Litopenaeus Vannamei And Its Microbial Diversity During Repeated Freeze-Thaw

Pacific white shrimp is loved by consumers and prone to corruption during storage due to its high moisture and high protein characteristics.Frozen storage is a commonly used preservation method in aquatic products,but in the process of storage and transportation,the imperfection of the cold chain causes the repeated freeze-thawing?F-T?cycles of shrimp,which aggravates the deterioration of its quality.How to protect the muscle quality and nutritional value of the Pacific white shrimp in the inevitable repeated F-T process has become one of the problems to be solved.In this paper,on the basis of a clear understanding of law in quality change of Pacific white shrimp during repeated F-T process,sodium tripolyphosphate and the mixture of sorbitol and sucrose were taken as contrast to study the effect of carrageenan oligosaccharide on the muscle nutritional value,flavor,water,protein and microbial diversity in shrimp during F-T cycles.The protection mechanism of carrageenan oligosaccharide as antifreeze and water retention agent on shrimp was preliminary discussed,which could provide the theoretical reference for the development of healthy and efficient antifreeze agent and the preservation of Pacific white shrimp.There are five chapters on Litopenaeus vannamei were carried on in this paper:Firstly,The moisture migration,protein degradation,microstructure and quality of Litopenaeus vannamei with different freeze-thawing?F-T?cycles were determined by LF-NMR,MRI and SEM,combined with SDS-PAGE,TPA,color difference,PPO activity,TVB-N value,TVC and sensory evaluation.The results showed that F-T cycles caused the significant increase of transverse relaxation time T₂₂ and T₂₃ and the decreased brightness of pseudo-color diagram after 3-4 F-T cycles,which means the water mobility increased and immobilized water was shifted to free water.The texture of samples was declined especially at 1-2 F-T cycles.The rapid growth of PPO activity caused the decrease of brightness?L^*?and the increase of redness?a^*?after 3 F-T cycles.Meanwhile,F-T cycles accelerated the protein aggregation before 4 F-T cycles and denaturation after 4 cycles.Therefore,repetitive F-T cycles could accelerate the changes of protein,microstructure,water distribution,quality deterioration and the increase of TVB-N,TVC and sensory score explained the quality changes of shrimp became obvious after 3 F-T cycles and unacceptable after 6 cycles.Secondly,the anti-freezing effect of different antifreeze agents on Litopenaeus vannamei with repeated F-T cycles and the influence of repeated F-T or different antifreeze agents on the nutritional value and flavor of shrimp were explored to identify the advantages and disadvantages of these antifreeze agents,and then revealed the advantages of carrageenan oligosaccharide.Shrimp samples treated with sterile water?CK?,sodium tripolyphosphate?ST?,mixture of sucrose and sorbitol?SS?and carrageenan oligosaccharides?CO?were subjected to different F-T cycles.The anti-freezing effect of antifreeze agents were evaluated by combining WHC,Ca²⁺-ATPase activity with MRI and FTIRI.The changes of nutritional value and flavor were studied by basic nutrition analysis supplemented with amino acid automatic analyzer,electronic nose and tongue.The results showed that with the increase of number of F-T cycles,the WHC,Ca²⁺-ATPase activity and the content of water,crude protein,crude fat in different groups decreased,indicating that repeated F-T accelerated protein frozen denaturation,water loss or lipid oxidation and different antifreezes could in different extent protect protein or muscle quality,especially carrageenan oligosaccharide followed by sugar and sorbitol mixture.In addition,LF-NMR pseudo-color images and FTIRI can more intuitively reflect the changes of water and protein content in shrimp with F-T cycles,and the results of them were consistent with the traditional chemical indexes.The analysis of amino acid showed that the content of total amino acid,essential amino acid and umami amino acid decreased,and the content of bitter amino acid increased.These changes were not obvious in CO or SS groups,and the content of Thr and Ser in the two groups showed a slight upward trend.The PCA analysis diagram of electronic tongue and nose showed that with the F-T cycles increased,the difference in taste and odor components of shrimp compared with fresh sample increased,but the difference in CO group was no significant,while in ST group was obvious after four F-T cycles and SS treatment made the flavor of samples be changed.Therefore,in terms of anti-freezing effect,the CO group was extremely significant,followed by SS and ST.The CO and SS treatments had better effect on the nutritional composition and amino acids.In terms of the influence on sensory taste and smell,the CO group was the best,followed by the ST group,while the SS group had a significant influence on it.Thirdly,the effects of carrageenan oligosaccharide on lipid