Separation method and application of actinomycetes for degrading allicin in garlic wastewater

Abstract

The invention provides a separation method and application of actinomycetes for degrading allicin in garlic wastewater. The separation method comprises the following steps: taking a soil sample near a water outlet of a garlic factory, culturing through an enrichment culture medium, gradually increasing the proportion of filtered garlic wastewater, culturing for multiple rounds, transferring to a separation solid culture medium, culturing, and carrying out streak purification to obtain a single bacterial colony. Compared with the prior art, the method has the advantages that the actinomycetes capable of effectively degrading the allicin are obtained by separating the garlic industrial wastewater for the first time, and the actinomycetes can reduce the content of the allicin in the garlic wastewater and can play a role in degrading in a relatively wide temperature range.

Description

Technical Field

[0001] The invention relates to the field of garlic wastewater treatment, and more particularly to a method for separating actinomycetes for degrading allicin in garlic wastewater and application thereof. Background Art

[0002] my country is the world's largest producer, consumer, and exporter of garlic, consistently ranking first in fresh garlic production. Furthermore, the garlic product industry, including garlic powder, black garlic, garlic cloves, and garlic slices, is also booming. Garlic processing generates significant amounts of industrial wastewater, which contains significant amounts of allicin, garlic oil, and other sulfur-containing compounds. These compounds are the source of odor and are highly bactericidal and persistent pollutants, killing conventional microorganisms and making them difficult to degrade through conventional microbial fermentation to meet emission standards. Currently, methods for treating allicin in industrial wastewater primarily include precipitation, extraction recovery, hydrolysis and acidification, and contact oxidation.

[0003] Actinomycetes, a group of microorganisms with significant ecological and industrial value, demonstrate unique advantages in soil ecosystems and pollution control. In soil environments, the vast majority of actinomycetes are saprophytes. Leveraging their well-developed hyphal networks, they penetrate deep into the interstices between soil particles. They secrete extracellular enzymes to decompose complex organic matter in plant and animal debris, releasing mineral nutrients such as nitrogen, phosphorus, and potassium. These enzymes significantly promote the formation of soil aggregates, improve soil aeration and water retention, and lay the foundation for sustainable agricultural development.

[0004] In the field of industrial wastewater treatment, the functional diversity of actinomycetes is particularly prominent: (1) Actinomycetes have a broad spectrum of pollutant degradation: their metabolic system can produce specific enzyme systems such as phenol degradation enzymes, dehalogenases, and nitrile hydrolases, and have high efficiency in decomposing phenolic compounds, polycyclic aromatic hydrocarbons (PAHs), halogenated hydrocarbons and other traditional biologically difficult-to-degrade organic matter, breaking through the metabolic bottleneck of conventional bacteria for complex pollutants.

[0005] (2) Actinomycetes have multifunctional synergistic processing capabilities: In addition to carbon degradation, actinomycetes can also enhance denitrification efficiency through ammonification and nitrification-denitrification pathways, or use sulfur oxidoreductase to achieve biological desulfurization of sulfides, and even simultaneously remove heavy metal ions through the adsorption of extracellular polymers, forming an integrated processing capability of "denitrification-carbon removal-desulfurization-detoxification".

[0006] (3) Actinomycetes have environmental adaptability and engineering advantages: filamentous mycelium can easily form dense biofilms, and its ability to resist shock loads is significantly better than that of unicellular bacteria. It can still maintain stable metabolic activity when water quality fluctuates (such as high salt, low pH or toxic substance shock); it has a wide temperature resistance range and is suitable for wastewater treatment scenarios in highly polluting industries such as chemical and pharmaceutical industries.

