Food applications of bioactive biomaterials based on gelatin and chitosan.

Food packaging must guarantee the products' quality during the different operations including packing and maintenance throughout transportation and storage until to consumption. Thus, it should satisfy, both, food freshness and quality preservation and consumers health safety. Natural bio-sourced polymers have been explored as safe edible materials for several packaging applications, being interestingly carrier of bioactive substances, once added to improve films' properties. Gelatin and chitosan are among the most studied biomaterials for the preparation of edible packaging films due to their excellent characteristics including biodegradability, compatibility and film-forming property. These polymers could be used alone or in combination with other polymers to produce composite films with the desired physicochemical and mechanical properties. When incorporated with bioactive substances (natural extracts, polyphenolic compounds, essential oils), chitosan/gelatin-based films acquired various biological properties, including antioxidant and antimicrobial activities. The emerging bioactive composite films with excellent physical attributes represent excellent packaging alternative to preserve different types of foodstuffs (fruits, meat, fish, dairy products, …) and have shown great achievements. This chapter provides the main techniques used to prepare gelatin- and chitosan- based films, showing some examples of bioactive compounds incorporated into the films' matrix. Also, it illustrates the outstanding advantages given by these biomaterials for food preservation, when used as coating and wrapping agents.

Techno-functional characterization of gelatin extracted from the smooth-hound shark skins: Impact of pretreatments and drying methods.

Gelatin derived from marine by-products could be an interesting alternative to classic mammalian gelatin. The pretreatment and extraction conditions could influence the size of the resulting peptide chains and therefore their techno-functional properties. Thus, it is important to optimize the production process to get a gelatin for the appropriate applications. Skin pretreatment was done by microwaves or oven-drying and the extracted gelatin was dried by spray- or freeze-drying. Freeze-dried gelatin extracted from untreated skin (FGUS) had the highest gelatin yield (10.40%). Gelatin proximate composition showed that proteins were the major component (87.12-89.95%), while lipids showed the lowest contents (0.65-2.26%). Glycine showed the highest level (299-316/1000 residues) in the extracted gelatins. Proline and hydroxyproline residues of gelatins from untreated skin were significantly higher than those from pretreated skin-gelatin. FTIR spectra were characterized by peaks of the amide A (3430-3284 cm-1), B (3000-2931 cm-1), I (1636-1672 cm-1), II (1539-1586 cm-1) and III (1000-1107 cm-1). Spray-drying decreased the gelling properties of gelatins, since it reduced gelling and melting temperatures compared to freeze-drying. Skin pretreatment significantly reduced the gel strength of gelatin by about 50-100 g depending on the gelatin drying method. The FGUS showed better surface properties compared to other gelatins. The highest emulsion activity index (39.42 ± 1.02 m2/g) and foaming expansion (172.33 ± 2.35%) were measured at 3% FGUS. Therefore, the promising properties of freeze-dried gelatin derived from untreated skin, gave it the opportunity to be successfully used as a techno-functional ingredient in many formulations.

Development of active edible coatings based on fish gelatin enriched with Moringa oleifera extract: Application in fish (Mustelus mustelus) fillet preservation.

An edible coating was developed using gelatin extracted from the skin of gray triggerfish (Balistes capriscus) and applied to the fillet of the smooth-hound shark (Mustelus mustelus). Moringa oleifera leaf extract was added to gelatin coating solution to improve its preservative properties. The phenolic profiles and antioxidant and antibacterial activities of M. oleifera extracts were determined. Phenolic acids constituted the largest group representing more than 77% of the total compounds identified in the ethanol/water (MOE/W) extract, among which the quinic acid was found to be the major one (31.48 mg/g extract). The MOE/W extract presented the highest DPPH• scavenging activity (IC50 = 0.53 ± 0.02 mg/ml) and reducing (Fe3+) power (EC0.5 = 0.57 ± 0.02 mg/ml), as well as interesting inhibition zones (20-35 mm) for the most tested strains. Coating by 3% of gelatin solution significantly reduced most deterioration indices during chilled storage, such as malondialdehyde (MDA), total volatile basic nitrogen (TVB-N), weight loss, pH, and mesophilic, psychrophilic, lactic, and H2S-producing bacterial counts. Interestingly, coating with gelatin solution containing MOE/W extract at 20 µg/ml was more effective than gelatin applied alone. Compared with the uncoated sample, gelatin-MOE/W coating reduced the weight loss and MDA content by 26% and 70% after 6 days of storage, respectively. Texture analysis showed that the strength of uncoated fillet increased by 46%, while the strength of fillet coated with gelatin-MOE/W only increased by 12% after 6 days of storage. Fish fillet coated with gelatin-MOE/W had the highest sensory scores in terms of odor, color, and overall acceptability throughout the study period.

