Mellein, a strain of amygdalin-degrading bacteria syy40, culture method and application thereof
By isolating and culturing Pseudomonas SYY40 from the root soil of peach trees, the problem of efficient degradation of amygdalin pollution was solved, achieving rapid, safe and low-cost degradation of amygdalin with a 100% degradation rate.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG UNIV OF CHINESE MEDICINE JINHUA RES INST
- Filing Date
- 2025-06-06
- Publication Date
- 2026-07-07
AI Technical Summary
Existing technologies for treating amygdalin contamination suffer from low degradation efficiency and the risk of secondary pollution due to traditional physicochemical methods, while biodegradation methods are difficult to purify and costly. There is a lack of efficient, safe, and low-cost microbial degradation technologies.
A strain of Pseudomonas SYY40 was isolated and cultured from the soil of peach tree roots. Through enrichment and domestication, a strain capable of efficiently degrading amygdalin was obtained, and rapid degradation was achieved through shaker culture.
It achieves a 100% degradation rate of amygdalin, with the degradation time completed within 48 hours, avoiding secondary pollution and being inexpensive.
Smart Images
Figure CN120665749B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial pollution degradation and environmental pollution technology, specifically to a mandelin-degrading bacterium SYY40, its culture method, and its application. Background Technology
[0002] Amygdalin, also known as enzymatic glycoside, is mainly found in plants of the Rosaceae family, such as peach, apricot, and plum. Amygdalin is a cyanogenic diglycoside with the molecular formula C1. 20 H 27 NO 11 The structural formula of amygdalin is phenylhydroxyethylcyano-β-D-glucose-6-β-D-glucosinolate, which is readily soluble in water. Under the action of relevant enzymes or microorganisms, amygdalin can decompose to produce arbutin, which further produces toxic substances such as amygdalin, benzaldehyde, and hydrogen cyanide. Benzaldehyde is easily oxidized to benzoic acid, and when benzoic acid and hydrogen cyanide accumulate in the soil to a certain level, they can affect plant growth, thus causing continuous cropping obstacles. Therefore, if plant waste containing amygdalin (such as almond shells) is not properly disposed of, amygdalin may enter the soil or water bodies and release cyanide through decomposition, posing a threat to the ecosystem and human health.
[0003] Traditional physicochemical degradation methods (such as boiling in hot water) suffer from low degradation efficiency, incomplete degradation, and a tendency to cause secondary pollution. Early biodegradation methods (such as Aspergillus niger extracellular enzymes) faced challenges such as difficulty in obtaining and purifying them, and high costs. In contrast, microbial degradation and remediation technology offers advantages such as high degradation rate, rapid and efficient operation, low cost, and no secondary pollution, making it a more environmentally friendly solution.
[0004] However, current research on the degradation mechanism and application of amygdalin-degrading bacteria is still relatively limited, and further exploration and development of efficient, safe, and low-cost microbial degradation technologies are needed. Summary of the Invention
[0005] This invention provides a mandelin-degrading bacterium SYY40, its culture method, and its application. Through enrichment culture, domestication, isolation, and purification, a strain of bacteria with tolerance and degradation ability to mandelin in peach root soil is obtained, thereby solving a key problem in the treatment of mandelin pollution.
[0006] To achieve the above objectives, the technical solution of the present invention is as follows:
[0007] A strain of amygdalin-degrading bacteria SYY40, characterized in that the amygdalin-degrading bacteria SYY40 is named Pseudomonas sp. SYY40, and was deposited at the China General Microbiological Culture Collection Center on May 19, 2025, with the culture accession number CGMCC NO.34596.
[0008] Preferably, the 16S rDNA sequence of the amygdalin-degrading bacterium SYY40 is shown in SEQ ID NO.1.
[0009] The present invention also provides a method for culturing the above-mentioned amygdalin-degrading bacteria SYY40, comprising the following steps:
[0010] S1. Enrichment and domestication of amygdalin-tolerant bacteria
[0011] The strains were screened from the suspension of peach root soil samples using a screening medium containing amygdalin, and then transferred and enriched in an inorganic salt medium containing amygdalin for at least three rounds of acclimatization.
[0012] S2. Degrading bacteria for the isolation and purification of amygdalin
[0013] Bacterial culture broth capable of degrading amygdalin was streaked onto LB agar plates for the isolation and purification of the strain, resulting in the amygdalin-degrading bacterium SYY40.
