Analysis And Comparison Of Key Influencing Factors Of Different Nisin Detection Methods

Nisin is a kind of cationic polypeptide produced by certain strains of Lactococcus lactis or Streptococcus uberis,which can effectively inhibit the growth of most Gram-positive bacteria.Nisin has been recognized as GRAS product by more than 80countries in the world.The appearance of many manufacturers has greatly satisfied the market demand of Nisin.The biological activity of Nisin is one of the most important factors to ensure the quality of Nisin products.Therefore,the purpose of this study is to analyze and discuss the main factors affecting the results of different Nisin titer testing methods,whereby developing a set of testing methods suitable for on-line monitoring of activity for Nisin manufacturers,and to provide guidance for the production of Nisin.Nisin A or Nisin Z standard was firstly dissolved in pH=2.5 hydrochloric acid solution,and the titers were then detected by agar diffusion method with the culture medium containing 1.3%or 1.4%agar.The concentration of indicator bacteria in the culture medium was adjusted to 1.0×10⁶ CFU/mL and then incubated 2.5 h for osmotic diffusion.The titer of standard products were determined as 1000±50IU/mg.When high performance liquid chromatography（HPLC）for Nisin detection was used,the ideal chromatograms were obtained under the following conditions:Agilent 1220 apparatus with reverse C18 column,4.6 mm x 150 mm and mobile phase consisting of 29%acetonitrile and 0.4 trifluoroacetic acid.For microbial turbidity analysis,the best gradient coloration results can be obtained when indicator bacterial suspension(OD₆₀₀ value=0.125)and 0.125%azure were added into the detection system,and cultured at 23℃.The final detection results were obtained using 595 nm filter in the enzyme label instrument which were more prominent to that of the agar diffusion results.The results showed that:（1）The selection of standard Nisin（Nisin A and Nisin Z）was the most important and often neglected factor in the detection of Nisin by agar diffusion method.When testing Nisin A,the standard Nisin A should be selected.And then,when testing Nisin Z,the standard Nisin Z should be selected.（2）The trend of detecting Nisin titer by HPLC is consistent with that by agar diffusion method,but the result is higher in the detection process.However,the control of sample size has a significant effect on the accuracy of the detection results when different sources of Nisin samples are detected by HPLC.In the range of 1000³000 IU/mg,20μL of Nisin should be selected;In the range of 5000¹0000 IU/mg,10μL of Nisin should be selected;the range of 10000¹00000 IU/mg,5μL of Nisin should be selected.（3）The azure concentration in the microbial turbidimetry-based reaction system has a significant effect on the detection results.When the concentration of azure solution is0.125%,the result is best.The titer of Nisin products can be predicted,and the efficiency of agar diffusion method for Nisin end product detection can also be improved.These three methods run through the whole production process of Nisin,and can achieve the purpose of comprehensive and precise monitoring of Nisin production by cooperating with each other.