and regulate the content of postharvest endogenous hormones in broccoli, thereby inhibiting postharvest yellowing and senescence. However, the effects of riboflavin on postharvest antioxidants and related metabolic genes in broccoli have not yet been reported. Summary of the Invention

[0005] To address the aforementioned issues, this study provides the efficacy and application of riboflavin in delaying the postharvest decline in antioxidant content in broccoli. For broccoli treated with riboflavin, the changes in the contents of ascorbic acid, glutathione, glucosinolates, and vitamin B2 during storage were measured. Real-time quantitative PCR was used to investigate the expression differences of these antioxidant-related metabolic genes between the riboflavin-treated group and the control group, providing a theoretical basis for riboflavin treatment in delaying the postharvest decline in antioxidant content in broccoli. The method for delaying the postharvest decline in antioxidant content in broccoli provided by this invention has a precise dosage, excellent effect, and ensures the quality of treated broccoli. Furthermore, riboflavin is inexpensive, and its use in treating broccoli will not significantly increase costs.

[0006] This invention provides the use of riboflavin in the preparation of formulations that delay the decline of antioxidants in postharvest broccoli.

[0007] In some methods, the concentration of riboflavin is 50–200 μM.

[0008] In some methods, the riboflavin is treated by soaking or spraying.

[0009] In some methods, the riboflavin treatment lasts for 5 to 15 minutes.

[0010] Preferably, the concentration of riboflavin is 100 μM, the treatment method is immersion, and the duration is 10 min.

[0011] In some methods, due to the photosensitivity of riboflavin, the entire treatment process is carried out in the dark. After treatment, the broccoli florets are allowed to air dry and then stored in a temperature- and humidity-controlled room at 15°C and 85–90% relative humidity, protected from light, for 5 days.

[0012] Furthermore, the antioxidants in the broccoli include any one or more of ascorbic acid, glutathione, glucosinolates, and vitamin B2.

[0013] Ascorbic acid (AsA), a powerful antioxidant, effectively scavenge free radicals in the body, alleviate oxidative stress, and protect cells from damage, thus playing an important role in the prevention of cardiovascular diseases and cancer. In this study, during the initial storage phase, the ascorbic acid (AsA) and reduced AsA content in broccoli showed a trend of first decreasing and then increasing, reaching their maximum values ​​in the riboflavin-treated group on the fourth day of storage. As the storage time increased, the total AsA and reduced AsA content in broccoli began to decrease again until the end of storage. However, throughout the entire storage period, the AsA content in the riboflavin-treated group was consistently higher than that in the untreated control group.

[0014] Glutathione (GSH), hailed as the "king of antioxidants," is one of the most important antioxidants in the human body. GSH is also a well-known antioxidant in plants, capable of reducing the accumulation of reactive oxygen species (ROS) under various abiotic stresses. In this study, the GSH and GSSG (oxidized glutathione) levels in the control group broccoli gradually decreased with prolonged storage, while the riboflavin-treated broccoli, by upregulating GSH and GSSG levels, provided an effective antioxidant defense mechanism, helping to extend the shelf life of broccoli and thus maintain its quality.

[0015] Glucosinolates, unique secondary metabolites in broccoli, are relatively stable and lack direct biological activity. However, they serve as important defense mechanisms for broccoli, and the active substances produced after hydrolysis offer numerous health benefits to humans. This study found that riboflavin treatment significantly promoted the accumulation of total glucosinolates and their components in broccoli throughout the storage period, with levels consistently significantly higher than the control group. The total glucosinolate content peaked on day 3 of storage, indicating that riboflavin treatment had the most significant promoting effect on glucosinolate synthesis during the early stages of storage.

[0016] Vitamin B2 (VB2) plays a crucial role in the metabolism of carbohydrates, fats, and proteins, helping to convert these nutrients into energy needed by the body. Furthermore, VB2 participates in hemoglobin synthesis and cellular redox reactions, which are essential for maintaining normal physiological functions. In this study, the VB2 content in broccoli tissues gradually decreased with increasing storage time, but this decline was significantly mitigated after riboflavin treatment, thus maintaining the nutritional value of the broccoli.

[0017] On the one hand, the present invention provides the use of riboflavin in the preparation of formulations that upregulate ascorbic acid-related synthesis genes or downregulate ascorbic acid-related degradation genes.

[0018] Furthermore, the ascorbic acid-related synthetic genes include any one or more of BoPGI, BoPMI, BoPMM, BoMIOX, and BoGGP, and the ascorbic acid-related degradation genes include BoAO.

[0019] The biosynthesis of AsA in plants mainly involves four pathways: L-galactose, D-galacturonic acid, L-glucose, and inositol. The L-galactose pathway is officially recognized as the primary biosynthetic pathway for AsA. Studies have found that using the gene GDP-l-galactose phosphorylase (GGP) increases AsA concentrations in tobacco and Arabidopsis thaliana. In this current study, riboflavin treatment upregulated most genes in the L-galactose pathway, leading to a corresponding increase in AsA levels. In this study, riboflavin treatment significantly upregulated the expression levels of BoPGI, BoPMI, BoPMM, BoMIOX, and BoGGP genes, while downregulating BoAO expression. BoPMI and BoPMM are key genes in the L-galactose pathway.

[0020] In another aspect, the present invention provides the use of riboflavin in the preparation of formulations that upregulate glutathione-related synthetic genes.

[0021] Furthermore, the glutathione-related synthesis genes include any one or more of BoGCS1, BoGS1, and BoGS3.

[0022] Glutamic acid ligase (GCS) is a key rate-limiting enzyme in GSH biosynthesis. It catalyzes the reaction of g-COOH and cysteine ​​to produce glutamate-NH- conjugated acid, while simultaneously consuming ATP to generate gamma-cysteine ​​(g-EC). Then, GSH synthase (GS) catalyzes the production of GSH from g-EC. In this experiment, riboflavin treatment increased the expression of three GSH biosynthesis genes, BoGCS1, BoGS1, and BoGS3, in broccoli, thereby inducing GSH accumulation.

[0023] The AsA-GSH cycle is an essential H2O2 scavenging system in plants. AsA and GSH are important non-enzymatic antioxidants in this cycle, and their redox states can represent the redox state in the cellular environment. GPX and APX are key enzymes in the AsA-GSH cycle metabolism. GPX plays a defensive role against oxidative damage in plant cells. APX can use AsA as an electron donor to reduce H2O2 to H2O and remove excess H2O2 from cells. The results showed that riboflavin treatment upregulated the expression of AsA-GSH cycle-related genes such as BoAPX1, thereby maintaining higher AsA and GSH levels during storage and extending the shelf life of postharvest broccoli.

[0024] In another aspect, the present invention provides the use of riboflavin in the preparation of formulations that upregulate glucosinolate-related synthetic genes or downregulate glucosinolate-related degradation genes.

[0025] Furthermore, the riboflavin-related synthesis genes include BoMYB28, BoCYP83A1, BoST5b, and BoFMO. GS-OX1 The glucosinolate-related degradation gene includes BoESP, BoAOP2, BoMYB51, or any one or more of these genes.

[0026] Studies have found that GRA, GBS, and NGBS are the main glucosinolate components in broccoli, accounting for 64%, 21%, and 7% of the total glucosinolates, respectively. This study suggests that preserving glucosinolates in postharvest broccoli is as important as maintaining its commercial quality. In this study, the total glucosinolate content in broccoli significantly increased after 3 days of riboflavin treatment at 15°C. In this study, when the control turned yellow, 100 μmol / L... -1 The riboflavin-treated broccoli maintained a better green color even on day 4. The expression of genes involved in the glucosinolate synthesis pathway, including AOP2, MYB28, ST5b, and FMO, was subsequently investigated. GS-OX1 BoFMO plays an important role in the formation of aliphatic glucosinolates in plants. Previous studies have shown that CaSO4 treatment promotes BoFMO. GS-OX1 The expression of BoMYB28 led to increased accumulation of glucosinolates in broccoli buds. A similar phenomenon was observed in broccoli buds treated with jasmonic acid, where upregulation of BoST5b also resulted in increased glucosinolates. Our results show that riboflavin treatment increased the expression of BoMYB28 and BoFMO. GS-OX1 The significant upregulation of gene expression could explain the elevated glucosinolate levels in broccoli. ESP typically leads to the loss of sulforaphane because it catalyzes the conversion of sulforaphane to inactive sulforaphane. In this study, riboflavin treatment upregulated BoMYB28, BoAOP2, BoCYP83A1, BoST5b, and BoFMO. GS-OX1 The expression of BoMYB51 was inhibited and the expression of BoESP was suppressed during storage, thereby maintaining the glucosinolate content of broccoli during room temperature storage.

[0027] In another aspect, the present invention provides the use of riboflavin in the preparation of formulations that upregulate vitamin B2-related synthetic genes.

[0028] Furthermore, the vitamin B2-related synthetic genes include any one or more of BoGCH1, BoPYRD, BoPYRR, BoPYRR2, BoRIB5, and BoRIB5.1.

