Comprehensive analysis of predominant pathogenic bacteria and viruses in seafood products.

Given the growing global demand for seafood, it is imperative to conduct a comprehensive study on the prevalence and persistence patterns of pathogenic bacteria and viruses associated with specific seafood varieties. This assessment thoroughly examines the safety of seafood products, considering the diverse processing methods employed in the industry. The importance of understanding the behavior of foodborne pathogens, such as Salmonella typhimurium, Vibrio parahaemolyticus, Clostridium botulinum, Listeria monocytogenes, human norovirus, and hepatitis A virus, is emphasized by recent cases of gastroenteritis outbreaks linked to contaminated seafood. This analysis examines outbreaks linked to seafood in the United States and globally, with a particular emphasis on the health concerns posed by pathogenic bacteria and viruses to consumers. Ensuring the safety of seafood is crucial since it directly relates to consumer preferences on sustainability, food safety, provenance, and availability. The review focuses on assessing the frequency, growth, and durability of infections that arise during the processing of seafood. It utilizes next-generation sequencing to identify the bacteria responsible for these illnesses. Additionally, it analyzes methods for preventing and intervening of infections while also considering the forthcoming challenges in ensuring the microbiological safety of seafood products. This evaluation emphasizes the significance of the seafood processing industry in promptly responding to evolving consumer preferences by offering current information on seafood hazards and future consumption patterns. To ensure the continuous safety and sustainable future of seafood products, it is crucial to identify and address possible threats.

Effect of modified washing process on water usage, composition and gelling properties of grass carp surimi.

BACKGROUND: Washing is an essential process in surimi production, from which a large amount of wastewater is generated. Due to the increasing pressure of environmental protection, it is an urgent technical requirement for surimi manufacturers to reduce water usage while maintaining the quality of surimi. In this study, composition, structure and gelling properties of grass carp surimi prepared with a modified washing process (MWP) were investigated. Intermediate dehydration with various compression ratios were utilized between two washing cycles. RESULTS: Water usage and wastewater discharge were reduced significantly by 33% and 38%, respectively, when MWP was applied. As the compression ratio increased, composition of fat, cathepsins, transglutaminase and heme proteins in surimi decreased gradually. Yield, protein content and the major protein pattern of surimi were not changed, but surface hydrophobicity gradually decreased. As the compression rate increased to 1:2.0, textural values and water holding capacity of the corresponding surimi gel decreased gradually, while whiteness increased and then remained unchanged. At a higher compression ratio (>1:1.5), aggregated network and excessive free water were observed in the surimi gel. Composition and gelling properties of the MWP surimi with a compression ratio of 1:1.2-1:1.5 were equal to those of the surimi prepared under conventional three-cycle washing. CONCLUSION: Results indicated that MWP demonstrated its great potential in surimi production by dramatically reducing the usage of cold water and discharge of wastewater without scarifying surimi quality. © 2022 Society of Chemical Industry.

Vibrational spectroscopy and biochemical changes in silver carp as related to quality of washed mince.

BACKGROUND: Biochemical and protein conformational changes in silver carp occurred during ice storage, affecting the physico-chemical and textural properties of its washed mince. Fourier-transform infrared spectroscopy (FTIR) and FT Raman could offer insightful molecular-level information that could be related to the freshness of fish and textural properties of washed mince. RESULTS: The K value increased from 15.8% to 85.0% after 14 days on ice. The surface hydrophobicity of silver carp muscle proteins increased during ice storage, and its thrice-washed mince showed the same trend. The yield and textural properties of washed mince continually decreased as the storage time was extended. Fourier-transform infrared spectroscopy revealed that, as storage time increased, the α-helix content of mince decreased, while the ß sheet content increased. Prolonged ice storage led to the exposure of buried aromatic amino acid residues and an increase in disulfide interchanges in mince and washed mince. Changes in the ∑ß sheet structure and Raman intensity at 828 cm-1 observed in mince correlated well with the K value. The α-helix content and Raman intensity of raw washed mince at 621 and 828 cm-1 showed a strong correlation with its textural properties. CONCLUSION: Silver carp should be processed to surimi within 7 days of ice storage to obtain a reasonably good yield and gel texture. Infrared and Raman spectroscopy can possibly be utilized to monitor freshness quality and protein conformational changes in silver carp and to estimate the textural properties of washed mince as affected by freshness. © 2019 Society of Chemical Industry.

