Isolation And Development Of Rapid Detection For Heat-resistant Bacillus In Milk Powder

Bacillus is common in milk powder, and has a serious impact on the product quality.Geobacillus stearothermophilus and Bacillus coagulans are also common flat sour spoilagebacteria in canned food, such as condensed milk. Heat-resistant bacillus can produce variousenzymes, such as protease and lipase, and reduce the sensory quality and nutrition of food. At thesame time, these microorganisms can also indirectly reflect the hygiene of the processing plant.Therefore, rapid detection and disinfection of these heat-resistant bacillus has an importantsignificance.Loop-mediated isothermal amplification (LAMP) is a new type of nucleic acid amplificationtechnology, which can achieve rapid amplification of nucleic acids by using4specific primers, andselectively using loop primers. This technology does not need the expensive PCR amplicationinstrument, and amplification detection can be completed even under the condition of constanttemperature. It can be widely applied to microbial nucleic acid as its high specificity, reactionefficiency and convenient operation.A strain of heat-resistant bacteria named KLDS9.1101was isolated and identificated frommilk powder samples, which was collected in a milk powder factory near Harbin. By themicroscopy examination, culture and determination of the growth curve, it proved to beGram-positive, and its optimum growth temperature was55℃. It belonged to Anoxybacillusflavithermus by16S rDNA sequence analysing in gene bank.Partial spo0A gene sequence of Anoxybacillus flavithermus, Geobacillus stearothermophilusand Bacillus coagulans was aligned. The result was used as target gene to design LAMP primers.Through culture, cells DNA was extracted as template. The temperature and the concentration ofbetaine、dNTPs and Mg2+were optimized. The best system for LAMP amplification: inner primersFIP and BIP1.6μmol/L, outer primers F3and B30.2μmol/L, loop primers LB and LF0.8μmol/L,2.5μL10×buffer,0.6mol/L betaine,1.0mmol/L dNTPs,8mmol/L MgSO4,2μL template, addingddH2O up to24ul. The react condition was manipulated as follow:95℃pretreatment5min, cooldown, then add1μL Bst DNA polymerase8U,63℃amplification for30~60min, finally10min at80℃to inactivate enzymes. The amplication result can be detected by gel electrophoresis, HNBand calcein. Amplification result can be obviously judged whether happened or not.Common Bacillus DNA were extracted as the LAMP template to confirm the specificity andsensitivity of LAMP. The results showed that only Anoxybacillus flavithermus, Geobacillus stearothermophilus and Bacillus coagulans DNA extract can cause amplification, while others didnot. It indicated that LAMP primers were designed well by choosing the spo0A gene as the target.Sensitivity test showed that the detection limit can reach8CFU/mL. LAMP detection was morespecific, rapider and more efficient than the traditional plate count method. Compared with PCR, itwas more sensitive and it did not need expensive PCR equipment.Anoxybacillus flavithermus was chosen as experimental strain, peracetic acid was chosen asthe disinfectant, and0.3g Na2S2O3+100mL PBS was chosen as neutralizing agent. Theexperimental results showed that peracetic acid can disinfect well. When bacillus was soaked andwashed in200mg/L peracetic acid disinfectant for3min or more, the killing logarithm was morethan6, and the disinfection purposes can be achieved.In summary, the LAMP method can be used to detect Anoxybacillus flavithermus,Geobacillus stearothermophilus and Bacillus coagulans rapidly in food, and peracetic acid can beused as disinfectant to disinfect heat-resistant bacillus such as Anoxybacillus flavithermuseffectively.