oxidation,protein oxidation or denaturation and moisture migration of Litopenaeus vannamei were further evaluated during repeated F-T cycles.Distilled water?CK?,sodium tripolyphosphate?ST?and carrageenan oligosaccharides?CO?were subjected to shrimp samples with different F-T cycles.In this study,lipid oxidation were evaluated by lipid content,TBARS,FFA and PV value,protein oxidative denaturation and degradation were evaluated by carbonyl content,total sulfhydryl content?SH?,surface hydrophobicity,SDS-PAGE combined with DSC and dynamic rheology.And LF-NMR was used for the determination and analysis of water migration.The results showed that with the increase of F-T cycle times,the lipid content,total sulfhydryl group and intracellular water decreased,and TBARS value,FFA content,PV,carbonyl group content and surface hydrophobicity increased significantly.And these changes were more obvious in CK and ST groups.The results of DSC,dynamic rheology and SDS-PAGE showed that carrageenan oligosaccharide could protect the structure and integrity of myosin significantly and delay the degradation and denaturation of sarcoplasmic protein in shrimp during F-T cycles.And the change of the indicators about lipid oxidation in the three groups were closely related to protein oxidation and water loss,indicating that there was a certain interaction between lipid oxidation,protein oxidation and water loss.As an antifreeze water retention agent,carrageenan oligosaccharide was more effective in controlling the quality changes of shrimp during repeated F-T cycles,which was mainly reflected in the protection of protein functional properties.Fourthly,In order to study the influencing mechanism of F-T cycles and carrageenan oligosaccharide on muscle protein of shrimp in depth,FT-IR and Raman spectra of myofibrillar protein in shrimp with different pretreatments?aseptic distilled water,tripolyphosphate and carrageenan oligosaccharide?after 0,2,4,6 F-T cycles were respectively collected by FT-IR complemented with Raman spectroscopy.The intensity changes of each characteristic peak in FT-IR and Raman first-order spectrogram indicated that F-T cycles aggravated the loss and structural damage of muscle protein in shrimp.For the amide I band(1600-1700 cm^-1)after gaussian fitting characterizing the primary chain conformation of secondary structure in the shrimp muscle protein,the results of FT-MIR showed that the secondary structure of fresh shrimp protein was mainly?-turn,followed by?-sheet.Combined with the result of Raman spectrum,the secondary structure changes of protein during F-T were mainly the decrease of?-helix and increase of random coil and carrageenan oligosaccharide could significantly inhibit the loss of?-helix in shrimp protein during F-T.The peak intensity ratio at 850 cm^-1 and 830 cm^-1 in Raman spectrum indicated the exposure of tyrosine,which showed an increasing trend during F-T.The peak strength changes of2935 cm^-1 and 1448 cm^-1 were on behalf of hydrophobic interaction strength of side chain amino acid,which increased during F-T.The changes in characteristic spectral bands of protein side chains in Raman spectra showed that F-T cycles made the hydrogen bond rupture of protein intramolecular or intermolecular and the exposure of tyrosine or aliphatic side chain amino acid residues.And carrageenan oligosaccharides could delay the rupture of hydrogen bond and exposure of the side chain hydrophobic group in muscle protein with F-T,further stabilize protein secondary structure,maintain protein function and protect the quality of shrimp under F-T cycles.Finally,the changes of microbial diversity in the Pacific white shrimp with sterile water?CK?or carrageenan oligosaccharide?CO?treatment during repeated F-T cycles were studied.TVB-N value,K value,ACP or ADA activity,biogenic amine and TVC,Pseudomonas and Shewanella were measured respectively to characterize the quality changes and the correlation between microorganisms and ATP degradation.Then combined with high-throughput sequencing analysis,the effects of repeated F-T and carrageenan oligosaccharide on microbial flora were studied.The results showed that CO treated samples maintained good quality,as evidenced by attenuating the production of TVB-N,retarding the increase of putrescine,cadaverine and tyramine,delaying the degradation of ATP,tryptamine and spermine,and inhibiting microbial growth.Meanwhile,the changes of ADA and ACP activity indicated that the effect of carrageenan oligosaccharide on ACP activity was significant and then the ATP degradation process was affected.The results of the high-throughput sequencing targeting on 16S rRNA genes showed that carrageenan oligosaccharides observably inhibited the growth of Moraxellaceae and Pseudomonadaceae and exhibited a slight effect on Shewanellaceae and Listeriaceae.In conclusion,combined with relative abundance of microbiota at the genus level,carrageenan oligosaccharides altered the bacterial communities and delayed quality deterioration of Penaeus vannamei during repeated F-T cycles.