CN113521115A - Aflatoxin detoxification composition and preparation method and application thereof - Google Patents
Aflatoxin detoxification composition and preparation method and application thereof Download PDFInfo
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- CN113521115A CN113521115A CN202110800794.XA CN202110800794A CN113521115A CN 113521115 A CN113521115 A CN 113521115A CN 202110800794 A CN202110800794 A CN 202110800794A CN 113521115 A CN113521115 A CN 113521115A
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Abstract
The invention discloses an aflatoxin detoxification composition, a preparation method and application thereof. According to the invention, the richness, diversity and flora structure information of microbial flora are obtained by 16S rRNA gene sequencing technology, and the result shows that mycotoxin breaks the balance of intestinal flora to cause intestinal flora disorder; the glycyrrhizic acid and lactobacillus casei are combined, and the proportion of the Rubrivivax and Brevundimonas diminuta is reduced by increasing the neosphingobium vesiculosum (Novosphingobium-capsulatum), so that the disordered intestinal flora environment is repaired, the growth condition of the broiler chicken is improved, and the production performance is improved.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to an aflatoxin detoxification composition and a preparation method and application thereof.
Background
The first occurrence of the term Mycotoxin (Mycotoxin) was that in 1960, turkeys in the suburban turkey farm in london, the year started to loss appetite and died two or three months later, and dissected to show death due to hemorrhage and necrosis of the liver and swelling of the kidneys. After the inspection and analysis, the cause of the turkey fatality is found to be that workers feed moldy peanut powder to the turkey, and the worker finally determines the turkey to be Aflatoxin (AF). The aflatoxin is a secondary metabolite of Aspergillus parasiticus and Aspergillus flavus, and is divided into more than ten kinds of toxins and more than 20 kinds of derivatives, which are respectively named as B1、B2、G1、G2And the like.
Aflatoxin B1(Aflatoxin B1,AFB1) Has the strongest toxicity and the widest distribution, not only causes immunosuppression and reduces the production performance and feed efficiency of animals, but also has stronger cancerogenic, teratogenic and mutagenic hazards and even causes the death of the animals. Researches show that bone cancer, rectal cancer, liver cancer, breast cancer and the like are all combined with AFB1In connection with this, it was classified as a class i carcinogen in 1993. Currently, about 25% of the world's food or feed is contaminated with mycotoxins to varying degrees, causing enormous economic losses to the animal husbandry every year.
The aflatoxin detoxification method comprises three physical, chemical and biological methods: the physical method comprises the following steps: including adsorption, ultraviolet irradiation, heating, water washing, light radiation, ion radiation, etc.; physical adsorbents such as montmorillonite are the most commonly used method, but are gradually eliminated due to the defects of desorbing and adsorbing nutrients. The chemical method comprises the following steps: including chlorination, oxidation, hydrolysis and ammoniation processes; because the chemical treatment has the disadvantages of environmental pollution, easy formation of other harmful substances and the like, the method is rarely applied in production.
In conclusion, the existing detoxification agent and method for aflatoxin have the defects of insecurity, harmful substance generation and environmental pollution, and further improvement is urgently needed.
Disclosure of Invention
Therefore, the invention provides an aflatoxin detoxification composition and a preparation method and application thereof.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for degrading aflatoxin B1The composition of (1), wherein the active ingredients are glycyrrhizic acid and lactobacillus casei.
In one embodiment of the invention, the glycyrrhizic acid accounts for 0.01-0.1% by mass, and the concentration of lactobacillus casei accounts for 1 × 106-1×108CFU/mL, and the balance of auxiliary materials or solvents.
In one embodiment of the invention, the composition comprises 0.04% by weight of glycyrrhizic acid and 1 × 10% by weight of lactobacillus casei7CFU/mL, and the balance of auxiliary materials or solvents.
The invention also provides the composition, the application of the composition in any one of the following (a) to (g),
(a) preparation of degraded AFB1The product of (1);
(b) preparing a product for improving the daily feed intake of chickens and the daily weight gain of the broilers;
(c) preparation for preventing or treating chicken AFB1Products with symptoms of intoxication;
(d) preparing a product for repairing chicken intestinal flora;
(e) preparing a product which increases the bursal neosphingosine bacteria and reduces the proportion of the red longevity bacteria and the shortwave monosomyia defectives;
(f) preparing a product for improving the activity of chicken jejunum protease;
(g) the product for improving the villus height of jejunum is prepared.
