Degradation Of Skatole That Caused Boar Taint By Lactic Acid Bacteria

Rearing entire males instead of castrated male pigs for meat production has a number of advantages including better feed conversion efficiencies, leaner meat, higher polyunsaturated fatty acid percent and reduction of suffering for the animal. The occurrence of boar taint is the biggest obstacle of the use of entire male pigs. Skatole (3-methylindole,3MI) is one of the two main compounds that have been held as responsible for boar taint. Studies on manipulation of boar taint through dietary composition and rearing condition modifications, selections of low level of boar taint compounds breed have been carried out. But these studies do not yet seem to fulfill the industrial specifications. Immunocastration control boar taint at the price of losing part of boar's desirable characteristics. So the efforts to manipulating boar taint in meat processing are fascinating. However all limited previous works focused on using traditional methods such as smoking, curing and spicing to mask the unpleasant odors or diluting the odors with normal meat. The handling aimed at skatole or skatole variation in the process has been ignored. The objective of this study was to degrade skatole with Lactic acid bacteria (LAB). Four parts of work were conducted and the corresponding results are as following:(1) Skatole's influence on the growth of LAB was studied and four LAB strains' degradability were compared. The growth of four strain (Lactobacillus bevis /Lac1.12, Lactobacilus lactis/Lac1.454, Lactobacillus plantarum/Lac102, Streptoco-cus lactis/Str6020) in two kinds of medium (MRS broth and modified MRS broth with reduced concentrations of nitrogen and carbon) with four levels (0,0.5,2.0 and 5.0μg/mL) of skatole concentration was checked in 72h by OD600 determination. The skatole reduction of four strains in MRS and modified MRS (MMRS) with three levels of skatole after 192h culture was compared. The binding or absorption capacity of strains was observed too. Results indicated that:0.5-2.0μg/mL skatole can lead to biomass reduction without apparent lag phase and a lag phase about 12h was observed when the concentration reached 5.0μg/mL. Lac1.12 was more tolerant than other three strains within the observed concentration. The inhibitory effect upon growth depends on both skatole concentration and strain. Str6020 could degrade skatole most effectively under all the condition observed with 22.17-28.05% skatole reduction. Broth content and skatole level have no significant influence on the utilization of Str6020 but for the other strains skatole reduction at 0.5μg/mL was significant less than that of 2.0 and 5.0μg/mL. Bacteria displayed sight binding or absorption capacity of 0.85-2.96%. No relationship has been found between the sensitivity and degradation of strains.(2) Phase I metabolites of skatole produced by Str6020 were isolated and identified. Culture samples were extracted with ethyl acetate then solvent was stripped by evaporation under a stream of nitrogen. The residue was dissolved in methanol for HPLC analysis. HPLC was performed on a LC-20A system connected to a RID-10A diode-array UV detector and a RF-10AX fluorescence detector. Ammonium acetate buffer-acetonitrile and phosphate buffer-acetonitrile were used as mobile phase respectively with running gradient elution. Metabolites were identified by comparison with corresponding authentic standard on the basis of the HPLC retention time and the UV spectral. MMRS amended with authentic standards was incubated with Str6020 to check if the strain can mineral these standards. Metabolites can be separated well with present HPLC analysis. Four peaks corresponding to the metabolites identified on the basis of their retention time as 3-methyl oxindole (3MOI),3-hydroxy-3-methylo-xindole (HMOI),2-aminoacet-ophenone (2AM) and indole-3-carbinol (I3C). But only the UV spectrum of 3MOI and HMOI identified on the basis of their retention time were identical with those of authentic standards. The I3C was also checked out in negative control culture with lower concentration.3MOI, HMOI, I3C and indole-3-carboxylic acid (13 CA) added to MMRS can be degraded by Str6020. On the basis of results from previous investigations 3MOI and HMOI was confirmed to be the metabolites. I3C and 2AM need further studies. I3C was not found. The appearance of 3MOI and HMOI substantiated that degradation was initiated by the addition of oxygen to position 2 of the pyrrole ring.(3) The influences of skatole on the cell growth, single DNA strand damage and cell cycle were compared between before and after fermentation. Human lung squamous cell line QG56 was exposed to 1.1-4.4μg/mL initial skatole with or without fermentation. Single DNA strand breaks were measured using alkaline single-cell gel electrophoresis (comet assay). Cell growth was measured using MTT colorimetric assays. Cell cycle distribution was analyzed by flow cytometry. Results indicated that fermentation decreased toxicity of skatole to QG-56 growth and the difference was statistically significant at the concentration of 1.1and 4.4μg/mL. No statistically significant difference DNA damage extent was observed as demonstrated by indexes such as Olive tail moment, comet length, comet tail length, tail DNA% and comet tail/head (length). No statistically significant change of cell cycle distribution was suggested by flow cytometry results. As to present studies no indicator suggested that fermentation would increase the toxicity of skatole to QG-56.(4) Screening of most important environment factors influencing skatole degradation was carried out. Skatole in constructed boar taint meat was degraded by Str6020. The most important factors influencing skatole degradation were screened by a two-level Plackett-Burman (PB) design with 11 parameters including inoculum size, pH, NaCl concentration, temperature, NaNO2 concentration and six dummy variables. Skatole was added to boar meat with an end concentration of 2.0μg/g to make constructed boar taint meat. The constructed meat was processed to sausage with Str6020 as the starter culture and the skatole contents were investigated before and after 144h fermentation at 25℃to calculate the skatole reduction in the processing progress. Results were as following:Temperature, NaNO2 concentration and NaCl concentration were the significant factors with contribution value of 65.29%,15.55% and 11.06% respectively and the contribution value added up to 91.90%.144h fermentation at 25℃decreased 12.65% skatole in the meat and the decreasing was mainly owed to Str6020.The degradation of skatole in medium or constructed boar meat by fermentation with LAB was explored for the first time. Str6020 having considerable degradability has been selected out. Selected metabolites of skatole produced by Str6020 fermentation were isolated and identified. Four metabolites of skatole were detected. Two metabolites were identified using retention time and UV spectral comparison. The growth of QG-56 cells was interfered by skatole with or absent Str6020 fermentation. The viability of cells was examined by MTT assay. The distribution of cell cycle phase was quantified and analyzed by flow cytometry. The extent of DNA damage was measured by using the alkaline comet assay. Dates were analyzed by SPSS 16.0 software. Either t-test or ANOVA was used. P<0.05 was considered to be statistically significant. No toxicity increasing was observed in the three aspects studied. Temperature, NaNO2 concentration and NaCl concentration were assured to be the key factors influencing the skatole degradation by PB experiment design method. The present work established foundation for optimizing skatole degradation fermentation conditions, exploring the mechanisms of skatole degradation and dealing with boar taint by fermentation with LAB. These results also promised new intention to control boar taint in meat processing. They provided preference for screening or cultivating new strains or new species to degrade skatole and systematic assessing skatole toxicity variation caused by degradation.