Low-salt Fish Research Articles

Functional and Mechanistic Dissection of Protein Glutaminase PG3 and Its Rational Engineering for Enhanced Modification of Myofibrillar Proteins.

Protein glutaminases (PG; EC = 3.5.1.44) are enzymes known for enhancing protein functionality. In this study, we cloned and expressed the gene chryb3 encoding protein glutaminase PG3, exhibiting 39.4 U/mg specific activity. Mature-PG3 featured a substrate channel surrounded by aromatic and hydrophobic amino acids at positions 38-45 and 78-84, with Val81 playing a pivotal role in substrate affinity. The dynamic opening and closing motions between Gly65, Thr66, and Cys164 at the catalytic cleft greatly influence substrate binding and product release. Redesigning catalytic pocket and cocatalytic region produced combinatorial mutant MT6 showing a 2.69-fold increase in specific activity and a 2.99-fold increase at t65°C1/2. Furthermore, MT6 boosted fish myofibrillar protein (MP) solubility without NaCl. Key residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108 were vital for PG3-myosin interaction, particularly Asn54 and Asn107. This study sheds light on the catalytic mechanism of PG3 and guided its rational engineering and utilization in low-salt fish MP product production.

An isolated salt-tolerant Tetragenococcus halophilus 2MH-3 improved the volatile flavor of low-salt fermented fish sauce by regulating the microbial community

The application of low-salt fish sauce is limited by its tendency to spoil easily and inadequate flavor generation. Herein, a salt-tolerant Tetragenococcus halophilus 2MH-3 strain with strong abilities of enzyme production and biogenic amine degradation was utilized as a starter for the production of low-salt fish sauce. Bacterial community analysis revealed discrepancies in microbiota between low-salt fish sauces fermented with (Th group) or without 2MH-3 (LF group). Staphylococcus was the primary genus in the Th group at 1 M fermentation (47.42 %), followed by Psychrobacter (10.82 %), while Tetragenococcus swiftly ascended to the dominant status with a relative abundance of 5.88 % after 3 M fermentation. Conversely, the abundance of Tetragenococcus throughout the LF fermentation period was no significant change. In Th group, 118 volatile components were detected with 21 high-concentration flavor compounds being the primary flavor components (OAV ≥ 1), which were basically produced by Alkaliphilus, Psychrobacter, Tetragenococcus, Bacteroides and Staphylococcus based on the co-occurrence heatmaps after PLS-DA evaluation. Furthermore, the co-occurrence network map demonstrated that the decrease in key biogenic amines such as histamine, putrescine, cadaverine and tyramine, the increase in bacterial diversity, as well as the increase in 21 core volatile flavor compounds (OVA ≥ 1.0), were mainly caused by the addition of T. halophilus 2MH-3 in the low-salt fish sauce. Therefore, T. halophilus 2MH-3 could be utilized as an underlying microbial starter in the industrialization of fish sauce.

Elevated levels of branched chain fatty acids in low-salt fish sauce by co-fermentation: flavor improvement and metabolism analysis.

Traditional fish sauce products rely on relatively long fermentation time and high salt concentration, resulting in inconsistent quality and health risks. Branched-chain fatty acids (BCFAs) are associated with nutritional benefits and health-care effects, mainly derived from food fermentation. This study aimed to screen BCFAs-producing bacteria with high protease and aminotransferase activity as starter cultures for fish sauce fermentation. The low-salt fish sauce products were obtained by co-fermentation with three chosen strains. Trichloroacetic acid (TCA)-soluble peptides and amino acid nitrogen concentrations were higher in the co-fermentation group (FH group). The organoleptic evaluation showed co-fermentation optimized flavor composition and endured with rich taste. The levels of BCFAs and branched-chain amino acids (BCAAs) significantly increased by co-fermentation. Volatile metabolomics analysis indicated that BCFAs, branched-chain esters, and pyrazines were the key flavor compounds in the co-fermented group. The co-fermentation system with selected strains to ferment low-salt fish sauce has the potential to increase BCFA content and improve flavor and nutrition. © 2024 Society of Chemical Industry.

