CN113521115B - 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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- CN113521115B CN113521115B CN202110800794.XA CN202110800794A CN113521115B CN 113521115 B CN113521115 B CN 113521115B CN 202110800794 A CN202110800794 A CN 202110800794A CN 113521115 B CN113521115 B CN 113521115B
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- A—HUMAN NECESSITIES
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
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- A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
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Abstract
The invention discloses an aflatoxin detoxification composition, a preparation method and application thereof, and the active ingredients of the composition are glycyrrhizic acid and lactobacillus casei. The richness, diversity and flora structure information of the microbial flora are obtained by a 16S rRNA gene sequencing technology, and the result shows that mycotoxin breaks the balance of intestinal flora and causes the disorder of the intestinal flora; and the combination of glycyrrhizic acid and lactobacillus casei can reduce the proportion of rhodochrous (Rubrivivax) and Brevibacterium deficiency (Brevundimonas-inter) by increasing the new sphingosine bacteria (Novosphingabium-capsuloum), and repair the disturbed intestinal flora environment, thereby improving the growth condition of broilers and improving the production performance.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an aflatoxin detoxification composition, a preparation method and application thereof.
Background
The first appearance of the word Mycotoxin (Mycotoxin) was that turkeys in the suburban turkey farm in london, united kingdom had just begun to die after two and three months in 1960, and anatomical findings were death due to liver hemorrhagic necrosis and kidney swelling. After inspection analysis, it was found that the cause of the turkey death was that workers fed the mildewed peanut meal to the turkey, which was ultimately identified as Aflatoxin (AF). Aflatoxins are secondary metabolites of aspergillus parasiticus, aspergillus flavus and the like, and are specifically divided into more than ten toxins and more than 20 derivatives, which are respectively named as B 1 、B 2 、G 1 、G 2 Etc.
Aflatoxin B 1 (Aflatoxin B 1 ,AFB 1 ) The toxicity and the distribution are the strongest, which not only leads to immunosuppression and reduces the animal productivity and feed efficiency, but also has stronger cancerogenic, malforming and outburst hazard and even causes animal death. Research shows that bone cancer, rectal cancer, liver cancer, breast cancer and the like are all similar to AFB 1 In connection with this, it was listed as a class i carcinogen in 1993. At present, about 25% of the grains or feeds in the world are contaminated by mycotoxins to different extents, and huge economic losses are caused for animal husbandry every year.
The detoxication method of aflatoxin comprises three methods of physics, chemistry and biology: the physical method comprises the following steps: including adsorption, ultraviolet irradiation, heating, water washing, light irradiation, ion irradiation, etc.; physical adsorbents such as montmorillonite are the most commonly used method, but are phased out due to the disadvantages of desorption and adsorption of nutrients. The chemical method comprises the following steps: including chlorination, oxidation, hydrolysis, and ammonification processes; the chemical treatment has the defects of environmental pollution, easy formation of other harmful substances and the like, and is rarely applied to production.
In view of the above, the existing detoxication agent and method for aflatoxin have the defects of unsafe and harmful substances generation and environmental pollution, and further improvement is 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 object, the present invention provides the following technical solutions:
the invention provides a method for degrading aflatoxin B 1 The active ingredients of the composition are glycyrrhizic acid and lactobacillus casei.
In one embodiment of the present invention, the glycyrrhizic acid has a mass fraction of 0.01-0.1% and the Lactobacillus casei has a concentration of 1×10 6 -1×10 8 CFU/mL, and the balance of auxiliary materials or solvents.
In one embodiment of the invention, the composition comprises 0.04% of glycyrrhizic acid by mass and 1×10 of lactobacillus casei by mass 7 CFU/mL, and the balance of auxiliary materials or solvents.
The invention also provides the use of said composition in any one of the following (a) - (g),
(a) Preparation of degraded AFB 1 Is a product of (a);
(b) Preparing a product for improving daily feed intake of chickens and daily weight gain of broilers;
(c) Preparation of a composition for preventing or treating chicken AFB 1 A product with toxic symptoms;
(d) Preparing a product for repairing intestinal flora of chickens;
(e) Preparing a product for increasing chicken cystic neosphingosine bacteria and reducing the proportion of rhodochrous and Brevundimonas deficiency;
(f) Preparing a product for improving the activity of chicken jejunum protease;
(g) The product for improving jejunum villus height is prepared.