[0029] Vitamin B2 is a precursor to the coenzymes FMN and FAD, which are essential for maintaining normal metabolism in organisms. FMN and FAD participate in many important processes in all organisms. In plants, pyrimidine deaminase, reductase, and phosphatase, decoded by genes PYRD, PYRR, and PYRP2, respectively, catalyze the conversion of the precursors guanosine triphosphate (GTP) and ribulose-5-phosphate (Ru5P) into 6,7-dimethyl-8-ribosepromethazine, which participates in riboflavin synthesis. In Saccharomyces cerevisiae, the deletion of a copy of the RIB5 chromosome leads to riboflavin malnutrition and loss of enzyme activity. FAD can be reduced to FMN under the catalysis of FAD pyrophosphatase, and subsequently, FMN is further hydrolyzed to riboflavin by FMN hydrolase (FHY). GCH is the rate-limiting enzyme, and mutations in its gene can lead to abnormal plant growth and development or even death. In this study, riboflavin treatment delayed the vitamin B2 content in postharvest broccoli by upregulating the expression of BoGCH1, BoPYRD, BoPYRR, BoPYRR2, BoRIB5, and BoRIB5.1.

[0030] The present invention has the following beneficial effects:

[0031] 1. The efficacy and application of riboflavin in delaying the decline of antioxidant content in broccoli after harvest were discovered;

[0032] 2. It was discovered that riboflavin can promote the expression levels of ascorbic acid synthesis-related genes BoPGI, BoPMI, BoPMM, BoMIOX, and BoGGP in postharvest broccoli, and can be used to prepare preparations that promote the expression of ascorbic acid synthesis-related genes BoPGI, BoPMI, BoPMM, BoMIOX, and BoGGP in postharvest broccoli;

[0033] 3. Riboflavin was found to inhibit the expression level of BoAO, a gene related to ascorbic acid degradation, in postharvest broccoli, and can be used to prepare a formulation that inhibits the expression of BoAO, a gene related to ascorbic acid degradation, in postharvest broccoli;

[0034] 4. It was discovered that riboflavin can promote the expression levels of glutathione synthesis-related genes BoGCS1, BoGPX2 and BoGGT1 in postharvest broccoli, and can be used to prepare preparations that promote the expression of glutathione synthesis-related genes BoGCS1, BoGPX2 and BoGGT1 in broccoli.

[0035] 5. Genes BoMYB28, BoCYP83A1, and BoFMO, which are related to riboflavin promoting glucosinolate synthesis in postharvest broccoli, were discovered. GS-OX1 The expression levels of these genes can be used to prepare the expression levels of BoMYB28, BoCYP83A1, and BoFMO, which are related to the synthesis of glucosinolates in broccoli. GS-OX1 Expression formulation;

[0036] 6. Riboflavin was found to inhibit the expression level of BoESP, a gene related to glucosinolate degradation, in postharvest broccoli, and can be used to prepare a formulation that inhibits the expression of BoESP in broccoli.

[0037] 7. Riboflavin was found to promote the expression levels of vitamin B2 synthesis-related genes BoPYRR, BoPYRR2, BoRIB5, and BoRIB5.1 in broccoli, and can be used to prepare preparations that promote the expression of vitamin B2 synthesis-related genes BoPYRR, BoPYRR2, BoRIB5, and BoRIB5.1 in broccoli. Attached Figure Description

[0038] Figure 1 Example 1: Effect of riboflavin treatment on the appearance of broccoli;

[0039] Figure 2 Example 1: Effect of riboflavin treatment on the color of broccoli;

[0040] Figure 3 Example 1: Effect of riboflavin treatment on the ultrastructure of broccoli chloroplasts;

[0041] Figure 4 Example 1: Effect of riboflavin treatment on weight loss rate of broccoli;

[0042] Figure 5 Example 2: Effect of riboflavin treatment on the senescence index of broccoli;

[0043] Figure 6 Example 2: Effect of riboflavin treatment on the senescence gene BoSAG12 in broccoli;

[0044] Figure 7 Example 3: Effect of riboflavin treatment on chlorophyll in broccoli;

[0045] Figure 8 Example 3: Effect of riboflavin treatment on chlorophyll degradation genes in broccoli;

[0046] Figure 9 Example 4: Effect of riboflavin treatment on total carotenoid content in broccoli;

[0047] Figure 10 Example 4: Effect of riboflavin treatment on the content of carotenoid components in broccoli;

[0048] Figure 11 Example 4: Effects of riboflavin treatment on carotenoid metabolism genes in broccoli;

[0049] Figure 12 Example 5: Effect of riboflavin treatment on MT content in broccoli;

[0050] Figure 13 Example 5 illustrates the effect of riboflavin treatment on the MT synthesis gene in broccoli.

[0051] Figure 14 Example 5 illustrates the effect of riboflavin treatment on the IAA content of broccoli;

[0052] Figure 15 Example 5: Effect of riboflavin treatment on the IAA metabolic gene in broccoli;

[0053] Figure 16 Example 5: Effect of riboflavin treatment on GA3 content in broccoli;

[0054] Figure 17 Example 5: Effect of riboflavin treatment on the GA3 metabolic gene in broccoli;

[0055] Figure 18 Example 5 illustrates the effect of riboflavin treatment on ethylene release from broccoli.

[0056] Figure 19 Example 5 illustrates the effect of riboflavin treatment on the ethylene synthesis gene in broccoli.

[0057] Figure 20 Example 5 illustrates the effect of riboflavin treatment on the 6-BA content of broccoli;

[0058] Figure 21 Example 5 illustrates the effect of riboflavin treatment on the 6-BA synthesis gene in broccoli;

[0059] Figure 22 Example 5: Effect of riboflavin treatment on BR content in broccoli;

[0060] Figure 23 Example 5 illustrates the effect of riboflavin treatment on the BR synthesis gene in broccoli.

[0061] Figure 24 Example 5 illustrates the effect of riboflavin treatment on the ABA content of broccoli;

[0062] Figure 25 Example 5: Effect of riboflavin treatment on ABA metabolism genes in broccoli;

[0063] Figure 26 Example 6: Effect of riboflavin treatment on total AsA and reduced AsA content in broccoli;

[0064] Figure 27 Example 6: Effect of riboflavin treatment on the AsA metabolic gene in broccoli;

[0065] Figure 28Example 6: Effect of riboflavin treatment on the content of oxidized and reduced GSH in broccoli;

[0066] Figure 29 Example 6: Effect of riboflavin treatment on GSH metabolism genes in broccoli;

[0067] Figure 30 Example 6: Effect of riboflavin treatment on the content of total glucosinolates and their components in broccoli;

[0068] Figure 31 Example 6: Effect of riboflavin treatment on broccoli glucosinolate synthesis genes;

[0069] Figure 32 Example 6: Effect of riboflavin treatment on VB2 content in broccoli;

[0070] Figure 33 Example 6 illustrates the effect of riboflavin treatment on the VB2 synthesis gene in broccoli. Detailed Implementation

[0071] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and are not intended to limit it in any way. All features disclosed in the embodiments of the present invention, or all steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0072] Example 1: Effects of riboflavin treatment on the quality of postharvest broccoli during storage

[0073] Fresh broccoli was sourced from a farm in Cixi City, Zhejiang Province, China. Harvesting took place 110 days after sowing. Ninety-six broccoli florets were selected based on strict criteria: uniform size, undamaged, unopened buds, and bright green color. The samples were then divided into two groups. One group was immersed in a 100 μM riboflavin solution for 10 minutes. This concentration was determined through preliminary testing. Initially, different riboflavin concentrations, such as 50, 100, 150, and 200 μM, and three treatment durations, 5, 10, and 15 minutes, were tested. The results showed that immersing the broccoli in a 100 μM riboflavin solution for 10 minutes had the most significant effect on delaying postharvest yellowing. Due to the photosensitivity of riboflavin, the entire treatment process was conducted in the dark. The other group was immersed in distilled water under the same conditions as the control group. After treatment, the broccoli florets were naturally dried and stored in a constant temperature and humidity chamber at 15°C and 85–90% relative humidity in the dark for 5 days. Subsequently, buds from broccoli florets were collected, frozen in liquid nitrogen, and stored at -80°C for further analysis. The entire experiment was repeated three times.

[0074] 1. Effects of riboflavin treatment on the appearance changes of postharvest broccoli

[0075] like Figure 1 As shown, under storage conditions of 15℃, broccoli gradually turned from green to yellow as the storage time increased. Specifically, the broccoli in the control group showed initial signs of yellowing on day 1, and this change gradually intensified in the following days. In contrast, the broccoli treated with riboflavin showed a significantly delayed process of turning from green to yellow, only showing slight yellowing symptoms on day 3. Particularly noteworthy is the most significant difference in the degree of yellowing between the two groups of broccoli on day 4 of storage, creating a stark contrast.

[0076] 2. Effects of riboflavin treatment on the color of postharvest broccoli

[0077] Color data of broccoli florets were determined using a Konica Minolta CM-26D spectrophotometer. The broccoli was divided into five sections (top, bottom, left, right, and center) with the center as the boundary, and measurements were taken on all five sides. Results are expressed as L*, a*, b*, and -a / b.

[0078] like Figure 2 As shown, the -a / b value of broccoli exhibited a continuous decreasing trend, while the values ​​of L* (brightness), a* (red-green value), and b* (yellow-blue value) gradually increased. This series of changes clearly reflects that the original green color of broccoli is gradually fading, replaced by an increase in yellow hue. In riboflavin-treated broccoli samples, the changes in these parameters were consistently more gradual than in the control group. Compared to the untreated control samples, the treatment group showed significantly smaller changes in the -a / b value (a decrease in this value indicates reduced green) and the L* value (an increase in brightness indicates a lighter or yellower color).

