A method for storing and preserving alisma flower moss

Abstract

This invention provides a method for storing and preserving Alisma plantago-aquatica flower buds. A synergistic preservation system is constructed by preparing an inactivated bacterial solution using Pantotheca acuminata and Vinyasa vinifera, and adding aminoethoxyvinylglycine to the bacterial solution culture medium. In this method, the mixed bacterial agent forms a biological protective layer on the surface of the flower buds, while the bacterial solution acts as a carrier for aminoethoxyvinylglycine, achieving slow release and efficient penetration of the active ingredients, inhibiting ethylene synthesis from within. This technology combines the advantages of biological control and physiological regulation, is green and leaves no chemical residues, significantly maintains cell membrane integrity, effectively preserves the product's color, texture, and nutrition, and extends its shelf life, opening up new avenues for the preservation of green agricultural products.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product preservation and processing technology, specifically relating to a method for storing and preserving Alisma plantago-aquatica flowers. Background Technology

[0002] Alisma orientale (Sam.) Juzep. is a perennial aquatic herb belonging to the genus Alisma in the family Alismataceae. The pharmacopoeia lists the dried underground tubers as the medicinal part of Alisma orientale, while the above-ground parts, such as the flower stalks, tender stems, and leaves, are used as vegetables and are collectively known as the "Four Treasures of Jiyang" along with lotus seeds, water spinach (commonly known as water spinach in various regions), and fairy grass. Different parts of Alisma orientale (tubers, flower stalks, stems, and leaves) contain amino acids, proteins, and fats. The flower stalks have the highest amino acid and protein content, followed by the tubers, indicating that the flower stalks have high edible value. The tubers have the highest total polysaccharide and total flavonoid content, followed by the flower stalks, while the flower stalks have the highest total polyphenol content, suggesting that they may have some medicinal value. The flower stalks of Alisma orientale have both edible and medicinal value, showing great development potential and warranting further research.

[0003] However, as a climacteric organ, Alisma plantago-aquatica flower buds exhibit extremely active postharvest metabolism, making them highly susceptible to yellowing, wilting, rotting, and quality deterioration during storage and transportation, leading to a rapid loss of commercial value. Currently, the preservation of such fresh-cut vegetables or flowers primarily relies on two main categories of conventional techniques: physical and chemical methods.

[0004] In physical preservation, low-temperature storage (such as 4°C refrigeration) is the most widely used basic method. It slows down aging by inhibiting tissue respiration and microbial activity. However, this technology has significant limitations: firstly, it can only delay, not fundamentally inhibit, metabolic processes, thus limiting its preservation effect; secondly, quality degradation accelerates when ambient temperature fluctuates or the product is removed from the low-temperature environment; more importantly, low temperatures themselves cannot effectively prevent programmed aging driven by endogenous hormones (such as ethylene), offering limited protection for cell membrane integrity. In chemical preservation, direct fumigation with ethylene receptor inhibitors such as 1-methylcyclopropene (1-MCP) or the application of chemical preservatives (such as chlorine dioxide and imidacloprid) are common synergistic measures. While 1-MCP can effectively block the effects of ethylene, its effectiveness is significantly affected by the enclosed environment, concentration, and processing time, and it is powerless against the aging process that has already begun. More importantly, although chemical preservatives can inhibit microorganisms, they are prone to leaving residues on the product surface, raising consumer concerns about food safety. They may also affect the original natural flavor of the product, which contradicts the current trend of green and healthy consumption.

[0005] The core reason why these existing technologies face difficulties is that they mostly employ a single mechanism of action, focusing either solely on external microbial control or targeting only a specific internal physiological process, failing to construct a synergistic, comprehensive protective system from the surface to the core. Maintaining the post-harvest quality of Alisma plantago-aquatica flower buds is essentially a dual process involving both microbial spoilage and its own physiological aging. Existing technologies often fall short in this regard: low temperatures and chemical preservatives primarily inhibit external microorganisms but are insufficient in delaying internal cell aging; while 1-MCP can inhibit ethylene, it does not directly contribute to inhibiting spoilage-causing microorganisms. These technological deficiencies make it difficult to achieve a qualitative breakthrough in preservation effects.

[0006] Improving the post-harvest preservation of Alisma plantago-aquatica flower buds is crucial for enhancing its economic value and promoting the standardized development of the industry. Post-harvest losses not only directly lead to economic losses but also hinder the commercialization and branding of this specialty agricultural product. Therefore, developing a new preservation technology that can simultaneously resist microbial spoilage and internal physiological aging while ensuring food safety is the core breakthrough for the industry's development.