In one embodiment of the invention, the application is manifested in the degradation of aflatoxin B1.
The invention also provides a method for preparing the composition, which comprises the step of mixing glycyrrhizic acid, lactobacillus casei viable bacteria and auxiliary materials to obtain the composition.
In one embodiment of the invention, the lactobacillus casei is inoculated into MRS liquid culture medium, and is subjected to constant temperature static culture for 24 hours at 37 ℃ to obtain lactobacillus casei bacterial liquid, the viable count of the lactobacillus casei is measured by a flat plate coating method by using bacterial strains, and the viable count of the lactobacillus casei is adjusted to 1 multiplied by 10 9 CFU/mL。
In one embodiment of the invention, the auxiliary material is a pharmaceutically acceptable carrier.
In the present invention, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, for example: 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; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters 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.
The 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 by mixing the active ingredient with one or more carriers and then forming it into the desired dosage form.
In the invention, liquorice is an old traditional Chinese medicine and is used as a medicinal plant for more than 2500 years. Glycyrrhizic acid is the effective component of Glycyrrhrizae radix, and belongs to triterpenoid saponins. The main components of glycyrrhizic acid are: glycyrrhizin, glycyrrhizin flavonoids, quercetin, etc. Glycyrrhizic acid has antiviral, anticancer, antiinflammatory, liver protecting, toxic materials removing, and immunity enhancing effects, and has been found to have biological functions such as protecting liver cells, antiinflammatory, and regulating immunity, and has been widely used for clinical treatment of various hepatitis and liver injury.
The invention has the following advantages:
experimental results in this example show that the combination of glycyrrhizic acid and Lactobacillus casei can increase daily weight gain, daily feed intake and jejunum protease activity of broiler chickens, and reduce AFB in tissues and serum 1 Content, relieving AFB of broiler chicken 1 Symptoms of poisoning.
The richness, diversity and flora structure information of the microbial flora are obtained by a 16S rRNA gene sequencing technology, and the result shows that mycotoxin breaks the balance of intestinal flora and causes the disorder of the intestinal flora; and the combination of glycyrrhizic acid and lactobacillus casei can reduce the proportion of rhodochrous (Rubrivivax) and Brevibacterium deficiency (Brevundimonas-inter) by increasing the new sphingosine bacteria (Novosphingabium-capsuloum), and repair the disturbed intestinal flora environment, thereby improving the growth condition of broilers and improving the production performance.
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 will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
Fig. 1 is a diagram of a change disease of a liver tissue structure of a broiler chicken according to an embodiment of the present invention, wherein, note a: a control group; b: negative control group (mildewed corn instead of normal corn); d: negative control group + glycyrrhizic acid + lactobacillus casei;
fig. 2 is a diagram of a change in jejunum tissue structure of a broiler chicken according to an embodiment of the present invention, wherein a: a control group; b: negative control group (mildewed corn instead of normal corn); d: negative control group + glycyrrhizic acid + lactobacillus casei.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test material of the present invention: mildew corn (AFB) commercially available 1 The content of aflatoxin B was 163. Mu.g/kg), and aflatoxin B was carried out as a substrate 1 And (5) screening degrading bacteria. Lactobacillus casei (GIM 1.159) and Bacillus subtilis (GIM 1.715) were purchased from the microorganism strain collection center of Guangdong province, and candida utilis (CGMCC 2.0615) was purchased from the China general microbiological culture collection center, and Lactobacillus acidophilus and Lactobacillus plantarum were selected and stored by the feed biotechnology laboratory of the agricultural university of Henan.
The microbial culture medium of the invention: LB medium: 1g of tryptone, 0.5g of yeast extract powder, 1g of sodium chloride, 100mL of water and pH of 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 medium: 1.5g of tryptone, 1g of yeast extract powder, 2g of glucose, 80 0.1mL,K2HPO 2g g of tween, 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 for 20min at 121 ℃ and 0.15MPa for standby after the volume is fixed.
