Amygdalin degrading bacterium SYY40 as well as culture method and application thereof

Abstract

The invention discloses a amygdalin degrading bacterium SYY40 and a culture method and application thereof, and relates to the technical field of microbial pollution degradation and environmental pollution, the amygdalin degrading bacterium SYY40 provided by the invention is identified as pseudomonas, is named as Pseudomonas sp.SYY40, is preserved in China General Microbiological Culture Collection Center on May 19, 2025, and has the preservation number of CGMCC NO.9687. The amygdalin degrading bacterium SYY40 has the preservation number of CGMCC NO.9687. The amygdalin degrading bacterium SYY40 has the preservation number of CGMCC NO.9687. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) The strain can realize high-efficiency removal of the mandelic acid, and the degradation rate of the mandelic acid to the mandelic acid within 48 hours reaches 100%.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial pollution degradation and environmental pollution, and in particular to an amygdaloid-degrading bacterium SYY40, a culture method and application thereof. Background Art

[0002] Amygdalin, also known as amygdalin, is mainly found in Rosaceae plants, such as peach, apricot and plum. Amygdalin is a cyanogenic disodium glycoside with a molecular formula of C 20 H 27 NO 11 , the structural formula is phenylhydroxyacetonitrile-β-D-glucose-6-β-D-glucoside, which is easily soluble in water. Amygdalin can be decomposed into prunin under the action of related enzymes or microorganisms, and further produces toxic substances such as amygdalin, benzaldehyde, and hydrocyanic acid. Benzaldehyde is easily oxidized to produce benzoic acid, and when benzoic acid and hydrocyanic acid accumulate to a certain amount in the soil, they will affect the growth of plants, thereby causing continuous cropping problems. Therefore, if plant waste containing amygdalin (such as almond shells) is not handled properly, amygdalin will enter the soil or water body and may release cyanide through decomposition, causing harm to the ecosystem and human health.

[0003] Traditional physical and chemical degradation methods (such as boiling water) suffer from low degradation efficiency, incomplete degradation, and the risk of secondary pollution. Previous biodegradation methods (such as extracellular enzymes from Aspergillus niger) have been difficult to obtain and purify, and have been costly. In contrast, microbial degradation and remediation technology offers the advantages of high degradation rates, rapid efficiency, low cost, and no secondary pollution, making it a more environmentally friendly solution.

[0004] However, the current research on the degradation mechanism and application of amygdaloid-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] The present invention provides a strain of amygdalin-degrading bacteria SYY40, a cultivation method thereof, and an application thereof. The strain having the ability to tolerate and degrade amygdalin in peach root soil is obtained through enrichment culture, domestication, separation, and purification, thereby solving the key problem in the treatment of amygdalin pollution.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A strain of amygdaloid-degrading bacteria SYY40, characterized in that the amygdaloid-degrading bacteria SYY40 is named Pseudomonas sp. SYY40, and was deposited in the General Microbiology Center of the China Culture Collection Administration on May 19, 2025, with a strain deposit number of CGMCC NO.34596.

[0008] Preferably, the 16S rDNA sequence of the amygdaloid-degrading bacterium SYY40 is as shown in SEQ ID NO.1.

[0009] The present invention also provides a method for culturing the amygdaloid-degrading bacteria SYY40, comprising the following steps:

[0010] S1. Enrichment and acclimation of amygdaloid-tolerant bacteria

[0011] Using a screening medium containing amygdalin, bacterial strains were screened from a suspension of a peach tree root soil sample, and the bacterial strains were transferred and enriched and cultured for at least three rounds using an inorganic salt medium containing amygdalin;

[0012] S2. Isolation and purification of amygdaloid-degrading bacteria

[0013] The bacterial culture liquid capable of degrading amygdalidine was streaked onto LB medium plates, and the strain was isolated and purified to obtain the amygdalidine single-degrading bacterium SYY40.

[0014] Preferably, the screening culture medium is an inorganic salt culture medium.

[0015] Preferably, the inorganic salt culture medium needs to be added with sterile trace element stock solution and FeSO4 before use, and the volume 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 components of the inorganic salt culture medium are 1.75g / LNa2HPO4·12H2O, 0.5g / LKH2PO4, 0.5g / L(NH4)2SO4, 0.1g / LMgCl2·6H2O, and 50mg / L CaCl2.

[0017] Preferably, the sterile trace element stock solution is 500 mg / L EDTA, 10 mg / L ZnSO4·7H2O, 3 mg / LMnCl2·4H2O, 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] The present invention also provides the use of the amygdaloid-degrading bacteria SYY40 in amygdaloid degradation.