[0014] Preferably, the screening medium is an inorganic salt medium.
[0015] Preferably, sterile trace element stock solution and FeSO4 are added to the inorganic salt culture medium before use, and the volume-to-mass ratio of the inorganic salt culture medium, sterile trace element stock solution and FeSO4 is 1L:0.8-1.2mL:0.15-0.25mg.
[0016] Preferably, the inorganic salt culture medium comprises 1.75 g / L Na2HPO4·12H2O, 0.5 g / L KH2PO4, 0.5 g / L (NH4)2SO4, 0.1 g / L MgCl2·6H2O, and 50 mg / L CaCl2.
[0017] Preferably, the sterile trace element stock solution is composed of 500 mg / L EDTA, 10 mg / L ZnSO4·7H2O, 3 mg / L MnCl2·4H2O, 30 mg / L H3BO3, 20 mg / L CoCl2·6H2O, 1 mg / L CuCl2·2H2O, 2 mg / L NiCl2·6H2O, and 3 mg / L Na2MoO4·2H2O.
[0018] This invention also provides the application of the aforementioned amygdalin-degrading bacteria SYY40 in amygdalin degradation.
[0019] The present invention also provides a microbial inoculant comprising the above-mentioned amygdalin-degrading bacteria SYY40 or the fermentation broth of amygdalin-degrading bacteria SYY40.
[0020] The present invention also provides a method for degrading amygdalin, wherein the amygdalin-degrading bacteria SYY40, or the amygdalin-degrading bacteria SYY40 obtained by the above-mentioned culture method, or the above-mentioned microbial agent, are activated and then inoculated into a basic culture medium containing amygdalin for culture. The shaking culture temperature is 25-35℃ and the rotation speed is 200-250 rpm, which can achieve rapid degradation and removal of amygdalin.
[0021] The sequence involved in this invention is as follows:
[0022]
[0023] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:
[0024] This invention isolates an amygdalin-degrading bacterium, SYY40, from a suspension of soil samples from peach tree roots. It was identified as a Pseudomonas sp. and named Pseudomonas sp. SYY40. This amygdalin-degrading bacterium SYY40 can efficiently degrade amygdalin, and the degradation rate of amygdalin reaches 100% within 48 hours. Attached Figure Description
[0025] Figure 1 This is a colony plate image of the amygdalin-degrading bacterium SYY40 used in this invention;
[0026] Figure 2 This is a microscopic image of the amygdalin-degrading bacteria SYY40 used in this invention.
[0027] Figure 3 This is a phylogenetic tree of the 16S rDNA of the amygdalin-degrading bacterium SYY40 in this invention;
[0028] Figure 4 This is a degradation curve of amygdalin degradation rate over time in an inorganic salt culture medium according to the present invention.
[0029] Figure 5 This is a graph showing the OD value changes of amygdalin growth in an inorganic salt culture medium according to the present invention. Detailed Implementation
[0030] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.
[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.
[0032] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.
[0033] Inorganic salt culture medium (liquid): 1.75g Na2HPO4·12H2O, 0.5g KH2PO4, 0.5g (NH4)2SO4, 0.1g MgCl2·6H2O, 50mg CaCl2, bring the volume to 1L with dd H2O, sterilize at 121℃ for 20min, and cool to room temperature for later use.
[0034] Trace element stock solution: 500mg EDTA, 10mg ZnSO4·7H2O, 3mg MnCl2·4H2O, 30mg H3BO3, 20mg CoCl2·6H2O, 1mg CuCl2·2H2O, 2mg NiCl2·6H2O, 3mg Na2MoO4·2H2O, bring to a final volume of 1L with dd H2O, autoclave at 121℃ for 20min, and cool to room temperature for later use.
[0035] LB solid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 1.5 g agar, sterilized at 121°C for 20 min, cooled to room temperature before use.
[0036] Preparation of amygdalin stock solution: Weigh 0.05g of amygdalin powder, transfer it to a 15mL centrifuge tube, and dilute to 10mL with dd H2O to obtain a 5000mg / L amygdalin stock solution. Store at 4℃ for use as a stock solution.
