Preliminary Test on a New Type of Antibacterial Substance

Lingxiao YI,Zhiyong CHEN,Wei DONG

1.International College,Hunan Agricultural University,Changsha 410128,China;2.Biological Science and Technology College,Hunan Agricultural University,Changsha 410128,China;3.Veterinary Medicine College,Hunan Agricultural University,Changsha 410128,China

Bacteria is one of the major disaster for agricultural production,and has brought great loss to the husbandry and plants cultivation.At present,people largely depend on antibacterial substance to prevent bacteria diseases.However,the overuse of antibacterial substance gives rise to the tolerance of medicines in some bacteria,and even results in super bacteria,which pose serious threat to the prevention of bacteria in plants and animals[1].Therefore,to develop new antibacterial medicine has become the focus of studies all over the world.

Extracting naturalanti-bacteria substances from animals,plants and microorganisms has become the essential way to find out new antibacterial medicine,because natural antibacterial medicine is highly efficient,causes no pollution or resistance to drugs in the plants.It is easy to produce and takes short time[2].In recent years,many experts have extracted natural antibacterial substances from many animals and plants[3-8],but there is not any report on extracting natural antibacterial substance from Escherichia coIl.Escherichia coli is a commonly used experimental bacteria,easy to cultivate.It will be of great theoretical significance if natural antibacterial substance can be extracted from Escherichia coIi.

In addition,in the current reports about the natural antibacterial substances,there is not any report concerning the prevention of blight in plants.In fact,the blight is very dangerous.The blight caused by Ralstonia solanacearum (Smith)Yabuuchi often occurs in the tropical and subtropical areas.It is one of the most dangerous plants diseases,while the prevention of blight still depends on antibiotics.Therefore,developing natural antibacterial substances to prevent blight is of essential application significance in agricultural production.

Escherichia coli is used as the supply of natural antibacterial substances in this experiment.Application of high-speed centrifugation and highperformance liquid chromatography to separation and purification obtained a new type of natural antibacterial substance fromEscherichiacolicell.Chemical method and mass spectrometry are applied to identify the composition.The aim is to develop a new natural antibacterial substance which is of application significance in agricultural production,and can replace the overuse of antibiotics.

Materials and Methods

Experimental materials

Culture medium and agentLB culture medium,1/2 MS culture medium,NA culture medium,solution I,solution II,ethane nitrate,concentrated sulfuric acid,ninhydrin,potassium iodide,picric acid,phosphomolybdate acid, silicotungstic acid, mercuric potassium iodide,sodium carbonate,ether,ammonia solution of silver nitrate,and sudan IV solution.The culture medium and solutions mentioned above were prepared and preserved in the laboratory.Wst-1 dyeing solution was frozen and preserved avoiding lights.

Plants seeds.The seeds ofArabidopsis thalianawere a gift from Professor Xia Shitou.

Experimental methods

Isolation and selection of antibacterial substance(1)The cultivation ofEscherichia coli:single bacteria are collected from the Escherichia coli on the no-bacteria operation platform,and are put in to the LB solution culture medium,and are cultivated in the 37℃constantly for 24 hours.(2)Breakdown ofEscherichia colicell is based on the Jiang Huanhuan’s method[13].(3)The grade centrifugation ofEscherichia colicell:we collectedEscherichia colicell solution,and recognized ten grades,1 000,2 000,3 000,4 000,5 000,6 000,7 000,8 000,9 000 and 10 000 r/min.At the same time,we collected the concentration of each centrifugation grade.(4)Selection of active antibacterial component.MTT method is applied to measure the antibacterial activity.Centrifugal concentration of each grade was parched at 50℃.We calculated the active bacteria and diluted them to 106 per ml.we added 100 μl of diluted bacteria liquid into the cell culture medium which had 96 holes,and then added another 15 μl of sediment water solution(1 mg/ml).We made three parallel holes in each sample,and we collected the cell culture medium one day after it being cultivated in 37℃constant temperature foster box.We added 10 μl Wst dyeing solution and then put them in the incubator for 1 hour.We detected theODvalue when the enzyme-labelling measuring instrument was at 490 nm,and calculated the averageODvalue in three average holes.The bacteria in the hole with lowODvalue was inhibited,and then we selected the sediment of anti-bacteria activity.(5)High performance liquid isolation: chromatographic column was unitary C18,5 μm,4.6 mm ×250 mm.The mobile phase was acetiontrile.The elution gradient was between 0 and 12 min.The acetiontrile was between 10% and 95% ;12-26 min,95% acetiontrile;26-30 min,acetiontrile 95% to 100%.