[0019] The present invention also provides a microbial agent comprising the amygdaloid-degrading bacteria SYY40 or a fermentation broth of the amygdaloid-degrading bacteria SYY40.

[0020] The present invention also provides a method for degrading amygdaloids, wherein the amygdaloid-degrading bacteria SYY40, or the amygdaloid-degrading bacteria SYY40 obtained by the above-mentioned culture method, or the above-mentioned microbial agent is activated and inoculated into a basic culture medium containing amygdaloids for culture. The culture temperature of the shaking incubator is 25-35°C and the rotation speed is 200-250 rpm, thereby achieving rapid degradation and removal of amygdaloids.

[0021] The sequence involved in the present 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] The present invention isolates a strain of amygdaloids degrading bacteria SYY40 from a suspension of a soil sample from the root system of a peach tree. The strain is identified as Pseudomonas sp. and named Pseudomonas sp. SYY40. The amygdaloids degrading bacteria SYY40 can efficiently degrade amygdaloids, and the degradation rate of amygdaloids reaches 100% within 48 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a colony plate image of the amygdaloid-degrading bacteria SYY40 of the present invention;

[0026] Figure 2 This is a microscopic image of the amygdaloid-degrading bacteria SYY40 of the present invention;

[0027] Figure 3 is the 16S rDNA phylogenetic tree of the amygdaloid-degrading bacterium SYY40 of the present invention;

[0028] Figure 4 The degradation curve diagram of the degradation rate of amygdalidine in the inorganic salt culture medium of the present invention over time;

[0029] Figure 5 This is a graph showing the OD value changes of amygdaloids growing in an inorganic salt medium according to the present invention. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0032] Unless otherwise specified, the reagents and materials used in the following examples can be obtained from commercial sources.

[0033] Inorganic salt culture medium (liquid): 1.75g ​​Na2HPO4·12H2O, 0.5g KH2PO4, 0.5g (NH4)2SO4, 0.1g MgCl2·6H2O, 50mg CaCl2, dilute to 1L with dd H2O, sterilize at 121℃ for 20min, and cool to room temperature for use.

[0034] Trace element stock solution: 500 mg EDTA, 10 mg ZnSO4·7H2O, 3 mg MnCl2·4H2O, 30 mg H3BO3, 20 mg CoCl2·6H2O, 1 mg CuCl2·2H2O, 2 mg NiCl2·6H2O, 3 mg Na2MoO4·2H2O, dilute to 1 L with dd H2O, autoclave at 121°C for 20 min, and cool to room temperature for use.

[0035] LB solid medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 1.5 g agar, sterilized at 121°C for 20 min, cooled to room temperature and used.

[0036] Preparation of amygdaloid stock solution: Weigh 0.05 g of amygdaloid powder, transfer to a 15 mL centrifuge tube, and dilute to 10 mL with dd H2O to obtain a 5000 mg / L amygdaloid stock solution. Refrigerate at 4°C for use as a stock solution.

[0037] Example 1 Isolation and screening of amygdaloid-degrading bacteria

[0038] A method for culturing amygdaloid-degrading bacteria strain SYY40 comprises the following steps:

[0039] S1. Enrichment and domestication of amygdaloid-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 of amygdalin, and then transferred and enriched for three rounds using an inorganic salt basic medium containing 50 mg / L of 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 amygdaloid-degrading bacteria:

[0043] The bacterial culture liquid capable of degrading amygdalidine was streaked onto LB medium plates, and the strain was isolated and purified to obtain amygdalidine-degrading single strain; the morphology of the purified strain SYY40 colony on the solid medium was as follows: Figure 1As shown. The colony is light yellow in color, with a beige center. The colony is approximately circular in shape, with a smooth and uniform surface. The mycelium in the center grows denser, forming a distinct aggregation effect, while the edges of the colony show a looser growth state. The colony boundary is clear and complete, with no obvious burr-like protrusions or irregular gaps. The morphology of the microscopic examination is as follows Figure 2 As shown, the bacteria are rod-shaped, about 1-3 μm in length and about 0.5 μm in width, and are independent of each other.

[0044] Example 2 Identification Method of Amygdaloid-Degrading Bacteria SYY40

[0045] The genomic DNA of the amygdaloid-degrading bacteria strain SYY40 was extracted using a bacterial genome extraction kit, and its 16S rRNA gene was amplified by PCR. The upstream and downstream primers of 16S rDNA were both universal bacterial primers.