The test instrument and the consumable of the invention: LDZX-330KBS autoclave (Shanghai Shen An medical instruments factory, shanghai, china), BVM-1000 biological purification bench (su zhou purification equipment limited, su zhou, china), 101 electrothermal blast dryer (beijing, beijing's wep instruments limited), elx800 microplate reader (Bio Tek Instruments, ins, USA), PHS-2C acidometer (Shanghai electrosurgery instruments, shanghai, china), AB204-N electronic balance (Shanghai meler-tolith instruments, shanghai, china), SG-200 plastic sealer (deluge, ningbo, china), PYX-DHS-BS double layer gas bath shaker (Jiangsu jie rayl electric appliance, su, china), PYX-DHS-BS-ii thermostated incubator (Shanghai medical instruments, shanghai, china), R1211 aflatoxin enzyme linked reaction assay kit was purchased from bio-R-Germany, germany.
Example 1 for degradation of aflatoxin B 1 Screening of bacterial species
1. Activation of bacterial species
The laboratory-preserved candida utilis was inoculated into YPD medium and cultured at 30 ℃ under shaking at 180r/min for 24 hours. Bacillus subtilis is inoculated into LB liquid medium and cultured for 24 hours at 37 ℃ under shaking at 180 r/min.
Lactobacillus (Lactobacillus casei, lactobacillus acidophilus, lactobacillus plantarum) was inoculated into MRS liquid medium, and cultured at 37℃for 24h. The number of viable bacteria is uniformly regulated to 1 multiplied by 10 after the number of viable bacteria is measured by a flat plate coating method for strains 9 Colony Formed Unit (CFU)/mL, the number of viable bacteria is expressed by natural log (lg), and the strain is stored in a refrigerator at 4 ℃ for later use.
1.2 screening for degradation of AFB 1 Is a single bacterium of (2)
1.2.1 test design and test grouping
The test culture medium is crushed moldy corn, 72g of moldy corn is weighed and placed in a 1L beaker during fermentation, 27mL of distilled water is measured and mixed with 1mL of single bacterial liquid uniformly, the mixture is added into the moldy corn and stirred uniformly, the mixture is packaged into self-sealing bags, the self-sealing bags are sealed by a sealing machine, each test group is three times, each test group is equal in weight, each test group is cultured for 3 days at the temperature of 30 ℃ and the temperature of 37 ℃ respectively, and a group of blank control groups (3 times) without inoculating bacteria are additionally arranged.
1.2.2 test index determination
AFB in post-fermentation mildewed corn 1 Is determined by: on 3d, 10g of corn samples were weighed in duplicate units into 100mL Erlenmeyer flasks containing 50mL of 70% methanol, and the samples were assayed for AFB using the kit with shaking for 30 minutes to extract toxins 1 Is contained in the composition.
2. Test results
2.1 different microorganism pairs aflatoxin B 1 Is to be degraded by
As can be seen from Table 1, lactobacillus casei group AFB 1 The content is significantly lower than other groups (P<0.05 Lactobacillus casei against AFB) 1 The degradation rate is significantly higher than that of other groups (P<0.05 A degradation rate of 76.26%; thus, lactobacillus casei was selected in the subsequent experiments.
TABLE 1 degradation of aflatoxin B1 by different microorganisms
Note that: the viable count of the microorganisms in the table was adjusted to 1X 10 7 CFU/mL。
This example screens for p-AFB from a plurality of microorganisms 1 Lactobacillus casei with higher degradation rate for AFB 1 The degradation rate reaches 76.26 percent.
