CN117431171A - Lactobacillus brevis for inhibiting mold and adsorbing and degrading aflatoxin B1 and application thereof - Google Patents

Abstract

The invention belongs to the field of microbial application technology, and particularly relates to a Lactobacillus brevis that inhibits mold and adsorbs and degrades aflatoxin B1 and its application. The Lactobacillus brevis ND‑1 has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee on June 20, 2023. Its abbreviation is CGMCC, and the deposit number is NO. 27664. Lactobacillus brevis ND-1 provided by the present invention mainly removes AFB1 through physical adsorption of the cell wall, and its detoxification efficiency in PBS solution is 71.38%. Strain DN‑1 has good growth rate and acid production performance and can be used as an alternative strain for silage inoculants. After washing 3 times, the detoxification rate remains highly efficient. The optimal conditions for strain DN‑1 to detoxify AFB1 are: inoculum volume 2.5%, initial pH 6.0, temperature 36°C, and culture time 4 hours.

Description

Lactobacillus brevis that inhibits mold and adsorbs and degrades aflatoxin B1 and its application

Technical field

The invention belongs to the field of microbial application technology, and particularly relates to a Lactobacillus brevis that inhibits mold and adsorbs and degrades aflatoxin B1 and its application.

Background technique

Aflatoxin B1 (AFB1) is a secondary metabolite mainly produced by fungi such as Aspergillus flavus and Aspergillus parasiticus, and is widely present in food and feed. AFB1 is the most stable and toxic mycotoxin discovered so far. Long-term intake can cause liver cancer and cause cancer in other organs such as kidneys, lungs, gastrointestinal tract, etc. It has been classified as a Class I carcinogen by the International Agency for Research on Cancer. . In recent years, reports of significant losses caused by AFB1 contamination in silage have also increased. Silage is the most important component of the diet of ruminants such as dairy cows and beef cattle, accounting for 50-75% of the total diet. It mainly uses lactic acid bacteria to produce organic acids under anaerobic conditions to reduce the feed pH, thereby inhibiting the proliferation of harmful bacteria, and has It has the advantages of long storage time and high nutritional value. Compared with other conventional feeds, silage is more susceptible to AFB1 contamination due to the high temperature and humidity of its storage environment. A survey showed that corn silage is the main source of detected toxins and has three times the impact on mycotoxin intake in dairy cows than other feeds. When ruminants such as dairy cows and beef cattle eat silage contaminated with AFB1, it will damage the body's health and easily cause problems such as reduced immunity, reduced milk production, reproductive and developmental disorders, and the harm will become greater as the feeding time is extended.

At present, the methods for removing AFB1 from food and feed are mainly divided into three methods: physical method, chemical method and biological method. Physical methods mainly include ultraviolet light, ionizing radiation, etc.; chemical methods use reagents that can destroy the structure of mycotoxins. Long-term use of these methods will promote the development of drug resistance in toxin-producing fungi, and problems such as high cost and damage to the environment and human health limit their application. Different from the first two methods, biological methods mainly achieve detoxification effects through the microorganisms themselves and their secretions, that is, the degradation and adsorption of microorganisms. Based on this green and safe characteristic, biological methods have been widely researched and applied. Lactic acid bacteria are often used as feed additives in silage production, and research on the removal of AFB1 by lactic acid bacteria continues to increase, indicating the great potential of lactic acid bacteria in removing AFB1. In terms of removing AFB1, Zhu et al. isolated a strain of Lactobacillus plantarum from fermented food, and the detoxification rate of AFB1 was 89.5%. Guan et al. isolated Lactobacillus rhamnosus XH753 from pasture and silage in southwest China. Inoculation of this strain into silage can prolong aerobic stability and reduce AFB1 content.

Lactic acid bacteria have been certified as a safe biological additive. Research on lactic acid bacteria as silage additives is also increasing. The sources of these lactic acid bacteria are usually fermented foods and animal intestines. However, screening of mycotoxin-contaminated silage and cow feces that consume mycotoxin-contaminated silage has detoxification properties. Research on AFB1 lactic acid bacteria has rarely been reported.