[0007] In this research process, selecting a sensitive, objective evaluation standard that can reveal the underlying mechanisms is crucial. Traditional evaluations of preservation effects often rely on macroscopic indicators such as color and firmness. While these indicators are intuitive, they are often subjective and typically only become apparent after irreversible deterioration in quality, failing to provide early warning and mechanistic analysis. A suitable core evaluation standard, like a precise "diagnostic tool," must be able to directly and quantitatively reflect the health status of the product at the cellular level. For example, cell membrane integrity is a core indicator for measuring cellular aging and resilience; its degree of integrity directly determines the product's preservation potential, making research results more convincing and innovative. Summary of the Invention

[0008] This invention discloses a method for storing and preserving Alisma plantago-aquatica flower buds. This method, through a series of synergistic processes, effectively extends the shelf life of Alisma plantago-aquatica flower buds while maintaining their commercial quality. The method includes the following steps:

[0009] S1, Screening and Grading

[0010] First, the Alisma plantago-aquatica flower stalks are screened, and all products that do not meet the basic requirements are eliminated. The basic requirements are: the flower stalks must be fresh, clean, odorless, intact, and free from rot, mold, wilting, pithy core, insect damage, and obvious mechanical damage; their maturity must be moderate. Then, the flower stalks that meet the basic requirements are classified into three grades—Special Grade, Grade 1, and Grade 2—based on their texture, color, uniformity of stem, and degree of mechanical damage.

[0011] The specific criteria for the classification are as follows:

[0012] Premium grade: crisp and tender texture, bright green color, uniform thickness and length of moss stems, no mechanical damage, and good integrity;

[0013] Grade 1: The texture is crisp and tender, the color is green, the moss stems are of uniform thickness and length, there is no mechanical damage, and the integrity is good;

[0014] Level 2: The texture is relatively crisp and tender, the color is still green, the moss stems are relatively uniform in thickness and length, there are a few minor mechanical damages, and the integrity is generally average.

[0015] Meanwhile, the following quality errors are allowed during grading:

[0016] Among the premium grade products, 5% may not meet the premium grade requirements, but must meet the first grade requirements;

[0017] Of the Grade 1 products, 10% are allowed to fail to meet the Grade 1 quality requirements, but must meet the Grade 2 requirements.

[0018] Of the grade II products, 10% may not meet the grade II requirements, but must meet the basic requirements.

[0019] S2, Preprocessing

[0020] Pre-treatment is performed on the graded Alisma plantago-aquatica flower stalks. Yellow leaves, diseased leaves, debris, and residual soil are manually removed from the flower stalks, and then the surface is rinsed with running water at a temperature of 10-15℃. After rinsing, the flower stalks are quickly sent to a pre-cooling device for pre-cooling. The operating temperature for pre-cooling is 0-5℃, and the goal is to reduce the core temperature of the flower stalks to 2-6℃ within 1-2 hours.

[0021] S3. Preservation treatment of fermentation products:

[0022] The pretreated flower buds were soaked in the inactivated microbial fermentation broth.

[0023] The preparation and inactivation process of the microbial fermentation broth is as follows:

[0024] a. Prepare a liquid culture medium containing 8-12g peptone, 4-6g yeast extract, 8-12g glucose, 4-6g sodium chloride, 0.8-1.2g dipotassium hydrogen phosphate and 0.15-0.25g magnesium sulfate per liter, and adjust the pH of the culture medium to 7.0±0.2.

[0025] b. Add aminoethoxyvinylglycine to the liquid culture medium to achieve a final working concentration of 28-36 mg / L in the culture medium.

[0026] c. Inoculate *Pantotheca cumulus* and *Venerendica venerealis* separately into the above-mentioned liquid culture medium containing aminoethoxyvinylglycine, and culture with shaking at 30±2℃ and 150±10 rpm for 24-48 hours until their respective bacterial concentrations reach 10⁻⁶. 8 -10 9 CFU / mL.

[0027] d. Inactivate the clump-forming Pantotheca acuminata culture from step c with the Vignelandia diffusa culture, and then mix the two inactivated cultures at a volume ratio of 1:1.

[0028] In step d, the inactivation treatment is thermal inactivation, and the conditions are to keep it in a water bath at 70-80℃ for 20-30 minutes.

[0029] Before soaking, add 0.01% Tween20 by volume to the inactivated microbial fermentation broth, and then dilute it 10-100 times with sterile distilled water to obtain the treatment solution.

[0030] Furthermore, the inactivated bacterial solution used in premium grade products is diluted 60-100 times with sterile distilled water, the inactivated bacterial solution used in first grade products is diluted 30-60 times with sterile distilled water, and the inactivated bacterial solution used in second grade products is diluted 10-30 times with sterile distilled water.

[0031] The specific conditions for the soaking treatment are as follows: the temperature of the treatment solution is controlled at 10-15℃, the soaking time is 8-12 minutes, and the standard dosage of the treatment solution is 4-6 kg of Alisma plantago-aquatica flower buds per 1 liter of treatment solution. The treated Alisma plantago-aquatica flower buds can be packaged and stored together with the treatment solution or drained of the surface treatment solution and stored in a plastic bag.