Example 2 use of detoxification composition
1 feeding test design and grouping
The experiment was performed on a Henan agricultural university Hechang test base with 200 healthy AA broilers 1d, randomly divided into 4 treatment groups of 5 replicates each, 10 chickens each. The feeding period was 21d. Adopting multilayer cage culture, freely feeding, and illuminating for 24 hours in the first week (23 hours of illumination and 1 hour of darkness after one week); the temperature and humidity are controlled according to the conventional feeding management flow; the immunization procedure was: at 7d, broilers were immunized with new-arm bivalent seedlings. The ration, feed formulation and nutrient levels were formulated according to the broiler feeding criteria of NRC (1994) and are specifically shown in table 2. The test groups were as follows:
group A: basic ration (positive control, AFB in ration) 1 3.04μg/kg)；
Group B: daily ration containing mildewed corn (mildewed corn replaces normal 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 0.04%)
Group D: negative pairGroup + glycyrrhizic acid + lactobacillus casei (drinking water, concentration of glycyrrhizic acid in drinking water is 0.04%, concentration of lactobacillus casei in drinking water is 1×10) 7 CFU/mL)
Table 2 composition and nutrient level of broiler 1-21d ration (%), air-dried basis
Note that: premix: each kilogram of daily ration contains: vitamin A12000 IU; vitamin E20 IU; vitamin D3 3000IU; vitamin K3.0 mg; vitamin B6.5 mg; vitamin B1.0 mg; vitamin B12.01 mg; iron (ferrous sulfate) 100mg; copper (copper sulfate) 8mg; 60mg of zinc (zinc oxide); iodine (calcium iodate) 0.45mg; selenium (sodium selenite) 0.35mg; 80mg of manganese (manganese sulfate); 10mg of calcium pantothenate and 0.15mg of biotin; 6mg of riboflavin; folic acid 1.25mg; nicotinic acid 35mg. Except that the crude protein, calcium and phosphorus indices are measured values, the remaining values are calculated values. The following is the same.
1.1 measurement index and method
1.1.1 measurement of the productivity, the influence of different treatment groups on the growth performance of broilers
Each repeat feed intake was recorded daily, weighing at 1d, 21d, 42d, respectively. The death status of each duplicate chicken was recorded. Average daily feed intake (Average daily feed intake, ADFI) and average daily gain (Average daily gain, ADG) were calculated.
As seen from table 3, group a had significantly higher growth performance than the other groups (P < 0.05), the other groups had insignificant differences in growth performance (P > 0.05), but group C and D had higher broiler weights, average daily gain, and feed ratios than group B (P > 0.05); group C mortality was 4% at maximum. The method shows that the mildewed corn reduces the growth performance of the broiler chickens, and the combination of glycyrrhizic acid and lactobacillus casei can improve daily gain and daily feed intake and relieve mycotoxin poisoning symptoms of the broiler chickens.
Table 3 effect of different treatment groups on 1-21d broiler growth performance (g, n=5)
1.1.2 determination of the nutrient metabolism Rate, influence of different treatment groups on the nutrient metabolism Rate of broilers
Collecting manure samples for 18-20d for three days by using a total manure collecting method, fixing nitrogen in the manure samples with 10% sulfuric acid every day, preserving at-20 ℃, uniformly mixing the manure samples for three days after the test is finished, drying and conditioning at 65 ℃ for 24 hours, and crushing to measure indexes of Crude Protein (CP), crude fat (EE), calcium (Ca) and phosphorus (P). Crude Protein (CP) is measured by adopting national standard CB/T6432-94; crude fat (EE) is measured by national standard GB/T6433-2006; calcium (Ca) is measured by adopting an ethylenediamine tetraacetic acid disodium complexometric titration method; phosphorus (P) was measured using national standard GB/T6437-2002.
As can be seen from table 4, the group a crude protein, crude fat, calcium and phosphorus metabolism rates were significantly higher than the other groups (P < 0.05), and the group D calcium metabolism rates were significantly lower than the other groups (P < 0.05). The mildew corns are shown to reduce the nutrient metabolism rate of broilers, and the addition of the mycotoxin antidote does not significantly improve the nutrient metabolism rate.
TABLE 4 influence of different treatment groups on the metabolic rate of broiler nutrients (%)
1.1.3 determination of the Activity of the digestive enzymes of the jejunum of broiler chickens
At 21d, 1 chicken was slaughtered for each repeat, the jejunal contents were placed into sterile centrifuge tubes and rapidly transferred to liquid nitrogen and finally placed into-80 ℃ for storage.
Measurement of protease Activity
Measured with reference to the national standard (SB/T10317-1999).
