CN103146633A

CN103146633A - Clone and culture of lead-addictive bacteria

Info

Publication number: CN103146633A
Application number: CN2013100745433A
Authority: CN
Inventors: 赵宏坤; 满兆红; 都启晶; 姜彦君; 范颖华; 李凤梅
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2013-03-08
Filing date: 2013-03-08
Publication date: 2013-06-12

Abstract

The invention obtains probiotic lactic acid bacteria with lead adsorption property through the research of the adsorption in vitro and in the body after separating and screening the lactic acid bacteria with lead resistance and lead adsorbability from the animal intestines and excrements. Due to the lead adsorption property of the obtained lactic acid bacteria, the rabbit in vivo experiments prove that the bacteria are capable of reducing the lead in the bodies and have a lead-excreting and detoxicating function for promoting the excretion of blood lead and visceral lead without complexing side effects, and thus resulting in no loss of microelements such as calcium, iron and zinc. The stain, which takes the lactic acid bacteria as one of the normal flora, is relatively obvious in physiological benefit and good in stability without pathogenicity, and is capable of reducing the lead content in the animal and human bodies by being regarded as feeds or food additives.

Description

Clone and the cultivation of plumbous bacterium had a liking in one strain

Technical field

The present invention relates to the screening of the plumbous bacterial strain of efficient adsorption, PCR method identifies, the purpose bacterium is the mensuration of adsorption efficiency in environment in vivo and in vitro, animal lead poisoning modeling, and the ICP-MS method is measured Pb ²⁺Content, the milk-acid bacteria adsorption efficiency that screens is high, and the detection method detection limit is low, and is highly sensitive.

Background technology

Environmental persistence pollutent especially heavy metal is on the rise to the pollution of air, water source and soil and becomes indisputable fact.The drinking-water in serious pollution zone, the farm crop such as vegetables, fruit and grain, and in animal-derived food, heavy metal content exceeds standard unavoidable.How impedance and reduce heavy metal in animal and human's body to accumulate with harm be the key subjects that need to be resolved hurrily.Therefore, our team has carried out this research.

Purpose screening technology and the Rapid Identification traditional with identifying employing combines, and uses the milk-acid bacteria selective medium, and adds therein the Pb of different concns ²⁺, preliminary screening goes out to have the bacterial strain of high resistance lead and high absorbability, through biochemical identification test kit and 16SrDNA sequence analysis, the bacterial strain of screening is identified.Universal primer is used in the 16SrDNA amplification, and pcr amplified fragment is about 1500bp, called after Q dk/m-1

In the purpose bacterium adsorption test in vivo and in vitro of screening, adsorption efficiency is high, vitro Adsorption test simulation gastrointestinal tract environment.And body internal adsorption test uses rabbit as subjects, is at first the lead poisoning modeling stage, after the modeling success, according to the weight of animals plumbous scavenging agent of feeding.

Compare with atomic absorption spectrometry, atomic fluorescence spectroscopy, atomic emission spectrometry, inductively coupled plasma mass spectrometry (ICP-MS) is measured Pb ²⁺Detection limit is low, sensitivity and precision is high, selectivity is good and can detect simultaneously multielement, is considered to the sensitiveest detection method at present, has been widely used in the analysis and research in the fields such as food, environment, medicine, biological sample.

Summary of the invention

The technical problem to be solved in the present invention is to filter out the milk-acid bacteria that a strain has the plumbous performance of efficient adsorption, for developing a kind of plumbous opposing probiotics preparation, effectively reduces Pb ²⁺Accumulation at animal and human's body provides tachnical storage.

Technical scheme of the present invention is to filter out the milk-acid bacteria of plumbous resistance in the body of animal, through a series of biochemical identification, utilizes universal primer to carry out pcr amplification and identifies.Add in animal-feed by fermentation culture, measure it to scavenging(action) plumbous in animal body, with the Pb in ICP-MS method mensuration blood, ight soil, muscle, viscera tissue ²⁺

Description of drawings

Accompanying drawing is the clone of plumbous resistance milk-acid bacteria of the present invention and cultivates schema, embodiment:

1. anti-plumbous lactic acid screening

Aseptic collection animal intestinal and faecal samples utilize traditional bacterium separation technology to exist

Separate in MRS milk-acid bacteria selective medium, at first containing different Pb ²⁺Conscientious anti-lead domestication in the MRS liquid nutrient medium of concentration is selected to filter out anti-lead concentration greater than the bacterial strain of 400mg/l.

2. the screening of plumbous efficient adsorption bacterial strain

The anti-plumbous bacterial strain of screening is carried out adsorption efficiency measure, the purpose bacterium is inoculated in contains Pb ²⁺Concentration is in the MRS liquid nutrient medium of 100mg/l 37 ℃, measures Pb after 140r/min vibration 6h ²⁺Content.