Development and characterization of fish gelatin-based biodegradable film enriched with Lepidium sativum extract as active packaging for cheese preservation.

The physical and functional properties of gelatin-based films enriched with organic extracts from Lepidium sativum seeds were studied. Gelatin was extracted from the skin of dogfish (Squalus acanthias) and the functional gelatin-based films were used to preserve cheese during chilled storage. Ethanol extract (LSE3) and gelatin-based film enriched with LSE3 at 20 µg/mL showed high antioxidant potential using various complementary methods. No significant difference was measured in the mechanical parameters of the enriched films in terms of thickness, tensile strength and elongation at break. LSE3 incorporation at the highest level slighltly decreased the film L∗ value from 90.30 ± 0.10 to 88.10 ± 0.12, while the b∗ value increased from 0.91 ± 0.07 to 8.89 ± 0.12. Wrapping the cheese with gelatin-based film enriched with 20 µg LSE3/mL reduced the syneresis by 40% and stabilized the color, peroxidation and bacteria growth as compared to the unwrapped sample after 6 days of storage. In addition, cheese wrapped with the active gelatin-based film showed the lowest changes in texture parameters. Overall results suggest the use of the enriched gelatin film as active packaging material to preserve cheese quality.

Microstructure and characteristic properties of dogfish skin gelatin gels prepared by freeze/spray-drying methods.

The effects of two pretreatments (microwaves or oven-drying) on the dogfish (Squalus acanthias) skin as well as two drying processes (freeze-drying or spray-drying) on the extracted gelatins were studied. Thus six types of gelatins were obtained, three of which were freeze-dried (FG) and the others were spray-dried (SG), from the untreated skin (US), microwaves-pretreated skin (MS) and oven-pretreated skin (OS). The highest yield (8.67%) was obtained for the OSFG, while the lowest one (3.06%) was measured for the OSSG. Interestingly, all gelatins exhibited relatively high protein (84.02-89.53%), and low lipid (0.50-1.71%) and ash (3.05-7.17%) contents. In addition, gelatins were analyzed by the Fourier transform infrared and the spectra displayed important differences in some specific peaks, particularly in the amide I, amide II and amide III. The gelatins extracted from the untreated skin, regardless the drying method, presented the highest foaming capacity. The textural profile analysis showed that USSG was the hardest (213.6 g) and the chewier (23.8 N × mm) gelatin. Moreover, analysis of thermal properties showed that USSG also has the highest glass-transition temperature. The interesting properties of gelatin extracted from dogfish skin encourage their future use as a functional ingredient in industrial food formulations.

Response-surface methodology for the production and the purification of a new H2 O2 -tolerant alkaline protease from Bacillus invictae AH1 strain.

This work deals with the optimization of the culture conditions of Bacillus invictae AH1 in order to increase the production level of the proteolytic activity. Response-surface methodology (RSM) was applied for the most significant fermentation parameters (concentration of wheat bran and K2 HPO4 /KH2 PO4 ) that were earlier identified by Plackett-Burman Design from seven possible factors. A central composite design was used and the quadratic regression model of producing active protease was built. A maximum protease activity was reached and validated experimentally, using a maximum wheat bran concentration (50 g/L) with increased K2 HPO4 /KH2 PO4 concentration (2.275 g/L). Protease production obtained experimentally coincident with the predicted value and the model was proven to be adequate. Interestingly, the use of RSM increased the protease production by four times (7,000 U/mL) using a low-cost substrate and a culture time of 40 hr, as compared to the standard culture conditions. In the second part of this study, a H2 O2 -tolerant alkaline protease produced from B. invictae AH1 with a molecular mass of about 41 kDa, noted P3, was purified by successive steps of ultrafiltration, gel filtration and ion exchange chromatography. The K m and Vmax values of the purified protease using casein, as substrate, were about 4 mg/mL and 27 µM/min, respectively. The highest enzyme activity was found at pH 9.0 and a temperature of 60°C. In addition, the enzyme showed a quasi-total stability against H2 O2 (5% for 1 hr) and against most of the tested solid and liquid detergents, suggesting its eventual use in bio-detergent formulations.