Anti-HIV-1 activity of phlorotannin derivative 8,4‴-dieckol from Korean brown alga Ecklonia cava.

8,4‴-dieckol is a natural product which has been isolated from brown alga, Ecklonia cava. This polyphenolic compound is a phlorotannin derivative with a broad range of bioactivities. Its inhibitory activity on human immunodeficiency virus type-1 (HIV-1) was tested and the results indicated that 8,4‴-dieckol inhibited HIV-1 induced syncytia formation, lytic effects, and viral p24 antigen production at noncytotoxic concentrations. Furthermore, it was found that 8,4‴-dieckol selectively inhibited the activity of HIV-1 reverse trancriptase (RT) enzyme with 91% inhibition ratio at the concentration of 50 µM. HIV-1 entry was also inhibited by 8,4‴-dieckol. According to data from this study, 8,4‴-dieckol is an effective compound against HIV-1 with high potential for further studies. These results suggest that it might be used as a drug candidate for the development of new generation therapeutic agents, although further studies on the mechanism of inhibition should be addressed.

Comprehensive review: by-products from surimi production and better utilization.

Over 1 million MT of surimi is produced globally, which theoretically would generate approximate 2 million MT of solid by-products and more than 1 million MT of wash water. Utilization of the by-products has increasingly become interested based on their nutritional, economical, and environmental issues. Surimi by-products represent an important source of valuable compounds such as functional protein, collagen, gelatin, fish oil, peptides, minerals, and enzymes. Better utilization of the by-products would make the surimi industry sustainable and profitable. This review paper characterizes sources and composition of the solid by-products and wash water generated from the surimi production as well as factors related to extraction and processing techniques. In addition, the potential food applications are explored including specialty foods and snacks, flavor ingredients, bioactive ingredients, and functional ingredients. Moreover, an outlook summarizing the challenges and prospects on the utilization of surimi by-products is provided.

Feeding Neurons Integrate Metabolic and Reproductive States in Mice.

Trade-offs between metabolic and reproductive processes are important for survival, particularly in mammals that gestate their young. Puberty and reproduction, as energetically taxing life stages, are often gated by metabolic availability in animals with ovaries. How the nervous system coordinates these trade-offs is an active area of study. We identify somatostatin neurons of the tuberal nucleus (TNSST) as a node of the feeding circuit that alters feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of TNSST neurons increased food intake across sexes, selective ablation decreased food intake only in female mice during proestrus. Interestingly, this ablation effect was only apparent in animals with a low body mass. Fat transplantation and bioinformatics analysis of TNSST neuronal transcriptomes revealed white adipose as a key modulator of the effects of TNSST neurons on food intake. Together, these studies point to a mechanism whereby TNSST hypothalamic neurons modulate feeding by responding to varying levels of circulating estrogens differentially based on energy stores. This research provides insight into how neural circuits integrate reproductive and metabolic signals, and illustrates how gonadal steroid modulation of neuronal circuits can be context-dependent and gated by metabolic status.

Feeding neurons integrate metabolic and reproductive states in mice.

Balance between metabolic and reproductive processes is important for survival, particularly in mammals that gestate their young. How the nervous system coordinates this balance is an active area of study. Herein, we demonstrate that somatostatin (SST) neurons of the tuberal hypothalamus alter feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of SST neurons increased food intake across sexes, ablation decreased food intake only in female mice during proestrus. This ablation effect was only apparent in animals with low body mass. Fat transplantation and bioinformatics analysis of SST neuronal transcriptomes revealed white adipose as a key modulator of these effects. These studies indicate that SST hypothalamic neurons integrate metabolic and reproductive cues by responding to varying levels of circulating estrogens to modulate feeding differentially based on energy stores. Thus, gonadal steroid modulation of neuronal circuits can be context dependent and gated by metabolic status.

Evaluation of Lipid Oxidation, Volatile Compounds and Vibrational Spectroscopy of Silver Carp (Hypophthalmichthys molitrix) During Ice Storage as Related to the Quality of Its Washed Mince.

