In order to improve the fermentation process of the emerging commercial low-salt shrimp paste, this study systematically analyzed the changes in the major quality indicators during the fermentation process. High-throughput sequencing technology was used to analyze the microbial community diversity and composition. The results showed that during the fermentation of shrimp paste, the pH, malondialdehyde (MDA) content, total volatile basic nitrogen (TVBN) content, amino acid nitrogen content, moisture content, salt content and biogenic amine content fluctuated in the range of 7.19–6.90, 3.09–5.56 mg/kg, 44.81–175.05 mg/100 g, 1.42–1.63 g/100 g, 59.19%–62.40%, 11.02%–12.04% and 0–113.29 mg/kg, respectively. The MDA content increased and then subsequently decreased. The contents of amino acid nitrogen, TVBN and biogenic amines all showed an increasing trend. At the middle and late stages of fermentation, the bacterial diversity indexes decreased significantly, and the fungal diversity indexes decreased slowly. At the early stage of fermentation (0–7 days), Streptococcus and Lactobacillus were the dominant bacterial genera. During the middle and late periods (34–145 days), Tetragenococcus was the dominant bacterial genus and was differential between the three stages of fermentation. Trichosporon was the dominant fungal genus at the early stage of fermentation. At the middle stage, Candida and Alternaria became the dominant genera. At the late stage, Candida became the dominant genus, and no significantly differential fungal genera were found between the three fermentation stages. The Spearman correlation heat map showed that compared with the fungus genera, the bacterial genera had a higher correlation with the physiochemical indicators, indicating that the bacterial community was dominant in the fermentation process. Tetragenococcus was positively correlated with the TVBN, amino acid nitrogen, and MDA contents; Streptococcus was significantly negatively correlated with the content of biogenic amines. Starting from the 64^th day, the quality indicators and microbial community of the shrimp paste remained basically stable, suggesting that shortening the fermentation time can be considered as an option to improve the fermentation process. These results provide a theoretical basis for improving the fermentation process of low-salt shrimp paste.

Natural chitin has a highly ordered crystal structure and strong intermolecular and intramolecular hydrogen bonds, and is insoluble in water, so it is difficult to enzymatically degrade. In order to improve the enzymatic degradation efficiency of chitin, combined treatment of ultra-micro grinding and high-pressure homogenization was used to modify chitin in this study. The physicochemical properties of modified chitin were determined, and its microstructure was characterized by Fourier transform infrared (FTIR) spectroscopy, elemental analysis, X-ray diffraction, thermogravimetric-differential scanning calorimetric (TG-DSC) analysis and scanning electron microscopy (SEM). Besides, non-specific enzymatic hydrolysis experiments were carried out to validate the effect of the combined treatment on the hydrolysis efficiency of chitin. The results showed that after the modification treatment, the average particle size of chitin decreased by 99.71%, the specific surface area increased by 85.11%, the pore volume increased by 29.03%, the viscosity-average molecular mass decreased by 85.80%, and the expansion ratio increased by 2.02 times as compared from tap density. Moreover, the intensity of the absorption peaks near 3448 and 3263 cm^-1 increased, whereas the absorption intensity of the absorption peak at 896 cm^-1 decreased. However, there was no obvious change in the degree of deacetylation. The crystallinity index on the (110) and (020) plane decreased by 17.49% and 1.31%, respectively. The thermal stability was destroyed. The structure became loose and porous. The modified chitin was more easily degraded by cellulase and papain. In addition, the combined treatment was more effective than either treatment alone. In summary, the combined treatment of ultra-micro grinding and high-pressure homogenization can effectively modify the physicochemical properties and microstructure of natural chitin, thus improving the enzymatic hydrolysis efficiency of chitin.

The metabolite profile of low-salt shrimp paste during low-temperature fermentation was investigated by metabolomics based on gas chromatography-mass spectrometry (GC-MS), and the differential metabolites were selected by multivariate statistical analysis combined with t-test and variable importance in projection (VIP) analysis. The results showed that the orthogonal partial least squares discriminant analysis (OPLS-DA) model clearly discriminated the metabolite profiles of shrimp paste samples fermented at 10 (low temperature) and 20 (control) ℃. A total of 44 metabolites were identified in the two samples, including 16 amino acids, 6 fatty acids, 2 amines, 8 organic acids, 3 alcohols, 3 nucleotides and 6 other compounds. By considering P < 0.05 and VIP > 1, 30 differential metabolites were obtained, including 7 amino acids, 2 esters, 2 purines, 8 organic acids, 2 alcohols and 9 other metabolites. The levels of L-serine, L-threonine, xanthine, hypoxanthine and L-glutamine in the low-temperature group were significantly higher than those in the control group, while the levels of nerazine acetate, zeaxanthin and melatonin were significantly lower (P < 0.01). These results provide a theoretical basis for further improving the production process of low-salt shrimp paste.

