Lactobacillus plantarum R4-30 and application thereof

Abstract

The invention discloses a mycotoxin degrading bacterium (Lactobacillus plantarum) R4-30 and application thereof, the strain is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC No: M2018437, and the strain is obtained by screening in Guangxi sugarcane tail leaf silage. The strain can inhibit the growth of aspergillus flavus and fusarium graminearum, so that the production of mycotoxin is reduced, and the mycotoxin can be degraded. The aflatoxin B1 degradation rate of the strain is as high as 91.21% when the bacterial concentration is 12 * 10 < 8 > cfu / ml, the pH value is 2 and the culture time is 16 days; when the bacterium concentration is 3 * 10 < 8 > cfu / ml, the pH value is 6 and the culture time is 12 days, the zearalenone degradation rate is as high as 94.56%; when the bacterial concentration is 12 * 10 < 8 > cfu / ml, the pH value is 2 and the culture time is 16 days, the degradation efficiency of AFB1 and ZEN in the mango peel fermented feed is remarkably improved, the degradation rates of AFB1 and ZEN under the conditions reach 91.2% and 58.16% respectively, the nutritional quality is high, the fermentation quality is good, chemical additives are not needed, and the method is suitable for green detoxification treatment of agricultural products and feed.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detoxification, and particularly relates to a mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 and its application. Background Art

[0002] Aflatoxin B1 (AFB1) and zearalenone (ZEN) are common mycotoxins in grains and feeds, with strong toxicity, carcinogenicity and immunosuppression, seriously threatening the health of humans and animals. Traditional physical and chemical detoxification methods have problems such as low efficiency, high cost and secondary pollution. The activity of strains is restricted by environmental conditions, the degradation efficiency is unstable, and there is a lack of systematic optimization of the synergistic effect of multiple factors, which limits industrial application. In order to improve the efficiency of degrading mycotoxins, the applicant screened Lactobacillus plantarum and found that a specific Lactobacillus plantarum strain R4-30 can synergistically degrade multiple toxins through multiple mechanisms such as surface adsorption, enzymatic hydrolysis and chelation of metabolites under specific bacterial concentration, pH and time conditions, breaking through the bottleneck of the existing technology. Summary of the Invention

[0003] In order to solve the problem of high mycotoxin content during feed stacking, the present invention provides a mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 and its application. The mycelial growth inhibition method was used to test Lactobacillus plantarum, and a Lactobacillus plantarum (Lactobacillus plantarum) R4-30 strain with high efficiency in degrading mycotoxins was screened out. This strain is green, non-toxic and safe, and can solve the problems of low efficiency and small applicability in degrading mycotoxins in the existing technology.

[0004] To achieve the above object, the technical solution of the present invention is: to provide a mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30, which is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC No: M2018437, the preservation date is July 2, 2018, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0005] The mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 is screened from the silage of sugarcane tail leaves in Guangxi, and can grow within the temperature range of 15-45°C, with a growth pH value range of 2.0-10.0 and an optimal pH of 8.0.

[0006] The method for the mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 to degrade mycotoxins includes the following steps:

[0007] (1) Mix the mycotoxin-degrading bacterium with the fermented material containing mycotoxin;

[0008] (2) Ferment under the conditions of different bacterial liquid concentrations of 3×10 8 cfu / ml, 6×10 8 cfu / ml, 9×10 8 cfu / ml, 12×10 8 cfu / ml, different pH values of 2, 4, 6, 8, and different fermentation times of 4 d, 8 d, 12 d, 16 d;

[0009] (3) Degrade mycotoxin synergistically through multiple mechanisms such as surface adsorption, enzymatic hydrolysis and metabolite chelation of the strain.

[0010] The mycotoxin includes aflatoxin B1 and zearalenone.

[0011] The fermented material is mango peel fermented feed.

[0012] The fermented feed is prepared by the method described above, and the degradation rate of aflatoxin B1 in the fermented feed reaches 91.20%, and the degradation rate of zearalenone reaches 94.56%.

[0013] While degrading mycotoxin, the fermented feed retains relatively high nutritional components, including crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), crude ash (Ash), crude fat (EE) and water-soluble sugar (WSC).

[0014] Application of the mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 in the preparation of fermented feed for degrading mycotoxin.