In one embodiment of the invention, the application is shown as degrading aflatoxin B1.
The invention also provides a method for preparing the composition, which comprises the step of mixing the glycyrrhizic acid, the live lactobacillus casei and auxiliary materials to obtain the composition.
In one embodiment of the invention, the lactobacillus casei is inoculated into MRS liquid culture medium, is statically cultured for 24 hours at the constant temperature of 37 ℃ to obtain lactobacillus casei liquid, the viable count of the lactobacillus casei is determined by using a plate coating method of the strain, and is adjusted to 1 × 109CFU/mL。
In one embodiment of the invention, the excipient is a pharmaceutically acceptable carrier.
In the present invention, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, such as: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, and the like. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.
Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient may be combined with one or more carriers and then formulated into the desired dosage form.
In the invention, the liquorice is an ancient Chinese medicament and is used as a medicinal plant for more than 2500 years. Glycyrrhizic acid is an effective component of liquorice and belongs to triterpenoid saponin compounds. The glycyrrhizic acid mainly comprises the following components: glycyrrhizin, glycyrrhetinic flavonoids, quercetin, and the like. Glycyrrhizic acid has the functions of resisting virus, resisting cancer, resisting inflammation, protecting liver, detoxifying and enhancing immunity, researches show that glycyrrhizic acid has the biological functions of protecting liver cells, resisting inflammation, regulating immunity and the like, and is widely applied to clinical treatment of various hepatitis and liver injury.
The invention has the following advantages:
the experimental result of the embodiment shows that the combination of glycyrrhizic acid and lactobacillus casei can improve the daily gain, daily feed intake and jejunum protease activity of broiler chickens and reduce AFB in tissues and serum1Content of the meat chicken AFB1And (4) toxic symptoms.
According to the invention, the richness, diversity and flora structure information of microbial flora are obtained by 16S rRNA gene sequencing technology, and the result shows that mycotoxin breaks the balance of intestinal flora to cause intestinal flora disorder; the glycyrrhizic acid and lactobacillus casei are combined, and the proportion of the Rubrivivax and Brevundimonas diminuta is reduced by increasing the neosphingobium vesiculosum (Novosphingobium-capsulatum), so that the disordered intestinal flora environment is repaired, the growth condition of the broiler chicken is improved, and the production performance is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
Fig. 1 is a diagram of a change disease of a broiler liver tissue structure provided by the embodiment of the invention, wherein the B: a control group; b: negative control group (normal corn replaced by moldy corn); d: negative control group + glycyrrhizic acid + lactobacillus casei;
fig. 2 is a disease chart of changes of jejunum tissue structures of broilers provided by an embodiment of the present invention, wherein a: a control group; b: negative control group (normal corn replaced by moldy corn); d: negative control group + glycyrrhizic acid + lactobacillus casei.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Test materials of the present invention: commercially available moldy corn (AFB)1163. mu.g/kg) as substrate for aflatoxin B1And (4) screening degrading bacteria. Lactobacillus casei (GIM1.159) and Bacillus subtilis (GIM1.715) are purchased from Guangdong province microorganism strain preservation center, Candida utilis (CGMCC2.0615) is purchased from China general microorganism strain preservation management center, and Lactobacillus acidophilus and Lactobacillus plantarum are screened and preserved by the feed biotechnology laboratory of the university of Henan agriculture.
The microorganism culture medium of the invention: LB culture medium: 1g of tryptone, 0.5g of yeast extract powder, 1g of sodium chloride and 100mL of water, wherein the pH value is 7.0-7.2;
YPD medium: 2g of peptone, 1g of yeast extract powder, 2g of glucose and 100mL of water, wherein the pH value is 7.0-7.2;
MRS culture medium: 1.5g of tryptone, 1g of yeast extract powder, 2g of glucose, 800.1 mL of tween, 2g of K2HPO, 0.5g of sodium acetate, 0.2g of ammonium citrate, 0.02g of magnesium sulfate, 0.005g of manganese sulfate, 100mL of water and pH of 6.2-6.6;
when the culture medium is solid, 2% agar is added, and the culture medium is sterilized at 121 deg.C and 0.15MPa for 20 min.