[0007] It is noteworthy that despite the aforementioned unique advantages of actinomycetes in industrial wastewater treatment, research on the treatment of wastewater from the garlic processing industry remains limited. High concentrations of allicin (a sulfur-containing organic compound with strong antibacterial properties) in garlic wastewater can easily inhibit the activity of traditional microorganisms. The unique sulfur metabolism pathways and stress resistance mechanisms of actinomycetes offer a new approach to addressing this challenge. Currently, there are no reports on the use of actinomycetes to specifically degrade allicin and reduce wastewater toxicity. Isolating an actinomycete capable of degrading allicin in garlic wastewater is expected to overcome current treatment bottlenecks and provide an innovative solution for the biological treatment of challenging wastewaters from the food processing industry. Summary of the Invention

[0008] In order to address the shortcomings of existing garlic wastewater treatment methods, the present invention provides a method for isolating actinomycetes that can be used to degrade allicin in garlic wastewater, and applies the method to the treatment of garlic wastewater, which can stably achieve the degradation of allicin in garlic wastewater. At the same time, the actinomycetes obtained by the present invention can degrade allicin in a wide temperature range.

[0009] To achieve the above object, the present invention provides a method for isolating actinomycetes for degrading allicin in garlic wastewater, comprising the following steps: (1) Take soil samples near the drainage outlet of the garlic factory, ventilate and dry them, grind them in a sterile mortar, and transfer them to enrichment culture medium. Incubate them in a shaking incubator at 28-32°C and 100-300 rpm for 1-3 days.

[0010] (2) The solution obtained in step (1) is transferred to a new culture medium at a 2-10% inoculum size, which includes 80-90% enrichment culture medium and 10-20% filtered garlic wastewater, and cultured in a shaking incubator at 28-32°C and 100-300 rpm for 1-3 days.

[0011] (3) The solution obtained in step (2) is transferred to a new culture medium at a 2-10% inoculum size, which includes 70-80% enrichment culture medium and 20-30% filtered garlic wastewater, and cultured in a shaking incubator at 28-32°C and 100-300 rpm for 1-3 days.

[0012] (4) The solution obtained in step (3) was transferred to a new culture medium at a 2-10% inoculum size, which included 50-60% enrichment culture medium and 40-50% filtered garlic wastewater, and cultured in a shaking incubator at 28-32°C and 100-300 rpm for 1-3 days.

[0013] (5) Take 100-300 μL of the solution obtained in step (4), transfer it to a separate solid culture medium, and culture it at 28-32°C for 7-14 days.

[0014] (6) The single clone grown in the isolated solid culture medium in step (5) is streaked and purified on a new isolated solid culture medium and cultured at 28-32°C for 7-14 days until a single colony grows.

[0015] Furthermore, in steps (1) to (4), the enrichment medium is prepared according to the following mass ratio: the mass ratio of humic acid: KCl: FeSO4·7H2O: MgSO4·7H2O: Na2HPO4: CaCO3: water is 1: (1.5-2.0): (0.01-0.03): (0.3-0.7): (0.3-0.7): (0.01-0.03): 1000, and the pH of the enrichment medium is between 7.0 and 7.5. After the preparation is completed, the enrichment medium is sterilized at 110-130°C for 15-30 minutes. After the temperature is reduced to 28-32°C, 40-60 mg of cycloheximide and 15-30 mg of nalidixic acid are added per 1L of water. Cycloheximide is dissolved in DMSO to prepare a 40-60 mg / mL cycloheximide solution; and nalidixic acid is dissolved in 0.2-0.4 M NaOH solution to prepare a 15-30 mg / mL nalidixic acid solution.

[0016] Actinomycetes have natural resistance to cycloheximide and nalidixic acid, so actinomycetes can selectively grow in culture media containing cycloheximide and nalidixic acid and inhibit the growth of other bacteria.

[0017] Preferably, the humic acid in the enrichment medium is boiled in a water bath for 20-40 minutes, filtered through gauze, and the filtrate is collected.

[0018] Preferably, in steps (1) to (4), the filtered garlic wastewater is garlic wastewater that has been filtered through a 0.22 μm sterilizing filter membrane without any other treatment.