[0079] 3. Effects of riboflavin treatment on the ultrastructure of chloroplasts in postharvest broccoli florets

[0080] Broccoli buds were prepared for observation under a transmission electron microscope (TEM). Three to four buds were taken from the top of the broccoli stem, cut in half, and placed in a 0.1 mol / L solution containing 2.5% glutaraldehyde and 2% paraformaldehyde. -1 Fix overnight at 4°C in pH 7.0 phosphate buffer (PB). After fixation, use 0.1 mol / L... -1The sample was rinsed three times with pH 7.0 PB buffer for 10 min each time. Next, the sample was fixed in a solution containing 1% OsO4 and 1.5% K4Fe(CN)6 for 1.5 h, followed by three more rinses with PB buffer for 10 min each time. Subsequently, the sample was dehydrated sequentially in 30%, 50%, 70%, and 95% ethanol for 15 min each step. Further dehydration included two treatments with 100% ethanol and two treatments with 100% acetone for 15 min each. Finally, the sample was treated at 32 °C with a mixture of acetone and epoxy resin (1:2, v / v) and a mixture of acetone and resin. After complete infiltration, the sample was placed in an embedding mold containing embedding resin and polymerized at 65 °C for 36 h. Subsequently, the sample was trimmed and sliced ​​using an ultramicrotome (Leica EMUC7, Austria, Germany) to obtain slices approximately 70–90 nm thick, which were then mounted on a carbon-coated copper grid (150 mesh). Finally, the slides were observed and photographed using a transmission electron microscope (H-7600, Hitachi, Japan).

[0081] Figure 3 In the figures, A and B are TEM images of broccoli florets on day 0, C and D are TEM images of broccoli florets from the control group on day 4, and E and F are TEM images of broccoli florets treated with riboflavin on day 4. Chl, CW, PM, ST, and E represent chloroplasts, cell walls, plasma membranes, stroma, and nuclear membranes, respectively. As shown in the figures, on day 0, the cell structure of fresh broccoli florets was intact, with cell walls and plasma membranes clearly visible. Chloroplasts were arranged along their long axis within the plasma membrane, their membrane structure intact, and stroma vacuoles were neatly arranged. However, by day 4, the number of chloroplasts in the control group broccoli florets was significantly reduced, and they swelled from spindle-shaped to round. The integrity of the chloroplast membrane system was completely disrupted, leading to its complete disintegration. Chloroplasts in the stroma tissue showed signs of breakage and dispersion, exhibiting elongated and attenuated filamentous structures. Some chloroplasts showed partial vacuolation or even complete disintegration. In contrast, although the number of chloroplasts in the riboflavin-treated florets was also reduced, the number of chloroplasts on day 4 was greater than that in the control group. The membrane system remains highly developed, exhibiting only slight swelling and shape changes. The chloroplast stroma is still visible and shows a regular arrangement.

[0082] 4. Effect of riboflavin treatment on postharvest weight loss rate of broccoli

[0083] The formula for calculating the weight loss rate using the weighing method is as follows:

[0084]

[0085] The change in weight loss of broccoli after riboflavin treatment and storage at room temperature for 5 days is as follows: Figure 4As shown in the figure, the weight loss rate in both groups initially showed an increasing trend throughout the storage period, with a slight decrease in the riboflavin group on day 5. However, the rate and magnitude of the increase in weight loss rate in riboflavin-treated broccoli were significantly lower than those in the control group. Particularly on day 5, the weight loss rate in the riboflavin-treated group was significantly different from that in the control group, demonstrating a clear inhibitory effect. The weight loss rate in the control group was as high as 4.75%, while that in the riboflavin-treated group was only 1.49%, a highly significant difference.

[0086] In conclusion, the broccoli treated with riboflavin maintained a brighter green color, showing a more significant green retention effect compared to the control group. Furthermore, riboflavin effectively delayed chloroplast degradation, thus preserving the green color of the broccoli. In addition, riboflavin significantly inhibited the increase in weight loss during storage, indicating enhanced water retention capacity and benefiting post-harvest preservation of broccoli.

[0087] Example 2: Effects of riboflavin treatment on postharvest senescence in broccoli

[0088] 1. Effects of riboflavin treatment on the senescence index of postharvest broccoli

[0089] Visual evaluation was performed using a senescence index, based on browning and discoloration observed on the surface of eight broccoli florets from each replicate. For each broccoli, senescence was scored on a five-point scale: 0 = no open buds, no browning, no mold, and vibrant color; 1 = slightly open buds, yellowing but no mold; 2 = clearly open buds, mold, and yellowing; 3 = fully open buds, yellowing, mold, yellowish-black color, and a pungent odor; 4 = shriveled, rotten buds, and a strong pungent odor. The senescence index was calculated using the following formula:

[0090]

[0091] like Figure 5 As shown, the senescence index of broccoli exhibits a steady and continuous upward trend during storage. This increase not only reflects the gradual degeneration of broccoli cell structure but also signifies the loss of nutritional value and a decline in edible quality. Treatment with riboflavin significantly slows down the senescence process of broccoli.

[0092] 2. Effects of riboflavin treatment on senescence genes in postharvest broccoli

[0093] Weigh 0.1g of frozen broccoli sample and extract total RNA from the broccoli using a plant RNA miniprep kit (Aowei Biotechnology Co., Ltd.). cDNA was reverse transcribed using HiScript IIQ RT SuperMix (Novizan, Nanjing, Jiangsu, China).

[0094] Specific primers for the aging gene BoSAG12 were designed using Beacon Designer 7, with BoACT as an internal reference gene. Experiments were performed using a Bio-Rad CFX96 real-time PCR system (Bio-Rad, Hercules, California, USA). Primer sequences are shown in Table 1. Reaction conditions followed the instructions for the ChamQ Universal SYBR qPCR Master Mix (Novizan, Nanjing, Jiangsu, China), and four biological replicates were performed. Relative expression levels were measured using a 2-1 ratio. -ΔCT The method is used for calculation and analysis.

[0095] Experimental data were processed using GraphPad Prism 9 software and are presented as mean ± standard deviation (n=3). Multiple t-tests were used to compare the significance of the control and treatment groups (*P<0.05, **P<0.01, ***P<0.001).

[0096] Table 1 Primer sequences for the aging gene BoSAG12

[0097]

[0098]

[0099] The expression changes of the aging-related gene BoSAG12 in broccoli treated with riboflavin and stored at room temperature for 5 days are as follows: Figure 6 As shown. This trend is related to... Figure 5 The trends in the aging index of broccoli shown correspond to these trends. BoSAG12, a known aging marker gene, directly reflects the aging process of broccoli through changes in its expression level. For example... Figure 6 As shown, the expression level of the BoSAG12 gene remained at a relatively low level in the early stages of storage, but gradually increased with the passage of time. Figure 5 In the study, it was observed that the senescence index of broccoli steadily increased with the increase of storage time, a trend that corresponds to the increasing trend of BoSAG12 gene expression.

[0100] In conclusion, riboflavin treatment significantly inhibited the increase of the senescence index during broccoli storage, reflecting the slowing down of the senescence process of broccoli, which is of great significance for extending the shelf life of postharvest broccoli.

[0101] Example 3: Effect of riboflavin treatment on chlorophyll in broccoli during storage

[0102] 1. Effects of riboflavin treatment on chlorophyll content in broccoli during storage

[0103] 0.1 g of broccoli floret powder, ground in liquid nitrogen, was added to 1.5 mL of pre-cooled 95% ethanol and centrifuged at 10000 × g for 10 min. The precipitate was discarded, and the supernatant was collected. Its absorbance was measured at 649 nm and 665 nm wavelengths, with 95% ethanol used as a blank. The total chlorophyll content, chlorophyll a content, and chlorophyll b content were calculated, expressed on a fresh weight (FW) basis. The chlorophyll calculation formula is:

[0104] Chlorophyll content (mg / g) = (C×V×n) / W;

[0105] C = 18.16 × A 649 +6.63×A 665 ;

[0106] In the formula, C is the total chlorophyll concentration (mg / L); V is the extraction volume (L); n is the dilution factor; and W is the fresh weight of the sample (g).

[0107] like Figure 7 As shown, the gradual decline in chlorophyll content in broccoli florets was effectively mitigated after riboflavin treatment. Riboflavin, an important water-soluble vitamin, possesses significant antioxidant and free radical scavenging capabilities. During storage, riboflavin may slow down chlorophyll degradation by participating in the antioxidant system within broccoli floret cells, protecting chloroplasts from oxidative stress. Experimental data showed that compared to the untreated control group, the total chlorophyll, chlorophyll a, and chlorophyll b contents in treated broccoli florets decreased more slowly during storage, maintaining relatively high levels. Riboflavin treatment may slow down chlorophyll degradation during postharvest storage of broccoli by regulating the expression of genes related to chlorophyll degradation.

[0108] 2. Effects of riboflavin treatment on the expression of chlorophyll-related degradation genes during broccoli storage

[0109] The methods for total RNA extraction and cDNA synthesis were the same as those for real-time quantitative PCR in Example 2. Primer sequences for chlorophyll degradation-related genes in broccoli are shown in Table 2.

[0110] Table 2 Primer sequences of broccoli chlorophyll degradation-related genes

[0111]

[0112] like Figure 8As shown, the expression levels of BoNOL, BoPAO, BoSGR1, BoSGR2, BoCLH1, BoCLH2, BoNYC, and BoRCCR initially increased and then decreased. Transcripts of BoPAO, BoSGR1, and BoNYC peaked on day 2 of storage, while BoNOL, BoSGR2, and BoRCCR peaked on day 3. On day 4, the expression levels of BoCLH1 and BoCLH2 were highest. BoPPH expression decreased throughout the storage process. Riboflavin treatment reduced BoNOL expression on days 2 and 3, and reduced BoRCCR expression on days 1 and 3. Throughout the storage process, lower transcript levels of BoSGR1, BoCLH2, and BoPPH were observed in treated florets. Riboflavin treatment reduced the expression of BoPAO, BoSGR2, BoCLH1, and BoNYC throughout the storage process, except for day 5.