The test instrument and consumables of the invention: LDZX-330KBS autoclave (shanghai shenan medical instrument factory, shanghai, china), BVM-1000 biological purification workstation (suzhou purification equipment ltd, suzhou, china), 101 electric hot blast drying cabinet (beijing zhongwei Instruments ltd, beijing, china), Elx800 microplate reader (Bio Tek Instruments, Ins, USA), PHS-2C acidimeter (shanghai Instruments electro-scientific Instruments ltd, shanghai, china), AB204-N electronic balance (shanghai mettler-toli Instruments ltd, shanghai, china), SG-200 plastic sealer (deli group ltd, ningbo, china), PYX-DHS-BS double-layer air bath shaker (jiangjinjie ruier electrical appliance, junsu, china), PYX-DHS-BS-ii jump box (shanghai shenjian medical instrument ltd, shanghai, China), the R1211 aflatoxin enzyme-linked reaction detection kit is purchased from Bayer, Germany (R-BiopHarm, Germany).
Example 1 degradation of aflatoxin B1Screening of strains
1. Activation of bacterial species
Inoculating laboratory-preserved Candida utilis into YPD culture medium, and performing shaking culture at 30 deg.C and 180r/min for 24 hr. Inoculating the Bacillus subtilis to LB liquid culture medium, and performing shaking culture at 37 ℃ and 180r/min for 24 h.
Inoculating lactobacillus (Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus plantarum) into MRS liquid culture medium, and standing at 37 deg.C for 24 hr. The viable count of the strain is determined by plate coating method and then uniformly adjusted to 1 × 109Colony Formed Unit (CFU)/mL, viable count expressed as log natural number (lg), and the strain was stored in a refrigerator at 4 ℃ for later use.
1.2 screening for degraded AFB1Of (2)
1.2.1 test design and test grouping
The test medium is crushed moldy corn, 72g of moldy corn is weighed and placed in a 1L beaker during fermentation, 27mL of distilled water is weighed and mixed uniformly with 1mL of single fungus solution, the mixture is added into the moldy corn and stirred uniformly, the mixture is subpackaged into self-sealing bags, a sealing machine is used for sealing, each group of the test group is repeated for three times, the weight of each sample is equal, the test group is cultured for 3 days at the temperature of 30 ℃ and 37 ℃ respectively, and a blank control group (3 repetitions) without inoculation of bacteria is additionally arranged.
1.2.2 test index determination
AFB in fermented maize1The determination of (1): at 3d, 10g of corn samples were weighed in repeat units into 100mL Erlenmeyer flasks containing 50mL of 70% methanol, shaken for 30 minutes to extract toxins, and AFB in the samples was measured using the kit1The content of (a).
2. Test results
2.1 Aflatoxin B by different microorganisms1Degradation effect of
As can be seen from Table 1, Lactobacillus casei group AFB1The content is significantly lower than that of other groups (P)<0.05), i.e. Lactobacillus casei vs. AFB1The degradation rate is obviously higher than that of other groups (P)<0.05), the degradation rate is 76.26%; thus, lactobacillus casei was selected in subsequent experiments.
TABLE 1 degradation of aflatoxin B1 by different microorganisms
Note: the number of viable bacteria of the microorganisms in the table was adjusted to 1X 107CFU/mL。
This example screens a number of microorganisms for AFB1Lactobacillus casei, P.AFB, with higher degradation rate1The degradation rate reaches 76.26 percent.