Contents of the invention

In view of the technical problems existing in the removal of aflatoxin B1, the present invention proposes a Lactobacillus brevis that inhibits mold and adsorbs and degrades aflatoxin B1 and its application.

In order to achieve the above objects, the technical solutions adopted by the present invention are:

The present invention provides Lactobacillus brevis ND-1. The Lactobacillus brevis ND-1 has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee on June 20, 2023, and its abbreviation is It is CGMCC, and its address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The collection number is CGMCC No. 27664.

The Lactobacillus brevis ND-1 is used in the preparation of feed additives that inhibit aflatoxin B1.

The Lactobacillus brevis ND-1 is used in preparing feed additives for Lactobacillus brevis that adsorb and degrade aflatoxin B1.

The present invention also provides a feed additive containing the above-mentioned living cells or heat-killed cells of Lactobacillus brevis ND-1.

Compared with the existing technology, the advantages and positive effects of the present invention are:

Lactobacillus brevis ND-1 provided by the invention mainly removes AFB1 through physical adsorption of the cell wall, and its detoxification efficiency in PBS solution is 71.38%. Strain DN-1 has good growth rate and acid production performance, and can be used as an alternative strain for silage inoculant. After washing 3 times, the detoxification rate remains highly efficient. The optimal conditions for strain DN-1 to detoxify AFB1 are: inoculum volume 2.5%, initial pH 6.0, temperature 36°C, and culture time 4 hours.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present invention more clearly, the drawings needed to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. Those of ordinary skill in the art can also obtain other drawings based on these drawings without exerting any creative effort.

Figure 1 Detoxification efficiency chart of AFB1 of different strains;

Figure 2 is a diagram of the colony morphology and Gram stain of Lactobacillus brevis ND-1. Figure 2a is the colony morphology, Figure 2b is the Gram stain result, Figure 2c is the mold inhibition diagram, and Figure 2d is No inhibition control chart;

Figure 3 is the phylogenetic tree of Lactobacillus brevis ND-1;

Figure 4 is a graph showing the growth rate of Lactobacillus brevis ND-1;

Figure 5 is a diagram of acid-producing ability of Lactobacillus brevis ND-1;

Figure 6 is a diagram showing the effects of different active components of the strain and different treatments on the detoxification efficiency of aflatoxin B1. Figure 6a is a diagram of the detoxification efficiency of AFB1 by the strain supernatant, lysed contents and inactivated cells.

Figure 6b shows the effect of Lactobacillus brevis ND-1 treated with urea, lysozyme and acid on the detoxification efficiency of AFB1;

Figure 7 shows the AFB1 desorption rate after repeated washing of the bacterial cell-AFB1 complex with water and methanol solutions;

Figure 8a is a graph showing the detoxification efficiency of Lactobacillus brevis (Lactobacillus brevis) ND-1 against AFB1 as the inoculum amount changes;

Figure 8b is a graph showing the detoxification efficiency of Lactobacillus brevis ND-1 against AFB1 over time;

Figure 8c is a graph showing the detoxification efficiency of Lactobacillus brevis (Lactobacillus brevis) ND-1 against AFB1 as the pH value changes;

Figure 8d is a graph showing the detoxification efficiency of Lactobacillus brevis ND-1 against AFB1 as the temperature changes.

Detailed ways

In order to understand the above objects, features and advantages of the present invention more clearly, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that, as long as there is no conflict, the embodiments of the present application and the features in the embodiments can be combined with each other.

Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described here. Therefore, the present invention is not limited to the specific embodiments disclosed below. limit.

Example, this example provides a Lactobacillus brevis ND-1

1Materials and methods

1.1 Sample collection

The samples were collected from farms with serious mold pollution around Tongliao City, mainly including corn silage, FTMR, and cow manure fed the above-mentioned feeds.