Volatile Flavor Improvement and Spoilage Microorganism Inhibition in Low-Salt Fish Sauce (Yulu) by Salt-Tolerant Bacillus subtilis

Use of low-salt fish sauce (Yulu) is limited due to its perishable property and rapid loss of unique flavor. In this study, a salt-tolerant strain, Bacillus subtilis B-2, with high protease production was used as a microbial starter for low-salt Yulu fermentation. A total of 133 volatile compounds were detected through HS-SPME-GC-MS. Most aldehydes, alcohols, ketones, furans, and hydrocarbons reached their maximum at 15 d, while most esters, aromatic compounds, acids, nitrogen compounds, and sulfur compounds peaked at 45 d. The 16S rRNA gene high-throughput sequencing showed that Bacillus remained in high abundance during fermentation, reaching 93.63% at 45 d. The characteristic volatile flavor was obviously improved while the microbial contamination was significantly reduced in low-salt Yulu fermented with B. subtilis, compared with those without a microbial starter. The correlation network map suggested that the significant decrease in Pseudomonas, Achromobacter, Stenotrophomonas, Cyanobium, Rhodococcus, Brucella, Tetragenococcus, and Chloroplast contributed most to the decreasing richness and evenness of the microbial community, while Bacillus was the only genus playing a key role in the inhibition of spoilage microorganisms and improvement of volatile flavor after B. subtilis addition. This study suggests the potential use of salt-tolerant B. subtilis as a special starter for industrial Yulu fermentation in the future.

Novel insight into flavor and quality formation in naturally fermented low-salt fish sauce based on microbial metabolism

Low-salt fermentation is an effective way to shorten the fermentation time of fish sauce. In this study, the changes of microbial community, flavor, and quality during the natural fermentation of low-salt fish sauce were studied, followed by the elucidation of flavor and quality formation mechanisms based on microbial metabolism. The 16S rRNA gene high-throughput sequencing showed that both richness and evenness of microbial community were reduced during fermentation. The microbial genera, including Pseudomonas, Achromobacter, Stenotrophomonas, Rhodococcus, Brucella, and Tetragenococcus were more suitable for the fermentation environment, and obviously increased along with the fermentation. There were a total of 125 volatile substances identified by HS-SPME-GC–MS, of which 30 substances were selected as the characteristic volatile flavor substances, mainly including aldehydes, esters, and alcohols. Large amounts of free amino acids were produced in the low-salt fish sauce, especially umami and sweet amino acids, as well as high concentrations of biogenic amines. Correlation network constructed by the Pearson’s correlation coefficient showed that most characteristic volatile flavor substances were significantly positively correlated with Stenotrophomonas, Achromobacter, Rhodococcus, Tetragenococcus, and Brucella. Stenotrophomonas and Tetragenococcus were significantly positively correlated with most free amino acids, especially umami and sweet amino acids. Pseudomonas and Stenotrophomonas were positively correlated with most biogenic amines, especially histamine, tyramine, putrescine, and cadaverine. Metabolism pathways suggested that the high concentrations of precursor amino acids contributed to the production of biogenic amines. This study indicates that the spoilage microorganisms and biogenic amines in the low-salt fish sauce need to be further controlled, and the strains belonging to Tetragenococcus can be isolated as potential microbial starters for the production of low-salt fish sauce.

Microbial community changes induced by a newly isolated salt-tolerant Tetragenococcus muriaticus improve the volatile flavor formation in low-salt fish sauce

Application of low-salt fish sauce is restricted by its easy spoilage and poor flavor formation. In this study, a salt-tolerant strain Tetragenococcus muriaticus with powerful salt tolerance was isolated as starter for the low-salt fish sauce fermentation. The 16S rRNA gene high-throughput sequencing showed that Staphylococcus was the main microbial genus at the beginning of fermentation, reaching 73.75%, followed by Tetragenococcus (16.36%) and Pseudomonas (6.68%), while Tetragenococcus quickly took over the dominant position with the relative abundance over 56.9% after 10 d fermentation and peaked at the end of fermentation. There were a total of 144 volatile compounds identified by HS-SPME-GC-MS, among which aldehydes, esters, alcohols, heterocycles, ketones, and acids were the main volatile flavor compounds and were mainly produced by Tetragenococcus, Synechococcus, Rhodococcus, Stenotrophomonas, Achromobacter, and Brucella based on the correlation network maps constructed by the Pearson’s correlation coefficient after O2PLS evaluation. The Sobs, ACE, Chao, and Shannon indexes of microbial community were significantly reduced, while the Simpson index was markedly enhanced in the low-salt fish sauce fermented with T. muriaticus compared with them in the fish sauce without starter addition. The addition of T. muriaticus also obviously improved the types and concentrations of main volatile flavor compounds in the low-salt fish sauce. The correlation network map showed that Tetragenococcus was the only genus that played a crucial role in the spoilage microorganism inhibition and volatile flavor improvement in the fish sauce. T. muriaticus can be developed as a potential microbial starter for the industrial production of low-salt fish sauce.