The injection volume was 30 μl,and the flow speed was 1 ml/min.The detected wavelength was 254 nm.One tube of solution was taken every two minutes,and we collected 15 tubes,2 ml in each tube for nine times.

Identification of antibacterial active substanceWe identified the antibacterial active substance based on the following method.

(1)We applied Molish test to identify sugar,and referred the Li Xiaodong and Jia Lin’s approach[14-15].

(2)We identified protein based on ninhydrin testand Xu Wenyan’s method[16].

管理會(huì)計(jì)需要對(duì)財(cái)務(wù)基礎(chǔ)信息收集，并進(jìn)行再加工，結(jié)合當(dāng)下醫(yī)院政策環(huán)境信息，及時(shí)發(fā)現(xiàn)并反饋醫(yī)院運(yùn)行過(guò)程中的問(wèn)題，幫助管理層實(shí)施管理控制建言獻(xiàn)策，因此，具有完整知識(shí)結(jié)構(gòu)和較強(qiáng)學(xué)習(xí)能力的財(cái)務(wù)人員可以更好地適應(yīng)管理會(huì)計(jì)工作的要求。而現(xiàn)階段，醫(yī)院財(cái)會(huì)人員在學(xué)歷、職稱和專業(yè)素質(zhì)能力上都與管理會(huì)計(jì)要求有一定差距，且由于醫(yī)院人事制度改革效果尚不能顯現(xiàn)，醫(yī)院在崗人員對(duì)新的財(cái)務(wù)管理知識(shí)未表現(xiàn)出積極態(tài)度，所以醫(yī)院面對(duì)核算向管理轉(zhuǎn)型時(shí)，強(qiáng)化財(cái)務(wù)人員梯隊(duì)迫在眉睫。

(3)The identification of ketone and aldehyde was based on Zhao Lian’s approach[17].

(4)The identification of lipid was based on the approach developed by Wang Xiuliet al[18].

(5)The identification of biological alkaline component:firstly,we collected four types of acid infusion (1 ml each),and added iodine,potassium iodide,picric acid reagent,phosphormolybdic acid reagent,and silicotungstic acid reagent.If there is sediment in four reagents,it is obvious there is alkaloid in the sample and then we move on to the next experiment.Secondly,we collected other acid infusion,added Na2CO3and put them in the separate funnel.We added 10 ml ether,put them in still,and then collected the acid liquid.We collected four portions of acid liquid and conducted the following sediment reaction:a.Mayer reagent:putting Mayer reagent into acid liquid results in white sediment. b. Silicotungstic acid reagent:putting silicotungstic acid reagent into acid liquid produces light yellow or pale white sediment.The acid liquid reacted with the abovementioned three kinds of reagents and produced sediments.

(6)Energy dispersive analysis:we used the antibacterial active substance of the crude Escherichia coli before the high performance liquid isolation for energy dispersive analysis.

(7)Mass spectrometric detection:we used the antibacterial active substance of the Escherichia coli after the high performance liquid isolation for mass spectrometric detection.

Detection of the antibacterial activity outside the antibacterial active substance(1)Identification of the minimum inhibition concentration of antibacterial active substance:we collected pathogen,Escherichiacoli,pasteurellosis bacillus,aureus staphylococcus,streptococcus,strain blight for cultivation,and diluted the culture medium to 106 per ml.Then,we diluted the solution with normal saline.After that,we collected 0.1 ml bacterial liquid onto the solid culture medium.After 24 hours of constant temperature culture,we calculated the number of fungi groups.We added 1 ml culture medium into five fungi tubes,and added 20 μg/ml of solution into No.1 tube.We added 1 ml LB solution culture medium in the tube and took the 100 μl fungi solution as control.We put the diluted solution into 35℃constant temperature box for 24 hours to observe the number of fungi in the tube and to determine the minimum antibacterial concentration.(2)the determination ofminimum antibacterial concentration.We collected 100 μl of above-mentioned media and set them under 37℃to observe the growth of antibacterial groups.The one with less than five fungi was the minimum antibacterial concentration of the medicine.

