Chitooligosaccharide from Pacific White Shrimp Shell Chitosan Ameliorates Inflammation and Oxidative Stress via NF-κB, Erk1/2, Akt and Nrf2/HO-1 Pathways in LPS-Induced RAW264.7 Macrophage Cells.

Chitooligosaccharide (COS), found in both insects and marine sources, has several bioactivities, such as anti-inflammation and antioxidant activities. However, the mechanism of shrimp shell COS on retardation of inflammatory and antioxidant effects is limited. Therefore, the aim of this study is to examine the mechanism of the aforementioned activities of COS in LPS-activated RAW264.7 macrophage cells. COS significantly improved cell viability in LPS-activated cells. COS at the level of 500 µg/mL could reduce the TNF-α, NO and IL-6 generations in LPS-activated cells (p < 0.05). Furthermore, COS could reduce ROS formation, NF-κB overactivation, phosphorylation of Erk1/2 and Akt and Nrf2/HO-1 in LPS-exposed cells. These results indicate that COS manifests anti-inflammatory activity and antioxidant action via NF-κB, Erk1/2, Akt and Nrf2/HO-1 signaling with an increasing relevance for inflammatory disorders.

Oral Administration of Ethanolic Extract of Shrimp Shells-Loaded Liposome Protects against Aß-Induced Memory Impairment in Rats.

Alzheimer's disease is characterized by a progressive loss of memory and cognition. Accumulation of amyloid-beta (Aß) in the brain is a well-known pathological hallmark of the disease. In this study, the ethanolic extract of white shrimp (Litopenaous vannamei) shells and the ethanolic extract-loaded liposome were tested for the neuroprotective effects on Aß1-42-induced memory impairment in rats. The commercial astaxanthin was used as a control. Treatment with the ethanolic extract of shrimp shells (EESS) at the dose of 100 mg/kg BW showed no protective effect in Aß-treated rats. However, treatment with an EESS-loaded liposome at the dose of 100 mg/kg BW significantly improved memory ability in Morris water maze and object recognition tests. The beneficial effect of the EESS-loaded liposome was ensured by the increase of the memory-related proteins including BDNF/TrkB and pre- and post-synaptic protein markers GAP-43 and PSD-95 as well as pErk1/2/Erk1/2 in the cortex and hippocampus. These findings indicated the neuroprotective effects of the EESS-loaded liposome on Aß-induced memory impairment in rats. It produced beneficial effects on learning behavior probably through the function of BDNF/TrkB/pErk1/2/Erk1/2 signaling pathway and subsequently the upregulation of synaptic proteins. The present study provided evidence that the neuroprotective property of the ESSE-loaded liposome could be a promising strategy for AD protection.

Development of Hydrolysis and Defatting Processes for Production of Lowered Fishy Odor Hydrolyzed Collagen from Fatty Skin of Sockeye Salmon (Oncorhynchus nerka).

Lipid oxidation has a negative impact on application and stability of hydrolyzed collagen (HC) powder from fatty fish skin. This study aimed to produce fat-free HC powder from salmon skin via optimization of one-step hydrolysis using mixed proteases (papain and Alcalase) at different levels. Fat removal processes using disk stack centrifugal separator (DSCS) for various cycles and subsequent defatting of HC powder using isopropanol for different cycles were also investigated. One-step hydrolysis by mixed proteases (3% papain and 4% Alcalase) at pH 8 and 60 °C for 240 min provided HC with highest degree of hydrolysis. HC powder having fat removal with DSCS for 9 cycles showed the decreased fat content. HC powder subsequently defatted with isopropanol for 2 cycles (HC-C9/ISP2) had no fat content with lowest fishy odor intensity, peroxide value, and thiobarbituric acid reactive substances than those without defatting and with 1-cycle defatting. HC-C9/ISP2 had high L*-value (84.52) and high protein (94.72%). It contained peptides having molecular weight less than 3 kDa. Glycine and imino acids were dominant amino acid. HC-C9/ISP2 had Na, Ca, P, and lowered odorous constituents. Combined processes including hydrolysis and defatting could therefore render HC powder free of fat and negligible fishy odor.

