Enumeration Of Psychrotrophic Bacteria Colony Count In Raw Milk By A Rapid Polymerase Chain Reaction (PCR) Method

Psychrotrophic microorganisms play a leading role in spoilage of refrigerated milk and milk products.During cold storage after raw milk collection,psychrotrophic bacteria are dominating flora,and their extracellular enzymes,mainly proteases and lipases,contribute to the spoilage of dairy products with clot,bitter and fat oxidation flavor.There are two methods for enumerating the psychrotrophic microorganisms in raw milk,IDF Standard 101A and 132A.The IDF Standard 101A is one that calculate the number of colonies obtained on plates chosen at dilution levels after incubated at 6.5℃for 10 days to give a significant result,while the IDF Standard 132A so does at 21℃for 25h.IDF Standard 101A contributes to more accurately in result but IDF Standard 132A is more shortly in test period.Although some reports indicated that there were some new technologies applied in the enumeration of psychrotrophic microorganisms in raw milk,they had some disadvantages such as demanding higher cost or harsh test conditions.Recently,polymerase chain reaction(PCR) technology has a very broad range of application in food industry for its advantage of high-speed,efficiency and fitting for food industry.However,the PCR technology has some weaknesses that the general PCR could only achieve qualitative analysis and the real-time PCR,which could contribute to action from both qualitative analysis and quantitative analysis,is too expensive to food industry.The object of this study is aim to overcome the weaknesses above-mentioned about PCR technology,which take place through a combination of the PCR and the conventional instrument analysis to obtaine an easy,rapidly and inexpensive enumerating method of psychrotrophic bacteria in raw milk for dairy,industry.The relationship of two international standard methods,IDF standard 101A method and 132A method,was investigated by coinstantaneous enumerating the psychrotrophic microorganisms in the same raw milk sample and comparing the enumerating results.Then enumerating method of psychrotrophic microorganisms based on polymerase chain reaction was developed.Raw milk was pre-treatmented with three methods:immobilization with metal hydroxides method,citric acid and sodium hydroxide method and trichloroacetic acid method.The optimal one was shown to be trichloroacetic acid method.Two new methods,16S rRNA-PCR method and Apr-PCR method, were established to enumerate the psychrotrophic microorganisms in raw milk.SYBR Greenâ… ,a double strand DNA(dsDNA)-binding dye,was used to measure the variation of DNA template quantitatively between pre-PCR and post-PCR,which was based on the phenomenon that fluorescence intensity would be enhanced significantly when the dsDNA were mixed with SYBR Greenâ… .Some experiments were carried out to optimize the PCR conditions of the two methods. Enumerating resulte form two menthods were compared to that of IDF Standard 132A method.Some results in this study were as follows.(1) A comparison was made between IDF Standard 101A and IDF Standard 132A when milk sample was enumerated coincidently by two methods.A linear recession equation was obtained to illustrate the relationship of the two menthods,which was logy = 0.6303logx + 2.1215(y:IDF Standard 101 A;x:IDF Standard 132 A(25 h)),and the modified method was logy = 1.4314logx -2.2988(y:IDF Standard 101 A;x:IDF Standard 132 A(48 h)).(2) Milk samples were pretreated with three methods,immobilization with metal hydroxides method,citric acid and sodium hydroxide method and trichloroacetic acid method.The results showed that trichloroacetic acid method was best one.When DNA was extrated with four methods from analysis samples,boiling water method,lysozyme method,proteinase k method and kit method,the best choice was proteinase k method.(3) According to the fact that Pseudomonas spp.was the most frequently reported psychrotrophic microorganisms in raw milk,two new methods,16S rRNA-PCR method and Apr-PCR method, were employed to enumerate Pseudomonas spp..For 16S rRNA-PCR method,the optimum concentration of Mg²⁺ was 1 mM.The optimum 16S rRNA PCR conditions consisted of 35 cycles at 94℃for 1 min,64℃for 1 min,and 72℃for 1 min,followed by a hold at 72℃for 10 min.The optimum Apr-PCR conditions consisted of 40 cycles at 94℃for 1 min,55℃for 30 s,and 72℃for 30 s,followed by a hold at 72℃for 10 min. The results showed that the optimum fluorescence analysis condition was that 5μL PCR products was added to 990μL TE buffer,thenlμL SYBR Greenâ… was added.Analysis results with16S rRNA-PCR method showed that the detection limit was less than 10³ CFU/mL,linear range of detection was 10³Ã¯Â½Å¾10⁸ CFU/mL.Analysis results with Apr-PCR method results showed that the detection limit was less than 10⁴ CFU/mL,linear range of detection was 10⁴～10⁸ CFU/mL.(4) Two new methods waere applied to enumerate psychrotrophic microorganisms in raw milk that were collected in dairy around Harbin City.Comparison with the results form two methods and that from IDF Standard 132A,the results obtained with 16S rRNA-PCR method had no significant difference from that with IDF Standard(P＞0.05),while the results obtained with Apr-PCR method showed a siginifant difference from that with IDF Standard(P＜0.05).This indicated that 16S rRNA-PCR method developed in this study could be applied to enumerate psychrotrophic microorganisms in raw milk instead of IDF Standard.