1.2 Main reagents and culture media

0.01M phosphate buffered saline (PBS), AFB1 standard (Shanghai Yuanye Biotechnology Co., Ltd.), bacterial biochemical identification tube, AFB1 enzyme-linked immunosorbent assay (ELISA) detection kit (Shanghai Youyou Biotechnology Co., Ltd.) MRS Liquid culture medium (Beijing Jinclon Biotechnology Co., Ltd., pH is 6.2±0.1); MRS agar medium (Beijing Jinclon Biotechnology Co., Ltd., pH is 6.2±0.2); Potato dextrose agar medium (PDA), will be cultured The base was sterilized at 121°C for 30 minutes; Aspergillus flavus and Aspergillus parasiticus standard strains were purchased from Shanghai Yuanye Biological Co., Ltd.

1.3 Strain screening

1.3.1 Isolation and purification of lactic acid bacteria

Weigh 5g of the processed samples on the ultra-clean workbench and add them to an Erlenmeyer flask containing 95mL of sterile water, seal it, and place it in a constant temperature shaker at 37°C and 180r/min for 3 hours of shaking and enrichment. Take 1 ml of supernatant from different samples and dilute it to 10-5 to inoculate it into MRS liquid medium and culture it at 37°C for 48 hours. At the end of the culture, add 100 μL of each culture solution to 900 μL of sterile water, vortex, and dilute it to three gradients of 10-8, 10-9, and 10-10. Take 100 μL of each bacterial solution of different gradients and apply it to MRS. In solid medium, culture at 37°C for 48 hours. Pick a single colony according to the colony shape, color, size, etc. and streak it on the culture medium. Purify it three times by this method. The purified strain is mixed with an equal volume of glycerol and stored in a -20°C refrigerator for later use.

1.3.2 Screening of detoxified AFB1 lactic acid bacteria

The isolated and purified lactic acid strain was cultured in MRS liquid culture medium until the bacterial cell concentration reached 2.0×1010CFU/mL. The culture medium was centrifuged at 10000r/min for 10 minutes and the supernatant was discarded. The bacterial pellet was washed with 5 ml of PBS. After two times, it was resuspended in the PBS solution containing AFB1, and the PBS solution containing AFB1 without bacteria was used as a blank control. After culturing for 2 hours at 37°C, centrifuge at 10,000 r/min for 10 minutes (10°C). Collect 500 μL of the supernatant and store it in a -20°C refrigerator for detection by the ELISA kit. Calculate the strain detoxification efficiency according to the method provided by the ELISA kit.

1.3.3 Screening of lactic acid bacteria that inhibit toxin-producing fungi

The double-layer plate method was used to determine the inhibition of toxin-producing fungi by the selected lactic acid bacteria with detoxification effect. First pour the MRS solid culture medium into a petri dish as the lower plate. After solidification, use an inoculating loop to dip the lactic acid bacteria liquid activated to the logarithmic phase into two parallel lines of 2-3cm on the plate. Incubate at 37°C for 48 hours until growth occurs. Bacteria lawn. Pour the PDA culture medium containing fungal spores at a concentration of 106/ml into the culture medium, cover it on the lower culture medium containing the lactic acid bacteria lawn, culture it at 30°C for 72 hours, and detect the size of the inhibition zone around the lactic acid bacteria growth strip. , select lactobacilli with good antibacterial effects for later-stage testing.

1.4 Strain identification

1.4.1 Identification of colonies and bacteria

The morphology of the isolated lactic acid bacteria colonies was observed, and a single colony was picked for Gram staining and observed with an oil microscope to observe the strain morphology. Pick bacterial colonies and add 3% hydrogen peroxide solution dropwise. The presence of bubbles is considered positive, and the absence of bubbles is considered negative. Physiological and biochemical tests are performed on the selected strains.