EXAMPLE 2 use of the detoxification composition
1 feeding test design and grouping
The test was carried out at the schchang test base of the university of agriculture in the south of Henan, selecting 200 healthy AA broilers of 1 day, randomly dividing the broilers into 4 treatment groups of 5 replicates each, each of which was 10 chickens. The breeding period is 21 d. Adopting multi-layer cage culture, feeding freely, illuminating for 24h in the first week (after one week, illuminating for 23h, and darkness for 1 h); controlling the temperature and the humidity according to a conventional feeding management flow; the immunization program is as follows: and (5) immunizing the broiler chickens for 7 days by using the new-branch combined vaccine. The daily ration is prepared according to the feeding standard of broiler chickens NRC (1994), and the feed formula and the nutrient level are shown in table 2. The experimental groups were as follows:
group A: basal diet (Positive control, AFB in diet)1 3.04μg/kg)；
Group B: daily ration containing mildewed corn (normal corn replaced by mildewed corn, negative control group, AFB in daily ration)1 36.25μg/kg)；
Group C: negative control group + glycyrrhizic acid (drinking water, concentration in drinking water is 0.04%)
Group D: negative control group + glycyrrhizic acid + lactobacillus casei (drinking water, glycyrrhizic acid concentration in drinking water is 0.04%, lactobacillus casei concentration in drinking water is 1 × 10%7CFU/mL)
TABLE 2 broiler 1-21d diet composition and Nutrition levels (%, air-dried basis)
Note: premixing: each kilogram of daily ration contains: vitamin A12000 IU; vitamin E20 IU; vitamin D33000 IU; vitamin K31.0 mg; vitamin B63.5mg; vitamin B12.0 mg; vitamin B120.01mg; iron (ferrous sulfate) 100 mg; copper (copper sulfate) 8 mg; zinc (zinc oxide) 60 mg; iodine (calcium iodate) 0.45 mg; selenium (sodium selenite) 0.35 mg; manganese (manganese sulfate) 80 mg; 10mg of calcium pantothenate and 0.15mg of biotin; 6mg of riboflavin; folic acid 1.25 mg; nicotinic acid 35 mg. The values are calculated, except for the crude protein, calcium and phosphorus indicators which are actual values. The same applies below.
1.1 measurement index and method
1.1.1 measurement of Productivity, influence of different treatment groups on growth performance of broiler chickens
The weight was measured at 1d, 21d, 42d, respectively, and the amount of food intake was recorded every day for each repetition. The mortality status of each duplicate chicken was recorded. Average Daily Feed Intake (ADFI) and Average Daily Gain (ADG) were calculated.
As can be seen from Table 3, the growth performance of the group A is significantly higher than that of the other groups (P <0.05), the difference of the growth performance of the other groups is not significant (P >0.05), but the weight of the broilers, the average daily gain and the feed-meat ratio of the groups C and D are higher than that of the group B (P > 0.05); group C mortality was 4% maximum. The mildew corn can reduce the growth performance of the broiler chicken, and the combination of the glycyrrhizic acid and the lactobacillus casei can improve the daily gain and the daily feed intake and relieve the mycotoxin poisoning symptom of the broiler chicken.
Table 3 effect of different treatment groups on growth performance of 1-21d broilers (g, n ═ 5)
1.1.2 determination of nutrient metabolism rate, influence of different treatment groups on nutrient metabolism rate of broiler chickens
Collecting feces samples for 18-20d three days by using a total feces collection method, fixing nitrogen by using 10% sulfuric acid every day, then storing at-20 ℃, uniformly mixing the feces samples for three days after the test is finished, drying at 65 ℃ and dampening for 24h, and then crushing to determine indexes of Crude Protein (CP), crude fat (EE), calcium (Ca) and phosphorus (P). The Crude Protein (CP) is measured by using national standard CB/T6432-94; the crude fat (EE) is measured by adopting the national standard GB/T6433-2006; calcium (Ca) is measured by ethylene diamine tetraacetic acid disodium complexation titration; phosphorus (P) is measured by using the national standard GB/T6437-2002.
As can be seen from table 4, the group a crude protein, crude fat, calcium and phosphorus metabolic rates were significantly higher than those of the other groups (P <0.05), and the group D calcium metabolic rate was significantly lower than those of the other groups (P < 0.05). The mildew corn reduces the nutrient metabolism rate of the broiler chicken, and the addition of the mycotoxin antidote does not obviously improve the nutrient metabolism rate.
Table 4 influence of different treatment groups on broiler nutrient metabolism rate (%)
1.1.3 determination of chicken jejunum digestive enzyme activity
At 21 days, 1 chicken was selected repeatedly and slaughtered, the jejunum contents were taken into a sterile centrifuge tube and rapidly transferred to liquid nitrogen, and finally stored at-80 ℃.
Determination of protease Activity
Reference is made to the national Standard (SB/T10317-1999).
The enzyme activity unit is as follows: 1mg tyrosine was produced per minute as 1 protease activity unit (U).
Calculating the formula: the enzyme activity (U/g) is measured as tyrosine content/reaction time × 4 × sample dilution/sample weight.
(2) Determination of Amylase Activity
The measurement was carried out by the DNS method.