Changes in the lipid oxidation of silver carp (Hypophthalmichthys molitrix) stored in ice for 14 days and that of its respective washed mince were evaluated. Total lipid, phospholipid, polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid (MUFA) contents of the skin, belly flap and mince decreased as the storage time in ice increased. The washing process decreased the lipid contents but concentrated their phospholipid counterparts. The fish belly flap exhibited the highest thio-barbituric acid reactive substances (TBARS) value, while the mince had the lowest. 1-Hexanol, 1-octen-3-ol, and 1-hexanal were key volatile compounds detected in the belly flaps of fish stored for 7-14 days. Hexanal was the only major volatile compound found in washed mince prepared from fish stored for an extended period in ice, but in a much lower amount compared with that in the belly flap. FTIR (Fourier transform infra-red) spectra revealed a decrease in the number of cis double bonds, methylene groups and phosphate groups in lipids extracted from fish stored in ice for 7-14 days as compared with those extracted from fresh fish. Principle component analysis (PCA) revealed that the FT-Raman band at 1747 cm-1 could be a potential marker for tracking the degree of lipid oxidation in the belly flap of silver carp stored in ice. In addition, IR bands indicating phosphate group (925, 825 cm-1) in oil extracted from washed mince were correlated with the extent of the lipid oxidation of the raw material.

Impact of acidity regulator and excipient nutrients on digestive solubility and intestinal transport of calcium from calcium phosphate and carbonate.

The aim of the current study was to investigate the effect of an acidity regulator (SPORIX®), lactose, and vitamin D3 as excipient ingredients on digestive solubility and intestinal transport of calcium from four different calcium materials (tricalcium phosphate (TCP), fish bone (FB), nano-fish bone (NFB), and algae calcium (AC)) through an in vitro digestion model system combined with Caco-2 cells. The concentration of ionized calcium (Ca2+) in an aqueous fraction after in vitro digestion increased with the addition of SPORIX®, and it was further enhanced by adding SPORIX® + lactose + vitamin D3 into TCP, FB, NFB, and AC, respectively. In particular, FB with SPORIX® + lactose + vitamin D3 enhanced calcium ionization to 33.89 ± 0.69 mg g-1, which was about 11.76 times higher than that of FB only. In the case of intestinal cellular uptake of calcium, there was no significant difference in all the tested calcium materials with SPORIX® + lactose + vitamin D3. However, the absolute amount of intestinal transport of calcium in FB (43.95 ± 3.29 µg) was significantly higher than other calcium materials with the addition of SPORIX® + lactose + vitamin D3 (p < 0.05). This study suggests that the co-consumption of SPORIX®, lactose, and vitamin D3 with FB could enhance the calcium bioavailability by lowering pH as well as improving calcium intestinal transport by modulating the paracellular and transcellular uptake mechanism.

Hypothalamic estrogen receptor alpha establishes a sexually dimorphic regulatory node of energy expenditure.

Estrogen receptor a (ERa) signaling in the ventromedial hypothalamus (VMH) contributes to energy homeostasis by modulating physical activity and thermogenesis. However, the precise neuronal populations involved remain undefined. Here, we describe six neuronal populations in the mouse VMH by using single-cell RNA transcriptomics and in situ hybridization. ERa is enriched in populations showing sex biased expression of reprimo (Rprm), tachykinin 1 (Tac1), and prodynorphin (Pdyn). Female biased expression of Tac1 and Rprm is patterned by ERa-dependent repression during male development, whereas male biased expression of Pdyn is maintained by circulating testicular hormone in adulthood. Chemogenetic activation of ERa positive VMH neurons stimulates heat generation and movement in both sexes. However, silencing Rprm gene function increases core temperature selectively in females and ectopic Rprm expression in males is associated with reduced core temperature. Together these findings reveal a role for Rprm in temperature regulation and ERa in the masculinization of neuron populations that underlie energy expenditure.

Estrogen-sensitive medial preoptic area neurons coordinate torpor in mice.