Protein-polyphenol conjugates: Preparation, functional properties, bioactivities and applications in foods and nutraceuticals.

Protein is a crucial nutritional ingredient in the daily human diet. Polyphenols (PPNs) are the abundant phytochemicals in plants, which are associated with health promotion as well as affect functionality in food systems. Both ingredients possess different types of functionalities (crosslinking, gelling, emulsifying, film-forming, etc.) and bioactivities (antioxidant, antimicrobial, anti-inflammatory, etc.). In the past decade, various methods have been implemented to enhance the functionalities and bioactivities of foods. Conjugation or grafting methods has been introduced widely. Conjugations of PPNs with proteins through various methods have been performed for the synthesis of the protein-polyphenol conjugate. Those potential grafting methods are alkaline associated, free-radical mediated, enzyme catalyzed, and chemical coupling methods. Several factors such as reaction conditions, type of proteins, and PPNs also influenced the conjugation efficiency. Various technologies, e.g., mass spectroscopy, fluorescence spectroscopy, UV spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and sodium dodecyl sulfate polyacrylamide gel electrophoresis have been used to elucidate conjugation and structural alternation of proteins and some properties of resulting conjugates. The prepared protein-PPN conjugates have been documented to enhance the bioactivities and functional properties of an initial protein. Moreover, conjugates have been employed in emulsions or as nanoparticles for nutraceutical delivery. Edible-films for food packaging and hydrogels for controlled drug release have been developed using protein-PPN conjugates. This chapter focuses on the methodologies and characteristics of protein-PPN conjugates and their applications in various food systems and nutraceutical field.

Impact of Hydrolyzed Collagen from Defatted Sea Bass Skin on Proliferation and Differentiation of Preosteoblast MC3T3-E1 Cells.

Osteoporosis is a serious problem affecting health of the elderly. Drugs (bisphosphonates) applied for treatment are often accompanied by adverse side effects. Thus, fish byproduct-derived peptides, particularly hydrolyzed collagen (HC) from defatted sea bass skin, could be a safe source of anti-osteoporosis agents. This study aimed to examine the effects of HC on proliferation and differentiation of preosteoblast cells. HC prepared using papain before Alcalase hydrolysis was determined for molecular weight (MW) distribution. Thereafter, the resulting HC (50-800 µg/mL) was added to the cell. Proliferation, alkaline phosphatase activity (AP-A) and mineralization of cells were investigated. Moreover, the expression of runt-related transcription factor 2 (RUNX2) and the p-Akt/Akt pathway were also determined using Western blot. The results showed that HC had an MW < 3 kDa. HC (50-200 µg/mL) could promote cell proliferation. Nevertheless, HC at 100 µg/mL (HC-100) had enhanced AP-A and increased mineralization during the first 7 days of culture. Moreover, HC-treated cells had higher calcium depositions than the control (p < 0.05). Additionally, cells treated with HC-100 had higher levels of RUNX2 and p-Akt expressions than control (p < 0.05). Therefore, HC could be a promising functional ingredient to promote osteoblast proliferation and differentiation, which could enhance bone strength.

Effects of sonication and ultrasound on properties and bioactivities of liposomes loaded with hydrolyzed collagen from defatted sea bass skin conjugated with epigallocatechin gallate.