Assessment of Cholesterol, Glycemia Control and Short- and Long-Term Antihypertensive Effects of Smooth Hound Viscera Peptides in High-Salt and Fructose Diet-Fed Wistar Rats.

In this study, the antihypertensive activity of Purafect®-smooth hound viscera protein hydrolysate (VPH) and its peptide fraction with molecular weight (MW) below 1 kDa (VPH-I) was investigated. In addition, the lipase inhibitory activity, as well the anticoagulant potential, in vitro, were assessed. The antihypertensive effects of VPH and VPH-I were studied during 24 h (short-term effect) and 30 days (long-term effect) using high-salt (18% NaCl) and -fructose (10%) diet (HSFD)-induced hypertension. Data showed that, 4 h post-administration of VPH and VPH-I (200 mg/kg BW), the systolic blood pressure of rats was reduced by about 6 and 9 mmHg, respectively. These effects were similar to that obtained with Captopril (~9 mmHg at t = 4 h). On the other hand, exposing the rats to daily to HSFD, coupled to the administration of viscera peptides, was found to attenuate hypertension. In addition, the proteins' treatments were able to correct lipid and glycemic disorders, by reducing the total cholesterol and triglyceride contents and resorting to the plasma glucose level, compared to the HSFD group. Overall, the present findings demonstrated the preventive effect of VPH-peptides from hypertension complications, as a result of their biological properties.

Extraction, structural characterization, and thermal and biomedical properties of sulfated polysaccharides from razor clam Solen marginatus.

In this study, the antioxidant, antibacterial and anticoagulant activities of sulfated polysaccharides extracted from Solen marginatus flesh were investigated via physicochemical characterization of the crude polysaccharide SM-CP and its deproteinized fraction (SM-DP); their total sugar contents were 47.15% and 66.01%. The results obtained via molecular weight evaluation showed that SM-CP mainly had a high molecular weight (1075 kDa), whereas SM-DP had a lower molecular weight (almost 237.9 kDa); in addition, thermal analysis (differential scanning calorimetry and thermogravimetry) was conducted; the results indicated that SM-CP was thermally more stable as its degradation temperature was 307 °C, whereas SM-DP was thermally less stable, with the degradation temperature of 288 °C. Moreover, the results obtained via the investigation of biological properties revealed that the extracted polysaccharides exhibited strong antioxidant and anticoagulant activities. Subsequently, SM-CP was fractionated using the DEAE-cellulose column. The peak (FII) eluted at high NaCl concentrations indicated highest anticoagulant activity as designated by the prolongation of the activated partial thromboplastin time (over 120 s), prothrombin time (28 s) and low level of fibrinogen (0.7 g l-1). The overall data demonstrated the significant therapeutic potential of the polysaccharides extracted from razor clam flesh.

Characterization and assessment of antioxidant and antibacterial activities of sulfated polysaccharides extracted from cuttlefish skin and muscle.

In this study, novel polysaccharides extracted from cuttlefish skin (CSP) and muscle (CMP), by precipitation with cetylpyridinium, were characterized and their antioxidant and antibacterial activities were investigated. CMP showed the highest amounts of sulfated groups (6.6%), uronic acids (9.2%) and proteins (3.7%). Infrared spectroscopic analysis indicated the presence of sulfonyl (OSO) and acetyl (CH3CO-) groups for both CSP and CMP. In addition, CSP showed the presence of glucuronic acid (GlcA) and galacturonic acid (GalA) as major components, while CMP showed highest amount of GalA in its monosaccharide composition. Sulfated polysaccharides were found to display important antibacterial activity against several Gram+ and Gram- bacteria. In addition, they exhibited strong antioxidant activities as showed by various in vitro tests. Fractionation of cuttlefish polysaccharides, by DEAE-cellulose column showed one peak during the buffer elution phase and three major fractions for CMP and two peaks for CSP during the linear gradient of NaCl. The last eluting sulfated fraction from each sample, characterized by the highest negative charge, was found to exhibit the best antioxidant and antibacterial activities. The obtained results demonstrated that cuttlefish polysaccharides and their fractions could serve as natural antioxidant and antibacterial agents.