1.4.2 Identification of strain 16S rRNA

Extract the DNA of the target strain according to the instructions of the bacterial DNA extraction kit, using the universal primers provided by the kit: 27F (5′-AGAGTTTGATCMTGGCTCAG-3′), 1492R (5′-TACGGYTAC CTGTTACGACTT-3′). 50 μL PCR reaction system: 5.0 μL 10×Ex Taq buffer, 4.0 μL 2.5mM dNTP Mix, 2.0 μL 10p Primer 1, 2.0 μL 10p Primer 2, 0.5 μL 5u Ex Taq, 2.0 μL Template, 34.5 μL ddH2O. PCR reaction conditions: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 1 minute and 30 seconds, a total of 24 cycles; final extension at 72°C for 10 minutes. The PCR products were sent to Beijing Meiyouan Biotechnology Co., Ltd. for sequencing, and the sequencing results were submitted to NCBI for BLAST analysis, and the N-J method in MEGA software was used to construct a phylogenetic tree.

1.5 Determination of biological characteristics of strains

The highly degraded strain was activated and inoculated into MRS liquid culture medium, and cultured at a constant temperature of 37°C. , measure the OD600 value of the strain fermentation broth at 36h, take the strain culture time as the abscissa and the OD600 value as the ordinate, and draw the strain growth curve. Cultivate the strain according to the above method, measure the pH of the bacterial solution at 0, 2, 4, 6, 8, 12, 16, 24, 36, 48, and 72 hours. Take the strain cultivation time as the abscissa and the pH value as the ordinate, and draw the product Acid rate curve.

1.6 Study on the adsorption and degradation of AFB1 by bacterial strains

The strain DN-1 with the highest ability to detoxify AFB1 was selected as the subsequent test strain. The strain was inoculated into MRS liquid culture medium and activated until the bacterial concentration was 2.0×1010CFU/ml. The bacterial solution was divided into three equal volumes. One part is stored in a refrigerator at 4°C for later use. One part of the DN-1 culture medium is centrifuged at 10,000 r/min for 20 minutes. The supernatant is filtered with a 0.22 μm water filter and stored for later use. The bacterial cells are precipitated with sterile PBS. After washing twice, resuspend in PBS solution, use a hand-held ultrasonic cell disrupter to disrupt the cells, centrifuge at 4°C and 10,000 r/min for 20 min, and filter the supernatant with a 0.22 μm water-based filter to obtain the cell contents. The last portion was sterilized in a high-pressure steam sterilizer at 121°C for 20 minutes. Centrifuge the bacterial liquids of each group above for 10 minutes at 10000r/min, discard the supernatant, wash the bacterial pellet twice with PBS solution, and then resuspend the bacterial cells with an equal amount of sterile PBS containing AFB1 (10 μg/mL). An equal amount of sterile PBS containing AFB1 (10 μg/mL) was used as a blank control, and cultured at a constant temperature of 37°C for 2 h. Three replicates were set for each sample. After the culture, the supernatant was collected by centrifugation for detection of AFB1 content.

1.7 Study on different treatments and compound stability

The strain pellet was inoculated into PBS solution containing AFB1 and then treated with HCI, urea and lysozyme in different ways. After treatment, incubate at 37°C for 2 hours and then centrifuge to collect the supernatant to measure the detoxification rate.

The strains were divided into living cell treatment group and inactivated cell treatment group. After the detoxification test, centrifuge at 10000r/min for 10min to obtain the precipitate. The bacteria were resuspended with 0.5mL PBS (pH7.4) solution and methanol solution respectively, and shaken for 5min to extract the bacteria. AFB1 adsorbed by the body was centrifuged at 10000 r/min for 20 min to collect the supernatant for detection. Repeat the above steps three times.

1.8 Effect of culture conditions on detoxification efficiency of strain AFB1

To study the impact of different culture conditions on the degradation efficiency of strain AFB1, the following culture conditions were set: inoculum volume (2%, 4%, 6%, 8%, 10%); temperature (22, 27, 32, 37, 42°C) ; Initial pH (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0); Culture time (1, 2, 4, 8, 12, 16, 20, 24, 28h). Three replicates were set for each sample to detect changes in AFB1 content.