[0019] Furthermore, in step (5) and step (6), the separation solid culture medium is prepared according to the following mass ratio: the mass ratio of peptone: KCl: FeSO4·7H2O: MgSO4·7H2O: Na2HPO4: CaCO3: agar: water is 5: (1.5-2.0): (0.01-0.03): (0.3-0.7): (0.3-0.7): (0.01-0.03): (15-30): 1000, and the pH of the separation solid culture medium is between 5.5–6.5. After the preparation is completed, the separation solid culture medium is sterilized at 110-130°C for 15-30 minutes. After the temperature is reduced to 28-32°C, 40-60 mg of cycloheximide, 15-30 mg of nalidixic acid and 400-600 mg of allicin are added to each 1L of water. Cycloheximide is dissolved in DMSO to prepare a 40-60 mg / mL cycloheximide solution; nalidixic acid is dissolved in 0.2-0.4 M NaOH solution to prepare a 15-30 mg / mL nalidixic acid; and allicin is dissolved in DMSO to prepare an 80-120 mg / mL allicin solution.

[0020] The present invention also provides an actinomycete for degrading allicin, which is separated and obtained by the above method.

[0021] The present invention also provides an application of actinomycetes in degrading allicin in garlic wastewater.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention is the first to use garlic industrial wastewater to isolate an actinomycete that can effectively degrade allicin, and the actinomycete can be used in the treatment of garlic wastewater.

[0023] (2) The actinomycetes obtained by the present invention for degrading allicin in garlic wastewater can effectively reduce the allicin content in garlic wastewater. At the same time, the actinomycetes obtained by the present invention can degrade allicin in a wide temperature range. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a picture of the actinomycete plate of Example 1 of the present invention.

[0025] Figure 2 This is a species analysis diagram of the actinomycetes in Example 1 of the present invention.

[0026] Figure 3 Liquid chromatogram of allicin content in wastewater after actinomycete treatment provided as a test example. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions of the present invention are further described below through specific embodiments. Those skilled in the art should know that the embodiments are only for helping to understand the technical content and technical effects of the present invention and should not be regarded as limiting the present invention.

[0028] Example 1

[0029] A method for isolating actinomycetes for degrading allicin in garlic wastewater comprises the following steps: (1) Take 1 g of soil sample near the drain outlet of the garlic factory, ventilate and dry it, grind the soil sample in a sterile mortar and pestle, and transfer it to 100 mL of enrichment culture medium. Incubate it in a shaking incubator at 30°C and 200 rpm for 2 days.

[0030] (2) The solution obtained in step (1) was transferred to a new 100 mL culture medium at a 5% inoculum volume, which included 90 mL of enrichment medium and 10 mL of filtered garlic wastewater, and cultured in a shaking incubator at 30°C and 200 rpm for 2 days.

[0031] (3) The solution obtained in step (2) was transferred to a new 100 mL culture medium at a 5% inoculum volume, which included 80 mL of enrichment medium and 20 mL of filtered garlic wastewater, and cultured in a shaking incubator at 30°C and 200 rpm for 2 days.

[0032] (4) The solution obtained in step (3) was transferred to a new 100 mL culture medium at a 5% inoculum volume, which included 60 mL of enrichment medium and 40 mL of filtered garlic wastewater, and cultured in a shaking incubator at 30°C and 200 rpm for 2 days.

[0033] (5) Take 200 μL of the solution obtained in step (4), transfer it to a separate solid culture medium, and culture it at 30°C for 7 days.

[0034] (6) The single clone grown in the isolated solid culture medium in step (5) was streaked and purified on a new isolated solid culture medium and cultured at 30°C for 7 days until a single colony grew.

[0035] In steps (1) to (4), the enrichment medium contains 1 g of humic acid, 1.7 g of KCl, 0.01 g of FeSO4·7H2O, 0.5 g of MgSO4·7H2O, 0.5 g of Na2HPO4, 0.02 g of CaCO3, and 1 L of water. After preparation, the medium is sterilized at 121°C for 20 min, then the temperature is lowered to 30°C and 50 mg of cycloheximide and 20 mg of nalidixic acid are added. The mother solutions of cycloheximide and nalidixic acid are 50 mg / mL DMSO and 20 mg / mL 0.3 M NaOH, respectively.