Bioactive potential and structural characterization of sulfated polysaccharides from Bullet tuna (Auxis Rochei) by-products.

The present study deals with the isolation of sulfated polysaccharides (Ps) from the Bullet tuna by-products (head, skin and bones). Results of chemical characterization revealed that Ps-bones showed the highest total sugar, uronic acid and sulfate group contents. Tuna extracted-Ps contained a mixture of neutral sugars, with high amounts of glucuronic and galacturonic acids and presented different molecular weights. The presence of sulfate groups in different Ps was confirmed by FTIR analysis. Interestingly, Ps-bones showed the highest antioxidant activity among all the extracted Ps. Moreover, results revealed that all polysaccharides displayed varying degrees of antibacterial activity. Ps-bones exhibited high and wide spectrum of activities, inhibiting the growth of all bacteria tested. Ps-bones incorporated during fillet cooking offered an excellent protection of fish fillet by avoiding pH change, color loss, lipid oxidation and spoilage. Overall, the results demonstrated that Ps could be potentially used as natural antioxidant and antibacterial agents.

Structural, antioxidant and antibacterial activities of polysaccharides extracted from olive leaves.

In the present study, hot-water extraction procedure was used to recover polysaccharides from olive leaves. Primary structural characteristics were determined by nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR), Fourier Transform-Infrared spectroscopy (FT-IR) and X-ray diffractometry analysis. Emulsifying capacity, Zeta (ζ) potential and differential scanning calorimetric (DSC) of olive leaf polysaccharides (OLP) were investigated. Antioxidant and antibacterial activities were then examined. Infrared spectroscopy data revealed that OLP were constituted of functional groups OH, CO and CH which were specific to polysaccharides. Results of ζ-potential showed that OLP possessed an anionic structure and exhibited donated electron capacities. OLP displayed strong DPPH-radical scavenging activity (IC 50=34.80µg/mL). They showed also important reducing power and ß-carotene bleaching inhibition activities. Besides, OLP have attractive antibacterial activity against S.enterica and M.luteus with inhibition zones of 23.5 and 21.5mm, respectively.

In silico analysis and molecular docking study of angiotensin I-converting enzyme inhibitory peptides from smooth-hound viscera protein hydrolysates fractionated by ultrafiltration.

Smooth-hound viscera hydrolysates (SHVHs) were prepared by treatment with Neutrase (SHVH-N) and Purafect (SHVH-P). Hydrolysates were then separated according to their molecular weight, using the ultra-filtration membrane system, into 5 fractions (≥50, 50-5, 5-3, 3-1 and ≤1kDa). Fractions showed different amino acid compositions and angiotensin I-converting enzyme (ACE) inhibitory potentials. The SHVH-P-FV (≤1kDa) and SHVH-N-FIV (3-1kDa) fractions showed the best ACE-inhibitory activities with IC50 values of 53.31 and 75.05µg/ml, respectively. According to their high ACE-inhibitory potential, FIV and FV were fractionated by RP-HPLC and then analyzed by LC-MS/MS to identify peptide sequences. A systematic peptidomic study resulted in the identification of numerous novel sequences. Furthermore, in silico data, based on the molecular docking simulation, showed that GPAGPRGPAG, AVVPPSDKM, TTMYPGIA, and VKPLPQSG could bind ACE active site with low interaction scores. Indeed, they share hydrogen bonds and Van der Waals and electrostatic interactions with ACE catalytic pockets.

Proteolytic and amylolytic enzymes from a newly isolated Bacillus mojavensis SA: Characterization and applications as laundry detergent additive and in leather processing.