Hydrolyzed collagen (HC) from defatted sea bass skin conjugated with 3% epigallocatechin gallate (EGCG) was prepared and the resulting HC-EGCG conjugate at various levels (0.25%-2%, w/v) was loaded into liposome. The obtained liposomes were subjected to sonication (S). Liposome loaded with 1% conjugate showed the highest encapsulation efficiency (EE) (p < .05). When the ultrasound-assisted process (UAP) at different amplitudes (20% and 40%) and times (2, 5, 10, and 15 min) were implemented, the highest EE of conjugate-loaded liposome was found at 20% amplitude for 2 min (p < .05). When S-liposome and UAP-liposome were lyophilized, decreasing EE of both samples was observed (p < .05). Lyophilized UAP-liposome had higher stability than lyophilized S-liposome during storage at 25℃ for 28 days. Additionally, antioxidant activity in the gastrointestinal track model system (GIMs) and digest obtained from GIMs were higher for UAP-liposome (p < .05). Therefore, liposome can be used for the delivery of conjugate. PRACTICAL APPLICATIONS: HC from defatted sea bass skin is considered to possess several bioactivities, especially skin nourishment and bone strengthening. Nevertheless, antioxidant activity, related to the treatment of several ailments, is still low for HC. Thus, grafting of HC with polyphenol such as EGCG via free radical method can be used for the enhancement of the antioxidant activity of HC. Although the resulting conjugate has augmented activity, it is unstable during storage and in the gastrointestinal digestion system. Liposome is a promising means to stabilize the conjugate under harsh condition, especially with the aid of the UAP. Thus, liposome loaded with conjugate having the reduced size has higher antioxidant activity with increased stability, which can have a wider range of applications.

Effect of hydrolyzed collagen from defatted Asian sea bass (Lates calcarifer) skin on fibroblast proliferation, migration and antioxidant activities.

Hydrolyzed collagen from the defatted Asian sea bass (Lates calcarifer) (Asbs-HC) had high hydrophobic amino acids and imino acids. When fibroblast cell was treated with Asbs-HC, there was no cytotoxicity at any concentrations (25-1000 µg/mL). Asbs-HC at 1000 µg/mL exhibited the highest cell proliferation and cell migration (p < 0.05), indicating wound healing ability. Antioxidative activities of Asbs-HC at different concentrations were determined. ABTS radical scavenging activity (ABTS-RSA) and oxygen radical absorbance capacity (ORAC) increased when Asbs-HC levels augmented up to 1 mg/mL (p < 0.05). Decreased activities in scavenging DPPH radical and chelating metal were found at higher levels of Asbs-HC (0.5 and 1 mg/mL) (p < 0.05). Molecular weight (MW) of peptides in Asbs-HC ranged from 406 to 16,120 Da. Peptide containing MW of 406 Da rendered the highest scavenging activity towards ABTS radical. Thus, Asbs-HC could be applied as antioxidant, skin nourishment and wound healing agents for food/drink fortification.

In vitro antioxidant and wound-healing activities of hydrolyzed collagen from defatted Asian sea bass skin as influenced by different enzyme types and hydrolysis processes.

Hydrolyzed collagen (HC) from defatted Asian sea bass skin was prepared by different enzymatic hydrolysis processes. For one-enzyme hydrolysis, papain (0.3 unit per g dry matter, DM) at 40 °C for 90 min or Alcalase (0.2 or 0.3 unit per g DM) at 50 °C for 90 min were used. The two-enzyme hydrolysis was accomplished with papain at 0.3 unit per g DM (P0.3), followed by Alcalase hydrolysis at 0.2 or 0.3 units per g DM (A0.2 or A0.3, respectively). HC prepared using the P0.3 + A0.3 process showed higher peptide yield, recovery and imino acid content in addition to stronger ABTS, DPPH radical scavenging activities and ferric reducing antioxidant power than other hydrolysis processes. HC obtained from the P0.3 + A0.3 process (at 125-500 µg mL-1) induced MRC-5 fibroblast proliferation and augmented migration and lamellipodia formation in the cells. Peptides with average molecular weight of 750 Da exhibited the highest ABTS radical scavenging activity while the 4652 Da fraction had the lowest. Thus, HC can be considered as a suitable ingredient to formulate functional products for skin nourishment and wound healing.

Conjugate between hydrolyzed collagen from defatted seabass skin and epigallocatechin gallate (EGCG): characteristics, antioxidant activity and in vitro cellular bioactivity.