Influences of antibacterial active materials on Arabidopsis thalianaWe putthe sterilizedArabidopsis thalianainto 1/2 MS culture media,and expose them at 25℃for four weeks.The 1/2 MS culture media is set into three groups,A group as blank control,B group with 2% antibacterial active substances,and C group 5% antibacterial active substances.The robustArabidopsis thalianawas put into these three culture media to observe its growth.

Results and Analyses

Centrifugation of antibacterial active substance and MTT detection results

Escherichia colialkaline solution went through ten different gradients to get ten different sediments which were treated with antibacterial active substance with MTT.Results suggested that there was no antibacterial active substance in the sediments at 6 000 r/min gradient centrifugation,and at 7 000 r/min gradient centrifugation.The bacteria concentration was low and transparent.TheODvalue was the minimum at 490 nm,which suggested that the bacteria of this group can restrain the bacteria growth.Fig.1 shows the detection results.

The sediment was parched at 50℃through 7 000 r/min of centrifugation toobtain thebrownparticles(Fig.2).

Isolation of high performance liquid isolation of antibacterialactive substance and MTT detection results

The water solution of the sediments,which was obtained at 7 000 r/min gradient centrifugation was treated with high performance liquid isolation,and we carried out MTT antibacterial active substance detection.Results suggest that the average value of the 15 and 16thmin was the minimum,and the number of active bacteria was the least,which suggested that the antibacterialactivity was the strongest(Fig.3).

Identification of antibacterial active substance

We identified sugar,protein,ketones,lipid,and alkaline in the antibacterial active substance obtained by 7 000 r/min gradient.Molish experiment did not show purple red ring,and proved that there was no sugar in the antibacterial active substance.Ninhydrin chromogenicreaction did not show blue purple,which suggested that there was no protein in the antibacterialactive substances.Silver experiment proved that there was no ketone and aldehyde in the antibacterial active substances.The biological alkaline identification suggested that there were sediments in three kinds of reactions in the four kinds of biological alkaline identification reagents,and there were sediments in two kinds of reactions among the four kinds of reagents,which proved that there was biological alkaline in the antibacterial active reagents(Fig.4).

Bacteriostasis effect of antibacterial active substance

According to the comparison of the control group with a series of bacteriostasis experiments on the antibacterial active substances,it was found that the antibacterial substances have distinct inhibition effects onEscherichia coli,Streptococcus,Pasteurellosis bacillus,and blights.The minimum antibacterial concentration to aureus staphylococcus,Escherichia coli,Streptococcus,Pasteurellosis bacillus,and blights were 2.5,5,0.625,0.625,1.25 μg/ml.The maximum antibacterialconcentration was 5,5,1.25,2.5,2.5 μg/ml(Fig.5-Fig.8).

Influences of antibacterial active substances on the growth of plants

We added different concentrations of antibacterial active substances in the culture medium ofArabidopsis thalianaand observed the growth ofArabidopsis thalianaafter one and two weeks after inoculation.It is obvious that the antibacterial active substance did not affect the growth ofArabidopsis thaliana(Fig.9).

Conclusions

Extracting natural antibacterial active substance has become a new trend in developing antibacterial medicine to prevent bacterial-related diseases.Extracting antibacterial active substance fromEscherichia coliis a new way to find antibacterial medicine.MTT approach was applied to detect the culture solution ofEscherichia coli,fungi,Escherichia coliultrasonic breakdown andEscherichia colisaline cracking breakdown,but there was no antibacterial activity,which suggested that only through alkaline breakdown can the antibacterial activity in theEscherichia colibe activated.The antibacterial activity of the active substance was high,and reached the antibacterial concentration of routine antibiotics.The experimental results show that the antibacterial active substance is a broadspectrum,high efficiency,safe antibacterial natural product,with the prospect of development and utilization of alternative antibiotics.

The antibacterial active substance has been primarily identified as a kind of biological alkaline,which fits the result of the active substance extracted through the alkaline breakdown method.However,what is the substance?This study suggested that the elements in the active substance ranked gradually from C,O,Na to N,and the spectrum identification continued to be molecule compounds.At present,the author is carrying out isolation purification and structural analysis.Besides,its antibacterial mechanism requires further studies.

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