[0032] S4. Low-temperature storage

[0033] Store in a low-temperature environment at 4±1℃.

[0034] The advantages and beneficial effects of this invention are as follows:

[0035] 1. The beneficial effects of this invention lie in providing a green, safe, and efficient comprehensive preservation method for Alisma plantago-aquatica flower buds. Compared with conventional technologies that rely on a single action, this invention utilizes an inactivated fermentation broth of Pantotheca acuminata and Nitrostrophus venereum. This fermentation broth contains various metabolites produced by the bacteria during their growth, as well as adsorbed or bound aminoethoxyvinylglycine. The inactivated bacterial cells and their metabolites can form a uniform protective film on the surface of the flower buds, reducing moisture evaporation through physical covering and, to some extent, hindering the infection of putrefactive microorganisms; simultaneously, the intracellular components and their metabolites released after bacterial lysis can directly inhibit the synthesis and action of ethylene. More importantly, the aminoethoxyvinylglycine added during the fermentation stage is enriched or evenly distributed in the fermentation broth by the bacteria, and can adhere to and be slowly released during the treatment of the flower buds, forming a continuous "miniature drug reservoir" that efficiently inhibits ethylene synthesis from within. This method of utilizing inactivated fermentation products achieves a synergistic effect of physical barrier, component preservation, and internal anti-aging. This technology eliminates the risk of live microbial growth and leaves no harmful chemical residues, meeting the requirements for green agricultural products. It can fully maintain the color, texture, and flavor of the flower buds. Through the sensitive indicator of relative conductivity, it has been confirmed that this invention can significantly maintain cell membrane integrity, effectively delaying aging at the cellular level, ultimately achieving a substantial extension of storage life and maximizing commercial value.

[0036] 2. Differentiated treatment is applied based on the quality and storage tolerance of different grades of flower buds. Premium grade buds, with their superior quality and minimal damage, require only a lower concentration of bacterial solution to form effective protection, avoiding over-treatment and resource waste. Second-grade buds, due to minor mechanical damage, are more susceptible to microbial contamination and require a higher concentration of bacterial solution to form a denser protective film on their surface, ensuring antibacterial effects. This tiered treatment method achieves efficient and precise utilization of resources, achieving optimal cost while maintaining overall preservation effectiveness. Compared to conventional technologies such as physical preservation (e.g., low-temperature storage) or chemical preservation (e.g., 1-MCP fumigation, application of chemical preservatives), the inactivated bacterial agent fermentation product preservation technology employed in this invention represents a greener, safer, and more controllable development direction.

[0037] 3. First, this invention creates a multi-layered synergistic preservation mechanism. Inactivated bacterial cells and their extracellular polysaccharides and other metabolites can form a biophysical protective film on the surface of the flower stalks, effectively slowing down water evaporation (anti-wilt) and acting as a physical barrier to prevent the contact and colonization of external putrefactive microorganisms. Simultaneously, various metabolically active components in the fermentation broth, such as enzymes, antimicrobial peptides, and plant hormone analogs, may directly inhibit the growth of putrefactive bacteria or induce the flower stalks' own defense system, enhancing their resistance. The aminoethoxyvinylglycine added during fermentation is effectively integrated into the fermentation products and can continue to act on the flower stalk tissue after treatment, inhibiting ethylene biosynthesis from within. This synergistic system, consisting of "physical coverage," "metabolic component antibacterial / resistance induction," and "internal physiological regulation," is far more effective than single-mechanism preservation methods.

[0038] 4. Secondly, this technology is safe and stable. Using inactivated bacterial solutions completely avoids the biosafety uncertainties that live bacteria may present, as well as the fluctuations in effectiveness caused by differences in colonization capabilities on different product surfaces. No active microorganisms proliferate on the treated product surface. Furthermore, the components in the fermentation broth originate from natural microbial metabolism, and after degradation, there are no toxic or harmful residues, meeting the safety production requirements for green agricultural products.

[0039] 5. Finally, this technology offers a more comprehensive and lasting effect. It utilizes the rich collection of metabolites accumulated by the microorganisms during fermentation. These components work together to not only inhibit spoilage but also synergistically slow down the physiological aging process of the flower stalks through multiple pathways, including delaying moisture loss, inhibiting ethylene production, and maintaining cell membrane stability. This allows the treated flower stalks to better retain their natural color, crisp texture, and flavor, thus maintaining a near-fresh commercial value even in the later stages of storage.