Enzyme activity unit: 1mg tyrosine produced per minute was 1 protease activity unit (U).
The calculation formula is as follows: enzyme activity (U/g) =tyrosine content/reaction time x 4 x dilution of sample/sample weight.
(2) Determination of amylase activity
Measured by a DNS method.
Enzyme activity unit: the amount of enzyme used to produce 1mg of glucose per minute is one enzyme activity unit (U).
The calculation formula is as follows: amylase activity (U/g) =sample reducing sugar content x total volume of sample dilution x dilution fold/sample weight/enzyme volume used for assay/5 min.
Influence of different treatment groups on jejunum digestive enzyme activity of broiler chickens
As can be seen from table 5, group D protease activity was significantly higher than group a (P < 0.05); the differences in amylase force were not significant for each group (P > 0.05). Indicating that the combination of glycyrrhizic acid and lactobacillus casei improves the jejunum protease activity of the broiler chickens.
TABLE 5 influence of different treatment groups on jejunal enzyme activity of 1-21d broilers (U/g, n=5)
1.1.4 determination of serum Biochemical index
The chickens were sacrificed at 21d for blood collection, one for each repeat. Each blood sample is taken by 5mL, and the blood is stood at normal temperature to separate out serum, and then the serum is sent to a second people hospital in Zhengzhou city for detection, and the glutamic-pyruvic transaminase (ALT), alkaline phosphatase (ALP), glutamic-pyruvic transaminase (AST), lactic Dehydrogenase (LDH), total protein, albumin and globulin are measured by using a full-automatic blood biochemical automatic analyzer. Glucose, total cholesterol. Triglyceride, high density lipoprotein, low density lipoprotein content.
As can be seen from table 6, group B aspartate transferase (AST) was significantly higher than group A, D (P < 0.05), group a Lactate Dehydrogenase (LDH) was significantly higher than group B, D (P < 0.05), group D low density lipoprotein cholesterol (LDL-C) was significantly higher than group B (P < 0.05), the difference from group a was not significant (P > 0.05), and the differences from other indicators were not significant (P > 0.05). It is shown that the combination of glycyrrhizic acid and lactobacillus casei reduces aspartate transferase (AST), increases low density lipoprotein cholesterol (LDL-C) in serum, and relieves the damage of mycotoxin.
Table 6 effect of different treatment groups on 1-21d broiler serum biochemical index (n=5)
1.5 fecal matter and AFB in organs 1 Content determination, influence of different treatment groups on blood biochemical indexes of broiler chickens, and influence of different treatment groups on AFB (alpha-fetoprotein) in tissues and organs of broiler chickens 1 Influence of the content
Taking tissue samples of the same parts of liver, pectoral muscle and jejunum of each treatment group of broiler chickens, serum and feces, and measuring AFB in the samples by using Bayer hair kit 1 Is contained in the composition.
As is clear from Table 7, AFB was found in the stool, serum, liver, pectoral muscle, jejunum of group B 1 The highest residual amount of AFB in group D serum and liver 1 The content is lower than that of group B (P)>0.05). Indicating that the combination of glycyrrhizic acid and Lactobacillus casei reduced AFB 1 Deposition within the body.
Table 7 1-21d AFB in tissues and organs of broiler chickens 1 Content (μg/kg, n=5)
Note that: "-" indicates undetected. The following is the same.
The results of this example show that: the combination of glycyrrhizic acid and lactobacillus casei can improve daily gain of broiler chickens, daily feed intake and jejunum protease activity, and reduce AFB in tissues and serum 1 Content, relieving AFB of broiler chicken 1 Symptoms of poisoning.
Example 3 Effect of composition for detoxification on liver and jejunum tissue structures of broiler chickens and microflora
Test reagent: formalin solution.
1 acquisition of test Material
In the 21d slaughter test, 6 chickens were slaughtered (male and female halves) in 3 treatment groups (A, B, D), the same jejunum was taken at about 1.5cm, the same liver was taken at the same site and the weight was 5g, and the tissue was immersed in 10% formalin after washing with physiological saline. The jejunal contents were placed in 2mL sterile cryopreservation tubes and stored in liquid nitrogen.