The enzyme activity unit is as follows: the enzyme amount per minute for producing 1mg of glucose is one enzyme activity unit (U).
Calculating the formula: amylase activity (U/g) is sample reducing sugar content x total sample diluent volume x dilution factor/sample weight/volume of enzyme used for assay/5 min.
Influence of different treatment groups on digestive enzyme activity of jejunum of broiler chickens
As can be seen from table 5, the protease activity of group D was significantly higher than that of group a (P < 0.05); the amylase activity was not significantly different among groups (P > 0.05). The glycyrrhizic acid and lactobacillus casei combination is proved to improve the activity of the chicken jejunum protease.
TABLE 5 influence of different treatment groups on the jejunum enzyme activity of 1-21d broilers (U/g, n ═ 5)
1.1.4 measurement of serum Biochemical indicators
The chickens were killed at 21d and bled, one for each repeat. 5mL of blood is collected for each blood, kept stand at normal temperature to precipitate serum, sent to a second hospital in Zhengzhou city for detection, and used for measuring alanine Aminotransferase (ALT), alkaline phosphatase (ALP), aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH), total protein, albumin and globulin by using a full-automatic blood biochemical automatic analyzer. Glucose, total cholesterol. Triglyceride, high density lipoprotein, and low density lipoprotein.
As can be seen from Table 6, aspartate transferase (AST) in group B is significantly higher than A, D (P <0.05), Lactate Dehydrogenase (LDH) in group A is significantly higher than B, D (P <0.05), low-density lipoprotein cholesterol (LDL-C) in group D is significantly higher than B (P <0.05), and the difference between the group A and the group D is not significant (P >0.05), and the difference between the other indexes in each group is not significant (P > 0.05). The combination of glycyrrhizic acid and lactobacillus casei is proved to reduce aspartate transferase (AST), increase low density lipoprotein cholesterol (LDL-C) in serum and relieve the harm of mycotoxin.
TABLE 6 Effect of different treatment groups on biochemical indices of 1-21d broiler serum (n ═ 5)
1.5 AFB in feces and organs1Content determination, influence of different treatment groups on biochemical indexes of blood of the broiler chicken, and AFB (immune deficiency syndrome) of the broiler chicken in various tissues and organs1Influence of the amount
Taking tissue samples of the same parts of the liver, the pectoralis muscle and the jejunum of broilers in each treatment group, serum and feces, and measuring AFB in the samples by using a baifa kit1The content of (a).
As can be seen from Table 7, AFB was found in feces, serum, liver, pectoralis, and jejunum of group B1Highest residual amount of (A), AFB in group D serum and liver1The content is lower than that of group B (P)>0.05). Shows that the combination of glycyrrhizic acid and lactobacillus casei reduces AFB1Deposition within the body.
TABLE 71-21 d broiler chicken tissue and organ AFB1Content (μ g/kg, n ═ 5)
Note: "- -" indicates no detection. The same applies below.
The results of this example show that: the glycyrrhizic acid and lactobacillus casei can improve daily gain, daily feed intake, jejunum protease activity of broiler chicken, and reduce AFB in tissue and serum1Content of the meat chicken AFB1And (4) toxic symptoms.
Example 3 Effect of the composition for detoxification on broiler liver and jejunum tissue Structure and microflora
Test reagents: formalin solution.
1 Collection of test materials
For the 21d slaughter test, 6 chickens were slaughtered (half of a cock and a half of a mother) per group in 3 treatment groups (A, B, D), the jejunum was taken at the same position of about 1.5cm, the liver was taken at the same position and the weight was 5g, and the tissue was washed with physiological saline and then soaked in 10% formalin. The jejunum contents were placed in 2mL sterile cryovials and stored in liquid nitrogen.
1.1 tissue section preparation and microscopic examination
After the sample is fixed, the sample is sent to a pathology research and development room of the university of agriculture in Henan.
2.1 Effect of different treatment groups on the liver and jejunum tissue Structure of broiler chickens
The tissue section of the liver of the broiler chicken is shown in figure 1, and the liver cell morphology of the control group A is normal. Mycotoxin group B has liver hemorrhage and liver lobular gap broadening. The bleeding area of group D was reduced, and the gap between liver lobules was narrowed, which was close to that of the control group.