[0040] 6. The key to this invention lies in the delivery of aminoethoxyvinylglycine via an inactivated fermentation broth. Traditional methods of treating with aminoethoxyvinylglycine involve direct spraying, whose effectiveness is easily affected by rinsing and is difficult to sustain. In this invention, aminoethoxyvinylglycine is added during the bacterial culture stage, allowing it to be adsorbed, absorbed, or bound to metabolites by the bacteria. When this inactivated fermentation broth containing aminoethoxyvinylglycine is used to treat flower stalks, the components in the fermentation broth form a stable protective layer on the flower stalk surface. This protective layer acts like a "slow-release drug reservoir," allowing the aminoethoxyvinylglycine to penetrate the flower stalk tissue more persistently and slowly, thereby achieving sustained and efficient inhibition of ethylene synthesis.

[0041] 7. Therefore, this improvement brings about a synergistic effect from both the surface and the interior. The inactivated fermentation broth constructs a physical defense line on the outside, reducing water loss and blocking microorganisms, while its various active ingredients may play a role in inhibiting bacteria and inducing resistance; meanwhile, the aminoethoxyvinylglycine carried by the fermentation broth plays a role inside the flower stalks, delaying the aging process of the cells themselves. This multi-layered protection from the surface to the interior results in a significant synergistic effect in preservation, which can more fundamentally delay the physiological metabolism of the flower stalks, maintain their cell vitality and tissue structure, and ultimately achieve a significant extension of the storage period.

[0042] 8. Furthermore, subsequent relative conductivity testing directly reflects the integrity of the cell membrane. The fundamental cause of post-harvest vegetable quality deterioration is the disintegration of cell structure and loss of function, all of which begin with damage to the cell membrane. Under aging, stress, or microbial attack, the cell membrane transforms from a selectively permeable barrier into a porous, leaky structure, leading to the leakage of large amounts of electrolytes such as potassium and calcium ions from the cell. The measurement of relative conductivity precisely assesses the severity of cell membrane damage by quantifying the degree of this electrolyte leakage. The lower the measured relative conductivity value, the more intact the cell membrane structure is, and the better the cell's health. This indicator can provide early, sensitive, and objective warnings of quality changes. Compared to sensory evaluations (such as color and shape) or conventional physicochemical indicators (such as firmness and weight loss rate), which typically show significant changes only in the later stages of quality deterioration, the disruption of cell membrane integrity is an early event in the entire aging process. Therefore, changes in relative conductivity can reflect the differences in the effectiveness of different preservation treatments earlier and more sensitively. The relative conductivity of the flower buds treated by this invention is significantly lower than that of other preservation processes, which shows that this invention can effectively delay the aging process at the most basic cellular level. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, the reagents and materials are commercially available. The *Pantotheca acuminata* strain XK-JZ-1089 was purchased from Shanghai Xuanke Biotechnology Co., Ltd.; the *Venerendaeda* strain bio-16308 was purchased from Beijing Bio-Biobio Biotechnology Co., Ltd.

[0044] First, in any specific implementation of this invention, the following two pre-processing steps must be performed:

[0045] (a) Screening and grading

[0046] The stored Alisma plantago-aquatica flower stalks were manually screened, and all products that did not meet the basic requirements were strictly rejected. The basic requirements are: the flower stalks are fresh, clean, and odorless; they are intact and free from any rot, mold, wilting, or pithiness; they are free from pests; they have no obvious mechanical damage; and they are at a moderate maturity.

[0047] Subsequently, flower stalks that meet the basic requirements are graded according to their texture, color, uniformity of stem, and degree of mechanical damage, with the specific standards as follows:

[0048] Premium grade: crisp and tender texture; bright green color; uniform thickness and length of stems; no mechanical damage and good integrity.

[0049] Grade 1: crisp and tender texture; greenish color; uniform thickness and length of moss stems; no mechanical damage and good integrity.

[0050] Grade II: The texture is relatively crisp and tender; the color is still green; the thickness and length of the moss stems are relatively uniform; a small amount of minor mechanical damage is allowed, and the integrity is generally good.

[0051] During the grading process, the following quality tolerances are permitted: 5% of the products in the premium grade are allowed to fail to meet the premium grade requirements but must meet the first grade requirements; 10% of the products in the first grade are allowed to fail to meet the first grade requirements but must meet the second grade requirements; and 10% of the products in the second grade are allowed to fail to meet the second grade requirements but must meet the basic requirements.

[0052] (ii) Pretreatment

[0053] The graded Alisma plantago-aquatica flower stalks undergo pretreatment. Yellow leaves, diseased leaves, debris, and residual soil are manually removed from the stalks. Then, the surface of the stalks is rinsed with running water at 10°C to remove attached dust, impurities, and microorganisms. After rinsing, the stalks are quickly transferred to a pre-cooling device and pre-cooled at an ambient temperature of 5°C, with the goal of reducing the core temperature of the stalks to 6°C within one hour. After pre-cooling, the surface moisture of the stalks is drained.

[0054] Only after completing the above steps can subsequent bacterial solution preservation and storage be carried out.