Homeotherms maintain a stable internal body temperature despite changing environments. During energy deficiency, some species can cease to defend their body temperature and enter a hypothermic and hypometabolic state known as torpor. Recent advances have revealed the medial preoptic area (MPA) as a key site for the regulation of torpor in mice. The MPA is estrogen-sensitive and estrogens also have potent effects on both temperature and metabolism. Here, we demonstrate that estrogen-sensitive neurons in the MPA can coordinate hypothermia and hypometabolism in mice. Selectively activating estrogen-sensitive MPA neurons was sufficient to drive a coordinated depression of metabolic rate and body temperature similar to torpor, as measured by body temperature, physical activity, indirect calorimetry, heart rate, and brain activity. Inducing torpor with a prolonged fast revealed larger and more variable calcium transients from estrogen-sensitive MPA neurons during bouts of hypothermia. Finally, whereas selective ablation of estrogen-sensitive MPA neurons demonstrated that these neurons are required for the full expression of fasting-induced torpor in both female and male mice, their effects on thermoregulation and torpor bout initiation exhibit differences across sex. Together, these findings suggest a role for estrogen-sensitive MPA neurons in directing the thermoregulatory and metabolic responses to energy deficiency.

Impacts of environmental conditions on the sorption of volatile organic compounds onto tire powder.

A series of batch tests were performed and the impacts of environmental conditions and phase change on the sorption of volatile organic compounds (VOCs) were investigated. Benzene, trichloroethylene, tetrachloroethylene, and ethylbenzene were selected as target VOCs. Sorption of VOCs onto tire powder was well demonstrated by a linear-partitioning model. Water-tire partition coefficients of VOCs (not tested in this study) could be estimated using a logarithmic relationship between observed water-tire partition coefficients and octanol-water partition coefficients of the VOCs tested. The target VOCs did not seem to compete with other VOCs significantly when sorbed onto the tire powder for the range of concentrations tested. The influence of environmental conditions, such as pH and ionic strength also did not seem to be significant. Water-tire partition coefficients of benzene, trichloroethylene, tetrachloroethylene, and ethylbenzene decreased as the sorbent dosage increased. However, they showed stable values when the sorbent dosage was greater than 10 g/L. Air-tire partition coefficient could be extrapolated from Henry's law constants and water-tire partition coefficient of VOCs.

Combined effect of pH and heating conditions on the physical properties of Alaska pollock surimi gels.

Physical properties of Alaska pollock surimi paste were investigated as affected by pH (4.0 and 6.0-10.0) and heating conditions (slow and fast). The highest values of gel strength and deformability, as shown by breaking force and penetration distance, were obtained at pH 7.5-8.0, while the lowest values were at pH 10.0 followed by pH 6.0 and pH 6.5, respectively. Two-step slow heating process increased the breaking strength value nearly two times higher than one-step fast heating. The effect of pH was strikingly high at pH 7.5 when gels were prepared using 2-step heating, indicating the pH dependence of endogenous transglutaminase. However, the highest gel strength was obtained at pH 8.0 when gels were prepared in fast heating. Whiteness value (L - 3b*) increased significantly (p < .05) as pH increased from 6.0 to 6.5, but thereafter decreased significantly (p < .05) as pH increased. L* value (lightness) and b* value (yellowness) continuously decreased as the pH is shifted from 6.0 to 10. Fast heated gels showed the lowest yellowness, resulting in whiter appearance, probably due to the effect of reduced browning reaction. PRACTICAL APPLICATIONS: The uniqueness of this study was to measure the combined effect of pH and heating conditions on the gel texture and color. There were various studies dealing with pH or heating conditions independently. As the primary character for surimi seafood is gel texture and color. The highest values of gel strength and deformability, as shown by breaking force and penetration distance, were obtained at pH 7.5-8.0, while the lowest values were at pH 10.0 followed by pH 6.0 and pH 6.5, respectively. Two-step slow heating process increased the breaking strength value nearly two times higher than one-step fast heating. Whiteness value (L - 3b*) increased significantly as pH increased from 6.0 to 6.5, but thereafter decreased significantly as pH increased. L* value (lightness) and b* value (yellowness) continuously decreased as the pH is shifted from 6.0 to 10. Fast heated gels showed the lowest yellowness, resulting in whiter appearance.

FT-IR and Raman spectroscopies determine structural changes of tilapia fish protein isolate and surimi under different comminution conditions.