3. the biochemical identification of purpose bacterial strain

Adopt milk-acid bacteria biochemical identification test kit to carry out biochemical identification, the bacterium of picking one ring activation

Strain is inoculated in Reagent Tube, inserts on cystose, cultivates 24h in 37 ℃ of incubators.

4. the milk-acid bacteria genome extracts

The purpose bacterium that preserves is activated in LB, adopt the CTAB/NaCl method to extract milk-acid bacteria

DNA。By the SDS lysing cell, proteasome degradation albumen, CTAB Polysaccharide removing composition.

5. amplimer

Universal primer is adopted in the 16SrDNA amplification, and forward primer is 27f:

5 '-AGAGTTTGATCCTGGCTCAG-3 '; Reverse primer 1541r:5 '-AAGGAGGTGATCCAGCC-3 '.

6.PCR amplification

Carry out pcr amplification under the PCR reaction conditions of optimizing, the DNA product that to obtain one section size be 1500bp.

Reaction conditions is 94 ℃ of denaturations, 5min; Sex change: 94 ℃, 1min, annealing: 58 ℃, 1min, extend: 72 ℃, 2min, 30 circulations; Extend: 72 ℃, 10min, 4 ℃ of preservations.

25ul PCR reaction system, 10 * PCR Buffer, 2.5 μ L, the whole amount of substance of dNTP is 2pmol, and the whole amount of substance of Mg2+ is 32.5pmol, and 1.25U Taq enzyme, primer 1, the whole amount of substance of primer 2 primer are 5pmol.

7. vitro Adsorption test

The absorption environment of simulation simulated gastric fluid and simulated intestinal fluid carries out Pb ²⁺Adsorption test.

8. animal vivo test

The selection rabbit is subjects, the poisoning modeling test of advanced quadrat, and after the modeling success,

Carry out plumbous antagonistic effect, every morning the 9:00 5ml streptococcus acidi lactici fermented solution of feeding, cell concentration reaches 10 ⁹Cfu/ml, the tap water of control group fed same dose.Carry out the arteria auricularis blood sampling in every 3 days, measure blood lead content, and gather the excretion that ight soil is measured lead.Feed after end, put to death and gather blood, ight soil, muscle, bone, internal organ resistance tissue test lead amount, the interior elimination efficiency of the body of judgement streptococcus acidi lactici fermented solution.

9. Specimen eliminating

Each sample all adopts Wet, and accurately weighing sample 0.1000-0.5000g, use

Mixing acid (nitric acid: perchloric acid=5:1) clear up.

10.ICP-MS measure Pb ²⁺Concentration

7700X inductively coupled plasma mass spectrometry (ICP-MS) sensitivity and tolerance range is high, detection limit is low is considered to the sensitiveest detection method at present, has been widely used in the analysis and research in the fields such as food, environment, medicine, biological sample.

By ICP-MS measure dye plumbous before blood lead concentration be 3.1304mg/l, lead poisoning modeling success blood lead concentration is 7.2992mg/l, feeding, blood lead concentration is reduced to 2.136mg/l after the milk-acid bacteria of this laboratory screening, in body, clearance rate is 70.74% with comparing after modeling, and after the antagonist of feeding blood lead concentration lower than the concentration of dying before plumbous, therefore, the anti-plumbous bacterial strain of this strain can effectively be removed the heavy metal lead in body.

In addition, the mensuration of internal organs lead content shows, compares with control group, and in liver, lead content reduces the most obviously, also decrease in kidney, but not obvious.

Claims

1. the milk-acid bacteria with heavy metal lead adsorptivity of Screening and Identification, to screen from the enteron aisle of chicken and movement thereof by traditional bacteria screening method to obtain, screen from animal intestinal due to this bacterial strain and obtain, good stability, no pathogenicity, can be in the animal intestinal advantage such as grow decided at the higher level but not officially announced, reduce entering that in enteron aisle, heavy metal lead is absorbed and accumulates in the in-vivo tissue internal organs by body;

Method is as follows:

Step 1, the domestication of anti-plumbous bacterial strain, tradition screening

Step 2, the plumbous bacterial strain screening of efficient adsorption

Step 3, design of primers

Step 4, pcr amplification

Step 5, the preparation of adsorption liquid

Step 6, the experiment of animal body internal adsorption

Step 7, the ICP-MS method is measured lead content

The characteristics of this bacterial strain be anti-lead concentration high, stablize, adsorption efficiency is high, safe.