[0113] Example 4: Effect of riboflavin treatment on carotenoids in broccoli during storage

[0114] 1. Effect of riboflavin treatment on total carotenoid content in broccoli during storage

[0115] Add 5 mL of anhydrous ethanol (containing 0.1% AsA, w / v) to a 50 mL centrifuge tube and pre-cool on ice for 30 min. Randomly weigh approximately 1 g of sample that has been ground into a uniform powder using liquid nitrogen and add it to the pre-cooled centrifuge tube. Mix thoroughly using a vortex mixer (40 s) and then place in a -20°C freezer for overnight extraction. Preheat a water bath to 95°C and add 300 μL of 80% potassium hydroxide solution (cover the tube opening with 4 layers of plastic wrap before screwing on the cap). Vortex thoroughly and place in a water bath for saponification in the dark for 45 min. Immediately after the saponification reaction is completed, transfer the centrifuge tube to an ice bath for rapid cooling. Perform gradient extraction using petroleum ether with a boiling range of 60–90°C (5 mL × 3), vortexing for 30 s to ensure complete emulsification, followed by high-speed centrifugation at 4°C (6000 × g, 10 min). Transfer the upper organic phase to a clean centrifuge tube and concentrate it by nitrogen purging at a constant temperature of 35°C until the solvent is completely evaporated. Accurately add 3 mL of an equal volume mixture of dichloromethane and methanol (v:v = 1:1) to the dried sample, vortex to redissolve, and then filter through a 0.22 μm organic filter membrane. Take 1 mL of the filtrate and measure the absorbance at 450 nm using a UV-Vis spectrophotometer. Calculate the total carotenoid content using the following formula:

[0116] ρ = A 450 ×v×f×10×1000×m -1 ×2500 -1 ;

[0117] In the formula:

[0118] ρ: Mass concentration of carotenoids (μg·g⁻¹);

[0119] A450: Absorbance of the carotenoid extract at 450 nm;

[0120] v: Volume of carotenoid extract (mL) (3mL);

[0121] f: Dilution factor of carotenoid extract (10 times);

[0122] m: Sample mass (g);

[0123] 2500: Average absorbance of 1% carotenoids at the maximum absorption wavelength.

[0124] A separate 1 mL filtrate was used to detect carotenoid components using a Waters Alliance E2695 system (Milford, Massachusetts, USA). The photodiode array (PDA) detector and C30 column (250 × 4.6 mm, 3 μm; YMC Co, Japan) were set to 40 °C and 10 μL / s. -1 Flow rate. The mobile phase consisted of methanol (eluent A), methyl tert-butyl ether (eluent B), and H₂O (eluent C), with the following gradient program: time 0, 86% A, 10% B, 4% C; 15 min, 71% A, 15% B, 4% C; 50 min, 6% A, 90% B, 4% C; 53 min, 86% A, 10% B, 4% C. The absorbance was monitored at 450 nm and the carotenoid content was calculated.

[0125] like Figure 9 As shown, the total carotenoid content of broccoli gradually increased throughout the storage period, with the difference in total carotenoid content gradually increasing from day 2, reaching its maximum on day 3. These results indicate that riboflavin treatment reduced the total carotenoid level of broccoli during storage, which explains why riboflavin treatment delayed the yellowing of broccoli during storage.

[0126] 2. Effects of riboflavin treatment on the content of carotenoid components in broccoli during storage

[0127] like Figure 10As shown, the main carotenoids in broccoli include zeaxanthin, lutein, β-carotene, and β-cryptoxanthin. Throughout the storage period, riboflavin treatment significantly affected carotenoid content. Except for day 2, the zeaxanthin and lutein contents in the treated group were significantly lower than those in the control group; β-cryptoxanthin showed the same trend starting from day 2. Although there was no significant difference in β-carotene between the two groups in the early stages of storage, the content in the treated group decreased significantly by day 5. Notably, except for β-cryptoxanthin, the other three carotenoids (zeaxanthin, lutein, and β-carotene) in the control group showed a continuous upward trend, consistent with the pattern of total carotenoid changes. Among the four carotenoid components, the contents of the control group were generally higher than those of the treated group, with relatively smaller differences in β-carotene between groups.

[0128] 3. Effects of riboflavin treatment on the expression of carotenoid-related metabolic genes during broccoli storage

[0129] The methods for total RNA extraction and cDNA synthesis were the same as those for real-time quantitative PCR in Example 2. Primer sequences for broccoli carotenoid metabolism-related genes are shown in Table 3.

[0130] Table 3 Primer sequences for genes related to broccoli carotenoid metabolism

[0131]

[0132] Figure 11 The expression of carotenoid metabolism genes was shown. After riboflavin treatment, BoPSY1 expression reached its peak on day 4 and was significantly different from the control group. The transcriptional levels of BoPDS5 and BoZISO fluctuated considerably during the later stages of storage, consistently lower in the riboflavin-treated group than in the control group, with highly significant differences in transcriptional levels on days 4 and 5, respectively. The expression of BoZEP, BoZDS1, BoLCY1, and BoVDE1 initially increased and then decreased. The expression of BoCCD1, a carotenoid degradation-related gene, initially increased and then decreased with storage time, but remained higher in the riboflavin-treated group than in the control group throughout the entire storage period. Throughout the storage process, except for day 5, riboflavin treatment reduced the expression of BoPSY1, BoCHY1, and BoLCY1.

[0133] Analysis of the results from Examples 3 and 4 revealed that riboflavin treatment significantly delayed postharvest chlorophyll degradation and the accumulation of carotenoid components in broccoli. Riboflavin treatment downregulated the expression of chlorophyll degradation genes, including BoNOL, BoPAO, BoSGR1 / 2, BoCLH1 / 2, BoNYC, BoRCCR, and BoPPH. This is likely a key reason for the delayed chlorophyll degradation in riboflavin-treated broccoli. Furthermore, riboflavin treatment delayed the biosynthesis of four carotenoid components by downregulating the expression of synthesis genes BoPSY1, BoPDS5, BoZISO, BoCHY1, BoZEP, BoZDS1, BoLCY1, and BoVDE, and upregulating the expression of degradation gene BoCCD1, thereby alleviating yellowing in postharvest broccoli. These findings further elucidate the regulatory mechanism of pigment metabolism in postharvest broccoli and suggest that riboflavin treatment may be an effective green conservation strategy that can improve the commercial value of broccoli and extend its postharvest shelf life.

[0134] Example 5: Effects of riboflavin treatment on endogenous hormones in broccoli during storage

[0135] 1. Effects of riboflavin treatment on melatonin levels in broccoli during storage

[0136] Melatonin (MT) is a hormone produced by animals and plants in response to changes in the light and dark cycle. In plants, MT is involved in various biological processes, such as seed germination, flowering, and fruiting, as well as plant growth and development. MT functions by interacting with MT1, MT2, and MT3 receptors. MT1 and MT2 receptors are G protein-coupled. In contrast, the MT3 receptor is a nuclear hormone receptor that binds to DNA and regulates gene expression. It is known to interact with several other signaling pathways, including the nitric oxide pathway, the calcium pathway, and the cyclic adenosine monophosphate pathway. These pathways are involved in various cellular processes, such as cell division, apoptosis, and stress responses. In plants, MT has been found to play a role in various processes, including regulating nutrient use efficiency. Nutrient use efficiency is the ability of plants to efficiently absorb and utilize soil nutrients. Furthermore, MT regulates the expression of stress response genes. As a transcription factor, it regulates the activity of various genes involved in stress responses. These are key factors in crop production because they affect crop yield and quality.

[0137] Melatonin content was determined using a kit (Jiangsu Enzyme-Linked Immunosorbent Assay Co., Ltd., Nanjing, China). Total RNA extraction and cDNA synthesis methods were the same as in Example 2. Primer sequences for broccoli MT metabolism-related genes are shown in Table 4.

[0138] Table 4 Primer sequences for broccoli MT metabolism genes

[0139]

[0140] like Figure 12 As shown, the MT content in both the control and treated broccoli samples initially increased during the first 3 days of storage, then decreased until the end of storage. Throughout the storage period, the MT content in riboflavin-treated broccoli was significantly higher than that in the control group. Figure 13 As shown, compared with the control group, the BoTDC1 transcript level in the treated broccoli was significantly increased throughout the storage process. Riboflavin induced BoASMT expression on days 2, 3, 4, and 5. After 2 days of storage, the transcriptional levels of BoSNAT2 and BoCOMT1 in the treated group were significantly higher than those in the control group.

[0141] It has been reported that exogenous MT treatment can inhibit chlorophyll degradation and slow down the yellowing of apple leaves and rosette leaves. In barley leaves, MT can alleviate chlorophyll degradation caused by power outages. Previous studies have shown that MT can regulate nitrogen and sucrose metabolism and delay the yellowing of broccoli during storage. Therefore, it can be concluded that the increased MT content observed in riboflavin-treated broccoli in this study is due to the upregulation of biosynthetic genes such as BoTDC1, BoASMT, BoSNAT2, and BoCOMT1, which play an important role in delaying chlorophyll degradation.