1.1 tissue section preparation and microscopic observations
After the sample is fixed, the sample is sent to a pathology teaching and research room of Henan agricultural university for detection.
2.1 Effect of different treatment groups on liver and jejunum tissue structures of broilers
The tissue section of the liver of the broiler chicken is shown in fig. 1, and the liver cells of the control group A are normal in morphology. Mycotoxin group B liver bleeding and liver lobular gap broadening. Group D bleeding sites decreased and hepatic lobular spaces narrowed, approaching the control group.
The jejunum tissue sections are shown in FIG. 2, and the intestinal villi morphology of control group A is good. The group B mycotoxins have short intestinal villi, and peripheral villi fibrosis and disorder. Group D had longer villi and reduced peripheral fibrotic symptoms of villi. As can be seen from table 8: group D villus height was significantly higher than groups a and B (P < 0.05), and group a intestinal villus height was significantly higher than group B (P < 0.05). It is shown that the combination of glycyrrhizic acid and lactobacillus casei not only can repair liver and intestinal injury, but also can improve jejunum villus height.
TABLE 8 influence of different treatment groups on jejunal villus height (mm) of 1-21d broiler chickens
1.2 determination of intestinal microbiota of broiler chickens, influence of different treatment groups on intestinal microbiota of broiler chickens
Jejunal content samples were sent to Qingdao Paeno biosystems, inc. for determination and analysis of microbial diversity.
The sample sequencing procedure was as follows:
(1) First, the original data under high-throughput sequencing is primarily screened according to the sequence quality.
(2) And (3) carrying out library and sample division on the original sequence of the quality primary screen according to index and Barcode information, and removing the Barcode sequence.
(3) Sequence denoising or classification operation unit (Operational taxonomic unit, OTU) clustering was performed according to QIIME2 dada2 analysis procedure.
(4) The specific composition of each sample at the different species taxonomic level is shown.
(5) According to the distribution of OTU in different samples, the Alpha diversity level of each sample is evaluated, and whether the sequencing depth is proper or not is reflected by a sparse curve.
(6) At the OTU level, calculating the distance matrix of each sample, and measuring the beta diversity difference and the difference significance among different samples by means of sequencing and clustering and by combining a corresponding statistical test method.
(7) At the aspect of species taxonomy composition, the species abundance composition difference between different samples is further measured by various sequencing, clustering and modeling means and by combining corresponding statistical inspection methods, and searching for a marker species is attempted.
1.3 data analysis
The experimental data were analyzed using SPSS 20.0 statistical software and multiple comparisons were performed on each group using Duncan's method, with P <0.05 being significant differences, and results expressed as mean.+ -. Standard deviation. The intestinal tract microbiota system counting method adopts mothur to calculate the diversity index and the relative abundance of species, uses R language tool to make community histogram and graph, uses One-way ANOVE to analyze the difference of sample components.
1.3.1 Alpha Diversity and species abundance cumulative analysis, the differential microbial abundance of jejunal content Alpha Diversity refers to an index of species in local uniform habitat in terms of Richness (Richness), diversity (Diversity), uniformity (Evenness) and the like, also known as intrahabitat Diversity (Within-habitat Diversity). The Chao1 and sampled patterns index represents species richness, shannon and Simpson indices represent species diversity, the Faith PD index represents evolution-based diversity, the Pielou eveness index represents uniformity, and the Good coverage index represents coverage. As can be seen from table 9: the difference between the quality sequences of each group is not significant (P > 0.05); group a Shannon index is significantly higher than group B, D (P < 0.05), group D Pieloue index is significantly lower than group A, B (P < 0.05); group a Chao1, sampled patterns, 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 over 99 percent.