The present work aims to study the simultaneous production of highly alkaline proteases and thermostable α-amylases by a newly isolated bacterium Bacillus mojavensis SA. The optimum pH and temperature of amylase activity were 9.0 and 55°C, respectively, while those of the proteolytic activity were 12.0 and 60°C, respectively. Both α-amylase and protease enzymes showed a high stability towards a wide range of pH and temperature. Furthermore, SA crude enzymes were relatively stable towards non-ionic (Tween 20, Tween 80 and Triton X-100) and anionic (SDS) surfactants, as well as oxidizing agents. Both activities were improved by the presence of polyethylene glycol 4000 and glycerol. Additionally, the crude enzymes showed excellent stability against various solid and liquid detergents. Wash performance analysis revealed that the SA crude enzymes exhibited a remarkable efficiency in the removal of a variety type of stains, such as blood, chocolate, coffee and oil. On the other side, SA proteases revealed a potential dehairing activity of animal hide without chemical assistance or fibrous proteins hydrolysis. Thus, considering their promising properties, B. mojavensis SA crude enzymes could be used in several biotechnological bioprocesses.

Preventive effect of goby fish protein hydrolysates on hyperlipidemia and cardiovascular disease in Wistar rats fed a high-fat/fructose diet.

This study was carried out to investigate the hypolipidemic, cardioprotective and anticoagulant properties of fish goby protein hydrolysates (GPHs) in rats fed a high fat and fructose diet (HFFD). Wistar rats were fed with HFFD for 2 months, coupled with the oral administration of GPHs and undigested goby protein (UGP). Compared with the standard diet, HFFD induced dyslipidemia and liver structure alterations, and increased pancreatic lipase activity. In addition, HFFD caused a significant increase in body weight. Interestingly, administration of UGP and GPHs to HFFD fed rats was efficacious in lowering serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-c) as well as hepatic TC and TG, and increased the serum high density lipoprotein cholesterol (HDL-c) content. Moreover, all treatments significantly decreased the atherogenic index and coagulant factor levels (thrombin and prothrombin). UGP and GPH administration also significantly decreased pancreatic lipase activity, which mitigates lipid accumulation. Similarly, UGP and its hydrolysates showed cardioprotective potential revealed by decreasing the risk of atherogenic and coronary artery disease and improving the liver architecture. The ex vivo plasma clotting test showed that GPHs exert a great therapeutic anticoagulant potential. The overall results demonstrated that GPH supplementation can counteract high-fat/fructose diet-induced obesity.

Novel bioactive peptides from enzymatic hydrolysate of Sardinelle (Sardinella aurita) muscle proteins hydrolysed by Bacillus subtilis A26 proteases.

Sardinelle protein hydrolysate (SPH), prepared by treatment with Bacillus subtilis A26 proteases, was found to exhibit antibacterial, antioxidant and ACE-inhibitory activities. SPH, with a degree of hydrolysis of 4%, was fractionated by size exclusion chromatography on a Sephadex G-25 into five major fractions (F1-F5). F2, which exhibited the highest antibacterial and ACE-inhibitory activities, and F4, which exhibited the highest antibacterial and antioxidant activities, were further fractionated by reverse phase-high performance liquid chromatography (RP-HPLC) and then analysed using nano-ESI-LC-MS/MS to identify the sequences of peptides. Eight peptides were identified in the sub-fraction F2-A, nine peptides in the sub-fraction F4-B, and 45 peptides in F4-C. Identified peptides were found to share sequences with previously described bioactive peptides based on Biopep database. The results of this study suggest that SPH is a good source of natural bioactive peptides. Hence, it can be used as a potential ingredient in nutraceutical field.

Gelatin based bio-films prepared from grey triggerfish' skin influenced by enzymatic pretreatment.

Gelatins from grey triggerfish skin were extracted with different methods. The treatment by pepsin (PG) improved the yield of extraction when compared with untreated gelatin (UG) and acidic gelatin (AG). The outputs of gelatins AG, UG and PG, obtained respectively, with acitic acid, glycine buffer and glycine buffer added with 5U of pepsin/g of the skin beforehand treated by alkali, were 6.9%, 7.9% and 9.7%, respectively. The enzymatic treatment of the alkali-pretreated skin of grey triggerfish altered the electrophoresis profile, biophysical, gellification, rheological and thermal properties of the prepared gelatins extracted under acidic condition. However, the untreated gelatin obtained without pepsin exhibited the highest transition and enthaply temperatures. In addition, the properties of the prepared films were interconnected to their microstructure as demonstrated by scanning electron microscopy. Furthermore, films with PG and UG had a regular surface and a more condensed structure, whereas films prepared with AG had rougher surface.