Tilapia proteins refined by pH shift and water washing were chopped under various comminution conditions and their structural changes were investigated using Fourier transform infrared (FT-IR) and Raman spectroscopies. Both techniques revealed the degree of unfolding in protein structure increased when fish protein isolate (FPI) and surimi were chopped at 25°C for 18min compared to samples chopped at 5°C for 6min. Results indicated both hydrophobic interactions and disulfide bonds were significantly enhanced during gelation. FPI and surimi gels prepared at 25°C for 18min exhibited higher ß-sheet contents and more chemical bonds such as hydrophobic interactions and disulfide bonds than those at 5°C for 6min. Results suggested that controlling comminution is important to improve gel qualities in FPI and surimi from tropical fish like tilapia. Moreover, FT-IR and Raman spectroscopies are useful complementary tools for elucidating the change in the structure of protein during comminution and gelation.

Lymph node standardized uptake values at pre-treatment 18F-fluorodeoxyglucose positron emission tomography as a valuable prognostic factor for distant metastasis in nasopharyngeal carcinoma.

OBJECTIVE: The aim of the present study was to evaluate prognostic values of pre-treatment fluorine-18 fludeoxyglucose (18F-FDG) positron emission tomography (PET) parameters for predicting the distant metastasis (DM) of nasopharyngeal cancer. METHODS: 73 patients diagnosed with nasopharyngeal cancer with regional lymph node (LN) involvement, who underwent pre-treatment 18F-FDG PET evaluation between January 2005 and December 2012, were retrospectively reviewed. We assessed the 18F-FDG PET parameters of the primary tumours (T-) and regional LNs (N-). For patients with bilateral retropharyngeal, bilateral neck and/or supraclavicular LN involvement, we also assessed the 18F-FDG PET parameters of the farthest LN station [N(f)-]. The following 18F-FDG PET parameters were evaluated: maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), peak standardized uptake value (SUVpeak), metabolic tumour volumes (MTVs) (MTV30-MTV70, which were calculated as the tumour volume with 30%, 40%, 50%, 60% and 70% of the SUVmax as the threshold, respectively) and total lesion glycolysis (TLG) (TLG30-TLG70, which were determined by the product of each MTV and the corresponding SUVmean within that MTV). Distant metastasis-free survival (DMFS) rates were estimated from the date of the start of radiotherapy to the date of DM or last follow-up by the Kaplan-Meier method. Univariate and multivariate analyses were performed to identify prognostic factors for DMFS. The median follow-up period was 53 months (range 12-110 months). RESULTS: Most patients (95%) received concurrent chemoradiotherapy. The major failure pattern was DM (15 of all patients, 21%) and the 5-year DMFS was 79%. In univariate analysis, the T-SUVmax, T-SUVmean, T-SUVpeak, N-SUVmax, N-SUVpeak, N(f)-SUVmax and N(f)-SUVpeak were significant prognostic factors for DMFS. In multivariate analysis, the T-SUVmax, T-SUVpeak, N(f)-SUVmax and N(f)-SUVpeak were significant prognostic factors for DMFS. Of these parameters, the N(f)-SUVmax (hazard ratio = 6.524; p = 0.001) and N(f)-SUVpeak (hazard ratio = 5.399; p = 0.001) were the strongest prognostic factors for DMFS. CONCLUSION: In patients with nasopharyngeal cancer with LN involvement, the standardized uptake value parameter of the farthest LN station seems to be an important 18F-FDG PET parameter for predicting DM. Further studies are needed to validate its clinical significance. Advances in knowledge: We found that pre-treatment 18F-FDG PET parameters of primary tumours and regional LNs (the SUVmax and SUVpeak of the primary tumour and the farthest LN station) were significant prognostic factors for DMFS in patients with nasopharyngeal carcinoma with LN involvement.

Raman spectroscopy determines structural changes associated with gelation properties of fish proteins recovered at alkaline pH.

Structural changes of alkali-treated rockfish protein isolate (AKPI) during frozen storage were elucidated using a Raman spectrometer and scanning electron microscope (SEM). The results were compared to conventional surimi (CS). No significant textural difference was noted between AKPI stored at pH 5.5 and 7.0. The strongest texture was found for AKPI frozen with cryoprotectants and CS, while the weakest texture was observed in AKPI frozen without cryoprotectants. SEM revealed the most discontinuity in gels of AKPI with no cryoprotectants and a more aggregated microstructure after storage at pH 5.5 than at neutral pH. Raman spectral analysis demonstrated refolding of AKPI by pH readjustment to 7.0, although the refolded structure was not identical to that before the pH shift. CS showed higher alpha-helix content (approximately 50%) than AKPI (approximately 20-30%). Frozen storage induced a decrease and an increase in the alpha-helix content of CS and AKPI samples, respectively. AKPIs were slightly less stable than CS during frozen storage.