[0142] 2. Effects of riboflavin treatment on endogenous auxin levels during broccoli storage

[0143] Natural auxins are a group of heterogeneous aromatic carboxylic acids, important plant hormones that play a crucial role in almost all processes related to plant growth and development. Charles Darwin and his son Francis deduced their existence as a mediator of phototropism in grass coleoptiles. However, endogenous IAAs were first chemically identified in the 1930s as indole-3-acetic acid (IAA). Subsequent extensive research into different aspects of the biology of endogenous IAAs has revealed that their functions are far more broad, currently associated with several processes in the plant life cycle. These processes include embryogenesis, lateral root formation, apical dominance, vascular differentiation, fruit development, and many other developmental processes.

[0144] The content of IAA was determined using a kit (Jiangsu Enzyme-Linked Immunosorbent Assay Co., Ltd., Nanjing, China). The methods for total RNA extraction, cDNA synthesis, and real-time quantitative PCR were the same as in Example 2. Primer sequences for broccoli IAA metabolism-related genes are shown in Table 5.

[0145] Table 5 Primer sequences for broccoli IAA metabolic genes

[0146]

[0147] like Figure 14As shown, the endogenous IAA content in broccoli florets in both the control and treatment groups gradually increased during the first 3 days of storage, and then decreased. Throughout the storage period, the IAA content in the treatment group was significantly higher than that in the control group. Figure 15 As shown, riboflavin treatment increased the transcription level of BoMES2 during the storage period except for day 5, but significantly increased the transcription level of BoMES12 only on day 3. Riboflavin treatment significantly reduced the expression of the BoIAMT gene in untreated broccoli throughout the storage period. Compared with the control group, the transcription level of BoDAO was lower in the treated florets after 3 days of storage. Riboflavin treatment continuously suppressed the gene expression levels of BoGH3.6 and BoGH3.12. Compared with the untreated control group, the transcription levels of BoTAR4 and BoSAUR32 in broccoli treated with riboflavin were significantly increased. Riboflavin treatment induced the expression of BoSAUR36 and BoSAUR67 on days 1 and 2.

[0148] IAA is the most important auxin in higher plants, playing a crucial role in postharvest product maturation and senescence. Exogenous IAA increases GA levels in white clover, affecting chlorophyll catabolism and maintaining leaf chlorophyll content. In this study, riboflavin-treated broccoli showed increased transcription levels of IAA biosynthetic genes such as BoTAR4, BoMES2, and BoMES12, as well as early accessory nutrient response genes such as BoSAUR32, BoSAUR36, and BoSAUR67, while the transcription levels of degradation genes such as BoDAO, BoIAMT, BoGH3.6, and BoGH3.12 were downregulated. This indicates that the increase in IAA after riboflavin treatment is associated with the inhibition of chlorophyll degradation in broccoli.

[0149] 3. Effects of riboflavin treatment on gibberellin levels during broccoli storage

[0150] Gibberellins (GAs) are crucial plant hormones involved in plant growth and development, widely used in agriculture, forestry, and horticulture. Among GAs, GA3 is the most widely used. Currently, GA3 has been applied in numerous studies related to microalgae cultivation. GA3 has been shown to shorten the lag phase of algal cell growth, promote cell entry into the exponential growth phase, and stimulate the accumulation of pigments and proteins. Furthermore, GA3 can promote microalgae growth by regulating nutrient absorption and utilization, particularly by modulating carbon metabolism. Regarding enhancing microalgae's stress resistance, studies have shown that under abiotic stress, GA3 can promote the activity of antioxidant enzymes and the accumulation of AsA and GSH in microalgae, thereby improving their adaptability to abiotic stress.

[0151] The GA3 content was determined using a kit (Jiangsu Enzyme-Linked Immunosorbent Assay Co., Ltd., Nanjing, China). The total RNA extraction, cDNA synthesis, and real-time quantitative PCR methods were the same as in Example 2. The primer sequences for GA3 metabolism-related genes in broccoli are shown in Table 6.

[0152] Table 6 Primer sequences for GA3 metabolism-related genes in broccoli

[0153]

[0154] like Figure 16 As shown, the GA3 content in broccoli increased throughout the storage process, and the GA3 content in the riboflavin-treated group continued to increase compared to the control group. Figure 17 As shown, compared with untreated broccoli, the riboflavin-treated group exhibited higher levels of BoKAO1 transcripts on days 1 and 2. After 1 day of storage, treatment upregulated the transcriptional levels of BoGA20OX1 and BoGA20OX2, while BoGA20OX3 expression was upregulated during the first 4 days of storage. Throughout the storage process, the transcriptional levels of BoGA2OX2 and BoGA2OX6 decreased with continued treatment.

[0155] GA3 treatment delayed yellowing in bananas and broccoli and maintained high chlorophyll content post-harvest. Previous studies have shown that GA3 treatment can maintain chlorophyll content in okra post-harvest by reducing the transcriptional levels of genes involved in chlorophyll degradation. In this study, riboflavin upregulated the expression of GA3 synthesis genes BoKAO1, BoGA20OX1, BoGA20OX2, and BoGA20OX3, while inhibiting the expression of negative regulatory genes BoGA2OX2 and BoGA2OX6, leading to increased GA3 content and signal transduction. This indicates that the increase in GA3 content and the potential interaction between GA3 and IAA after riboflavin treatment are related to the inhibition of chlorophyll degradation in broccoli.

[0156] 4. Effect of riboflavin on ethylene during the storage period of broccoli

[0157] Ethylene is found in all parts of plants, including stems, leaves, flowers, roots, seeds, and fruits. It is a plant growth regulator widely used to accelerate the ripening of flowers and fruits. The molecular formula C2H4 or H2C=CH2 represents the colorless, flammable gas ethylene. Ethylene consists of four hydrogen-bonded atoms connected to carbon atoms via double bonds. The six hydrogen single bonds (H and CH) form at an angle close to 120°, or 117.4°, creating a hybrid carbon sp. 2Ethylene is the second most basic unsaturated hydrocarbon. Ethylene production is induced during plant development, such as germination, fruit ripening, leaf abscission, and flower senescence. Ethylene can accelerate plant senescence and can also regulate its production in plants under stress conditions, such as during biotic and abiotic stresses.

[0158] The method for determining ethylene content was as follows: Two broccoli florets were placed in a 10L sealed container. After standing at room temperature for 3 hours, 5 mL of gas sample was drawn from the top of the container using a syringe and injected into a gas chromatograph equipped with a flame ionization detector (FID, GC-2014C, Shimadzu, Japan). The chromatographic program used nitrogen as the carrier gas and also employed N2 (64 mL min). -1 H2 (40 mL min) -1 ) and air (500mL / min) -1 A specific flow rate was maintained. The inlet temperature was kept at 200°C, and the detector temperature was set at 260°C. The ethylene standard solution used for quantitative analysis was 200 μL / L. -1 50μLL -1 10μL -1 5μL -1 and 2μLL -1 Quantitative analysis was performed using ethylene standard solutions. Four biological replicates were performed for each sample group.

[0159] The methods for total RNA extraction and cDNA synthesis were the same as those for real-time quantitative PCR in Example 2. Primer sequences for broccoli ethylene metabolism-related genes are shown in Table 7.

[0160] Table 7 Primer sequences for broccoli ethylene metabolism-related genes

[0161]

[0162] like Figure 18 As shown, ethylene production in broccoli gradually increases during storage; after 1 day of storage, the use of riboflavin can reduce ethylene production. Figure 19 As shown, the transcriptional levels of BoACO2 and BoSAM were reduced in riboflavin-treated broccoli during the first 3 and 4 days of storage. BoACO1 expression was lower in the treated florets on days 2, 3, and 4, and the transcriptional levels of all three BoACS were also reduced after 1 day of storage.

[0163] Ethylene is one of the earliest discovered plant hormones, widely involved in organ abscission, seed germination, flowering induction, leaf elongation, and senescence. Researchers have discovered an ethylene-responsive factor, CitERF13, in citrus, whose transcriptional level is closely related to the fruit's chlorotic response to ethylene. Other researchers have found that chlorine dioxide, as a plant growth regulator, can effectively inhibit ethylene-induced chlorophyll degradation by suppressing ethylene production. After storage at 20°C for 2 days, ethylene treatment accelerates chlorophyll degradation in citrus because chlorophyllase increases chlorophyll a formation. Furthermore, pears treated with 1-MCP reduce ethylene production by inhibiting PcACS2 expression, thereby delaying chlorophyll degradation. Recently, studies have shown that salicylic acid treatment can delay chlorophyll loss and slow chlorosis in broccoli by inhibiting ethylene production. In this study, it was observed that riboflavin treatment significantly downregulated the transcriptional levels of ethylene biosynthesis genes (including BoACO2, BoACO1, BoACS1, BoACS2, BoACS7, and BoSAM), resulting in reduced ethylene production in the treated broccoli. This is crucial for reducing chlorophyll degradation in riboflavin-treated broccoli.

[0164] 5. Effect of riboflavin on 6-benzylaminopurine levels during the storage period of broccoli

[0165] Cytokinins (CKs) have a profound impact on almost all biological events in plant development and defense, including floral organ maturation and abscission, leaf / flower senescence, and responses to various biotic and abiotic stresses. Previous experiments have shown that these plant hormones play beneficial roles in physiological and molecular processes, as well as in the defense against biotic pathogens. Cytokinins are now known to regulate the metabolism and transport of amino acids and carbohydrates, which are essential for plant growth, as well as several macronutrients, including nitrogen, phosphorus, sulfur, and iron. 6-Benzylaminopurine (6-BA) is a plant growth regulator in broccoli that inhibits the breakdown of chlorophyll, nucleic acids, amino acids, and proteins, delays senescence, and maintains green color, thus improving broccoli quality. It can regulate physiological processes such as plant cell division, seed germination, photosynthesis, senescence, and stress resistance.