2 Results and discussion

2.1 Isolation and screening of AFB1 virus-free strains

In recent years, the detoxification of AFB1 by lactic acid bacteria has received widespread attention, and a variety of lactic acid bacteria have been confirmed to have efficient AFB1 detoxification capabilities. After continuous isolation in this experiment, a total of 60 lactic acid strains were isolated from the samples, 8 of which had the ability to detoxify AFB1. The ability of these 8 strains to detoxify AFB1 was obviously different, as shown in Figure 1. Among them, strain DN-1 showed the strongest detoxification activity (71.38%) compared with other strains, QZ-11 (64.43%), FY-12 (53.13%), FT-9 (20.88%), DN-3 (45.53%), DN-7 (10.57%), FT-1 (4.45%), FJ-4 (33.47%).

The detoxification efficiency of the strains selected in this test is higher than that of the above-mentioned strains. This may be due to differences in bacterial species and types of mycotoxins. In addition, different culture conditions will also lead to certain differences.

2.2 DN-1’s inhibitory ability against toxin-producing fungi

Subsequently, eight strains were tested for fungi inhibition. The results showed that the DN-1 strain and the FY-12 strain showed good inhibitory capabilities against Aspergillus flavus and Aspergillus parasiticus, forming an inhibition zone near the fungus. However, since the AFB1 detoxification rate of FY-12 strain was significantly lower than that of DN-1 strain (p<0.05), DN-1 strain was selected for subsequent research.

Table 1 Screening of lactic acid bacteria with fungal inhibitory activity

Note: Strong inhibition+++(formation of strong inhibition zones around the LAB culture); inhibition++(formation ofinhibition zones around the LAB culture); some inhibition+(LAB were not overlaidby the fungi); absenceofinhibition-(LAB were completely overlaidby fungal growth).

2.3 Identification of bacterial strains

The colonies of the DN-1 strain were milky white, with a smooth and moist surface, translucent, and a convex center. The colony size was 0.2 to 0.6 mm, and there were no spores (Figure 2a). DN-1 was identified as a Gram-positive bacillus (Fig. 2b). The biochemical characteristics of DN-1 are listed in Figure 2,

The obtained 16S rRNA gene sequences were analyzed by BLAST search in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Lactobacillus brevis 16S sequencing results:

AGTCGAACGAGCTTCCGTTGAATGACGTGCTTGCACTGATTTCAACAATGAAGCGAGTGGCGAACTGGTGAGTAACACGTGGGAAATCTGCCCAGAAGCAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAACAAAATCCGCATGGATTTTGTTTGAAAGGTGGCTTCGGCTATCACTTCTGGATGATCCCGCGGCGTATTAGTTAGTTGGTGAGGTAAAGGCCCACCAAGACGATGATACGTAGCCGACCTGAGAG GGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAATGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACACCTTTGAGAGTAACTGTTCAAGGGTTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTTCCGGATTT ATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTAGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGA GTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAATCTTAGAGATAAGACGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTT GGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTCAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTTGCGAAGTCGTGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCG CGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGATAACCTTCGGGAGTCAGCCGTCTAAGGTGA

The results showed that the 16S rRNA sequence similarity between strain DN-1 and Lactobacillus brevis reached more than 99%. At the same time, the phylogenetic tree results also showed that DN-1 and Lactobacillus brevis were in the same branch (Figure 3), so it was identified as Lactobacillus brevis.

Table 2. Physiological and biochemical experimental results of strain DN-1

Note: "+", positive; "-", negative.