[0055] Example 1

[0056] Preparation of bacterial culture: Prepare a liquid culture medium containing 10g peptone, 5g yeast extract, 10g glucose, 5g sodium chloride, 1.0g dipotassium hydrogen phosphate, and 0.2g magnesium sulfate per liter, adjusting the pH to 7.0. After autoclaving at 121℃ for 15 minutes, cool to below 50℃ and aseptically add aminoethoxyvinylglycine to a final concentration of 32mg / L. Inoculate *Pantotheca acuminata* and *Azotobacter venereum* at an inoculum rate of 1% (v / v) into this medium and incubate at 30℃ and 150rpm for 36 hours until the bacterial concentration reaches 5×10⁻⁶. 8 CFU / mL. The two bacterial solutions were inactivated by treating them separately in a 75°C water bath for 25 minutes. The two inactivated bacterial solutions were then mixed at a 1:1 volume ratio.

[0057] Preservation treatment: Add 0.01% (v / v) of Tween 20 to the above-mentioned inactivated bacterial solution, then dilute it 80 times with sterile distilled water to obtain a treatment solution. Immerse the pretreated premium Alisma plantago-aquatica flower buds in this treatment solution, keeping the temperature of the treatment solution at 12℃, and soak for 10 minutes. The dosage of the treatment solution is 1 liter per 5 kg of flower buds.

[0058] Storage: Store in a cold storage at 4℃.

[0059] Example 2

[0060] Preparation of bacterial culture: Prepare a liquid culture medium containing 10g peptone, 5g yeast extract, 10g glucose, 5g sodium chloride, 1.0g dipotassium hydrogen phosphate, and 0.2g magnesium sulfate per liter, adjusting the pH to 7.0. After autoclaving at 121℃ for 15 minutes, cool to below 50℃, and aseptically add aminoethoxyvinylglycine to a final concentration of 28mg / L. Inoculate both bacterial strains at 1% (v / v) and incubate at 28℃ and 140rpm for 24 hours until the bacterial concentration reaches 1×10⁻⁶. 8 CFU / mL. The two bacterial solutions were inactivated by treating them separately in a 75°C water bath for 30 minutes. The two inactivated bacterial solutions were then mixed at a 1:1 volume ratio.

[0061] Preservation treatment: Add 0.01% (v / v) of Tween 20 to the above-mentioned inactivated bacterial solution, then dilute it 50 times with sterile distilled water to obtain a treatment solution. Immerse the pretreated Grade 1 Alisma plantago-aquatica flower buds in this treatment solution, keeping the temperature of the treatment solution at 10℃, and soak for 8 minutes. The dosage of the treatment solution is 1 liter per 6 kg of flower buds.

[0062] Storage: Store in a cold storage at 3℃.

[0063] Example 3

[0064] Preparation of bacterial culture: Prepare a liquid culture medium containing 10g peptone, 5g yeast extract, 10g glucose, 5g sodium chloride, 1.0g dipotassium hydrogen phosphate, and 0.2g magnesium sulfate per liter, adjusting the pH to 7.0. After autoclaving at 121℃ for 15 minutes, cool to below 50℃, and aseptically add aminoethoxyvinylglycine to a final concentration of 36mg / L. Inoculate both bacterial strains at 1% (v / v) and incubate at 32℃ and 160rpm for 48 hours until the bacterial concentration reaches 1×10⁻⁶. 9 CFU / mL. The two bacterial solutions were inactivated by treating them separately in a 75°C water bath for 25 minutes. The two inactivated bacterial solutions were then mixed at a 1:1 volume ratio.

[0065] Preservation treatment: Add 0.01% (v / v) of Tween 20 to the above-mentioned inactivated bacterial solution, then dilute it 20 times with sterile distilled water to obtain a treatment solution. Immerse the pretreated secondary Alisma plantago-aquatica flower buds in this treatment solution, keeping the temperature of the treatment solution at 15℃, and soak for 12 minutes. The amount of treatment solution used is 1 liter per 4 kg of flower buds.

[0066] Storage: Store in a cold storage at 5℃.

[0067] Comparative Example 1

[0068] Preservation treatment: Place the pre-cooled flower buds into a sealed fumigation chamber, use 1-MCP fumigant, prepare a concentration of 1μL / L according to the product instructions, and fumigate at room temperature for 24 hours.

[0069] Storage: After fumigation, pack the premium Alisma plantago-aquatica flower buds into a preservation bag and store them in a cold storage at 4℃.

