Research On Preparation And Preliminary Antibacterial Mechanisms Of Compound Preservative Of Litopenaeus Vannamei

The paper studied the antibacterial effects and mechanism of tea polyphenols,chitosan, phytic acid, nisin and the extractives of Houttuynia cordata Thunb.,Scutellaria baicalensis Georgi and Taraxacum officrnale on the specific spoilageorganisms (SSO) of Litopenaeus vannamei. The antibacterial experiment in vitro wasused to optimize the ratios of compound preservative on the basis of the contrast testof single preservative. The antibacterial mechanism of the compound preservative andits fresh keeping effect during low temperature storage (4℃) were also studied. Thestudy is more targeted in inhibiting bacteria and keeping fresh according to the storingproperty, which greatly improves the quality of Litopenaeus vannamei during storage.The compound preservative improves some disadvantages of previous compoundpreservatives with wide bacterial spectrum and poor in keeping fresh.This study includes several main parts as follows:1. The screening of several single preservativeShewanella, Acinetobacter and Pseudomonas fluorescens are the main SSOs inLitopenaeus vannamei. The paper determines the antibacterial effects of teapolyphenols, chitosan, phytic acid, nisin and the extractives of Houttuynia cordataThunb, Scutellaria baicalensis Georgi and Taraxacum officrnale on the SSOs by usingELIASA to determine the OD value in the600nm and spreading suspension ofbacteria on the solid medium. The results show that tea polyphenols, phytic acid andthe extractives of Taraxacum officrnale have strongger antibacterial effects on thethree bacteria tested. The bacterial inhibition rate of0.10%tea polyphenols,0.10%phytic acid and5%extractive of Taraxacum officrnale on Shewanella, Acinetobacterand Pseudomonas fluorescens are92.56%,90.48%and90.90%;93.35%,94.90%and23.98%;99.13%,94.29%and5.41%respectively. The minimal bactericidal concentrations of the three preservatives are0.10%,0.10%and1.25%respectively.So the chosen concentration ranges of tea polyphenols, phytic acid and the extractiveof Taraxacum officrnale are0.02%~0.08%,0.04%~0.08%and0.4%~1.0%,respectively.2. Optimization of proportion of compound preservativeOn the basis of the study of several single preservatives, the formulation of teapolyphenols, phytic acid and the extractive of Taraxacum officrnale is made throughL9(33) orthogonal test. The OD values of the three bacteria in the600nm are used toevaluate the antibacterial effect, and to optimize the ratio of compound preservatives.The optimum concentrations of preservatives are0.05%of tea polyphenols,0.06%ofphytic acid and1.00%of the extractive of Taraxacum officrnale (Content of totalflavonoids is0.12%) respectively. And the total dose is1.11%. Then the compoundpreservative is used to deal with Litopenaeus vannamei. The prawns were first putinto the preservative solution, then taken out, drained, packed with PE and storedunder4℃. According to the values of pH, TVB-N and total bacterial count, thefreshness of Litopenaeus vannamei treated with the compound preservative is betterthan control group significantly(P<0.05). The sensory scores of treated group arehigher than that of control group and the shelf life can be prolonged for2~3days.3. Research on antibacterial mechanisms of compound preservativeAs the main SSOs of Litopenaeus vannamei, Shewanella, Acinetobacter andPseudomonas fluorescens are used to study the antibacterial mechanisms ofcompound preservative. The minimal inhibitory concentration of compoundpreservative is determined by the method of punching on solid culture medium. Theantibacterial mechanisms of compound preservative for the three bacteria areexplained by antibacterial kinetics, bacteria growth curve, analysis of cell membranepermeability, AKP enzyme activity and observation of the ultrastructure underscanning electron microscope. The results show that the minimal inhibitoryconcentrations of compound preservative on Shewanella, Acinetobacter andPseudomonas fluorescens are0.13%,0.13%and0.26%respectively. After treatedwith compound preservative, the cell gets smaller, cytoplasm leaks from the bacteria, the conductivity of inoculums increases and the activity of AKP enzyme increases.The growth of Shewanella, Acinetobacter and Pseudomonas fluorescens are inhibitedobviously (P<0.05) and exponential phase doesn’t occur. Antibacterial activity reachmaximum value at7h,6h and9h respectively, and is more than0.8. Conductivity andAKP enzyme activity increase obviously (P<0.05). The results show that the internalenvironment of cell and cell membrane permeability are damaged by the compoundpreservative. The compound preservative inhibits normal growth and metabolism, andhas bacteriostatic and bactericidal effects.