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  • The effect of biological mycotoxin removement agent on aflatoxin B1

    [Abstract] The aflatoxin B1, binding or degrading ability of the biological mycotoxin removement agent was studied,and the ratio of the aflatoxin B1 discarded with the feces compared to the total intaken amount was also detected. When added 1‰ biological agent, 50.44% aflatoxin B1 was discarded with the feces, while added 2‰ biological agent, 72.30%  aflatoxin was discarded efficiently with the feces.

    At present, the proportion of contaminated mycotoxins in grain feeds in the world is as high as 25%. In addition to causing economic losses to livestock production, some mycotoxins are also carcinogenic or teratogenic. From the perspective of agriculture and health, the naturally polluted aflatoxins are mainly aflatoxin B1 (AFB1),which is composed of Aspergillus' Aspergillus flavus and secondary metabolites produced by Aspergillus,parasiticus mainly contaminates corn, peanuts, nuts, etc. For mildly mildewed feed and raw materials, different methods can be used for detoxification treatment according to specific conditions. Detoxification methods generally include physical detoxification, chemical detoxification, enzymatic hydrolysis, and adsorption (Chen Xuefeng et al., 2007) .

    Biological mycotoxin removement agent are safe and have no side effects. Through the biotransformation of certain microorganisms, they can destroy the chemical structure of mycotoxins or reduce the toxicity of mycotoxins, and have attracted widespread attention. Numerous studies have shown that lactic acid bacteria can control the growth of other microorganisms and inhibit the growth of spoilage bacteria and pathogenic microorganisms.

    At present, most studies are limited to laboratory conditions, and there are few reports on the anti-mildew and detoxification effects of lactic acid bacteria preparations in animals. This test combines in vitro and in vivo tests to detect the biological mycotoxin removement agent effect of biological detoxifiers on aflatoxin B1, and combines biological detoxification and physical adsorption detoxification to provide technical support for the development of mildew feed detoxifiers .

    1 Materials and methods

    1.1 Test material: biological mycotoxin removement agent, the main components are active Lactobacillus acidophilus, Lactobacillus acidophilus cell wall and hydrated aluminosilicate.

    1.2 Main test instruments and equipment: Multiskan MK3 microplate reader, AFBI detection kit (Beijing Institute of Nutritional Source Biology), and forced feeding equipment.

    1.3 Experimental animals and grouping: 60 healthy broiler chickens of similar weight and 40 days of age were selected as experimental animals and randomly divided into 5 groups with 3 replicates in each group and 4 chickens in each replicate.

    Corn to be tested: the moldy corn was dried at 60°C, smashed through a 40-mesh sieve, accurately weighed 50g, packaged and sealed in a plastic bag, and stored in a 4°C refrigerator for later use.

    1.4 Test method:

    1.4.1 Test method for in vitro adsorption of biological mycotoxin removement agent AFB1: take a certain amount of biological mold remover 0.00mg (blank control group), 0.25mg, 0.50mg, 0.75mg, 1.00mg, add 5mL sterile saline, biological mold remover The concentration of the agent was 0.00‰ (blank control group), 0.50‰, 1.00‰, 1.50‰, 2.00‰, shake well, and add 100 μL aflatoxin B1 benzene-acetonitrile solution (AFB1 concentration is 100 μg/mL). The mixture was incubated at 37 °C for 1 h at 180 r/min. Centrifuge at 10,000 r/min for 10 min at 4°C to separate the unbound mycotoxins (still in solution) from the bound mycotoxins (in the adsorbent), add 5 mL of normal saline to the pellet and repeat the centrifugation once, discard the The amount of aflatoxin B1 in the supernatant obtained by 2 centrifugation was determined with a kit. The formula for calculating the adsorption rate of aflatoxin B1 by the biological mycotoxin removement agent is: Adsorption rate=(1-Amount of AFB1 in sample supernatant/Amount of AFB1 in supernatant of empty group)×100%.

    1.4.2 Test of total collection of feces to detect the detoxification effect of biological mycotoxin removement agent in vivo. The test is divided into three stages: preliminary trial period, formal trial period and body condition recovery period. The formal trial period includes fasting emptying period, gavage, and fecal and urine excrement collection.There are three processes and the process is shown in Table 1.

    Table 1: feeding time and feeding method in each experimental period

    Preliminary trial period

    Fasting emptying period

    Gavage

    Fecal and urine excrement collection

    Body condition recovery period

    Time

    3 days

    48 hours

    time by individual

    48 hours

    10 days - 14 days

    Feeding method

    Mildew-free cornmeal

    10% glucose

    Mildew cornmeal 50g

    Free drinking water

    Complete nutrient compound feed for broilers


             Immediately after the forced feeding, the fecal collection bottle was installed, and the excrement was collected in a repeating group for 48 hours, once every 1to2 hours. The average total amount of airdried excrement of the chickens in each replicate group was pulverized, passed through a 40-mesh sieve, mixed evenly, and put into a sampling bag for sealing.

    Extraction was carried out according to the AFB1 extraction method (GB/T5009.22-1996, 1998) with the fat content of GB samples less than or equal to 3%. The content of AFB1 was determined using AFB1 detection kit (Beijing Institute of Nutritional Source Biology), and the operation method was in accordance with the instructions.