Physicochemical and Antioxidant Properties of Fish Sauce Prepared by Virgibacillus sp. Starter Cultures Addition and Reduced Salt Process

ABSTRACT Antioxidant activity and peptide profiles of fish sauce samples prepared under varied conditions, namely traditional process, starter culture addition, and a reduced salt process, were determined. Samples were desalted using the HiTrap column, resulting in fraction F1, F2, and F3. The commercial fish sauce (HS-Com) contained smaller molecular weight peptides, and low salt fish sauce (LS) contained higher ratio of large peptides. For F1, high salt fish sauce with Virgibacillus sp. starter culture (HS+Vir), showed the highest 2, 2ʹ-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity and ferric reducing antioxidant power (FRAP) value. In contrast, the highest antioxidant activities were observed in the HS-Com in F3. The addition of Virgibacillus sp. with either full or reduced salt resulted in more antioxidant peptides. The de novo peptide sequencing revealed that proline (P) was predominantly found in the F1 fraction of LS, LS+Vir, and HS-Com. All samples were rich in glutamic acid (E).

Dynamics of water mobility, salt diffusion and hardness changes in bighead carp fillets during low-salting

Low-salt fish as a traditional preserved food has gained growing popularity for the reduction in sodium consumption. Water transport, salt diffusion, and hardness changes of bighead carp fillets were investigated by low-field nuclear magnetic resonance (LF-NMR), electric resistance, and texture profile analysis (TPA) under salting concentration of 15 mg/g and 30 mg/g. The decrease of P21 (T21 population) by LF-NMR analysis of G0 (control) was 12.1% while that of G1 (15 mg/g) and G2 (30 mg/g) was 2.2% and 5.3%, respectively. Meanwhile, the diffusion of salt was fast in the first 30 min, and the higher the salt concentration, the lower the resistance value. Furthermore, the higher hardness in G1 was observed, reaching 2.17 times that of control. At the end of salting, the proportion of P21 (Y1) and change of hardness (Y3) showed negative linear relationship with salt content (from 15 mg/g to 60 mg/g): Y1 = −0.0094 x + 0.9702 and Y3 = −0.1752x + 2.3544, and the resistance change rate (Y2) showed a positive correlation: Y2 = 5.9722x + 0.5625. The established model could effectively predict the hardness and water mobility of bighead carp fillets during the early stage of salting process.

Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L-1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The Km and Vmax values of the purified protease for casein were calculated to be 4.89 mg mL-1 and 1111.11 U mL-1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry.

Using pH-Shifting Process to Recover Proteins for Low Salt Gel Products from Silver Carp

The pH-shifting processes (include acid and alkali process) and conventional water-wash process was applied to recover protein from bony silver carp muscle. And the new process was evaluated to obtain proteins for low salt (0% and 1% NaCl, w/w) fish products. The alkali isolated protein (AK) exhibited the best solubility at all salt concentrations. Furthermore, the AK had the highest gel strength and water binding capacity at low salt concentrations (0% and 1%). Low salt products of AK containing 1% salt showed similar mechanical properties to the water wash surimi (WM) obtained with 2% salt. The results obtained showed that the alkali process is a good method to recover functional proteins from boney silver carp muscle for low salt fish products.

Food hydrocolloids as additives to improve the mechanical and functional properties of fish products: A review

The restructuring process offers to fish processors the opportunity to obtain new products, taking advantage of both low-value fish species and remains from filleting and other processing operations. However, mechanical and functional properties of restructured products depend on the biochemical and physicochemical properties of muscle proteins, mainly myosin and actomyosin. In this regard, the biochemistry of fish muscle is different from that of mammals and birds. Therefore, fish products must be processed in a different way from red meat or poultry. The main fish products are surimi and restructured products. Fish products can be improved or modified by using hydrocolloids (carbohydrates and protein) as additives. In this review, the modern technology to obtain these products, the applications of hydrocolloids in fish products, and the implications of the increasing demand for healthy, low-salt fish products are discussed.

Effect of salt and MTGase on the production of high quality gels from farmed sea bass

In order to obtain low salt fish products, the effects of the addition of 0.5% (w/w based on whole product, wp) MTGase and different levels (0.25%, 0.50%, 1.00% and 2.50%, w/w wp) of sodium chloride salt to heat-induced gels from farmed sea bass ( Dicentrarchus labrax) trimmings were tested and compared with gels without salt and/or without MTGase. MTGase and salt addition, both, had a favourable effect on texture. However, only salt incorporation augmented water holding capacity (WHC). A synergistic effect was detected between MTGase and salt for several important textural properties, particularly, gel strength, which reached 64.3 ± 8.1 N mm, a value comparable to those of best quality surimi. Moreover, MTGase required addition of salt to the product for its effect to be felt, however only a minimal amount (0.25%, w/w wp) sufficed. In fact, the addition of MTGase enabled reduction in salt content to 1.0% (w/w wp), without significant loss of textural and overall quality.