[0036] In steps (1) to (4), the filtered garlic wastewater is the garlic wastewater without other treatment that has been filtered through a 0.22 μm sterilizing filter membrane.

[0037] In steps (5) and (6), the solid culture medium for separation is 5 g of peptone, 1.7 g of KCl, 0.01 g of FeSO4·7H2O, 0.5 g of MgSO4·7H2O, 0.5 g of Na2HPO4, 0.02 g of CaCO3, 20 g of agar, and 1 L of water. After preparation, sterilize at 121°C for 20 min. After the temperature is lowered, add 50 mg of cycloheximide, 20 mg of nalidixic acid, and 500 mg of allicin. The mother solutions of cycloheximide and nalidixic acid are 50 mg / mL DMSO and 20 mg / mL 0.3M NaOH, respectively, and the mother solution of allicin is 100 mg / mL DMSO. After mixing, a solid plate is prepared.

[0038] The present invention carried out sequencing analysis on the single bacterial colony obtained in Example 1, and the results are shown in the attached figure. Figure 2 As shown, the sequence identity between it and Kitasatospora HP106 reached 99.60%, confirming that the strain belongs to Kitasatospora, a type of actinomycetes.

[0039] Two test cases and one control case were used to test the allicin degradation ability of the strain obtained in Example 1. The conditions for Test Example 1 were as follows: the strain obtained in Example 1 was added to untreated garlic wastewater at an inoculum of 0.1 g / L and incubated in a shaking incubator at 30°C and 200 rpm for 8 h.

[0040] The conditions of Test Example 2 are as follows: the strain obtained in Example 1 was added to untreated garlic wastewater at an inoculum amount of 0.1 g / L, and cultured on a shaking table at 37° C. and 200 rpm for 8 h.

[0041] The control group consisted of only garlic wastewater (without strains and culture medium components) to exclude non-biodegradable factors such as adsorption or volatilization.

[0042] The allicin content in the test and control samples was determined by liquid chromatography using a C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile and water containing 0.1% trifluoroacetic acid. Gradient elution conditions were: 0–3 min, 5% acetonitrile and 95% water containing 0.1% trifluoroacetic acid; 3–7 min, 35% acetonitrile and 65% water containing 0.1% trifluoroacetic acid; 7–20 min, 35% acetonitrile and 65% water containing 0.1% trifluoroacetic acid; 20–30 min, 5% acetonitrile and 95% water containing 0.1% trifluoroacetic acid. The detection wavelength was 210 nm.

[0043] like Figure 3As shown, after adding the bacteria prepared in Example 1, the allicin content in the wastewater was significantly lower than that in the blank group, indicating that the strain can decompose allicin and has application value in the subsequent treatment of garlic wastewater. It can be seen from Test Example 1 and Test Example 2 that the bacteria obtained by the present invention can have high activity at 30-37°C, while most strains used for allicin degradation can only have activity below 35°C. The bacteria of the present invention can still have the effect of decomposing allicin at 37°C, indicating that the bacteria of the present invention can survive in a wider temperature range, and also shows that it has the possibility of survival in a more harsh environment, showing more value for its next application research.