Conjugation between peptides and polyphenols, especially epigallocatechin gallate (EGCG) using covalent grafting, is a promising method that can modify peptides or augment their antioxidant activities. Moreover, the resulting conjugates can be intensively served as functional ingredient or supplement. Thus, the objectives of the present study were to investigate the grafting between hydrolyzed collagen (HC) from defatted seabass skin and EGCG and to study characteristics as well as bioactivities of the obtained HC-EGCG conjugate. Levels of EGCG used (1-5%, w/w) affected surface hydrophobicity (SH) and antioxidant activities of the conjugates. Overall, the addition of EGCG at 3% to HC (HC-3% EGCG) increased SH, ABTS radical scavenging and metal chelating activities (p < 0.05). FTIR spectra of HC-3% EGCG revealed the interaction between HC and EGCG via H-bonding and covalent interaction. Sephadex G-25 fraction of conjugate with molecular weight (MW) of 2771 Da rendered the highest redox ability. When HC-3% EGCG was applied in fibroblast (MRC-5) and keratinocyte (HaCaT) cells, all levels tested (125-1000 µg mL-1) had no toxicity on both cells. Higher proliferation of both cells were attained with increasing levels of HC-3% EGCG, particularly at 500 and 1000 µg mL-1 (p < 0.05). Moreover, both levels used had cytoprotective ability against reactive oxygen species (ROS) as evidenced by lowered ROS and cell death detected as compared to those found in cells induced with H2O2 or AAPH alone (p < 0.05) for both cells. HC-3% EGCG could serve as an effective antioxidant for application in foods or as supplement for skin nourishment.

Effect of stabilizing agents on characteristics, antioxidant activities and stability of liposome loaded with hydrolyzed collagen from defatted Asian sea bass skin.

Poor stability of fish hydrolyzed collagen (HC) hampers its applications, especially as food ingredients. The use of liposome as a vesicle can be a potential means to enhance bioactivities and stability of HC. HC from defatted Asian sea bass skin at different levels (0.25%-2%, w/v) were loaded into liposomes prepared from soy phosphatidylcholine (SPC) with various stabilizers (cholesterol (CHO) or glycerol (GLY)). The highest encapsulation efficiency (EE) was found in SPC-CHO-0.5%HC (P < 0.05) (85.42%), while liposome stabilized with GLY had the highest EE (74.54%) for SPC-GLY-0.25%HC (P < 0.05). After lyophilization, SPC-CHO-0.5%HC had higher EE than SPC-GLY-0.25%HC (P < 0.05). Increasing particle size and decreasing negative surface charge were found for both lyophilized samples. Lyophilized SPC-CHO-0.5%HC exhibited higher stability than lyophilized SPC-GLY-0.25%HC during storage at 25 °C for 28 days. Also, higher antioxidant activities in gastrointestinal track model system was found for SPC-CHO-0.5%HC. Thus, SPC-CHO liposome could be used as a promising carrier of HC.

Hydrolyzed collagen from porcine lipase-defatted seabass skin: Antioxidant, fibroblast cell proliferation, and collagen production activities.

Defatting of seabass skins using porcine pancreas lipase (PPL) at 25 or 50 units/g dry matter) for 1-3 hr at 30ºC was investigated. Treatment of seabass skin with PPL (25 unit/g dry matter) for 3 hr removed 83.81% lipids when compared to 57.27% using isopropanol. Hydrolysis of PPL-treated skin by papain (0.3 unit/g dry matter) (PPL-papain-3 process) at 40ºC for 90 min provided hydrolyzed collagen (HC) with higher yield, α-amino group content, ferric-reducing antioxidant power, and metal chelating activity than other treatments (p < 0.05). There was no difference in fishy odor between HC from PPL-papain-2 and PPL-papain-3 processes (p > 0.05). All the HC (50-250 µg/ml) samples stimulated L929 fibroblast cell proliferation and also induced collagen production in a dose-dependent manner. Also, all HC contained peptides with molecular weight of 406-11,860 Da. Gly and imino acids were dominant amino acids in HC prepared with PPL-papain-3 process. PRACTICAL APPLICATIONS: Seabass skin is a potential raw material for the production of hydrolyzed collagen (HC). However, seabass skin contains a large amount of lipids, including polyunsaturated fatty acids. These unsaturated lipids are oxidized during processing, particularly during hydrolysis at high temperature. This leads to the development of undesirable odor, especially fishy odor. Therefore, seabass skin defatting is an important step for improving the quality of the resulting HC. The use of lipase is an alternative method that can be used to remove lipids in skins without using solvents. HC from defatted skins will contain bioactive peptides and therefore, can be used as a food supplement or for skin nourishment.