[0166] The content of 6-BA was determined using a kit (Jiangsu Enzyme-Linked Immunosorbent Assay Co., Ltd., Nanjing, China). The methods for total RNA extraction, cDNA synthesis, and real-time quantitative PCR were the same as in Example 2. The primer sequences for broccoli 6-BA metabolism-related genes are shown in Table 8.

[0167] Table 8 Primer sequences for broccoli 6-BA metabolism-related genes

[0168]

[0169] like Figure 20As shown, the 6-BA content in riboflavin-treated broccoli and control broccoli significantly increased during the first two days of storage, then decreased, and then increased again on the last day. The 6-BA content in riboflavin-treated florets was consistently higher than that in the control group. Figure 21 As shown, the transcript level of the cytokinin receptor BoAHK5 was significantly higher than that of the untreated control group throughout the storage process. Riboflavin treatment enhanced BoAHP1 expression during broccoli storage, except on days 1 and 3. The expression of BoARR2 and BoARR10 increased with treatment throughout the storage process.

[0170] Studies have shown that 6-BA is involved in chlorophyll metabolism in postharvest fruits and vegetables. For example, 6-BA treatment of broccoli florets can reduce chlorophyll degradation by lowering chlorophyllase levels; 6-BA treatment can inhibit the increase of ethylene content, thus maintaining a high chlorophyll content in Chinese cabbage leaves. In mango fruits, application of 6-BA can reduce the activity of ACS and ACO, thereby delaying chlorophyll degradation and reducing ethylene production. In addition, 6-BA can also delay the yellowing of postharvest leeks and cucumbers and maintain chlorophyll content. Therefore, in this study, the higher chlorophyll content in broccoli treated with riboflavin may be due to the activation of cytokinin receptors BoAHK5, BoAHP, and BoARRs during storage, thereby increasing the 6-BA content and delaying the decrease in chlorophyll content in broccoli.

[0171] 6. Effects of riboflavin on brassinolide during the storage period of broccoli

[0172] Brassinosteroids, a type of polyhydroxy steroid, are defined as the sixth class of plant hormones. They are widely found in plants and are natural, non-toxic, harmless, and highly effective plant hormones, representing the sixth largest class of plant hormones. They possess antioxidant properties, can delay plant senescence, and play an important role in regulating plant growth and maintaining fruit quality attributes. Brassinosteroids (BR) are the most active brassinosteroids and have been widely used as plant growth regulators.

[0173] The BR content was determined using a kit (Jiangsu Enzyme-Linked Immunosorbent Assay Co., Ltd., Nanjing, China). The total RNA extraction and cDNA synthesis methods were the same as those used in Example 2. The primer sequences for broccoli BR metabolism-related genes are shown in Table 9.

[0174] Table 9 Primer sequences for broccoli BR metabolism-related genes

[0175]

[0176] like Figure 22As shown, during the first 3 days of storage, the BR content in both the control and riboflavin-treated groups increased sharply, and then decreased. Throughout the storage process, the BR content in the riboflavin-treated group remained higher than that in the control group. Figure 23 As shown, riboflavin treatment increased the transcriptional levels of the BR synthesis genes BoDET1 and BoDWF5 throughout storage. On days 1 and 4, compared to the untreated control group, the treated florets showed higher transcript abundance of BoBZR1 / 2, while BoBZR3 gene expression was significantly induced by riboflavin treatment throughout storage. Riboflavin upregulated the transcripts of BoCYP85A1, BoSMO1, and BoBSK throughout storage.

[0177] The role of riboflavin (BR) in chlorophyll degradation and senescence after harvest of various horticultural products has been widely reported. Exogenous EBR treatment has been shown to improve cell membrane integrity and maintain chlorophyll content, thus delaying yellowing of daylily buds. EBR treatment delayed postharvest chlorosis in cabbage leaves by downregulating genes related to chlorophyll catabolism. Studies have found that exogenous BR spraying upregulates the expression of chlorophyll-related biosynthetic genes, increasing chlorophyll content in celery. This study shows that, compared with the control group, riboflavin-treated broccoli florets showed a significant increase in endogenous BR content, and the transcriptional levels of BR-related biosynthetic genes BoDWF5, BoDET1, BoBZR1 / 2 / 3, BoCYP85A1, BoSMO1, and BoBSK were also significantly increased, thereby improving chlorophyll content.

[0178] 7. Effect of riboflavin on abscisic acid during the storage period of broccoli

[0179] Abscisic acid (ABA) is a key signaling molecule involved in various stress responses. ABA is a small sesquiterpene molecule that regulates plant growth and resilience. ABA is synthesized via a carotenoid pathway initiated in chloroplasts, where carotenoids are converted to ABA through several steps in the cytoplasm and plastids. When ABA levels in plant cells exceed a certain threshold, it is metabolized by uridine diphosphate glycosyltransferase into ABA-GE (ABA glucose ester) and stored in vacuoles. Conversely, β-glucosidase homologues can convert ABA-GE back to ABA in stressed plants. Excessive ABA inhibits photosynthesis and nutrient uptake, leading to seed dormancy and premature leaf senescence, severely impacting plant development. Therefore, ABA catabolism plays a crucial role in plant growth and defense responses.

[0180] The abscisic acid content was determined using a kit (Jiangsu Enzyme-Linked Immunosorbent Assay Co., Ltd., Nanjing, China). The total RNA extraction and cDNA synthesis methods were the same as those used in Example 2. The primer sequences for broccoli abscisic acid metabolism-related genes are shown in Table 10.

[0181] Table 10 Primer sequences for genes related to broccoli abscisic acid metabolism

[0182]

[0183] like Figure 24 As shown, the ABA content of broccoli in the riboflavin-treated group gradually increased during the first 3 days of storage and then decreased at the end of storage; similarly, the ABA content of the control group gradually increased during the first 3 days of storage, decreased on the 4th day, and then increased on the last day of storage. The ABA content of broccoli in the riboflavin-treated group was lower than that in the control group throughout the entire storage process. Figure 25 As shown, the transcription levels of BoAAO1, BoNCED2, and BoNCED3 in the untreated broccoli gradually increased during storage; while the expression of BoABA2 in the same group of broccoli was inhibited by the riboflavin-treated group during storage, except for days 2 and 5; in addition, the riboflavin-treated group also inhibited the BoABF4 gene; the expression level of BoCYP707A2 was higher than that of the control group throughout the storage of broccoli; except for day 1 of storage, the transcription level of BoCYP707A3 in the riboflavin-treated group was significantly higher than that in the control group.

[0184] Mounting evidence supports the role of ABA in plants. In many plant species, endogenous ABA levels increase during leaf senescence, while exogenous ABA application can induce SAG expression and accelerate leaf yellowing and senescence. ABF has been identified as a putative transcriptional regulator of NYE1, which has been identified as a key regulator of Chl degradation during green organ maturation and senescence in Arabidopsis. NCED is a key rate-limiting enzyme in ABA synthesis. In this study, riboflavin treatment significantly reduced endogenous ABA levels in broccoli and significantly increased the transcriptional levels of the ABA-related degradation gene BoCYP707A2 / 3, while downregulating ABA synthesis genes BoAAO1, BoNCED2 / 3, and positive response signals BoABA and BoABF4, thereby maintaining chlorophyll content and inhibiting carotenoid synthesis.

[0185] In the riboflavin-treated group, the contents of GA3 and 6-BA significantly increased and peaked on day 2 of storage, while the concentrations of MT, IAA, and BR showed the highest accumulation levels on day 3. Notably, the synthesis of ethylene and ABA was significantly inhibited on day 3, with their concentrations significantly lower than those in the control group. These temporal dynamics indicate that days 2 and 3 of storage are key time points for postharvest physiological regulation of broccoli. Further research suggests that riboflavin may trigger a cascade regulatory effect by activating the biosynthetic pathways of GA3 and 6-BA: on the one hand, it positively regulates the synthesis and accumulation of MT, IAA, and BR, and on the other hand, it inhibits the biosynthetic pathways of ethylene and ABA through antagonism. This synergistic and antagonistic relationship of the hormone network suggests that GA3 and 6-BA may act as core signaling nodes, delaying the postharvest senescence process of broccoli by regulating the dynamic balance of downstream hormones. Specifically, the early peaks of GA3 and 6-BA may inhibit ethylene and ABA signaling by activating the antioxidant system or regulating the expression of senescence-related genes (such as ethylene synthesis genes); while the subsequent accumulation of MT, IAA, and BR may jointly maintain tissue physiological homeostasis through mechanisms such as synergistically enhancing cell membrane stability and delaying chlorophyll degradation. This study provides new experimental evidence for elucidating the riboflavin-mediated plant hormone interaction network and its mechanism of action in postharvest preservation, and lays a theoretical foundation for developing hormone-regulated fruit and vegetable storage technologies.

[0186] In summary, exogenous riboflavin treatment significantly regulates the levels of endogenous MT, IAA, GA3, ethylene, 6-BA, BR, and ABA in broccoli during storage. The results indicate that riboflavin treatment affects the levels of endogenous plant hormones by regulating the metabolic genes of MT, IAA, GA3, 6-BA, BR, and ABA. Riboflavin can interact with these hormones, influencing their biosynthesis and signal transduction, ultimately synergistically extending the post-harvest shelf life of broccoli.