Effect of electrodialysis pretreatment on physicochemical properties and morphology of spray-dried-fish sauce powder

Fish sauce is a popular Asian food ingredient having specific flavor that gives pleasure to the senses of taste. However, fish sauce contains high-salt content, which is nowadays not desirable by health-conscious consumers. Recently, it has been shown that electrodialysis succeeds in reducing the salt content of fish sauce without affecting its important characteristics over a specific range of residual salt content. In this study the effect of electrodialysis pretreatment prior to spray drying to produce low-salt fish sauce powder, which is a more shelf-stable and convenient product compared with liquid fish sauce, was investigated. Product recovery, morphology and selected physicochemical properties, i.e., moisture content, water activity, hygroscopicity, as well as X-ray diffraction (XRD) patterns of the fish sauce powder were determined. The ED-treated-fish sauces of various salt contents (in the range of approximately 2–25% w/w) were spray dried at inlet drying air temperatures of 120–180 °C. It was found that the feed salt concentration and inlet air temperature had significant effects on all observed variables. This is due to the fact that the feed salt concentration strongly affected water removal from the ED-treated-fish sauce during spray drying, leading to significant alterations of the ED-treated-fish sauce powder properties.

Biochemical changes associated with fast fermentation of squid processing by-products for low salt fish sauce

In order to enhance the economical values of squid processing by-products and reduce the environmental problems caused by the wastes from squid processing, the possibility of utilizing squid processing by-products for low salt fish sauce production was investigated. Low salt fish sauce was prepared experimentally from squid processing by-products, according to three different manufacturing techniques (A, B and C) with or without the autolysis process and the addition of flavourzyme, soybean koji. Fish sauce products with similar quality were obtained at 48 °C after 30 days fermentation. The content of total soluble nitrogen in three fish sauce A, B and C were 2.135 ± 0.038%, 1.958 ± 0.041% and 2.038 ± 0.043%, respectively. The content of formaldehyde nitrogen in three fish sauce A, B and C were 1.028 ± 0.038 g/100 ml, 1.000 ± 0.046 g/100 ml, 1.127 ± 0.043 g/100 ml, respectively. The salt content in three fish sauce A, B and C were 8.842 ± 0.138%, 9.058 ± 0.142%, 8.764 ± 0.129%, respectively. At the same time, changes in total soluble nitrogen, conversion of nitrogen, pH, formaldehyde nitrogen, total titration acid, total volatile base nitrogen, salt concentration, protease activity, total plate counts of fish sauce were observed during fermentation. The results suggested that total soluble nitrogen, conversion of nitrogen, formaldehyde nitrogen, total volatile base nitrogen and salt concentration increased throughout the fermentation period. However, pH, total titration acid, protease activity and total plate counts showed different changes in different fermentation stage. The result of amino acids analysis suggested that glutamic acid was the most prominent in three fish sauce samples. The results from quantitative descriptive analysis test showed that all fish sauce samples were tasted and no particularly strong or unpleasant flavor.

Development of Low-salt Fish Sauce Using High Levels of Yeast

Development of Low-salt Fish Sauce Using High Levels of Yeast

Use of dairy proteins and microbial transglutaminase to obtain low-salt fish products from filleting waste from silver carp ( Hypophthalmichthys molitrix)

Restructured fish products from the filleting waste from silver carp ( Hypophthalmichthys molitrix) were obtained using sodium caseinate (1%), whey protein concentrate (WPC) (1%) or MTGase (0.3%) at different levels of salt (0%, 1% or 2%). The restructured products were obtained by incubating at 40 °C for 60 min and then at 90 °C for 20 min. Changes in the mechanical properties were studied by measuring changes in the textural profile analyses, punch test and torsional test analyses and expressible water. The mechanical properties increased when the salt level was also increased. The mechanical properties of non-salted and low-salt products were increased by using dairy proteins. Sodium caseinate had a greater effect in improving mechanical properties than WPC. MTGase increased expressible water. The results obtained showed that the mechanical properties of low-salt restructured fish products can be improved by using dairy proteins combined with MTGase, with a slight increase in expressible water.