The tissue sections of jejunum are shown in fig. 2, and the intestinal villus morphology of control group a is good. Mycotoxin group B has short intestinal villi, and the villi end is fibrotic and disorganized. Group D had longer villi and reduced fibrosis at the periphery of the villi. From table 8, it can be seen that: group D had significantly higher villus height than groups a and B (P <0.05), and group a had significantly higher intestinal villus height than group B (P < 0.05). The glycyrrhizic acid and lactobacillus casei combination can repair the damage of the liver and the intestinal tract and can also improve the height of the villus of the jejunum.
TABLE 8 influence of different treatment groups on the height of the villi (mm) in the jejunum of 1-21d broiler chickens
1.2 determination of intestinal microbial flora of broiler chicken, influence of different treatment groups on intestinal microbial flora of broiler chicken
Samples of jejunal contents were sent to the peninsula pennay biol ltd for determination and analysis of microbial diversity.
The sample sequencing procedure was as follows:
(1) the raw data under high-throughput sequencing is first preliminarily screened for sequence quality.
(2) And dividing the library and the sample of the primary quality-screened original sequence according to index and Barcode information, and removing the Barcode sequence.
(3) Sequence denoising or classification operation unit (OTU) clustering is performed according to QIIME2 dada2 analysis flow.
(4) The specific composition of each sample at the level of different species taxonomies is shown.
(5) And (3) according to the distribution of the OTU in different samples, evaluating the Alpha diversity level of each sample, and reflecting whether the sequencing depth is proper or not through a sparse curve.
(6) And on the OTU level, calculating a distance matrix of each sample, and measuring beta diversity difference and difference significance among different samples by means of sequencing and clustering and combining a corresponding statistical test method.
(7) In the aspect of the composition of the species taxonomy, the species abundance composition difference between different samples is further measured by various sequencing, clustering and modeling means and by combining corresponding statistical test methods, and the seeking of the marker species is attempted.
1.3 data analysis
The test data were analyzed using SPSS 20.0 statistical software and multiple comparisons of groups were performed using the Duncan method, with P <0.05 being significantly different and the results being expressed as mean ± standard deviation. The intestinal microflora statistical method adopts the mothur to calculate the diversity index and the species relative abundance, uses an R language tool to make a community bar chart and a curve chart, and uses One-way ANOVE to analyze the difference of sample components.
1.3.1 cumulative analysis of Alpha Diversity and species abundance, the difference in microbial abundance of jejunal content Alpha Diversity is an index of species in local homogeneous habitat in terms of Richness (Richness), Diversity (Diversity) and uniformity (Evenness), also known as Within-habitat Diversity (Within-habitats). The Chao1 and underserved speces indices indicate species abundance, the Shannon and Simpson indices indicate species diversity, the Faith PD index indicates diversity based on evolution, the pilou evenness index indicates uniformity, and the Good coverage index indicates coverage. As can be seen from Table 9: each group of high-quality sequences has no significant difference (P > 0.05); the Shannon index of group a is significantly higher than that of group B, D (P <0.05), and the Pieloue index of group D is significantly lower than that of group A, B (P < 0.05); group A, Chao1, occupied scenes, Faith PD, were significantly higher than group B (P <0.05), group B was significantly higher than group D (P < 0.05); the coverage rate of each group is more than 99%.
TABLE 9 Alpha diversity analysis
1.3.1.1 flora at phylum level in different treatment groups
As can be seen from Table 10, at the phylum level, it is mainly composed of Proteobacteria (Proteobacteria), Firmicutes (Firmicutes), Bacteroides (Bacteroides) and Actinomycetes (Actinobacteria), with the group A accounting for about 99%, the group B accounting for about 98%, and the group D accounting for about 99%. Proteobacteria (Proteobacteria) and Firmicutes (Firmicutes) are the dominant phylum of the three groups. Group a Firmicutes (Firmicutes) was significantly higher than group B, D (P < 0.05); the relative content of Proteobacteria D (Proteobacteria) is significantly higher than that of the Proteobacteria B and the Proteobacteria A (P < 0.05); the content of Bacteroides (Bacteroides), Actinomycetes (Actinobacillus), Thielavia (Tenericutes), Deferribacteroides (Deferribacteria) and TM7 bacteroides (TM7) in group A is significantly higher than that in group B, D (P < 0.05); the curvularia viridis (Chloroflexi) of the D group is significantly lower than that of the A group (P <0.05), and has no significant difference with the B group (P > 0.05); group D cyanophyta (cyanobacter) was significantly higher than group B, D (P < 0.05). It was demonstrated that the combination of glycyrrhizic acid and Lactobacillus casei increased the number of Proteobacteria (Proteobacteria) and Cyanophyta (Cyanobactria).