[0187] Example 6: Effect of riboflavin treatment on antioxidants in broccoli during storage

[0188] 1. Effects of riboflavin on ascorbic acid levels in broccoli during storage

[0189] L-ascorbic acid (AsA) is better known as vitamin C. In plants and animals, AsA is an important antioxidant and cofactor for several enzymes, playing a key role in mitigating ROS effects through enzymatic and non-enzymatic detoxification. In plants, AsA is involved in photosynthesis, cell division, and cell differentiation, and is a key molecule in the regulation of growth and development. It also participates in plant signaling responses and stress adaptation pathways. The biosynthesis of AsA in vascular plants and algae occurs via the Smirnoff-Wheeler pathway. However, alternative synthetic routes, namely the galacturonic acid, L-gulose, and inositol pathways, are thought to contribute to maintaining the AsA pool. In plants, AsA is synthesized in the inner mitochondrial membrane space and is mainly distributed in the cytoplasm, with approximately 5% transported to the apoplast. The apoplast portion of AsA is crucial in oxidative stress signaling; its redox buffering capacity is attributed to the AsA pool, which is the first line of defense against various stresses. Furthermore, the content of AsA in the apoplast affects hormone homeostasis, growth, mitogen-activated protein kinase signaling cascades, and antioxidant enzyme activity, and plays an important role in the perception of plant physiological and biochemical responses under normal and abiotic stress conditions. As mentioned above, AsA has been described as a cofactor and co-substrate in the biosynthesis of enzymes and hormones such as ethylene and GA3. Tóth et al. pointed out that AsA is a determinant of the defense mechanisms of photosynthetic tissue against oxidative stress.

[0190] The method for detecting AsA content is as follows: Approximately 0.02 g of frozen sample ground in liquid nitrogen was extracted with 1 mL of 5% (w / v) trichloroacetic acid (TCA), vortexed for 40 s, and centrifuged at 12000 × g for 15 min at 4℃ to obtain the supernatant for AsA determination. The sample was centrifuged at 4℃, and 0.2 mL of the supernatant was taken and added sequentially to 0.1 mL of 0.4% phosphate ethanol solution, 0.2 mL of 0.5% phenanthroline ethanol solution, and 0.1 mL of 0.03% FeCl3 ethanol solution. After thorough mixing, the mixture was reacted at 30℃ for 60 min, and then the absorbance was measured at a wavelength of 534 nm. The AsA content in the sample was calculated according to the standard curve.

[0191] like Figure 26As shown, in the initial stage of storage, the total AsA and reduced AsA contents in broccoli exhibited a trend of first decreasing and then increasing. With the continuation of storage time, the contents of these two AsA types generally showed an initial decreasing trend, followed by a gradual increase in the levels of total AsA and reduced AsA in the riboflavin-treated group, reaching their maximum on the 4th day of storage. However, with further extension of storage time, the total AsA and reduced AsA contents in the riboflavin-treated broccoli began to decrease again until the end of storage, although the loss of total AsA and reduced AsA contents at the end of storage was relatively small compared to the initial stage. Furthermore, the total AsA and reduced AsA contents in the control group of broccoli fluctuated significantly during storage and decreased overall, indicating a significant loss. Throughout the entire storage period, the AsA content in the riboflavin-treated group of broccoli was consistently higher than that in the untreated control group.

[0192] The methods for total RNA extraction and cDNA synthesis were the same as those for real-time quantitative PCR in Example 2. Primer sequences for broccoli ascorbic acid metabolism-related genes are shown in Table 11.

[0193] Table 11 Primer sequences of genes related to ascorbic acid metabolism in broccoli

[0194]

[0195] like Figure 27 As shown, during broccoli storage, the expression levels of BoPGI, BoPMM, BoGGP, BoAO, and BoAPX1 exhibited a trend of first increasing and then decreasing, while the expression levels of BoPMI and BoMIOX gradually increased with prolonged storage time. Riboflavin promoted the transcriptional levels of AsA synthesis-related genes BoPGI, BoPMI, BoPMM, BoMIOX, and BoGGP during storage, while downregulating the expression of AsA degradation-related gene BoAO. In the first 4 days of storage, the transcript abundance of BoPGI and BoPMM increased, but then rapidly decreased. In riboflavin-treated broccoli, the expression level of BoAO was significantly downregulated from day 3 to day 5. Furthermore, under riboflavin treatment, the BoAPX1 content was consistently higher than that in the blank control group. BoAPX1, as a core enzyme in the ascorbic acid-glutathione cycle of the plant's antioxidant system, primarily functions to catalyze the oxidation of synthesized AsA to dehydroascorbic acid (MDHA), followed by AsA regeneration via a glutathione-dependent reduction pathway. This process scavenges intracellular reactive oxygen species (ROS) and prevents oxidative damage. The consistently higher BoAPX1 expression compared to the control suggests that riboflavin treatment enhances the plant's antioxidant defense capabilities.

[0196] 2. Effects of riboflavin on glutathione levels in broccoli during storage

[0197] Glutathione (GSH) is a ubiquitous, abundant, and indispensable tripeptide found in most organisms, including plants. It plays a crucial role in plant systems as an antioxidant, redox buffer, and detoxifier. Its importance extends beyond basic physiological processes, including responses to biotic and abiotic stresses, cell signaling, and the regulation of gene expression. Plants encounter various environmental stresses such as drought, salinity, heavy metals, and pathogens. GSH plays a key role in alleviating these stresses by detoxifying ROS, repairing damaged proteins, and regulating stress response signaling pathways. Under stress conditions, plants produce high levels of reactive oxygen species (ROS), leading to oxidative stress. GSH effectively scavenge these ROS to enhance resistance to adversity. It also regenerates other antioxidants, such as AsA, through the AsA-GSH cycle. This cycle is crucial for hydrogen peroxide and maintaining cellular redox homeostasis.

[0198] GSH and oxidized form (GSSH) were determined using a GSH and GSSG kit (Solecom Technology Co., Ltd., Ningbo, China).

[0199] like Figure 28 As shown, the contents of GSH and GSSG in untreated broccoli underwent a typical dynamic change during natural storage. Initially, the contents of GSH and GSSG in broccoli tissues showed a gradual upward trend with the extension of storage time. The contents of both GSH and GSSG reached their peak on day 4 of storage. Subsequently, with further extension of storage time, the contents of GSH and GSSG in broccoli tissues began to gradually decrease. Comparing riboflavin-treated and untreated broccoli, the effect of riboflavin treatment on GSH content was clearly observed. The results indicate that riboflavin treatment has a significant effect on the GSH content of broccoli. By upregulating the contents of GSH and GSSG, it provides an effective antioxidant defense mechanism for broccoli, helping to extend its shelf life and maintain its quality.

[0200] The methods for total RNA extraction and cDNA synthesis were the same as those for real-time quantitative PCR in Example 2. Primer sequences for glutathione metabolism-related genes in broccoli are shown in Table 12.

[0201] Table 12 Primer sequences for glutathione metabolism-related genes in broccoli

[0202]

[0203] like Figure 29As shown, the transcript levels of genes such as BoGCS1, BoGS1, BoGS3, and BoGPX2 all initially showed a trend of rising and then gradually decreasing to a stable state. When broccoli was treated with riboflavin, the expression of these three genes, BoGCS1, BoGPX2, and BoGGT1, was significantly upregulated throughout the storage period. Although BoGGT is involved in glutathione degradation, its upregulation may promote the glutathione cycle, recovering precursors such as cysteine ​​through degradation products, providing raw materials for rapid resynthesis, thus supporting the rapid turnover of the glutathione pool rather than simple consumption. BoGPX2, as a glutathione peroxidase, directly utilizes glutathione to reduce peroxides and is an antioxidant executor. Its upregulation indicates an increased demand for antioxidants, thereby increasing the consumption of reduced glutathione. Both glutathione and riboflavin were synergistically upregulated under riboflavin treatment, forming an adaptive response together with the upregulation of the synthetic gene BoGCS1: accelerating the synthesis-utilization-recycling cycle of glutathione, improving its turnover efficiency and antioxidant flux, and maintaining redox homeostasis. During days 3–5 of storage, the transcript levels of BoGS1 / 3 were higher, indicating that the expression of the BoGS1 / 3 gene was particularly active during this period, and it may be involved in the key physiological response of broccoli to riboflavin treatment.

[0204] 3. Effects of riboflavin on glucosinolates during the storage period of broccoli

[0205] Sulfur is an essential and widely used nutrient element in organisms, including plants. Sulfur-containing amino acids, methionine and cysteine, are components of almost all proteins, making sulfur a key component of protoplasm. Furthermore, sulfur is an important component of coenzyme A, thiamine, and biotin, playing a crucial role in the metabolism of carbohydrates, proteins, and lipids. Disulfide bonds in proteins play a structural and regulatory role in redox control. In summary, sulfur-containing metabolites play a vital role in various physiological processes. Studies have found that insufficient sulfur supply due to a reduction in essential sulfur-containing metabolites leads to a severe decline in growth, development, and stress tolerance. Therefore, ensuring an adequate sulfur supply is crucial for crop yield and quality.

[0206] The method for detecting glucosinolate content is as follows: Weigh 0.5g of frozen broccoli tissue (florets) into a 15mL centrifuge tube. Add 4mL of 70% methanol and 200μL of myrosinase (5×10⁻⁶). -3 mol L -1 Stir for 1 min, then incubate in a 70℃ water bath for 30 min. Extract the mixture using an ultrasonic bath for 15 min, then centrifuge at 3000×g for 15 min. Collect the supernatant, evaporate at 35℃, resuspend in 1 mL of ultrapure water, and filter through a 0.22 μm organic phase filter.