[0015] The screening and identification method of the mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 is characterized by including the following steps:

[0016] a Mix the collected sugarcane tail leaf silage with sterile Ringer's solution, soak, filter, plate on the improved MRS medium, and purify with the basic MRS medium until single colonies are obtained for primary screening;

[0017] The composition of the Ringer's solution is: 1 L of distilled water, 2.25 g of Nacl, 0.105 g of Kcl, Cacl 2 0.12 g, NaHCO 3 0.05 g;

[0018] The improved MRS agar medium consists of: calcium carbonate 5 g / L, peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, and the pH value is 6.5 ± 0.2. The basic MRS agar medium consists of: peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, and the pH value is 6.5 ± 0.2. After being prepared with distilled water, it is placed in an autoclave and sterilized at 121 °C for 20 min;

[0019] b Perform an antibacterial test on the obtained single colony with Aspergillus flavus and Fusarium graminearum for secondary screening;

[0020] c Identify the safety of the colony with the maximum inhibition rate for tertiary screening;

[0021] The safety identification method is to inoculate a single colony of the strain on a blood agar medium and culture it at 37 °C for 48 h;

[0022] d Sequence the 16S of the strain with a high degradation rate and construct a phylogenetic tree.

[0023] The antibacterial test adopts the mycelial growth inhibition method: When in use, the Lactobacillus plantarum bacterial suspension is mixed in the MRS medium, diluted in gradients, and then poured into a petri dish. Using the mold medium as a control group, a 0.5 cm diameter bacterial cake is inoculated into the center of the medium containing Lactobacillus plantarum, and it is placed in an incubator at 28 °C for 2 d. The diameter of the colony growth in the medium containing Lactobacillus plantarum and the colony diameter of the control group are measured by the cross method. The inhibitory effect of this Lactobacillus plantarum on molds is judged based on the difference between the two. The mycelial growth inhibition rate is calculated according to the following formula:

[0024]

[0025] The application of the mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 in degrading aflatoxin B1 and zearalenone in mango peel fermented feed is to ferment the strain and naturally mildewed mango residue under the conditions of a bacterial concentration of 12×10 8 cfu / ml, pH = 2, and a reaction time of 16 days.

[0026] The properties of Lactobacillus plantarum strain R4-30 are as follows:

[0027] This strain has the function of degrading Aspergillus flavus and zearalenone, and has a higher degradation efficiency under specific conditions. It can grow in the temperature range of 15-45 °C, and the growth pH value range is 2.0-10.0, with the optimal pH being 8.0. After culturing on MRS agar medium for 32 h, its colony characteristics are: milky white, round, with a diameter of about 1 mm, smooth surface, slightly protruding in the center, opaque, and the edge is complete. Its cell characteristics are: bacilli, with a length of about 1-2 μm. Gram staining is positive. Using primers (789f): 5'-TAGATACCCSSGTAGTCC-3' and (932r): 5′-AAGGGCGGGTAACGTCA-3′, the 16S rRNA gene sequence of this strain was amplified by PCR. After comparing with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), a phylogenetic tree was constructed using MEGA11 software. The results show that this strain belongs to the Lactobacillus plantarum sp. species. Based on the above properties and characteristics, this strain was identified as Lactobacillus plantarum and named Lactobacillus plantarum R4-30.

[0028] The present invention has the following beneficial effects:

[0029] 1. The present invention provides a specific mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30, which was screened from the silage of sugarcane tail leaves in Guangxi and has the ability to efficiently degrade aflatoxin B1 and zearalenone.

[0030] 2. The strain of the present invention degrades mycotoxins through the synergistic action of multiple mechanisms such as surface adsorption, enzymatic hydrolysis, and chelation of metabolites, breaking through the bottleneck of low degradation efficiency of a single mechanism in the prior art.

[0031] 3. The present invention optimized the fermentation conditions through orthogonal experiments, enabling this strain to exhibit high degradation efficiency under specific conditions. When the bacterial concentration is 12×10 8 cfu / ml, pH = 2, and the culture time is 16 days, the degradation rate of aflatoxin B1 is as high as 91.21%, and at this time the degradation rate of zearalenone is 58.16%; when the bacterial concentration = 3×10 8 cfu / ml, pH = 6, and the culture time is 12 days, the degradation rate of zearalenone is as high as 94.56%. The feed fermented under these conditions is green, non-toxic, and safe.

[0032] 4. While efficiently degrading mycotoxins, the present invention can also effectively retain the nutritional components of fermented feed, reduce nutrient loss, and has significant practicality and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the colony morphology of the mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 of the present invention.

[0034] Figure 2 is the safety test result of the mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 of the present invention.

[0035] Figure 3 is the phylogenetic tree of the mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 of the present invention based on 16S sequencing results. DETAILED DESCRIPTION OF THE INVENTION

[0036] The technical solution of the present invention will be further described below through examples.