[0044] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for isolating actinomycetes for degrading allicin in garlic wastewater, comprising the following steps: (1) Take soil samples near the drainage outlet of the garlic factory, ventilate and dry them, grind them in a sterile mortar, transfer them to enrichment medium, and culture them in a shaking incubator at 28-32°C and 100-300 rpm for 1-3 days; (2) The solution obtained in step (1) is transferred to a new culture medium at a 2-10% inoculum size, which includes 80-90% enrichment culture medium and 10-20% filtered garlic wastewater, and cultured in a shaking incubator at 28-32°C and 100-300 rpm for 1-3 days; (3) The solution obtained in step (2) was transferred to a new culture medium at a 2-10% inoculum size, which included 70-80% enrichment culture medium and 20-30% filtered garlic wastewater, and cultured in a shaking incubator at 28-32°C and 100-300 rpm for 1-3 days; (4) The solution obtained in step (3) was transferred to a new culture medium at a 2-10% inoculum size, which included 50-60% enrichment culture medium and 40-50% filtered garlic wastewater, and cultured in a shaking incubator at 28-32°C and 100-300 rpm for 1-3 days; (5) Take 100-300 μL of the solution obtained in step (4), transfer it to a separate solid culture medium, and culture it at 28-32°C for 7-14 days; (6) The single clone grown in the isolated solid culture medium in step (5) is streaked and purified on a new isolated solid culture medium and cultured at 28-32°C for 7-14 days until a single colony grows.

2. The method for separating actinomycetes for degrading allicin in garlic wastewater according to claim 1, wherein: The enrichment medium in steps (1) to (4) is prepared according to the following mass ratio: the mass ratio of humic acid: KCl: FeSO4·7H2O: MgSO4·7H2O: Na2HPO4: CaCO3: water is 1: (1.5-2.0): (0.01-0.03): (0.3-0.7): (0.3-0.7): (0.01-0.03): 1000, and the pH of the enrichment medium is between 7.0 and 7.5; after the preparation is completed, the enrichment medium is sterilized at 110-130°C for 15-30 minutes, and after the temperature is reduced to 28-32°C, 40-60 mg of cycloheximide and 15-30 mg of nalidixic acid are added per 1L of water.

3. The method for separating actinomycetes for degrading allicin in garlic wastewater according to claim 2, wherein: Cycloheximide is dissolved in DMSO to prepare a 40-60 mg / mL cycloheximide solution; nalidixic acid is dissolved in 0.2-0.4 M NaOH solution to prepare a 15-30 mg / mL nalidixic acid solution.

4. The method for separating actinomycetes for degrading allicin in garlic wastewater according to claim 2, wherein: The humic acid in the enrichment medium is boiled in a water bath for 20-40 minutes, filtered through gauze, and the filtrate is collected.

5. The method for separating actinomycetes for degrading allicin in garlic wastewater according to claim 1, wherein: In steps (1) to (4), the filtered garlic wastewater is the garlic wastewater without other treatment that has been filtered through a 0.22 μm sterilizing filter membrane.

6. The method for separating actinomycetes for degrading allicin in garlic wastewater according to claim 1, wherein: The separation solid culture medium in step (5) and step (6) is prepared according to the following mass ratio: the mass ratio of peptone: KCl: FeSO4·7H2O: MgSO4·7H2O: Na2HPO4: CaCO3: agar: water is 5: (1.5-2.0): (0.01-0.03): (0.3-0.7): (0.3-0.7): (0.01-0.03): (15-30): 1000, and the pH of the separation solid culture medium is between 5.5-6.5; after the preparation is completed, the separation solid culture medium is sterilized at 110-130°C for 15-30min, and after the temperature is reduced to 28-32°C, 40-60mg of cycloheximide, 15-30mg of nalidixic acid and 400-600mg of allicin are added to each 1L of water.

7. The method for separating actinomycetes for degrading allicin in garlic wastewater according to claim 6, wherein: Cycloheximide is dissolved in DMSO to prepare a 40-60 mg / mL cycloheximide solution; nalidixic acid is dissolved in 0.2-0.4 M NaOH solution to prepare a 15-30 mg / mL nalidixic acid; and allicin is dissolved in DMSO to prepare an 80-120 mg / mL allicin solution.

8. An actinomycete for degrading allicin, which is isolated and obtained by the method according to any one of claims 1 to 7.

9. Use of the actinomycete according to claim 8 in biodegradation of allicin.

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