[0070] Comparative Example 2

[0071] Preparation and treatment of bacterial culture: Prepare a liquid culture medium containing 10g peptone, 5g yeast extract, 10g glucose, 5g sodium chloride, 1.0g dipotassium hydrogen phosphate, and 0.2g magnesium sulfate per liter, adjusting the pH to 7.0. After autoclaving at 121℃ for 15 minutes, cool to below 50℃, and aseptically add aminoethoxyvinylglycine to a final concentration of 32mg / L. Inoculate the medium with Bacillus subtilis at an inoculum rate of 1% (v / v) and incubate at 30℃ and 150rpm for 36 hours until the bacterial concentration reaches 5×10⁻⁶. 8 CFU / mL. The above bacterial culture was inactivated by treating it in a 75°C water bath for 25 minutes.

[0072] Preservation treatment: Add 0.01% (v / v) of Tween 20 to the above-mentioned inactivated bacterial solution, then dilute it 80 times with sterile distilled water to obtain a treatment solution. Immerse the pretreated premium Alisma plantago-aquatica flower buds in this treatment solution, keeping the temperature of the treatment solution at 12℃, and soak for 10 minutes. The dosage of the treatment solution is 1 liter per 5 kg of flower buds.

[0073] Storage: Store in a cold storage at 4℃.

[0074] Comparative Example 3

[0075] Preparation and treatment of bacterial culture: Prepare a liquid culture medium containing 10g peptone, 5g yeast extract, 10g glucose, 5g sodium chloride, 1.0g dipotassium hydrogen phosphate, and 0.2g magnesium sulfate per liter, adjusting the pH to 7.0. After autoclaving at 121℃ for 15 minutes, cool to below 50℃, and aseptically add aminoethoxyvinylglycine to a final concentration of 32mg / L. Inoculate the culture medium with clumps of Pantothenia glutinosa at a 1% (v / v) inoculation rate and incubate at 30℃ and 150rpm for 36 hours until the bacterial concentration reaches 5×10⁻⁶. 8 CFU / mL. The above bacterial culture was inactivated by treating it in a 75°C water bath for 25 minutes.

[0076] Preservation treatment: Add 0.01% (v / v) of Tween 20 to the above-mentioned inactivated bacterial solution, then dilute it 80 times with sterile distilled water to obtain a treatment solution. Immerse the pretreated premium Alisma plantago-aquatica flower buds in this treatment solution, keeping the temperature of the treatment solution at 12℃, and soak for 10 minutes. The dosage of the treatment solution is 1 liter per 5 kg of flower buds.

[0077] Storage: Store in a cold storage at 4℃.

[0078] Comparative Example 4

[0079] Preparation and treatment of bacterial culture: Prepare a liquid culture medium containing 10g peptone, 5g yeast extract, 10g glucose, 5g sodium chloride, 1.0g dipotassium hydrogen phosphate, and 0.2g magnesium sulfate per liter, adjusting the pH to 7.0. After autoclaving at 121℃ for 15 minutes, cool to below 50℃, and aseptically add aminoethoxyvinylglycine to a final concentration of 32mg / L. Inoculate *Venelandia diffusa* into this medium at an inoculum rate of 1% (v / v) and incubate at 30℃ and 150rpm for 36 hours until the bacterial concentration reaches 5×10⁻⁶. 8 CFU / mL. The above bacterial culture was inactivated by treating it in a 75°C water bath for 25 minutes.

[0080] Preservation treatment: Add 0.01% (v / v) of Tween 20 to the above-mentioned inactivated bacterial solution, then dilute it 80 times with sterile distilled water to obtain a treatment solution. Immerse the pretreated premium Alisma plantago-aquatica flower buds in this treatment solution, keeping the temperature of the treatment solution at 12℃, and soak for 10 minutes. The dosage of the treatment solution is 1 liter per 5 kg of flower buds.

[0081] Storage: Store in a cold storage at 4℃.

[0082] Comparative Example 5

[0083] The difference between this comparative example and Example 1 is that in this comparative example, aminoethoxyvinylglycine is replaced with vitamin C; otherwise, it is the same as in Example 1.

[0084] Comparative Example 6

[0085] Preparation of bacterial culture: Prepare a liquid culture medium containing 10g peptone, 5g yeast extract, 10g glucose, 5g sodium chloride, 1.0g dipotassium hydrogen phosphate, and 0.2g magnesium sulfate per liter, adjusting the pH to 7.0. After autoclaving at 121℃ for 15 minutes, cool to below 50℃ and aseptically add aminoethoxyvinylglycine to a final concentration of 48mg / L. Inoculate *Pantotheca acuminata* and *Azotobacter venereum* at an inoculum rate of 1% (v / v) into this medium and incubate at 30℃ and 150rpm for 36 hours until the bacterial concentration reaches 5×10⁻⁶. 8 CFU / mL. The two bacterial solutions were inactivated by treating them separately in a 75°C water bath for 25 minutes. The two inactivated bacterial solutions were then mixed at a 1:1 volume ratio.