TABLE 10 relative abundance of intestinal flora (%) -at phylum-sorted levels for different treatments
1.3.1.2 genus-level flora of different treatment groups
From Table 11, it is understood that at the genus level, Lactobacillus (Lactobacillus), oxalic acid bacterium (Aquobacterium), and neosphingolipid (Novosphingobium) are the dominant bacteria. Lactobacillus (Lactobacillus) is the dominant bacterium in groups A and B, and neosphingolipid (Novosphingobium) is the dominant bacterium in group D. Group D Lactobacillus (Lactobacillus) was significantly lower than group A, B (P < 0.05); group a Streptococcus (Streptococcus) and oscillatoria (noscillus) were significantly higher than group B, D (P < 0.05); group D, Oxalobacter (Aquobacterium), and Aureobacter (Caulobacter) are significantly higher than group A (P <0.05), and numerically higher than group B (P > 0.05). The D groups of neosphingobacterium (Novosphingobium) and Azospirillum (Azospirillum) are significantly higher than the A, B group (P < 0.05). The B group erythromelas (Rubrivivax) was significantly higher than the a and D groups (P < 0.05). Acidovorax and Pseudomonas (Pseudomonas-Pseudomonas) were not significantly different in each group (P > 0.05). It is shown that the combination of glycyrrhizic acid and Lactobacillus casei increases the amount of the new genus Sphingobacterium (Novosphingobium) and decreases the amount of the genus Erythrochilus (Rubivivax).
TABLE 11 relative abundance of intestinal flora (%) -at the Classification level for different treatments
1.3.1.3 different treatment groups at species level of flora
As can be seen from Table 12, at the species level, neosphingobium vesiculosum (Novosphingobium-capsulatum), Lactobacillus vaginalis (Lactobacillus-vagianalis), Lactobacillus helveticus (Lactobacillus-helveticus) and Kurthia gibsonii (Kurthia-gibsonii) are the dominant bacteria. The cystic neosphingosine bacteria (Novosphingobium-capsulatum) in the group D is significantly higher than that in the groups A and B (P < 0.05); group D Lactobacillus helveticus (Lactobacillus-helveticus) and Lactobacillus salivarius (Lactobacillus-salivariaus) are significantly less than group A, B (P < 0.05); group A Kurthia gibsonii (Kurthia-gibsonii), Streptococcus lactis (Streptococcus-alactolyticus), Ruminococcus acis (Ruminococcus-gnavus) and Ralstonia murinus (Rothia-nasimum) were significantly higher than group B, D (P < 0.05). It was demonstrated that glycyrrhizic acid in combination with Lactobacillus casei increased the number of neosphingobium vesiculosum (Novosphingobium-capsulatum).
TABLE 12 relative abundance of intestinal flora (%) at the differential treatment versus classification level
1.3.1.4 correlation analysis of jejunum microbial flora and environmental factors of different treatment groups
Correlation analysis shows that the ADG, jejunum and liver toxin residual quantity and jejunum microbial flora have large correlation. Lactobacillus and Pseudomonas are positively correlated with toxin residue in liver and jejunum, and negatively correlated with ADG, pectoralis and serotonin residue. Due to the combination of glycyrrhizic acid and lactobacillus casei added in the group D, the growth and reproduction of lactobacillus in jejunum are inhibited, so that the number of lactobacillus is low. Toxin residues in serum and pectoralis are positively correlated with the abundance of the genera oxalate (Aquabacterium), neosphingobium (Novosphingobium), petiolus (Caulobacter) and erythropolis (rubivivax), and ADG is negatively correlated therewith.
The chicken fed with mildew corn can damage liver and jejunum of chicken, and reduce jejunum velvetThe height of the hairs. The addition of glycyrrhizic acid and Lactobacillus casei can relieve AFB1Damage to the liver and intestinal tract and inflammatory reactions.