[0037] Example 1

[0038] This example is a method for screening and identifying the mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 of the present invention, including the following steps:

[0039] (1) Mix the collected sugarcane tail green silage with sterile Ringer's solution, soak, filter, plate it on the modified MRS medium, and purify it with the basic MRS medium until single colonies are obtained for primary screening;

[0040] (2) Using the Aspergillus flavus and Fusarium graminearum media as the control group, inoculate a 0.5 cm diameter fungal plug into the center of the medium containing Lactobacillus plantarum, and place it in an incubator at 28 °C for 2 days;

[0041] Use the cross method to measure the diameter of the colony growth in the medium containing Lactobacillus plantarum and the colony diameter of the control group, and judge the inhibitory effect of the agent on the strain at different concentrations according to the difference between the two; the mycelial growth inhibition rate is calculated according to the following formula:

[0042]

[0043] The results of the mycelial growth inhibition method are listed in Table 1.

[0044] Table 1 Inhibition rates of Lactobacillus plantarum in Example 1 against Aspergillus flavus and Fusarium graminearum

[0045]

[0046] From the test results in Table 1, it can be seen that the Lactobacillus plantarum in Example 1 has a relatively high inhibition rate against Aspergillus flavus and Fusarium graminearum.

[0047] After measurement, those with relatively high inhibition rates were screened out for safety identification, as shown in Figure 2 .

[0048] The strain with the highest inhibition rate and safety was subjected to 16S sequence sequencing to construct a phylogenetic tree, as shown in Figure 3 , and it was preserved.

[0049] Using primers (789f): 5'-TAGATACCCSSGTAGTCC-3' and (932r): 5′-AAGGGCGGGTAACGTCA-3′, the 16S rRNA gene sequence of this strain was amplified by PCR. After comparison with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), a phylogenetic tree was constructed using MEGA11 software. The results showed that this strain belongs to the species Lactobacillus plantarum sp. Based on the above properties and characteristics, this strain was identified as Lactobacillus plantarum and named Lactobacillus plantarum R4-30. It was preserved in the China Center for Type Culture Collection, Hubei Province on July 2, 2018, with the preservation number: CCTCC No: M2018437. The preservation address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0050] Example 2

[0051] This example is the application of the mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 of the present invention, including the following steps:

[0052] (1) After the activation and expansion culture of Lactobacillus plantarum R4-30, the OD value of the bacterial liquid was measured at 600 nm with an enzyme-labeling instrument and diluted to the required concentration;

[0053] (2) Weigh 700 g of the sample into a fermentation tank and adjust the pH of the sample with 1 mol / L NaOH and 1 mol / L HCl;

[0054] (3) Add the bacterial liquid with a bacterial content of 3% to the fermentation tank, mix evenly and then seal it;

[0055] (4) After reaching the target time, open the tank to take out the sample, dry it and then crush it, and pass through a 40-mesh sieve.

[0056] The mycotoxin content was determined using an ELISA kit, and the results are listed in Table 2.

[0057] Table 2 Mycotoxin degradation rate in Example 2

[0058]

[0059] From the test results in Table 2, it can be seen that in the seventh group of Example 2 (bacterial concentration was 12×10 8 cfu / ml, pH = 2, time was 16 days), the degradation rate was the highest, the degradation rate of aflatoxin B1 was 91.20%, and the degradation rate of zearalenone was 58.16%; in the fourth group (bacterial concentration was 3×10 8 cfu / ml, pH = 6, culture time was 12 days), the degradation rate of zearalenone was the highest, up to 94.56%.

[0060] Example 3

[0061] This example is a method for determining nutritional components in the application of the mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 of the present invention. The method is as follows: After reaching the target time, open the can to take out the sample, dry and crush it, and then pass it through a 40-mesh sieve. The nutritional substance indexes mainly measure the contents of crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), crude ash (Ash), crude fat (EE), and water-soluble sugar (WSC). Among them, the CP content is determined by the Kjeldahl method, the NDF and ADF contents are determined by the Van Soest method for detergent fiber, the Ash content is determined by the direct ash method, the EE content is determined by the Soxhlet extraction method, and the WSC content is determined by the anthrone colorimetric method. The determination results are listed in Table 3.

[0062] Table 3 Nutritional quality in mycotoxin degradation in Example 3

[0063]

[0064] From the test results in Table 3, it can be seen that in the seventh group of Example 3 (bacterial concentration was 12×10 8 cfu / ml, pH = 2, time was 16 days), the loss rate of nutritional quality was relatively low and it was preserved best.

[0065] From the above experimental results, it can be seen that:

[0066] 1. The mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30 can degrade mycotoxins and can significantly degrade aflatoxin and zearalenone under the fermentation conditions of a bacterial concentration of 12×10 8 cfu / ml, pH = 2, and time of 16 days.