[0086] Preservation treatment: Add 0.01% (v / v) of Tween 20 to the above-mentioned inactivated bacterial solution, then dilute it 80 times with sterile distilled water to obtain a treatment solution. Immerse the pretreated premium Alisma plantago-aquatica flower buds in this treatment solution, keeping the temperature of the treatment solution at 12℃, and soak for 10 minutes. The dosage of the treatment solution is 1 liter per 5 kg of flower buds.

[0087] Storage: Store in a cold storage at 4℃.

[0088] Comparative Example 7

[0089] Preparation of bacterial culture: Prepare a liquid culture medium containing 10g peptone, 5g yeast extract, 10g glucose, 5g sodium chloride, 1.0g dipotassium hydrogen phosphate, and 0.2g magnesium sulfate per liter, adjusting the pH to 7.0. After autoclaving at 121℃ for 15 minutes, cool to below 50℃ and aseptically add aminoethoxyvinylglycine to a final concentration of 16mg / L. Inoculate *Pantotheca acuminata* and *Venerenella venereum* at an inoculum rate of 1% (v / v) into this medium and incubate at 30℃ and 150rpm for 36 hours until the bacterial concentration reaches 5×10⁻⁶. 8 CFU / mL. The two bacterial solutions were inactivated by treating them separately in a 75°C water bath for 25 minutes. The two inactivated bacterial solutions were then mixed at a 1:1 volume ratio.

[0090] Preservation treatment: Add 0.01% (v / v) of Tween 20 to the above-mentioned inactivated bacterial solution, then dilute it 80 times with sterile distilled water to obtain a treatment solution. Immerse the pretreated premium Alisma plantago-aquatica flower buds in this treatment solution, keeping the temperature of the treatment solution at 12℃, and soak for 10 minutes. The dosage of the treatment solution is 1 liter per 5 kg of flower buds.

[0091] Storage: Store in a cold storage at 4℃.

[0092] Comparative Example 1, using only 1-MCP fumigation, could temporarily block ethylene receptors, but lacked a physical protective layer, failing to effectively slow moisture loss and block microorganisms, leading to a sharp decline in quality later. Comparative Examples 2, 3, and 4 used Bacillus subtilis, Pantotheca acuminata, or Nitrogenobacter venereum, respectively. Their metabolic product systems were singular, unable to replicate the complex and efficient combination of metabolites produced by the synergistic fermentation of two specific strains, thus lacking synergistic effects in forming a protective film and physiological regulation. Comparative Example 5 replaced the key ethylene synthesis inhibitor aminoethoxyvinylglycine with vitamin C. The latter only has antioxidant effects and cannot inhibit ethylene synthesis at its source, thus failing to effectively delay the ethylene-driven maturation and aging process. Comparative Examples 6 and 7 demonstrated the importance of aminoethoxyvinylglycine concentration: excessively high concentrations (Comparative Example 6) may be toxic to the flower stalks, accelerating spoilage; excessively low concentrations (Comparative Example 7) are insufficient to effectively inhibit ethylene synthesis, resulting in a significant reduction in preservation effect.

[0093] In summary, only by using a specific combination of Pantothenic agglomerates and Venetian violaceum for fermentation, as described in the examples, and adding aminoethoxyvinylglycine at the optimal concentration (28-36 mg / L), can the best preservation effect be achieved through the synergistic effect of three mechanisms: inactivation of bacterial cells to form a protective film, bacterial metabolites to inhibit and stabilize bacteria, and aminoethoxyvinylglycine to precisely and continuously inhibit ethylene synthesis.

[0094] Experiment 1: Preservation effect test

[0095] (a) Nutritional quality determination

[0096] 1. Chlorophyll content: Refer to NY / T3082-2017 "Determination of Chlorophyll Content in Fruits, Vegetables and Their Products - Spectrophotometric Method" for the determination of total chlorophyll content. The unit is mg / 100g.

[0097] 2. L(+)-Ascorbic Acid Content: Ascorbic acid, as a growth regulator and highly effective antioxidant, is extremely important for plant growth and development and human health. However, ascorbic acid is easily oxidized to form colored substances. The determination was performed using the 2,6-dichlorophenolindophenol titration method in GB5009.86-2016, "National Food Safety Standard - Determination of Ascorbic Acid in Food". The Alisma plantago-aquatica flower bud sample was pulverized and extracted with metaphosphoric acid solution. The extract was then titrated with 2,6-dichlorophenolindophenol standard solution. The L(+)-ascorbic acid content was calculated based on the volume consumed, expressed in mg / 100.

[0098] 3. Soluble solids content: The soluble solids content was determined using NY-T2637-2014, "Determination of soluble solids content in fruits and vegetables - refractometer method". Samples of Alisma plantago-aquatica flower stalks were taken and tested on days 1, 3, 5, 7, 12, 16, and 21 during storage. Units are %.

[0099] 4. Total phenol content: The total phenol content was determined using the Folin-Ciocalteu method.

[0100] The results are shown in Tables 1, 2, and 3.