The allergenicity of the major allergen tropomyosin (TM) in commercial low-value shellfish and its processed products was evaluated, and different methods to reduce the allergenicity of TM in edible shellfish tissues were studied. The contents of TM in commercial low-value shellfish and its frozen, dried, smoked or steamed products were analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and shellfish with high allergenicity was selected to explore the effects of traditional (drying, steaming, high temperature and pressure, and quick-freezing) and emerging processing techniques (high hydrostatic pressure, ultrasound, and glycosylation) on reducing tropomyosin allergenicity in edible tissues. The results showed that allergenicity was common in commercial shellfish and its processed products, with TM content ranging from 18.94 to 22.51 ng/g. Among the four kinds of fresh shellfish tested, the TM content of Mactra veneriforimis was the highest, which was (22.51 ± 1.06) ng/g. Compared with untreated Mactra veneriforimis, the allergenicity of dried, steamed or glycosylated samples did not change significantly (P > 0.05). High temperature and pressure, quick-freezing, high hydrostatic pressure and ultrasonic treatment could significantly reduce TM allergenicity (P < 0.05), the effect of ultrasonic (500 W and 30 min) and high temperature and pressure sterilization treatment being more pronounced that of the other treatment, reducing TM allergenicity by 32.4% and 26.9%, respectively. This study provides a theoretical basis and technical support for the development of hypoallergenic processed shellfish products.

Chitin is the second most abundant natural polysaccharide on Earth, which can be deacetylated to produce chitosan. However, due to the high crystallinity and poor solubility of chitin, its efficiency of enzymatic deacetylation is very low. To investigate the effects of different treatments on the microstructure and enzymatic deacetylation efficiency of chitin, chitin was modified by ball milling, ultrasonic or hydrochloric acid treatment in this study. First, the optimum conditions of ball milling and ultrasonic treatments were determined. Then, the microstructure of modified chitins was characterized by Fourier transform infrared (FTIR) spectroscopy, elemental analysis, X-ray diffraction, thermogravimetric-differential scanning calorimetry (TG-DSC) and scanning electron microscopy (SEM). Finally, the deacetylation efficiency of chitin deacetylase from Rhodococcus sp.11-3 for chitin was measured. The results showed that the optimum conditions of ball milling and ultrasonic modification were 1800 r/min and 45 min, and 400 W and 45 min, respectively. Among the three treatments, ball milling had the best modification effect, resulting in an 88.14% reduction in the viscosity-average molecular mass of chitin. The intermolecular hydrogen bond network of chitin was destroyed and some glycosidic bonds were broken; the degree of deacetylation increased; the crystallinity decreased from 96.30% to 73.04%; thermal stability was destroyed; and the treated chitin showed a stacking structure and was more loose and porous. In addition, chitin deacetylase had higher deacetylation efficiency for ball-milled chitin, and the yield of acetic acid was 3.4 times higher than that from raw chitin. In conclusion, ball milling treatment can improve the enzymatic deacetylation efficiency of chitin by changing its physicochemical properties and microstructure.

In order to develop new shrimp paste products and expand the shrimp paste market, ginger, cinnamon or perilla was added before fermentation of low-salt shrimp paste in this study. The physicochemical indexes of low-salt shrimp paste were determined, and the effect of spice addition on the microbial community composition and diversity of shrimp paste was explored by high-throughput sequencing technology. The results showed that the contents of moisture and salt in shrimp paste were 51.34%–61.77% and 15.61%–17.71%, respectively. The addition of spices decreased the pH and amino nitrogen content of shrimp paste. The addition of cinnamon and ginger reduced the total volatile basic nitrogen (TVB-N) content by 30.41% and 10.28%, respectively, while the addition of perilla increased the TVB-N content by 9.98%. Compared with the control shrimp paste, the diversity of fungi and bacteria in shrimp paste added with perilla was higher. The addition of cinnamon and ginger decreased the richness of bacterial community and increased the richness of fungal community in shrimp paste. The analysis of differential flora showed that the addition of ginger and cinnamon increased the relative abundance of Tetragenococcus and Wallemia, but reduced the relative abundance of Staphylococcus by 99.32% and 98.14%, respectively. The relative abundance of Penicillium and Aspergillus in shrimp paste added with perilla was higher. Pearson correlation analysis showed that Staphylococcus, Wallemia, Rhodotorula and Penicillium had significant correlations with physicochemical indicators, indicating that these microorganisms play an important role in shrimp paste. To sum up, the addition of spices can change the microbial community composition of shrimp paste, enrich the types of shrimp paste and improve the safety of shrimp paste.