2.4 Determination of biological characteristics of strains

The growth curve of the DN-1 strain showed that 1-14h was the exponential growth phase, and the growth slowly entered the stationary phase after 14h (Figure 4). In addition, the pH of the DN-1 strain dropped rapidly during fermentation 0-16h, and the pH of the bacterial liquid was low (<4.0) at 16h. The pH dropped the fastest during fermentation 0-2h, and had dropped below 5.0 (Figure 5) . The DN-1 strain showed good growth rate and acid production ability, and had good silage potential. Growth rate and acid production performance are important indicators used to evaluate lactic acid bacteria for silage. The fast growth rate and acid production rate help lactic acid bacteria become the dominant strain in silage, promote lactic acid production, and quickly reduce pH, thereby inhibiting the rapid reproduction of other bacterial species. Lactobacillus plantarum and Lactobacillus brevis are the more common lactic acid bacteria additives used for silage. The former is a homofermentative lactic acid bacterium that can quickly produce lactic acid to reduce the pH value. The latter is a heterofermentative lactic acid bacterium that can produce a large amount of lactic acid and effectively inhibit the growth of mold.

2.5 Study on adsorption and degradation conditions

There are two main ways for strains to remove AFB1, through biodegradation or cell wall adsorption. In order to clarify the detoxification method, we can see from Figure 6a that the AFB1 detoxification rate of living cells of strain DN-1 is significantly higher than that of culture supernatant and Lysed content (p<0.01), viable cells removed 71.79% of AFB1 within 2h, while culture supernatant and lysed content only removed 12.20% and 7.79% respectively, which shows that strain DN- 1 rarely secretes active substances that degrade AFB1. At the same time, the degradation rate of the inactivated cell reaction system of strain DN-1 (74.68%) is higher than that of the living cell system, which shows that the detoxification method of strain DN-1 for AFB1 is mainly the adsorption of the cell wall, and the higher the concentration of the strain, the more adsorbed AFB1 more. Lactic acid bacteria remove AFB1 through adsorption. Inactivated cells have a higher detoxification rate. The reason may be that high-temperature inactivation leads to denaturation of cell wall proteins and changes in pores, thereby exposing more active adsorption sites. Studies have shown that the intestinal mucosa has little adsorption capacity for inactivated cells. Based on this, these cell-toxin complexes can be easily cleared from the intestines and will not remain in the intestines.

To further elucidate the role of the bacterial cell wall, living cells were treated with acid, urea, and lysozyme. The results showed that the ability of acid-treated cells to bind AFB1 increased to 78.35% compared with living cells. The degradation ability of cells was significantly reduced after treatment with urea and lysozyme (p<0.01) (Fig. 6b), which indicates that the cell wall is necessary for the adsorption process. Studies have found that components such as peptidoglycan and teichoic acid in the cell wall play an important role in binding mycotoxins, and the hydrophobic properties of the cell wall also play a key role. After urea treatment, the removal rate of AFB1 was only 16.27%, which may be due to the anti-hydrophobic property of urea that interferes with hydrogen bonding. In addition, the removal rate of AFB1 by lysozyme-treated cells decreased from 71.73% to 12.35%, which may be related to the structural integrity of the peptidoglycan three-dimensional network mentioned above.

2.7 Study on adsorption stability

In order to evaluate the stability of the complex formed between living cells and heat-killed cells and AFB1, the bacterial cell-AFB1 complex was washed repeatedly with water and methanol solutions, and the AFB1 desorption rate was detected. It can be seen from Figure 7 that the AFB1 release amount was the largest during the first washing process, which were 23.07%, 34.45%, 39.94% and 47.39% respectively. Heat-killed cells have higher release compared to viable cells. Compared with aqueous solutions, methanol solutions can release more AFB1. After 3 times of elution, the amount of bacterial AFB1 complex released tends to be balanced. Only a trace amount of AFB1 is released after further elution. The desorption rate of strain DN-1 is maintained at 40%-50%, indicating that it also has a higher adsorption rate. . Many studies have also proven that the binding of lactic acid bacteria to AFB1 is a reversible process.