Pacific whiting frozen fillets as affected by postharvest processing and storage conditions.

Whole fish and H&G (headed and gutted) fish were stored under refrigeration (<4 °C) for 0, 2, and 5 days and subsequently filleted and frozen at -18 °C and -80 °C. Frozen fillets were analyzed during 24 weeks of storage. The activity of trimethylamine-N-oxide demethylase (TMAOase) decreased more quickly at -18 °C than -80 °C. TMAOase reduction was distinctively noted at -18 °C storage. Formaldehyde (FA) induced by TMAOase increased at all treatments at -18 °C as frozen storage extended to 24 weeks, but it was near zero at -80 °C up to 12 weeks of storage. Textural toughening, low water retention ability, and low salt soluble protein resulted from the denaturation function of FA. A sudden decrease in surface hydrophobicity at 24 weeks, when stored at -18 °C, resulted from FA-induced unfolding and subsequent aggregation. FA concentration appeared to affect protein aggregations and textual toughening of fillets during frozen storage.

Salmon blood plasma: effective inhibitor of protease-laden Pacific whiting surimi and salmon mince.

The effect of salmon plasma (SP) from Chinook salmon on proteolytic inhibition was investigated. SP was found to inhibit both cysteine and serine proteases as well as protease extracted from Pacific whiting muscle. SP was found to contain a 55kDa cysteine protease inhibitor through SDS-PAGE inhibitor staining. Freeze dried salmon plasma (FSP) and salmon plasma concentrated by ultrafiltration (CSP) were tested for their ability to inhibit autolysis in Pacific whiting surimi and salmon mince at concentrations of 0.25%, 0.5%, 1%, and 2%. Pacific whiting surimi autolysis was inhibited by an average of 89% regardless of concentration while inhibition of salmon mince autolysis increased with concentration (p<0.05). CSP performed slightly better than FSP at inhibiting salmon mince autolysis (p<0.05). Serine protease inhibition decreased when SP heated above 40°C but was stable across a broad NaCl and pH range. Cysteine protease inhibitors exhibited good temperature, NaCl, and pH stability.

Textural and rheological properties of Pacific whiting surimi as affected by nano-scaled fish bone and heating rates.

Textural and rheological properties of Pacific whiting (PW) surimi were investigated at various heating rates with the use of nano-scaled fish bone (NFB) and calcium chloride. Addition of NFB and slow heating improved gel strength significantly. Activity of endogenous transglutaminase (ETGase) from PW surimi was markedly induced by both NFB calcium and calcium chloride, showing an optimal temperature at 30°C. Initial storage modulus increased as NFB calcium concentration increased and the same trend was maintained throughout the temperature sweep. Rheograms with temperature sweep at slow heating rate (1°C/min) exhibited two peaks at ∼ 35°C and ∼ 70°C. However, no peak was observed during temperature sweep from 20 to 90°C at fast heating rate (20°C/min). Protein patterns of surimi gels were affected by both heating rate and NFB calcium concentration. Under slow heating, myosin heavy chain intensity decreased with NFB calcium concentration, indicating formation of ε-(γ-glutamyl) lysine cross-links by ETGase and NFB calcium ion.

Effects of nano-scaled fish bone on the gelation properties of Alaska pollock surimi.

Gelation properties of Alaska pollock surimi as affected by addition of nano-scaled fish bone (NFB) at different levels (0%, 0.1%, 0.25%, 0.5%, 1% and 2%) were investigated. Breaking force and penetration distance of surimi gels after setting increased significantly as NFB concentration increased up to 1%. The first peak temperature and value of storage modulus (G'), which is known to relate to the unfolding and aggregation of light meromyosin, increased as NFB concentration increased. In addition, 1% NFB treatment demonstrated the highest G' after gelation was completed. The activity of endogenous transglutaminase (TGase) in Alaska pollock surimi increased as NFB calcium concentration increased. The intensity of myosin heavy chain cross-links also increased as NFB concentration increased indicating the formation of more ε-(γ-glutamyl) lysine covalent bond by endogenous TGase and calcium ions from NFB.