In order to study the effect of fermentation temperature on the flavor substances in low-salt shrimp paste, the natural fermentation of shrimp paste was carried out at 10, 15, 20 or 25 ℃. The contents of free amino acids, free fatty acids and volatile compounds in shrimp paste were determined by high performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-ion mobility spectrometry (GC-IMS), respectively. The results showed that 17 free amino acids and 26 free fatty acids were detected in each shrimp paste sample. The content of flavor amino acids was highest in shrimp paste fermented at 10 ℃, and the content of free fatty acids was highest in shrimp paste fermented at 20 ℃. A total of 69 volatile compounds were identified in all samples, including 19 alcohols, 13 aldehydes, 13 ketones, 12 esters, 2 alkenes, 2 ethers, 1 acid and 7 other compounds. The relative contents of alcohols and ketones were higher in shrimp paste fermented at 10 ℃ and 15 ℃. The relative contents of ammonia were higher in shrimp paste fermented at 20 or 25 ℃. Principal component analysis (PCA) showed that volatile compounds were significantly distinguishable in low-salt shrimp pastes fermented at different temperatures. A total of six differential volatile compounds (variable importance in projection (VIP) >1, P < 0.05) were identified by orthogonal partial least squares-discriminant analysis (OPLS-DA), namely 3-methyl-1-butanol (monomer and dimer), ethyl acetate dimer, ethyl acrylate, 2-methylbutanal dimer and ammonia. Cluster heatmap analysis showed that the flavor of low-salt shrimp paste fermented at 10 and 15 ℃ was similar, and the flavor of low-salt shrimp paste fermented at 20 and 25 ℃ was similar. The results of this study may provide a theoretical basis for promoting the thermostatic fermentation of commercialized low-salt shrimp paste.

Response surface methodology (RSM) was employed to optimize the hydrolysis conditions of acid-soluble collagen (ASC) from Takifugu rubripes skin for the preparation of anti-tyrosinase peptides. The anti-tyrosinase and antioxidant activity of ultrafiltration peptides with different molecular masses were tested before and after being subjected to simulated gastrointestinal digestion, and the moisture absorption and retention properties were also evaluated to explore their application potential. The results showed that neutral protease was the most suitable enzyme for releasing antityrosinase peptides from T. rubripes skin collagen. The optimum process conditions were as follows: substrate concentration 2.9 g/100 mL, hydrolysis time 7.8 h, and enzyme dosage 7862 U/g. The hydrolysate obtained under these conditions inhibited tyrosinase activity by (35.14 ± 0.03)%. The molecular mass of the hydrolysate mainly ranged from 0 to 5 kDa, and the fraction CP-1 (< 3 kDa), with the highest anti-tyrosinase activity, scavenged 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation and hydroxyl radicals with half maximal inhibitory concentration (IC₅₀) values of 9.51, 2.03 and 7.29 mg/mL, respectively. CP-1 maintained high anti-tyrosinase and antioxidant activity after in vitro simulated digestion. In addition, CP-1 had high moisture absorption and retention properties and therefore could be used as a substitute for glycerol. This study may provide a theoretical basis for the deep processing and application of collagen peptides from T. rubripes skin.

In order to investigate the effect of salt addition on the fermentation of shrimp paste, the physicochemical indexes, sensory attributes and bacterial communities of shrimp paste (noted as C10, C15, C20 and C25, respectively) with four salt concentrations (10%, 15%, 20% and 25%) were analyzed. The results showed that C15 had the highest sensory scores for smell, taste and overall acceptability. As fermentation proceeded, the increasing trend of total volatile basic nitrogen (TVB-N) and amino acid nitrogen (AAN) contents in shrimp paste was negatively correlated with salt addition. Malondialdehyde (MDA) content showed a fluctuating upward trend, and at the end of fermentation, C15 had the lowest MDA value. The pH of shrimp paste did not change significantly during the fermentation process, and no significant difference was observed among the four shrimp pastes (P > 0.05). Bacterial diversity and abundance were significantly lower in C10 and C15 than in C20 and C25 (P < 0.05). The dominant bacterial genera in C10 were Tetragenococcus and Staphylococcus. Tetragenococcus was the dominant genus in C15. Unclassified_f__Rhodobacteraceae, Planktotalea and Pandoraea were the dominant genera in unfermented shrimp paste, C20, and C25. Using Kruskal-Wallis H Test, 10 significant differential bacterial genera were selected among unfermented shrimp paste and the four fermented samples. Pearson correlation analysis showed that in C10, the relative abundance of Acinetobacter, Virgibacillus and unclassified_f__Bacillaceae was positively correlated with TVB-N. In C15, the relative abundance of Persicirhabdus showed a significant negative and positive correlation with TVB-N and AAN, respectively. In C20, the relative abundance of Sulfitobacter, Rubritalea, Ruegeria and Planktotalea showed a positive correlation with both TVB-N and MDA. In C25, the relative abundance of Sulfitobacter, Ruegeria, Ruegeria, Ascidiaceihabitans and Planktotalea was positively correlated with all AAN, TVB-N, and MDA. Overall, the key microorganisms responsible for the differences in physicochemical properties between low- (C10 and C15) and high-salt (C20 and C25) shrimp pastes were Acinetobacter, Mycobacterium, Rubritalea, Planktotalea, Sulfitobacter and Ascidiaceihabitans. This study provides a theoretical basis for the development and industrial production of salt-reduced shrimp paste.