Changes in microflora in the jejunal contents were studied using 16S rRNA gene sequencing. The results show that: mycotoxins break the balance of intestinal flora, resulting in intestinal flora disturbance;
the glycyrrhizic acid and lactobacillus casei are combined, and the proportion of the Rubrivivax and Brevundimonas diminuta is reduced by increasing the neosphingobium vesiculosum (Novosphingobium-capsulatum), so that the disordered intestinal flora environment is repaired, the growth condition of the broiler chicken is improved, and the production performance is improved.
The correlation between ADG, jejunum and liver toxin residual quantity and jejunum microbial flora is large; lactobacillus and Pseudomonas are positively correlated with toxic residues in liver and jejunum, and negatively correlated with residual amounts of ADG, pectoralis and serum toxic. The genus Oxalobacter (AquaBacterium) and the genus Neosphingobium (Novosphingobium) are positively related to the residual amount of toxins in serum and pectoral muscle, and negatively related to ADG. Acidovorax is positively associated with ADG.
The results of the embodiment show that the combination of glycyrrhizic acid and lactobacillus casei can improve the daily gain, daily feed intake and jejunum protease activity of broiler chickens and reduce AFB in tissues and serum1Content of the meat chicken AFB1And (4) toxic symptoms. The richness, diversity and flora structure information of microbial flora are obtained by a 16S rRNA gene sequencing technology, and the result shows that mycotoxin breaks the balance of intestinal flora to cause intestinal flora disorder; the glycyrrhizic acid and lactobacillus casei are combined, and the proportion of the Rubrivivax and Brevundimonas diminuta is reduced by increasing the neosphingobium vesiculosum (Novosphingobium-capsulatum), so that the disordered intestinal flora environment is repaired, the growth condition of the broiler chicken is improved, and the production performance is improved.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. Be used for degrading aflatoxin B1The composition of (1), wherein the active ingredients are glycyrrhizic acid and lactobacillus casei.
2. The composition of claim 1,
the glycyrrhizic acid is 0.01-0.1 wt%, and the concentration of lactobacillus casei is 1 × 106-1×108CFU/mL, and the balance of auxiliary materials or solvents.
3. The composition of claim 2,
the composition comprises 0.04% glycyrrhizic acid, and the concentration of Lactobacillus casei is 1 × 107CFU/mL, and the balance of auxiliary materials or solvents.
4. The composition according to any one of claims 1 to 3, for use in any one of the following (a) to (g),
(a) preparation of degraded AFB1The product of (1);
(b) preparing a product for improving the daily feed intake of chickens and the daily weight gain of the broilers;
(c) preparation for preventing or treating chicken AFB1Products with symptoms of intoxication;
(d) preparing a product for repairing chicken intestinal flora;
(e) preparing a product which increases the bursal neosphingosine bacteria and reduces the proportion of the red longevity bacteria and the shortwave monosomyia defectives;
(f) preparing a product for improving the activity of chicken jejunum protease;
(g) the product for improving the villus height of jejunum is prepared.
5. The use according to claim 4,
the application is shown in degradation of aflatoxin B1。
6. A method of preparing the composition of claim 1, wherein the glycyrrhizic acid, viable lactobacillus casei and adjuvants are mixed to obtain the composition.
7. The method of claim 6,
inoculating the lactobacillus casei into an MRS liquid culture medium, standing and culturing at a constant temperature of 37 ℃ for 24 hours to obtain lactobacillus casei liquid, measuring the viable count by using a plate coating method of the strain, and adjusting the viable count to 1 multiplied by 109CFU/mL。
8. The method of claim 6,
the auxiliary material is a pharmaceutically acceptable carrier.
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| CN112175865A (en) * | 2020-09-29 | 2021-01-05 | 中国农业科学院农产品加工研究所 | Brevundimonas diminuta, application thereof and method for degrading mycotoxin |
| CN115232761A (en) * | 2022-05-31 | 2022-10-25 | 吉林农业大学 | Lactobacillus plantarum capable of improving liver injury and intestinal bacteria imbalance caused by AFB1 |
| CN115261255A (en) * | 2022-05-16 | 2022-11-01 | 西北农林科技大学 | Novel sphingosine bacillus and application thereof |
| CN117925450A (en) * | 2023-12-26 | 2024-04-26 | 黄淮学院 | Composite freeze-dried bacterial powder for protecting liver injury of broiler chickens and application thereof |
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