Detection And Control Of Histamine-forming Bacteria In Ovalipes Punctatus And Utilization Of By-products

Ovalipes punctatus is the medium-sized edible crab with the highest resource density and the largest population in the East China Sea crabs.However,the problems existed in processing and utilization of O.punctatus are:?1?due to the easily perishable and abundant free histidine,O.punctatus will contain large amounts of histamine formed through the decarboxylation of free histidine by exogenous histidine decarboxylase released by microorganisms,which bring safety risks to the consumption of O.punctatus and its products;?2?a large amount of cooking liquor and by-products containing meat scraps were produced during processing due to serious loss of juice and low crabmeat-picking rate.These by-products have not yet been effectively utilized,resulting in high development costs,resources waste and environmental pollution.Therefore,it is of great practical significance to control the histamine formation in O.punctatus,and to explore and utilize the processing by-products of O.punctatus.In order to provide theoretical basis and technical support for the comprehensive development and utilization of O.punctatus,the present thesis investigated the causes of histamine formation and its control methods in O.punctatus,and studied the separation and identification technology of functional peptides,and further analyzed and verified the functional characteristics of functional peptides from enzymatic hydrolysate of by-products O.punctatus.The histamine-forming bacteria in the gills and guts of live O.punctatus were screened and identified,and the effects of temperature,initial pH value and NaCl contents on the growth characteristics and histamine production as well as the inhibitory effects of chitooligosaccharide?COS?on the bacteria were studied.One strain HFB was isolated,identified and named as Enterobacter aerogenes OP-G2 based on morphology and 16S rDNA sequence analysis.This strain could produce 46.96±1.94 mg/L histamine in TSBH medium after incubation at 30°C for 48 h.The growth and histamine formation of E.aerogenes OP2-56 in TSBH medium could be effectively controlled both by low-temperature??4°C?and high NaCl concentration??10%?,and the inhibitory rate of histamine production of the strain could be up to more than 92%.Meanwhile,the growth of the strain could be effectively inhibited?all above 30.58%?by COS higher than 0.5%,and the antibacterial rate of 1%COS at pH 6.0 against E.aerogenes OP2-56 could be up to 47.56%.The histamine formation of O.punctatus and its influencing factors in O.punctatus were investigated.The results showed that O.punctatus is a crab species prone to high histamine levels when stored at temperatures?20°C and ice and cold storage?4°C?could inhibit the histamine accumulation in whole,unprocessed O.punctatus.The histamine former E.aerogenes OP2-56 was isolated from spoiled O.punctatus and produced 147.61 ppm of histamine in TSBH medium after incubation at 37°C for 48 h.The effect of storage condition,initial pH and NaCl concentration on growth and histamine production of E.aerogenes OP2-56 in the O.punctatus meat and fresh O.punctatus infusion broth?OPI?inoculated with E.aerogenes OP2-56 were investigated.The results showed that the growth and histamine formation of E.aerogenes OP2-56 in OPI broth were effectively controlled either by low-temperature??4°C?and/or high NaCl concentration??10%?,and the inhibitory rate of histamine production of the strain could be up to more than 97%.These results suggested that the rapid cooling,storage and transportation temperature?4°C or salting with 10%NaCl may be an effective method to control the histamine accumulation of O.punctatus.The effect of combination of 10%of trisodium phosphate?TSP?dip for 10 min and spray with 5.0%of protamine on the bacterial growth and histamine production in fresh O.punctatus inoculated with E.aerogenes OP2-56 during 4 days of 10°C storage was investigated.The results showed that the bacterial growth and histamine production of E.aerogenes OP2-56 were effectively inhibited by TSP dip,protamine spray and the combined treatment with TSP dip and protamine spray,compare with the NaCl-aseptic water.However the highest inhibition rate of bacterial growth and histamine production of E.aerogenes OP2-56 was obtained by the combined treatment with TSP dip and protamine spray.The inhibitory rates for the strain and histamine accumulation were 37.72%and 72.92%,respectively after the combined treatment with TSP dip and