[0101] Table 1

[0102]

[0103] Table 2

[0104]

[0105] Table 3

[0106]

[0107] Experiment 2:

[0108] (ii) Conduct conductivity determination in accordance with ISO 17312:2005 General rules for the determination of electrical conductivity of plant materials:

[0109] 1. Sample preparation:

[0110] On days 1, 4, and 10 of storage, samples were randomly taken from the treatment groups of the above-described examples and comparative examples. The surface of the flower stalks was rinsed with deionized water, and then circular pieces of the same diameter were cut from the same part of the flower stalk stem using a clean punch (or cut into small strips of uniform length and width with a knife). After mixing thoroughly, 5.00 grams of sample were accurately weighed.

[0111] 2. Initial conductivity (C1) measurement:

[0112] Place the weighed sample into a clean 50 mL beaker (or Erlenmeyer flask) and add 25 mL of deionized water.

[0113] Place the beaker in a vacuum desiccator and evacuate for 5-10 minutes. Then slowly release the air. This step is to remove the air from the interstitial spaces so that the electrolytes can fully seep into the water. Let it soak for 30 minutes, shaking it occasionally. Use a conductivity meter to measure the conductivity of the soaking solution and record the value. This is the initial conductivity (C1), in mS / cm.

[0114] 3. Measurement of final conductivity (C2):

[0115] Seal the beaker (containing the sample and soaking solution) with sealing film and heat it in a boiling water bath for 15-20 minutes to completely kill the tissue cells and allow all electrolytes to leak into the water. After removing it, cool it to room temperature (you can rinse the outer wall of the beaker with running water to accelerate cooling) and shake it well. Measure the conductivity of the same soaking solution again with a conductivity meter and record the value. This is the final conductivity (C2), in mS / cm.

[0116] Relative conductivity RE (%) = (C1 / C2) × 100%

[0117] The results are shown in Tables 4 and 5 below:

[0118] Table 4

[0119]

[0120] Table 5

[0121]

Claims

1. A method for storing and preserving the flower buds of Alisma plantago-aquatica, characterized in that, Includes the following steps: S1. Screening and Grading: The Alisma plantago-aquatica flower buds are screened, and products that do not meet the basic requirements are removed. The basic requirements are that the flower buds are fresh, clean, odorless, intact and free from rot, mold, wilting, pithiness, insect damage and obvious mechanical damage, and of moderate maturity. Then, the flower buds that meet the basic requirements are divided into special grade, first grade and second grade according to texture, color, uniformity of stem and bud and degree of mechanical damage. S2. Pretreatment: Remove yellow leaves, diseased leaves, debris and residual soil from the flower stalks, rinse with running water at 10-15℃, and then pre-cool to reduce the temperature of the flower stalks to 2-6℃ within 1-2 hours. S3. Preservation treatment of fermentation products: Soak the pretreated flower buds in the inactivated and diluted microbial fermentation liquid. S4. Low-temperature storage: Store the treated flower buds in an environment with a temperature of 4±1℃; The preparation and inactivation process of the microbial fermentation broth in S3 is as follows: a. Prepare a liquid culture medium containing: 8-12g peptone, 4-6g yeast extract, 8-12g glucose, 4-6g sodium chloride, 0.8-1.2g dipotassium hydrogen phosphate and 0.15-0.25g magnesium sulfate per liter, with a pH of 7.0±0.2; b. Add aminoethoxyvinylglycine to the liquid culture medium to a final concentration of 28-36 mg / L; c. Inoculate *Pantotheca cumulus* and *Venerendica virgaurea* separately into the above-mentioned liquid culture medium and incubate at 30±2℃ and 150±10 rpm for 24-48 hours until the bacterial concentration reaches 10⁻⁶. 8 -10 9 CFU / mL; d. Inactivate the clump-forming Pantotheca acuminata culture from step c with the Vignelandia diffusa culture, and then mix the two inactivated cultures at a volume ratio of 1:

1. The inactivation process in step d is thermal inactivation, which is carried out in a water bath at 70-80°C for 20-30 minutes.

2. The method according to claim 1, characterized in that, In step S3, before soaking, 0.01% by volume of Tween20 is added to the microbial fermentation broth, and then diluted 10-100 times with sterile distilled water to obtain the treatment solution.

3. The method according to claim 2, characterized in that, The soaking conditions are as follows: the temperature of the treatment solution is controlled at 10-15℃, the soaking time is 8-12 minutes, and the amount of treatment solution used is 4-6 kg of Alisma plantago-aquatica per 1 liter of treatment solution; the treated Alisma plantago-aquatica and treatment solution are packaged together for storage or the surface treatment solution is drained and placed in a plastic bag for storage.

4. The method according to claim 1, characterized in that, In step S2, the operating temperature for pre-cooling is 0~5℃.

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