[0207] Chromatographic separation was performed using a Shimadzu LC-30A high-performance liquid chromatography system (Japan), equipped with a Waters ACQUITY UPLCBEH C18 column (2.1 × 10⁻⁶). -3 m×0.1m, 1.7×10 -6 (m)(Milford, Massachusetts, USA). Gradient UPLC elution was performed using 0.2% (v / v) formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B. The elution process was as follows: 0–0.5 min, 95% A; 3 min, 60% A; 3.5–4 min, 5% A; 4.5–6 min, 95% A, at a flow rate of 2 L / min. -1 ×10 -4 The injection volume was 2×10 -6 L. The experiment was performed using a triple quadrupole mass spectrometer (LCMS-8050, Shimadzu, Japan). Based on the relative response factor, the internal standard method was used to quantify the experiment, calculate the final glucosinolate content, mass spectrometry parameters, and relative response factor, and express them on a fresh weight (FW) basis.

[0208] like Figure 30 As shown, riboflavin treatment significantly promoted the accumulation of total glucosinolates and their components in broccoli throughout the storage period, and their content levels were consistently significantly higher than those in the control group. The total glucosinolate content peaked on day 3 of storage, indicating that riboflavin treatment had the most significant promoting effect on glucosinolate synthesis in the early stages of storage. Regarding the distribution of glucosinolate components, the trends of GRA and NGBS were basically consistent with those of total glucosinolates, with GRA being the main component of total glucosinolates, ranging from approximately 0.5 to 2.0 mmol / kg. -1 It accounts for the vast majority of total glucosinolates; while the content of NGBS is relatively low, ranging from approximately 0.02 to 0.13 mmol / kg. -1 The proportion of GBS in the glucosinolate fraction was significantly lower than that in GRA. GBS in the glucosinolate fraction showed a trend of first decreasing and then increasing on day 1 of storage, with a content ranging from approximately 0.04 to 0.36 mmol / kg. -1 .

[0209] The methods for total RNA extraction and cDNA synthesis were the same as those for real-time quantitative PCR in Example 2. Primer sequences for broccoli glucosinolate metabolism-related genes are shown in Table 13.

[0210] Table 13 Primer sequences for broccoli glucosinolate metabolism-related genes

[0211]

[0212] like Figure 31As shown, the expression level of the BoMYB28 gene gradually decreased during the storage of broccoli, only rebounding on the 5th day of storage. Compared with the control group without riboflavin treatment, the BoMYB28 expression level in the riboflavin-treated group remained at a higher level throughout the storage period, especially on the 2nd and 3rd days of storage, where the BoMYB28 expression level in the riboflavin-treated group showed a highly significant difference compared with the control group. In addition, riboflavin treatment also significantly affected BoCYP83A1, BoST5b, and BoAOP2, and the expression levels of these three genes were significantly upregulated at different time points (1–4 days, 1–4 days, and 2–4 days, respectively), especially BoFMO. GS-OX1 Throughout the storage process, riboflavin treatment continuously upregulated BoFMO. GS-OX1 Gene expression levels. Conversely, BoESP gene expression was suppressed by riboflavin treatment during storage. Furthermore, the expression changes of BoMYB51 after riboflavin treatment were complex. Specifically, only on day 2 of storage did the BoMYB51 expression level in the riboflavin-treated group show a significant difference compared to the control group; at other time points, the expression differences between the two groups were not significant. This indicates that the response of BoMYB51 to riboflavin treatment is time-specific and complex.

[0213] 4. Effects of riboflavin on endogenous vitamin B2 during broccoli storage

[0214] Vitamin B2 (Riboflavin, VB2), a natural antioxidant widely found in various fresh agricultural products, is a valuable source of vitamins, minerals, and natural antioxidants such as carotenoids and flavonoids. Specifically, VB2 plays a crucial role in the metabolism of carbohydrates, fats, and proteins, helping to convert these nutrients into energy needed by the body. Furthermore, VB2 participates in hemoglobin synthesis and cellular redox reactions, essential for maintaining normal physiological functions. VB2 is vital for the health of skin and mucous membranes; a deficiency can lead to inflammation in the mouth, lips, and tongue, such as angular cheilitis, cheilitis, and glossitis. VB2 also helps maintain normal eye function and is beneficial for vision health. Therefore, vegetables can be consumed to supplement VB2. In summary, VB2 is a vital nutrient for human health, and proper supplementation can maintain physiological functions and a healthy state.

[0215] The detection method for VB2 is as follows: Accurately weigh 0.5 g of sample, add 500 μL of 80% methanol extract containing 1% acetic acid, vortex mix for 40 s, and then sonicate for 15 min. After sonication, centrifuge at 12000 × g for 10 min, collect the supernatant, and filter it through a 0.22 μm aqueous filter membrane. Quantitative analysis is performed using the external standard method.

[0216] Chromatographic conditions: A Waters HSST3 column (50 × 2.1 mm, 1.8 μm) was used. Mobile phase A was ultrapure water containing 0.1% acetic acid, and mobile phase B was acetonitrile containing 0.1% acetic acid. The flow rate was set at 0.3 mL / min. -1 The column temperature was maintained at 40℃, and the injection volume was 2 μL. The elution gradient was as follows: 0.0–1.0 min, water / acetonitrile (90:10, v / v); 1.0–5.0 min, the water / acetonitrile ratio linearly changed from 90:10 to 10:90; 5.0–7.0 min, water / acetonitrile (10:90, v / v) was maintained; 7.1–9.0 min, the ratio was restored to water / acetonitrile (90:10, v / v). Mass spectrometry was performed using a Thermo QExactive high-resolution mass spectrometer. The calculation formula is as follows:

[0217] Content of each component in solid sample (ng g) -1 )=(C×V×F) / M;

[0218] like Figure 32 As shown, the VB2 content in broccoli tissues gradually decreased with increasing storage days. However, the decline in VB2 content was mitigated after riboflavin treatment. Throughout the storage period, the endogenous VB2 content of riboflavin-treated broccoli remained higher than that of the untreated control group, especially on days 2–5 of storage, where the difference was significant. In other words, riboflavin treatment effectively slowed down the degradation rate of important nutrients such as VB2 in broccoli during room temperature storage, thereby maintaining the nutritional value of broccoli.

[0219] The methods for total RNA extraction and cDNA synthesis were the same as those for real-time quantitative PCR in Example 2. Primer sequences for endogenous vitamin B2 metabolism-related genes in broccoli are shown in Table 14.

[0220] Table 14 Primer sequences for genes related to endogenous vitamin B2 metabolism in broccoli

[0221]

[0222] like Figure 33 As shown, during the storage of broccoli, the expression levels of the genes BoGCH1, BoPYRD, BoPYRR, BoPYRR2, BoRIB5, and BoRIB5.1 initially increased and then decreased. The results indicated that riboflavin treatment significantly promoted the transcriptional levels of these genes, and these effects were significantly different from the control group during days 2–5 of storage.

[0223] In addition, this invention also explored the effects of riboflavin treatment on phenolic antioxidants and antioxidant enzymes in postharvest broccoli, but riboflavin had no significant effect on the content of phenolic antioxidants and antioxidant enzymes in broccoli.

[0224] In summary, riboflavin treatment reduced the loss of nutritional quality in broccoli. Riboflavin increased AsA content during storage by regulating AsA-related genes; upregulated GSH content during storage by regulating GSH-related genes; maintained glucosinolate content in broccoli during room temperature storage by upregulating the expression of glucosinolate synthesis genes and inhibiting the expression of hydrolysis genes; and delayed the loss of vitamin B2 content in postharvest broccoli by upregulating the expression of vitamin B2 synthesis genes.

[0225] The application of this invention is not limited thereto. It can be extended to other applications, such as those related to environmental protection. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of this invention; therefore, the scope of protection of this invention should be determined by the scope defined in the claims.

Claims

1. The use of riboflavin in the preparation of formulations that delay the decline of antioxidants in postharvest broccoli.

2. The use as described in claim 1, characterized in that, The antioxidants in the broccoli include any one or more of ascorbic acid, glutathione, glucosinolates, and vitamin B2.

3. Use of riboflavin in the preparation of formulations that upregulate ascorbic acid-related synthesis genes or downregulate ascorbic acid-related degradation genes.

4. The use as described in claim 3, characterized in that, The ascorbic acid-related synthetic genes include any one or more of BoPGI, BoPMI, BoPMM, BoMIOX, and BoGGP, and the ascorbic acid-related degradation genes include BoAO.

5. Use of riboflavin in the preparation of formulations that upregulate glutathione-related synthetic genes.

6. The use as described in claim 5, characterized in that, The glutathione-related synthesis genes include any one or more of BoGCS1, BoGS1, and BoGS3.

7. Use of riboflavin in the preparation of formulations that upregulate glucosinolate-related synthetic genes or downregulate glucosinolate-related degradation genes.

8. The use as described in claim 7, characterized in that, The glucosinolate-related synthetic genes include BoMYB28, BoCYP83A1, BoST5b, and BoFMO. GS-OX1 The glucosinolate-related degradation gene includes BoESP, BoAOP2, BoMYB51, or any one or more of these genes.

9. Use of riboflavin in the preparation of formulations that upregulate vitamin B2-related synthetic genes.

10. The use as described in claim 9, wherein the vitamin B2-related synthetic genes include any one or more of BoGCH1, BoPYRD, BoPYRR, BoPYRR2, BoRIB5, and BoRIB5.1.

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