protamine spray.This result suggested that TSP dip and protamine spray has synergistic inhibitory effects on the inhibition of the bacterial growth and histamine accumulation in O.punctatus.The total phosphorus content in the meat of O.punctatus was increased after the combined treatment with TSP dip and protamine spray.The maximum increase content of total phosphorus was 106.01 mg PO₄^3-/100g,which was much lower than the safety limit specified in GB 2760-2014 standard,indicating the combined treatment with TSP dip and protamine spray could be a safe and effective method to inhibit the accumulation of histamine in meat of O.punctatus.To rapidly determine the histamine content in marine fish,a novel analytical method for rapid determination of histamine in marine fish consists of methanol extraction,manual spotting,thin-layer chromatograph?TLC?for less than 15 min,visualization by diazotized p-nitroaniline reagent,quantitation by image analysis with ImageJ software.Histamine can be successfully detected and completely separated from other biogenic amines and diazonium reagent's positive compounds.Moreover,the method does not require expensive reagent and instrument as well as any tedious sample pre-treatment.The characterization of this method revealed good specificity,linearity in the range of 100–1200 ng/spot?Adjusted R-square=0.9987?,precision?relative standard deviation 1.07%–2.76%?,accuracy?with a trueness of1.76%–6.22%?,recovery rate?93.78%–98.24%?,limit of detection?25.84 ng/spot?,and limit of quantitation?78.31 ng/spot?.The method is simpler,faster and less expensive than HPLC and previously reported TLC/HPTLC.Therefore,the method has the potential to be adapted by aquatic enterprises and aquatic market supervision commissions for monitoring the quality of seafood products with high histamine content during processing,storage and sales.In order to utilize the by-products of O.punctatus,by-products containing meat scraps was hydrolyzed by trypsin to obtain tryptic hydrolysates rich in antioxidant peptides.Antioxidant peptides in the hydrolysates were purified using ultrafiltration membrane system,Sephadex G-15 column and semi-preparative reversed-phase high-performance liquid chromatography?semi-preparative RP-HPLC?.Their peptide sequences were identified by ultra-performance liquid chromatography/electrospray ionization time-of-flight tandem mass spectrometry?UPLC-ESI-TOF/MS/MS?.Their structure and ABTS⁺·scavenging capacity were further verified by the synthetic peptides.Two peptides with high ABTS⁺·scavenging capacity were purified from hydrolysates,and their peptide sequences were Tyr-Glu-Gly?YEG?and Tyr-Glu?YE?,respectively.The molecular weight of synthetic peptides was367.35 u and 310.30 u,and their purity was 98.52%and 98.04%,respectively.The synthetic peptides YEG and YE have good ABTS⁺·scavenging capacity,with IC₅₀ values of 0.456mg/mL and 0.184 mg/mL,respectively.Furthermore,IC₅₀ value of synthetic peptide YE is30.30%higher than that of reduced L-Glutathione.In order to further utilize the by-products of O.punctatus,the by-products was hydrolyzed by a stepwise hydrolysis with trypsin and flavourzyme to obtain hydrolysates rich in umami peptides.The content of free amino acids,nucleotides,their related substances and minerals in the umami components obtained by ultrafiltration and gel chromatography were analyzed to determine the effect of the peptides on its characteristics.Then umami peptides in the umami components were further purified using the semi-preparative reversed-phase high-performance liquid chromatography?semi-preparative RP-HPLC?.Their peptide sequences were identified by ultra-performance liquid chromatography/electrospray ionization time-of-flight tandem mass spectrometry?UPLC-ESI-TOF/MS/MS?.Their structure and taste characteristic were further verified by the synthetic peptides.The P3fraction was obtained from hydrolysates using ultrafiltration membrane and gel chromatography,and the peptides?<1 kDa?in P3 fraction played a major role to its umami.Two peptides with high umami taste were purified from hydrolysates,and their peptide sequences were Glu-Lue-Ser?ELS?and Trp-Asp-Asp-Met?WDDM?,respectively.Their umami taste thresholds were 1.5 and 0.85 times that of MSG,respectively.The molecular weight of synthetic peptides was 347.36 u and 565.59 u,and their purity was 97.93%and98.42%,respectively.The umami thresholds of synthetic peptides YEG and YE were 2.6 and1.1 times that of MSG,and were 1.7 and 1.3 times those of their corresponding natural peptides,respectively.