TABLE 9 Alpha diversity analysis
1.3.1.1 different treatment groups were at the phylum level
As is clear from Table 10, at the gate level, it is mainly composed of Proteus (Proteus), fimbristylis (Firmides), bacteroides (Bacteroides) and Actinobacillus (Actinobacillus), and the group A accounts for about 99%, the group B accounts for about 98% and the group D accounts for about 99%. Proteus (Proteus) and Thick-walled (Firmics) are dominant bacteria in the three groups. Group a Firmicutes was significantly higher than group B, D (P < 0.05); group D Proteobacteria (Proteobacteria) relative content is significantly higher than group B and group a (P < 0.05); the content of bacteroides group A (bacterioides), actinomycota (actinomycetes), actinomycota (tendrils), deironickettsia (deisobacteriaceae), and TM7 (TM 7) is obviously higher than that of B, D groups P < 0.05); group D green curved mycota (Chloroflexi) was significantly lower than group a (P < 0.05), with no significant difference from group B (P > 0.05); group D Cyanobacteria (Cyanobacteria) was significantly higher than group B, D (P < 0.05). Illustrating that the combination of glycyrrhizic acid and Lactobacillus casei increases the numbers of Proteus (Proteus) and cyanobacterium (Cyanobacter).
Table 10 relative abundance of intestinal flora at the different treatment vs. portal classification level (%)
1.3.1.2 different treatment groups were bacterial groups at the genus level
As is clear from Table 11, lactobacillus (Lactobacillus), oxalic acid bacteria (Aquabacterium) and neosphingolipid bacteria (Novosphingabium) are dominant bacteria at the genus level. Lactobacillus (Lactobacillus) is the dominant bacterium in groups A and B, and Novosphingabium (Novosphingabium) 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 spiralis (Noscillospira) are significantly higher than group B, D (P < 0.05); group D oxalate bacteria (Aquabacterium), acetobacter (Caulobacter) were significantly higher than group A (P < 0.05), numerically greater than group B (P > 0.05). Group D neosphingobacteria (novospungbium), azospirillum (Azospirillum) were significantly higher than group A, B (P < 0.05). Group B rhodochrous (rubrovax) was significantly higher than groups a and D (P < 0.05). The difference between the Acidovorax and Pseudomonas (Pseudomonas-pseudomonas) groups was not significant (P > 0.05). It was demonstrated that the combination of glycyrrhizic acid and Lactobacillus casei increased the number of novel Sphingobium (Novosphingabium) and decreased the number of Rhodotorula (Rubrivivax).
Table 11 relative abundance of intestinal flora on the genus classification level for different treatments (%)
1.3.1.3 different treatment groups were bacterial groups at the seed level
As is clear from Table 12, at the seed level, there are mainly Sphingomonas capsulatum, lactobacillus vaginalis, lactobacillus helveticus and Ji Shiku T.kurthia-gibsonii. Group D cystic neosphingobacteria (novospungbium-capsulosum) were significantly higher than groups a and B (P < 0.05); group D Lactobacillus helveticus (Lactobacillus-helveticus) and Lactobacillus salivarius (Lactobacillus-salivarius) were significantly smaller than group A, B (P < 0.05); group a Ji Shiku termitid (Kurthia-gibsonii), streptococcus lactis (Streptococcus-lactis), ruminococcus livens (Ruminococcus ] -gnavus) and ross murine (Rothia-nasamuum) were significantly higher than group B, D (P < 0.05). Illustrating that the combination of glycyrrhizic acid and Lactobacillus casei increases the number of Sphingomonas capsulata (Novosphingabium-capsulosum).
Table 12 relative abundance of intestinal flora at different treatment vs. species classification levels (%)
Correlation analysis of jejunal microbiota and environmental factors for different treatment groups 1.3.1.4
The correlation analysis shows that the residual amounts of ADG, jejunum and hepatotoxin are related to jejunum microbial flora. Lactobacillus and Pseudomonas are positively correlated with toxin residues in liver and jejunum, and are negatively correlated with ADG, pectoral muscle and serum toxic residue. The combination of glycyrrhizic acid and lactobacillus casei added in group D inhibits the growth and reproduction of lactobacillus in jejunum, so that the lactobacillus amount is low. Toxin residues in serum and pectoral muscles are positively correlated with the abundance of oxalic acid bacteria (aquabacteria), neosphingobacteria (novospungbium), sessile (callober) and rhodochrous (rubrovivax), and ADG is negatively correlated therewith.
