Effects Of Protamine On Microorganisms And Biogenic Amines Of Esox Lucius During Low-temperature Storage

The Esox lucius is soft and tender,delicious and nutritious.It is a high-quality indigenous fish species unique to the Ulungu Lake in Xinjiang,known as the"soft gold"of fish.Its annual output can reach 1500tons,and the processing volume accounts for about 45%.However,If the conditions are not properly controlled during transportation and storage,it will easily lead to microbial growth and eventually lead to the increase of biogenic amine content and resulting in food safety problems;On the other hand,the by-products generated during the processing of local Esox lucius（such as fish spermary,scales,viscera,etc.）can reach 680 tons,while the high value-added components contained in them,such as protamine,active peptides,fish oil,etc.,are not fully utilized and discarded,resulting in serious waste of resources.Therefore,in this study,the spermary,a by-product of the processing of Xinjiang Esox lucius,was first taken as the experimental object.The technological conditions for the preparation of protamine were obtained by response surface method.On the basis of the establishment of eight biological amine detection methods and ultrasound-assisted enrichment process,the protamine was used in the storage and preservation of Esox lucius,and the changes of microorganism and biological amine under different storage temperatures were analyzed and studied,The purpose is to use protamine to control the decline of fish quality caused by microorganisms and biogenic amines during the low-temperature storage of Esox lucius,and provide theoretical basis for the development of Esox lucius industry.The main research results are as follows:1.The preparation method of protamine was obtained.Combined with the single factor and response surface test results,the order of protein yield was obtained as follows:ethanol dosage>sulfuric acid dosage>extraction times>sulfuric acid concentration.The best extraction conditions were:sulfuric acid concentration 0.50 mol/L,sulfuric acid dosage 2 m L/g,extraction times 3 times,ethanol dosage 3 m L/g,and protein yield 2.44%.After purification by Sephadex G-50 and CM Sepharose Fast Flow,Tricine-SDS-PAGE showed a single band.2.Eight pre-column derivatization HPLC methods for the determination of biogenic amines were established.Mobile phase A and B are water and acetonitrile respectively,and gradient elution is adopted,and the injection volume is 20μL.The flow rate is 0.8 m L/min,the column temperature is 30℃,and the detection wavelength is 254 nm.The pre-column derivatization method is as follows:the concentration of the derivatization agent（dansyl chloride）is 10 mg/m L,the derivatization time and temperature are 45 min and 50℃.3.A method of ultrasound-assisted enrichment of biogenic amines in samples was selected.Combined with single factor and orthogonal test results,The order of influence on the extraction effect of two biological amines（putrescine and cadaverine are the main biological amines produced during the storage of Esox lucius）was as follows:ultrasound time>ultrasound temperature>ultrasound power,and the optimal combination of conditions was A₁B₁C₁,namely,ultrasound time 10 min,ultrasound temperature 40℃,ultrasound power 100 W.4.The effect of protamine on microorganism and biogenic amine of Esox lucius during storage was analyzed.After being treated with protamine,the Esox lucius was stored at 4℃,-3℃and-18℃for 13days.The results of total bacterial count showed that with the extension of storage time,the growth of microorganisms in the 0.5%and 1%protamine treatment groups was significantly inhibited during storage.The results of microbial flora change during storage by high-throughput sequencing showed that the dominant flora of Fresh group was Delford（47.61%）and Pseudomonas（23.71%）;After 13 days of storage at-3℃and 4℃,the dominant bacteria in CK group were Pseudomonas,with 97.62%and 98.67%contents;In the 0%treatment group,the dominant bacteria were also Pseudomonas,with the content of 47.29%and73.75%;When 0.1%treatment group was stored at-3℃for 13 days,Delfordii was the dominant bacteria,with the content of 51.13%;when stored at 4℃for 13 days,the dominant bacteria were Pseudomonas and Acinetobacter,with the content of 66.70%and 11.29%;The dominant bacteria in the 0.5%treatment group were Pseudomonas,with the content of 99.73%and 84.47%;Pseudomonas in 1.0%treatment group was the dominant bacteria,with the content of 95.96%and 98.95%;The change of biogenic amine content is as follows:During low-temperature storage,the Esox lucius mainly produces cadaverine and putrescine.When the storage time is extended,the content of eight biogenic amines increases.Protamine treatment inhibited the production of eight biological amines during low temperature storage,of which 1%concentration of protamine had the best inhibition effect.