Effect of Pulsed Electric Field-Assisted Process in Combination with Porcine Lipase on Defatting of Seabass Skin.

Defatting of seabass skins using pulsed electric field (PEF)-assisted process at different electric field strengths (16 and 24 kV/cm) and times (36, 72, and 108 ms) in combination with porcine pancreas lipase (PPL) at 25 U/g dry matter was investigated. PEF-treated skin at 24 kV/cm for 72 ms followed by PPL treatment removed 86.93% lipids. PEF-treated skin was further optimized for lipid reduction by response surface methodology. Central composite design was adopted to establish treatments based on two independent variables, involving PPL concentration (30 to 55 U/g dry matter) and hydrolysis time (60 to 180 min). Second-order polynomial model was used for predicting the response. The highest lipid removal (91.96 ± 1.70%) was attained when the optimal condition (42.36 PPL units/g dry skin matter for 139.78 min) was used. The experiment value was in accordance with the predicted value. PEF-PPL-treated skin had lower monounsaturated and polyunsaturated fatty acids than the solvent-extracted skin (P < 0.05). When PEF-PPL-treated skin was hydrolyzed using papain at 0.30 U/g dry matter, lower fishy odor/flavor of resulting hydrolyzed collagen (PEF-PPL-HC) was found than other samples (P < 0.05). Lower total volatile compounds were also obtained in PEF-PPL-HC sample. Thus, the use of PEF pretreatment along with PPL before papain hydrolysis effectively prevented the formation of fishy odor/flavor in hydrolyzed collagen from seabass skin. PRACTICAL APPLICATION: Fishy odor/flavor caused by lipid oxidation of fish skin hydrolysates limits their applications in foods. Defatting process is the significant step for skin pretreatment. Although several methods could remove lipids from fish skins, either by lipase or solvent extraction, fishy odor/flavor is still detected in hydrolysate. Pulsed electric field-assisted process in combination with porcine lipase is another approach that can be used to enhance efficiency via electroporation, causing the loosened skin matrix and facilitating the migration of lipase into the skin. Consequently, the resulting hydrolysate might have the lowered fishy odor/flavor and could be used in foods, especially for fortification.

Ultrasound assisted extraction of antioxidative phenolics from cashew (Anacardium occidentale L.) leaves.

Optimization of ultrasound-assisted extraction (UAE) of antioxidative phenolics from the leaves of cashew (Anacardium occidentale L.) was carried out by response surface methodology along with the central composite design. Two independent variables were considered: amplitude (30-77%) and time (7-31 min). The highest extraction yield was 23.61% when the optimal extraction condition (77% amplitude for 31 min) was implemented. The extract containing total phenolic content of 579.55 mg GAE/g dry extract possessed radical scavenging activities and reducing power. The experiment values were in line with the predicted counterparts. Extract contained gallic acid, isoquercetin, tannic acid, quercetin, catechin, apigenin, hydroquinin, eriodictyol, and rutin. The extract with increasing levels inhibited AAPH-induced DNA damage to a higher extent. Thus, UAE was demonstrated to potentially increase the extraction efficacy of phenolics from cashew leaves and the extract could be applied as a natural antioxidant.