Isolation of polysaccharides from Malva aegyptiaca and evaluation of their antioxidant and antibacterial properties.

This study investigated the antioxidant and antibacterial properties of crude water-soluble polysaccharides extracted from the mallow (Malva aegyptiaca) by precipitation with cetylpyridinium chloride (P1) or ethanol (P2). The Polysaccharides fractions were characterized by FTIR spectroscopy, a monosaccharide composition, and antioxidant and antibacterial activities. P1 showed the highest total sugars (81.2%) and sulfated groups (2.9%) contents but with the lowest total proteins content (8.7%). The infrared spectroscopic spectra exhibited the typical bands and peak characteristic of polysaccharides. Monosaccharides analysis revealed the dominance of galactose (40.3-43.7%) and glucuronic acid (25.9-30.9%). Interestingly, P1 displayed an important antioxidant activity as evaluated by the (Fe2+) chelating activity (IC50=1.15mg/ml), (Fe3+) reducing power (EC50=1.22mg/ml), ß-carotene bleaching inhibition capacity (IC50=1.56mg/ml) and DPPH-radical scavenging activity (IC50=1.94mg/ml). Furthermore, P1 at 10mg/ml was highly active than P2 against several bacterial strains and especially the Gram-positive bacteria, where the inhibition rate ranged between 84.2 and 90.3%. Obtained results suggest that M. aegyptiaca polysaccharides precipitated by cetylpyridinium chloride could be used as a naural antioxidant and antibacterial agent.

Development and characterization of cuttlefish (Sepia officinalis) skin gelatin-protein isolate blend films.

In the present study, cuttlefish skin gelatin (CSG) and protein isolate (CSPI) blend films were prepared and their physicochemical and antioxidant properties were characterized. CSG (4%, w/v) was blended with CSPI (3%, w/v) at different ratios and 15% of glycerol (w/w, plasticizer/polymer dry matter). Results showed that increasing of CSPI content induced lower lightness but higher redness, yellowness and color difference values compared to CSG film. In addition, CSPI film had the lowest tensile strength (TS) and elongation at break (EAB) values. The FTIR spectra of blend films showed a shift in the position of peaks related to the amide-I group, suggesting that they were involved in the interaction between the biopolymers. The structural properties evaluated by DSC showed a total miscibility between both polymers. This result was also confirmed by the SEM study, revealing a compact and homogenous structure in the blend films. Moreover, the water contact angle decreased in blend films, especially for those having higher proportion of CSPI. The antioxidant activity of films was markedly favored by the CSPI incorporation. Therefore, CSPI addition into gelatin matrix allowed to improve the blend films properties, mainly governed by the CSG/CSPI ratio.

Octopus vulgaris protein hydrolysates: characterization, antioxidant and functional properties.

Composition, functional properties and in vitro antioxidant activities of octopus (Octopus vulgaris) protein hydrolysates (OPHs) were evaluated. OPHs were prepared by treatment with commercial Esperase (OPH-Esp), alkaline protease extract from Zebra blenny (Salaria basilica) (OPH-ZB) and enzyme preparation from Bacillus subtilis A26 (OPH-A26). OPHs showed different degrees of hydrolysis (DH from 17.6 to 21%), and hydrophobic/hydrophilic peptide ratio. The amino acid profiles of OPHs showed a high level of essential amino acids, and Lys was the most abundant amino acid. Enzymatic hydrolysis improved solubility significantly as well as emulsifying and foaming properties of octopus proteins. The emulsifying activity index of OPHs decreased with increasing concentrations. Conversely, the foaming abilities increased as the hydrolysate concentrations increased. For the antioxidant activities, five different in vitro assay systems were investigated. All hydrolysates displayed various degrees and dose dependant antioxidant activities. The highest DPPH scavenging activity and reducing power were achieved by OPH-A26. OPH-Esp displayed the highest ability to prevent the bleaching of ß-carotene, whereas OPH-ZB exhibited the highest protection against hydroxyl radical induced DNA breakage. The results suggested that OPHs could be used, as a promising source of functional peptides with antioxidant activities, to formulate functional foods.