The liver and jejunum of the broiler chickens can be damaged by feeding the mildewed corns to the broiler chickens, and the jejunum villus height is reduced. The addition of glycyrrhizic acid and Lactobacillus casei combination can alleviate AFB 1 Injury to the liver and intestinal tract and inflammatory response.
The changes in microflora in jejunal contents were studied using 16S rRNA gene sequencing. The results show that: mycotoxins disrupt the intestinal flora balance, resulting in a disturbance of the intestinal flora;
the combination of glycyrrhizic acid and lactobacillus casei can reduce the ratio of rhodochrous (Rubrivivax) to Brevundimonas deficiency (Brevundimonas-inter) by increasing the amount of new sphingosine bacteria (Novosphingabium-capsulosum), and repair the disturbed intestinal flora environment, thereby improving the growth condition of broilers and improving the production performance.
The residual amount of ADG, jejunum and liver toxins is related to jejunum microbial flora; lactobacillus and Pseudomonas are positively correlated with residual amounts of toxin in the liver and jejunum, and negatively correlated with residual amounts of toxin in ADG, pectoral muscle and serum. Oxalic acid bacteria (aquabacteria) and neosphingobacteria (Novosphingobium) are positively correlated with the residual amount of toxins in serum and pectoral muscles, and negatively correlated with ADG. Acidovorax (Acidovorax) is positively associated with ADG.
The results of this example show that the combination of glycyrrhizic acid and Lactobacillus casei can increase daily gain, daily feed intake and jejunum protease activity of broiler chickens, and reduce AFB in tissues and serum 1 Content, relieving AFB of broiler chicken 1 Symptoms of poisoning. The richness, diversity and flora structure information of the microbial flora are obtained by a 16S rRNA gene sequencing technology, and the result shows that mycotoxin breaks the balance of the intestinal flora and causes the disorder of the intestinal flora; and the combination of glycyrrhizic acid and lactobacillus casei can reduce the proportion of rhodochrous (Rubrivivax) and Brevibacterium deficiency (Brevundimonas-inter) by increasing the new sphingosine bacteria (Novosphingabium-capsuloum), and repair the disturbed intestinal flora environment, thereby improving the growth condition of broilers and improving the production performance.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (7)
1. Be used for degrading aflatoxin B 1 The active ingredients of the composition are glycyrrhizic acid and lactobacillus casei GIM1.159;
the glycyrrhizic acid has a mass fraction of 0.01-0.1%, and the Lactobacillus casei GIM1.159 has a concentration of 1×10 6 -1×10 8 CFU/mL, and the balance of auxiliary materials or solvents.
2. The composition of claim 1, wherein the composition comprises,
the composition has a mass fraction of glycyrrhizic acid of 0.04%, and a concentration of Lactobacillus casei of 1×10 7 CFU/mL, and the balance of auxiliary materials or solvents.
3. The composition of claim 1 or 2, for use in any one of the following (a) - (g),
(a) Preparation of degraded AFB 1 Is a product of (a);
(b) Preparing a product for improving daily feed intake of chickens and daily weight gain of broilers;
(c) Preparation of a composition for preventing or treating chicken AFB 1 A product with toxic symptoms;
(d) Preparing a product for repairing intestinal flora of chickens;
(e) Preparing a product for increasing chicken cystic neosphingosine bacteria and reducing the proportion of rhodochrous and Brevundimonas deficiency;
(f) Preparing a product for improving the activity of chicken jejunum protease;
(g) The product for improving jejunum villus height is prepared.
4. The use according to claim 3, wherein,
the application is shown by degradation of aflatoxin B 1 。
5. A process for preparing the composition of claim 1, wherein said glycyrrhizic acid, lactobacillus casei viable bacteria and adjuvants are mixed to obtain said composition.
6. The method of claim 5, wherein,
inoculating the Lactobacillus casei into MRS liquid culture medium, standing at 37deg.C for 24 hr to obtain Lactobacillus casei bacterial liquid, measuring viable count by plate coating method, and adjusting the viable count to 1×10 9 CFU/mL。
7. The method of claim 5, wherein,
the auxiliary materials are pharmaceutically acceptable carriers.
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