[0067] This strain can inhibit the growth of Aspergillus flavus and Fusarium graminearum and degrade mycotoxins. Under the fermentation conditions with a bacterial concentration of 12×10 8 cfu / ml, pH = 2, and a time of 16 days, it can significantly degrade Aspergillus flavus and zearalenone. The peptidoglycan, polysaccharides, and surface proteins in the cell wall of Lactobacillus plantarum contain abundant functional groups, which can fix Aspergillus flavus and zearalenone toxin molecules through electrostatic adsorption, hydrophobic interaction, or hydrogen bond binding. Moreover, the extracellular enzymes secreted by Lactobacillus plantarum can directly destroy the toxic groups of toxin molecules, achieving irreversible degradation. In addition, metabolites such as organic acids, bacteriocins, and hydrogen peroxide produced by Lactobacillus plantarum during fermentation can assist in the degradation of Aspergillus flavus and zearalenone toxins.

[0068] 2. Effects of Optimized Conditions on the Nutritional Quality of Fermented Feed

[0069] The applicant found that under the fermentation conditions with a bacterial concentration of 12×10 8 cfu / ml, pH = 2, and a time of 16 days, Lactobacillus plantarum R4-30 is more capable of decomposing anti-nutritional factors, synthesizing functional metabolites, and inhibiting nutritional losses caused by spoilage. While achieving efficient detoxification, it reduces the nutritional loss of the feed.

[0070] The above results show that the mycotoxin-degrading strain (Lactobacillus plantarum) R4-30 of the present invention can effectively degrade mycotoxins. Under the fermentation conditions with a bacterial concentration of 12×10 8 cfu / ml, pH = 2, and a time of 16 days, it can effectively degrade Aspergillus flavus and zearalenone toxins in fermented mango peel, and it is an ideal feed treatment technology that takes into account both safe detoxification and nutritional retention.

Claims

1. A mycotoxin-degrading bacterium (Lactobacillus plantarum) R4-30, characterized in that: The strain was deposited in China Center for Type Culture Collection with the accession number CCTCC No: M2018437 and the deposit date was July 2, 2018.

2. The mycotoxin degrading bacteria (Lactobacillus plantarum) R4-30 according to claim 1, characterized in that The strain was screened from sugarcane tail leaf silage in Guangxi, and can grow in the temperature range of 15-45°C, with a growth pH range of 2.0-10.0, and an optimum pH of 8.

0.

3. A method for degrading mycotoxins using the mycotoxin degrading bacteria (Lactobacillus plantarum) R4-30 according to claim 1 or 2, characterized in that: The following steps are involved: (1) mixing the mycotoxin-degrading bacteria with a fermentation material containing mycotoxins; (2) Fermentation was carried out under different bacterial concentrations, different pH values, and different fermentation times; (3) The mycotoxins are synergistically degraded through multiple mechanisms including surface adsorption, enzymatic hydrolysis and metabolite chelation of the strain.

4. The method according to claim 3, characterized in that The mycotoxins include aflatoxin B1 and zearalenone.

5. The method according to claim 3, characterized in that: The fermented material is mango peel fermented feed.

6. A fermented feed, characterized in that: The fermented feed is prepared by the method according to any one of claims 3 to 5, and the degradation rate of aflatoxin B1 in the fermented feed reaches 91.20%, and the degradation rate of zearalenone reaches 94.56%.

7. The fermented feed according to claim 6, characterized in that: The fermented feed retains relatively high nutritional components, including crude protein CP, neutral detergent fiber NDF, acid detergent fiber ADF, crude ash Ash, crude fat EE and soluble sugar WSC, while degrading mycotoxins.

8. Use of the mycotoxin degrading bacteria (Lactobacillus plantarum) R4-30 according to claim 1 or 2 in the preparation of fermented feed for degrading mycotoxins.

9. The method for screening and identifying mycotoxin degrading bacteria (Lactobacillus plantarum) R4-30 according to claim 1, characterized in that: The steps include: a. Mix the collected sugarcane tail leaf silage with sterile Ringer's solution, soak, filter, plate on modified MRS medium, and purify with basic MRS medium until a single colony is obtained for primary screening; The Ringer's solution is composed of: 1L of distilled water, 2.25g of Nacl, 0.105g of Kcl, 0.12g of Cacl2, and 0.05g of NaHCO3; The improved MRS agar medium is composed of: 5 g / L calcium carbonate, 10.0 g / L peptone, 10.0 g / L beef extract powder, 5.0 g / L yeast extract powder, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 14.0 g / L agar, 1.0 g / L Tween 80, and a pH value of 6.5±0.

2. The basic MRS agar medium consists of: peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5 ± 0.

2. After preparing with distilled water, put it in an autoclave and sterilize it at 121 ° C for 20 min; b. Conduct antibacterial tests on the obtained single colonies with Aspergillus flavus and Fusarium graminearum for secondary screening; c. The colony with the highest inhibition rate was subjected to safety identification and screened three times; The safety identification method is to take a single colony of the strain and inoculate it on a blood agar medium and culture it at 37°C for 48 hours; d The strains with high degradation rates were sequenced by 16S sequencing and the phylogenetic tree was constructed.

Images