[0046] Finally, the obtained PCR product was subjected to electrophoresis on a 1% agarose gel. Based on the electrophoresis results, the PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing to obtain its 16Sr DNA sequence (SEQ ID NO. 1). The sequence was then compared with the NCBI database. The comparison results showed that the bacteria belonged to the genus Pseudomonas. Its phylogenetic tree is as follows: Figure 3 As shown. This strain was identified as Pseudomonas sp. and named Pseudomonas sp. SYY40. The strain was deposited at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on May 19, 2025, with the accession number CGMCC NO. 34596.

[0047] Example 3 Degradation effect of strain SYY40 on amygdaloids in inorganic salt medium

[0048] In this example, amygdaloids were diluted in an inorganic salt medium, a final concentration of 50 mg / L of amygdaloids was added, and the strain SYY40 was inoculated at an inoculum volume ratio of 1% (i.e., 100 μL of bacterial solution was added to 10 mL of culture medium); at the same time, amygdaloids were added to the inorganic salt medium at a final concentration of 50 mg / L as a blank sterile control, and the culture was carried out in a constant temperature shaker at 30° C. and 220 rpm for 72 h. The degradation of amygdaloids by the strain SYY40 was detected by ultra-performance liquid chromatography, and the degradation rate was calculated. The amygdaloid degradation rate was calculated as follows: Degradation rate (%) = (C0-C X ) / C0×100%;

[0049] Among them, C0 and C X Represent the concentrations of control samples and microbial treated samples, respectively.

[0050] The experimental results showed that the degradation rate of amygdaloids by strain SYY40 reached 100% within 48 hours ( Figure 4 The OD600 of the culture medium was measured by a microplate reader to characterize the growth of the bacteria. The 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 above-described embodiments merely represent preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the spirit of the present invention, and such modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A strain of amygdaloid-degrading bacteria SYY40, characterized by: The amygdaloid-degrading bacteria SYY40 was named Pseudomonas sp. SYY40 and was deposited in the General Microbiology Center of the China Culture Collection Administration on May 19, 2025, with the culture collection number CGMCCNO.34596.

2. The amygdaloid-degrading bacterium SYY40 according to claim 1, characterized in that The 16S rDNA sequence of the amygdaloid-degrading bacteria SYY40 is shown in SEQ ID NO.

1.

3. A method for culturing the amygdaloid-degrading bacteria SYY40 according to claim 1 or 2, characterized in that: The steps include: S1. Enrichment and acclimation of amygdaloid-tolerant bacteria Using a screening medium containing amygdalin, bacterial strains were screened from a suspension of a peach tree root soil sample, and the bacterial strains were transferred and enriched and cultured for at least three rounds using an inorganic salt medium containing amygdalin; S2. Isolation and purification of amygdaloid-degrading bacteria The bacterial culture solution capable of degrading amygdaloids was streaked onto LB medium plates, and the strain was isolated and purified to obtain the amygdaloid-degrading single bacterium SYY40.

4. The culture method according to claim 3, wherein In step S1, the screening culture medium is an inorganic salt culture medium.

5. The culture method according to claim 4, characterized in that The inorganic salt culture medium needs to be added with sterile trace element stock solution and FeSO4 before use. The volume 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.

6. The culture method according to claim 5, characterized in that The components of the inorganic salt culture medium are 1.75g / LNa2HPO4·12H2O, 0.5g / L KH2PO4, 0.5g / L (NH4)2SO4, 0.1g / L MgCl2·6H2O, and 50mg / L CaCl2.

7. The culture method according to claim 5, characterized in that The sterile trace element stock solution is 500 mg / L ETA, 10 mg / L ZnSO4·7H2O, 3 mg / L MnCl2·4H2O, 30 mg / L H3BO3, 20 mg / L CoCl2·6H2O, 1 mg / LCuCl2·2H2O, 2 mg / LNiCl2·6H2O, and 3 mg / LNa2MoO4·2H2O.

8. Use of the amygdaloid-degrading bacterium SYY40 according to any one of claims 1 to 2 or 4 to 7 in amygdaloid degradation.

9. A microbial agent comprising the amygdaloid-degrading bacterium SYY40 according to claim 1 or 2 or a fermentation broth of the amygdaloid-degrading bacterium SYY40.

10. A method for degrading amygdaloids, characterized in that: The amygdaloid-degrading bacteria SYY40 described in claim 1 or 2, or the amygdaloid-degrading bacteria SYY40 obtained by the culture method described in claims 4 to 7, or the microbial agent described in claim 9 are activated and inoculated into a basic culture medium containing amygdaloids for culture. The shaking culture temperature is 25 to 35° C. and the rotation speed is 200 to 250 rpm, so as to achieve rapid degradation and removal of amygdaloids.

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