2. tame according to claim 1 and screening method, the purpose bacterium of screening is milk-acid bacteria, so opt lactic acid bacteria selective medium MRS agar, its method is at first to cultivate in containing the plumbous MRS liquid nutrient medium of lower concentration (50mg/l), increase successively again concentration and tame 100,150,200mg/l, the anti-plumbous bacterial strain of domestication are the white circular bacterium colony on leaded MRS agar plate; The anti-plumbous bacterial strain of separation and purification is seeded in respectively and contains Pb ²⁺In the liquid nutrient medium of 0,250,300,350,400mg/l, the Pb that the purpose bacterial strain can tolerate ²⁺〉=400mg/l.

3. screening method according to claim 1 is by measuring bacterial strain in substratum

And in Imitative gastroenteric environments to the absorption of lead, filter out the purpose bacterium of efficient adsorption, to be the anti-plumbous bacterial strain of screening be inoculated according to 1% inoculum size its method cultivates 18-24h in the MRS liquid nutrient medium and reach the growth logarithmic phase, get the centrifugal 10min of 2ml nutrient solution 12000r/min and collect thalline, bacterial sediment joins respectively the 10mlMRS liquid nutrient medium, in the liquid of 10ml Imitative gastroenteric environments, 37 ℃, centrifugal taking-up thalline after 140r/min shaking table absorption 2h, supernatant liquor is measured plumbous concentration after being settled to 50ml;

The hydrochloric acid 16.4ml of the compound method of simulated gastric fluid: 9.5%-10.%, thin up makes PH reach 3.0;

Add the 1.0g stomach en-in every 100ml solution, mixing is with the aseptic membrane filtration of 0.2um;

The compound method of simulated intestinal fluid: take potassium primary phosphate 6.8g, add water 500ml dissolving, the sodium hydroxide solution with 0.4% is transferred PH to 6.8, is diluted with water to 1000ml, adds 1.0g trypsinase mixing in every 100ml liquid, with the aseptic membrane filtration of 0.2um.

4. screening method according to claim 1, universal primer is adopted in the 16SrDNA amplification, and forward primer is 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 '; Reverse primer 1541r:5 '-AAGGAGGTGATCCAGCC-3 '.

5. PCR reaction conditions according to claim 1, is characterized in that under this reaction conditions, the expanding effect of goal gene is fine, and described reaction conditions is 94 ℃ of denaturations, 5min; Sex change: 94 ℃, 1min, annealing: 58 ℃, 1min, extend: 72 ℃, 2min, 30 circulations; Extend: 72 ℃, 10min, 4 ℃ of preservations; In 25ul PCR reaction system, 10 * PCR Buffer, 2.5 μ L, the whole amount of substance of dNTP is 2pmol, and the whole amount of substance of Mg2+ is 32.5pmol, and 1.25U Taq enzyme, primer 1, the whole amount of substance of primer 2 primer are 5pmol.

6. the preparation of adsorption liquid according to claim 1, method are to be inoculated in LB meat soup by 1% inoculum size, and 37 ℃, the concentration that the 140r/min shaking table is cultured to thalline is 10 ⁹Cfu/ml saves backup in 4 ℃.

7. animal body internal adsorption experiment according to claim 1, due to the rabbit strong adaptability, be difficult for sick, the factors such as reasonable price, therefore the selection rabbit is experimental subjects, its body weight is 2.21 ± 0.2kg, feed and observe healthy rear random packet a week, 6 every group, carry out the lead poisoning modeling, according to the 3mg/kg body weight, the feed plumbic acetate solution of 5mg/ml, in the modeling process, blood lead content is measured in arteria auricularis blood sampling in every 3 days, does not cause the considerable change of rabbit body weight, do not reduce its food utilization, stop plumbous contamination during to Pb-B＞600ug/l;

After the modeling success, appoint the lead stamp of so feeding to intend every day by the lead in diet and drinking-water absorption body, simultaneously, every morning, 9:00 was according to half of rabbit food consumption, feed plumbous scavenging agent (milk-acid bacteria adsorption liquid) 5ml/d of various dose of experimental group, the tap water of control group fed same dose, and then add sufficient feed, blood lead concentration is measured in each all arteria auricularis blood sampling in scavenging process, and measures the ight soil lead amount; Put to death half for every group after 3 weeks, gather blood, ight soil, liver, heart, kidney, brain, muscle, fat test lead content.

8. in Determination of Pb according to claim 1, sample adopts 7700X inductively coupled plasma mass spectrometry (ICP-MS) through after Wet, and ICP-MS method detection limit is low, disturb less, highly sensitive; The coefficient R of lead element calibration curve reaches 0.9999, and method detects and is limited to 0.02205ppb.