In addition to different washing solutions, factors affecting the stability of mycotoxin complexes also include the type of strain and the content of toxins bound to the strain. Different strains have specific binding sites for toxins. The more toxins bound to the same strain, the better the adsorption stability of the strain will be. The results show that the adsorption of AFB1 by DN-1 bacteria is a reversible process, and the strain still retains a high removal rate after being eluted three times. The stability of the bacterial AFB1 complex depends on the binding ability of the strain itself and is strain-specific. In this study, it was observed that the bacterial DN-1 adsorbed AFB1 has good stability.

2.8 Effects of cell concentration, temperature, pH, and time on AFB1 detoxification rate

The study found that when the inoculation amount of DN-1 strain was between 0.5% and 2.5%, the detoxification efficiency of AFB1 increased with the increase of the inoculum amount, reaching a maximum of 78.66%. The higher the strain concentration, the more detoxified AFB1. When the inoculation amount exceeded 2.5%, the detoxification rate decreased significantly (p<0.05) (Fig. 8a). This may be because when the strain reaches the optimal inoculum amount, continued addition of strains cannot fully utilize the energy in the culture medium to meet its own growth, resulting in a decrease in the detoxification rate of strain AFB1. Therefore, the optimal inoculation amount of strain DN-1 is 2.5%.

Figure 8b shows that within 0-4h, the detoxification rate of DN-1 increases with time and tends to be stable within 4-28h. There was no significant difference in AFB1 detoxification rate between 4h and 28h (p>0.05). The results showed that the optimal culture time of DN-1 strain was 4h. During the entire 28h test, more than 80% of AFB1 was adsorbed in the first 1h, which shows that the adsorption of AFB1 by strain DN-1 is a rapid process and can be completed in a short time.

As shown in Figure 8c, strain DN-1 can maintain high AFB1 detoxification activity in the acidic pH range. When the pH is 4.0-6.0, as the pH increases, the strain detoxification efficiency increases. When the pH was 6.0, the detoxification rate of AFB1 by strain DN-1 reached the maximum, which was 69.13%. When the pH was greater than 6.0, the degradation rate of AFB1 decreased significantly (p<0.05). Therefore, when the initial pH of the culture medium is 6.0, strain DN-1 can exert the maximum AFB1 degradation activity.

Figure 8d shows that when the temperature is at 36°C and 40°C, the DN-1 strain shows a higher detoxification rate, and the detoxification rates are: 73.84% and 73.27% respectively. There was no significant difference in AFB1 detoxification rate between the two groups (p>0.05).

3Conclusion

In this example, a strain of lactic acid bacteria DN-1 capable of efficiently detoxifying AFB1 was isolated, which was identified as Lactobacillus brevis. Strain DN-1 mainly removes AFB1 through physical adsorption of the cell wall, and its detoxification efficiency in PBS solution is 71.38%. Strain DN-1 has good growth rate and acid production performance, and can be used as an alternative strain for silage inoculant. After washing 3 times, the detoxification rate remains highly efficient. The optimal conditions for strain DN-1 to detoxify AFB1 are: inoculum volume 2.5%, initial pH 6.0, temperature 36°C, and culture time 4 hours.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any skilled person familiar with the art may make changes or modifications to equivalent changes using the technical contents disclosed above. The embodiments may be applied to other fields, but any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims (6)

1. A kind of Lactobacillus brevis (Lactobacillus brevis) ND-1, characterized in that Lactobacillus brevis (Lactobacillus brevis) ND-1 has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee on June 20, 2023, Its abbreviation is CGMCC, and its preservation number is NO.27664. 2. Use of Lactobacillus brevis ND-1 as claimed in claim 1 in the preparation of feed additives that inhibit aflatoxin B1. 3. The Lactobacillus brevis ND-1 according to claim 1 is used in the preparation of feed additives for Lactobacillus brevis that adsorbs and degrades aflatoxin B1. 4. A feed additive characterized by containing living cells of Lactobacillus brevis ND-1 according to claim 1. 5. A feed additive comprising heat-killed cells of Lactobacillus brevis ND-1 according to claim 1. 6. A feed additive comprising a mixed strain of live cells and heat-killed cells of Lactobacillus brevis ND-1 according to claim 1.