    2 Results and discussion:

    2.1 The effect of biological mycotoxin removement agent on the adsorption of aflatoxin B1 in vitro, as can be seen from Table 2, after adding the biological mycotoxin removement agent to the moldy feed, the aflatoxin B1 in each test group was adsorbed to varying degrees, and the biological the adsorption rates of AFB1 were 59.69%, 82.22%, 98.05% and ≥99.75% ,in the four test groups with 0.50‰, 1.00‰, 1.50‰, and 2.00‰ of the biological mycotoxin removement agent, and 2.00‰ of biological mycotoxin removement agent was added. The adsorption rate of aflatoxin B1 in the feed group was above 99.75%, and the aflatoxin in the feed had basically been completely adsorbed, while the adsorption rate of aflatoxin B1 in the biological mycotoxin removement agent group with 1.50‰ was as high as 98.05%. The biological mycotoxin removement agent effect is very significant.

    Table 2: The effect of biological mycotoxin removement agent on adsorption of aflatoxin B1 in vitro

    biological mycotoxin removement agent/

    AFB1 remaining amount(ng/g)

    Adsorption rate/%

    0.50

    806.2

    59.69

    1.00

    355.6

    82.22

    1.50

    19.05

    98.05

    2.00

    5.00

    ≥99.75

    Peltonen et al. (2001) cultured lactic acid bacteria and AFB1 at 37°C for 24 hours, and measured the amount of AFB1 by HPLC. The results showed that two strains of Lactobacillus amylovorus and one strain of Lactobacillus rhamnosus could remove more than 50% of AFB1. In addition, Li Zhigang et al. (2003) showed that the strength of lactic acid bacteria binding to aflatoxin B1 was 4% to 50%, and Lactobacillus casei subsp. casei CGMCC 11539 had the strongest ability to adsorb aflatoxin B1. The lactic acid bacteria cell wall in the biological mycotoxin removement agent also has a strong adsorption capacity for AFB1. Hasard et al. (2001) systematically studied 12 strains of lactic acid bacteria, among which L. rhamnosus GG had the strongest binding ability to AFB1, which was 78.9%. They speculate that dead cells can also bind to AFB1 in large quantities, and the inactivated cells after heating and acidification treatment have a stronger ability to remove AFB1 than live cells due to the weakened crosslinking of the peptidoglycan layer on the cell wall surface, which increases the pore size. Because the biological mycotoxin removement agent contains active lactic acid bacteria, it can not only absorb the mycotoxins in the feed, but also produce inhibitory complexes during the growth process of the lactic acid bacteria, which can inhibit the synthesis of toxins and degrade the produced toxins at the same time. The hydrated aluminosilicate in the biological mycotoxin removement agent has a lamellar structure and can tightly bind to AFB1. The comprehensive effect of the three active ingredients in the biological mycotoxin removement agent makes its mold removal effect remarkable. Especially adding 2.00‰ of the biological mildew remover, its detoxification effect is very ideal.

    2.2 The excretion effect of animal feces aflatoxin B1 with the addition of biological mycotoxin removement agent can be seen from Table 3. The total amount of aflatoxin B1 in the feces of the blank control group was 24.10 μg, accounting for 38.20% of the total amount of aflatoxin B1 in moldy corn. , a large amount of mycotoxin B1 was not excreted in the feces and remained in the animal body. In test group 1, after adding 1.00‰ of biological mycotoxin removement agent to moldy corn, the total amount of aflatoxin B1 in feces was 31.83 μg, accounting for 50.44% of the total amount of aflatoxin B1 in moldy corn. That is, adding 2.00‰ of biological mycotoxin removement agent to moldy corn has the best mold removal effect, the total amount of aflatoxin B1 in feces is 45.62μg, the excretion rate of AFB1 of Aspergillus flavus is 72.3%, and the mold removal effect is remarkable. The lactic acid bacteria cell wall in the biological mold remover can be closely combined with aflatoxin B1. The study of Hasard et al. (2001) showed that the heattreated lactic acid bacteria strain L. rhamnosus GG has a strong ability to bind to AFB1, and after 5 washings, there is still 79 % of AFB1 was bound to it, and only 21% of AFB1 was washed down. The binding force between hydrated aluminosilicate and AFB1 is also very strong. The negatively charged area in the middle of hydrated aluminosilicate can bind to AFB1, and because hydrated aluminosilicate is not absorbed by the body, the bound mycotoxins can be effectively taken out of the body. . The biological mycotoxin removement agent combines organic adsorption and physical adsorption, which significantly improves the excretion rate of mycotoxins with feces.

    Table 3: Determination of total amount of AFB1 in feces and moldy corn

    Group

    Biological mycotoxin removement agent/

    Absorbance

    Sampling volume/%

    AFB1 Total/μg

    AFB1excretion rate/%

    Blank control group

    0

    0.161

    37

    24.10

    38.20

    Test 1st group

    1.00

    0.158

    30

    31.83

    50.44

    Test 2nd group

    2.00

    0.153

    25

    45.62

    72.30

    Moldy corn

    0.178

    10

    63.10

    3 Conclusions

    The biological mold remover contains active lactic acid bacteria, lactic acid bacteria cell wall and hydrated aluminosilicate and other active ingredients, and has a very significant effect on mold removal of moldy feed. Performance, adding 1.00‰ of the feed can absorb trace mycotoxins that are not easily detected by the naked eye to achieve the purpose of preventing mycotoxins poisoning; and for mildly mildewed feed, adding 1.50‰ of the biological mold remover can achieve The effect of antimildew and detoxification can prevent livestock from being poisoned after eating mildewed feed; for feeds with severe mildew that can be detected with the naked eye, the mildewed feed should be dispersed first to reduce the concentration of mycotoxins, and then add 2.00‰ of biological Mildew remover for detoxification.

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