[0037] Example 1: Isolation and Screening of Amygdalin-Degrading Bacteria
[0038] A method for culturing amygdalin-degrading bacterium SYY40 includes the following steps:
[0039] S1. Enrichment and domestication of amygdalin-tolerant bacteria:
[0040] The strains were screened from the suspension of peach root soil samples using a screening medium (inorganic salt medium) containing an initial concentration of 50 mg / L amygdalin, and then subjected to three rounds of transfer and enrichment culture acclimatization using an inorganic salt basic medium containing 50 mg / L amygdalin.
[0041] Before use, add 1 mL / L of trace element stock solution and 0.2 mg / L of FeSO4 to the inorganic salt culture medium.
[0042] S2. Isolation and purification of amygdalin-degrading bacteria:
[0043] Bacterial cultures capable of degrading amygdalin were streaked onto LB agar plates for isolation and purification, yielding a single amygdalin-degrading strain. The morphology of the purified strain SYY40 colonies on solid culture medium is as follows: Figure 1As shown. The colonies are generally pale yellow with a creamy white center. The colonies are nearly circular, with a uniform and smooth surface. The hyphae grow more densely in the central area, forming a clear aggregation effect; while the colony edges show a more loose growth pattern. The colony boundaries are clear and intact, with no obvious spiky protrusions or irregular gaps observed. The morphology observed under a microscope is as follows. Figure 2 As shown, the bacteria are rod-shaped, with a length of about 1-3 μm and a width of about 0.5 μm, and the bacteria are independent of each other.
[0044] Example 2: Identification method of amygdalin-degrading bacteria SYY40
[0045] Genomic DNA of amygdalin-degrading bacterium SYY40 was extracted using a bacterial genome extraction kit, and its 16S rRNA gene was amplified by PCR. The upstream and downstream primers for the 16S rDNA were universal primers for bacteria.
[0046] Finally, the obtained PCR products were subjected to electrophoresis on a 1% agarose gel. Based on the electrophoresis results, the PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing to obtain its 16S rDNA sequence (SEQ ID NO.1). The sequence was then compared with the NCBI database, and the comparison results showed that the bacterium belongs to the genus *Pseudomonas*, and its phylogenetic tree is as follows. Figure 3 As shown. This strain was thus identified as *Pseudomonas* sp. and named *Pseudomonas* SYY40. The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 19, 2025, with accession number CGMCC NO. 34596.
[0047] Example 3: Degradation effect of strain SYY40 on amygdalin in inorganic salt culture medium
[0048] In this embodiment, a final concentration of 50 mg / L amygdalin was added to an inorganic salt culture medium after diluting the stock solution. Strain strain SYY40 was inoculated at a 1% (v / v) inoculation rate (100 μL of bacterial suspension per 10 mL of culture medium). Simultaneously, an inorganic salt culture medium with a final concentration of 50 mg / L amygdalin was added as a blank sterile control. The cultures were incubated at 30°C and 220 rpm for 72 h in a constant temperature shaker. The degradation of amygdalin by strain SYY40 was detected by ultra-high performance liquid chromatography (UHPLC), and the degradation rate was calculated using the following formula: Degradation rate (%) = (C0 - C...) / (C0 - C0 ... X ) / C0×100%;
[0049] Among them, C0 and C X These represent the concentrations of the control sample and the microbial-treated sample, respectively.
[0050] Experimental results showed that strain SYY40 achieved a 100% degradation rate of amygdalin within 48 hours. Figure 4 The growth of bacteria was characterized by measuring the OD600 of the culture medium using an ELISA reader. Strain SYY40 began to grow significantly after 24 hours of culture, reached the logarithmic growth phase at 30 hours, and ended the logarithmic growth phase at 60 hours. Figure 5 ).
[0051] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.
Claims
1. A mandelin-degrading bacterium, SYY40, characterized in that, The amygdalin-degrading bacterium SYY40 was named *Pseudomonas*. Pseudomonas sp. SYY40 was deposited on May 19, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34596.
2. The application of the amygdalin-degrading bacterium SYY40 as described in claim 1 in the degradation of amygdalin.
3. A microbial inoculant comprising the amygdalin-degrading bacteria SYY40 of claim 1 or the fermentation broth of amygdalin-degrading bacteria SYY40.
4. A method for degrading amygdalin, characterized in that, The amygdalin-degrading bacteria SYY40 of claim 1 or the microbial agent of claim 3 are activated and then inoculated into a basic culture medium containing amygdalin for cultivation. The culture temperature is 25~35℃ and the rotation speed is 200~250rpm, which can achieve rapid degradation and removal of amygdalin.