Functional Properties of Collagen Extracted from Catfish (Silurus triostegus) Waste.

Collagen is used for a variety of biomedical and pharmaceutical uses, such as osteoarthritis-related pain management, hypertension, tissue engineering, and human implants, and is generally derived from porcine or bovine. Collagen from these animals has limitations due to the risk of disease transmission and religious constraints. Therefore, this study investigated the extraction of collagen from catfish (Silurus triostegus) waste. Acid-solubilized collagen and pepsin-solubilized collagen were extracted from catfish skin, fin, head, bone, and muscle. SDS-PAGE patterns of the extracted collagen showed that the protein molecular weights ranged from 97 to 200 kDa and skin, bone, and fin collagen consisted of 2 distinct α chains, which is typical of type 1 collagen. The proximate composition (moisture, protein, fat, and ash) and yield of the obtained extracts were determined. Skin collagen extracts were selected for further investigation due to the high collagen yield. The effects of the pH and salt concentration on solubility, and the denaturation temperature, FTIR spectra, reverse-phase HPLC, and SEM analysis were investigated to characterize the collagen samples. Based on the characterization of catfish skin collagen, this waste material has potential for use in the pharmaceutical and food industries.

Physical and emulsion stabilizing properties of maltodextrin fatty acid polymers produced by lipase-catalyzed reactions in ethanol.

Maltodextrin (MD) fatty acid esters (MFAs) have amphiphilic properties and the enzymatic synthesis of these molecules has gained growing interest. Here, MFAs were synthesized in a food-grade ethanol system and the properties of the products were analyzed. A total of 6 different MFAs were produced with 2 different MD sources and 3 combinations of fatty acids (lauric, palmitic, and both) with yields ranging from 72.7 to 83.4%. With an increase in fatty acid carbon length, degree of substitution (0.026 to 0.016) and solubility (100.9% to 93.1%) were significantly decreased. The stability of emulsions formulated with MFAs was investigated and all emulsions formulated were stable except those containing the lowest concentration of MFAs esterified with palmitate. Notably, MD esterified with laurate showed an enhanced emulsion stabilizing ability as compared to commercial emulsifiers. In conclusion, the emulsion stabilizing ability of MFAs may have applications in the food industry.

Enzymatic synthesis of chlorogenic acid glucoside using dextransucrase and its physical and functional properties.

Chlorogenic acid, a major polyphenol in edible plants, possesses strong antioxidant activity, anti-lipid peroxidation and anticancer effects. It used for industrial applications; however, this is limited by its instability to heat or light. In this study, we for the first time synthesized chlorogenic acid glucoside (CHG) via transglycosylation using dextransucrase from Leuconostoc mesenteroides and sucrose. CHG was purified and its structure determined by nuclear magnetic resonance and matrix-associated laser desorption ionization-time-of-flight mass spectroscopy. The production yield of CHG was 44.0% or 141mM, as determined by response surface methodology. CHG possessed a 65% increased water solubility and 2-fold browning resistance while it displayed stronger inhibition of lipid peroxidation and of colon cancer cell growth by MTT assay, compared to chlorogenic acid. Therefore, this study may expand the industrial applications of chlorogenic acid as water-soluble or browning resistant compound (CHG) through enzymatic glycosylation.

Food waste conversion to microbial polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are biopolymers with desirable material properties similar to petrochemically derived plastics. PHAs are naturally produced by a wide range of microorganisms as a carbon storage mechanism and can accumulate to significantly high levels. PHAs are an environmentally friendly alternative to their petroleum counterparts because they can be easily degraded, potentially reducing the burden on municipal waste systems. Nevertheless, widespread use of PHAs is not currently realistic due to a variety of factors. One of the major constraints of large-scale PHA production is the cost of carbon substrate for PHA-producing microbes. The cost of production could potentially be reduced with the use of waste carbon from food-related processes. Food wastage is a global issue and therefore harbours immense potential to create valuable bioproducts. This article's main focus is to examine the state of the art of converting food-derived waste into carbon substrates for microbial metabolism and subsequent conversion into PHAs.

Synthesis and Functional Characterization of Caffeic Acid Glucoside Using Leuconostoc mesenteroides Dextransucrase.

Caffeic acid was modified via transglucosylation using sucrose and dextransucrase from Leuconostoc mesenteroides B-512FMCM. Following enzymatic modification, a caffeic acid glucoside was isolated by butanol separation, silica gel chromatography, and preparative HPLC. The synthesized caffeic acid glucoside had a molecular mass-to-charge ratio of 365 m/z, and its structure was identified as caffeic acid-3-O-α-d-glucopyranoside. The production of this caffeic acid-3-O-α-d-glucopyranoside at a concentration of 153 mM was optimized using 325 mM caffeic acid, 355 mM sucrose, and 650 mU mL-1 dextransucrase in the synthesis reaction. In comparison with the caffeic acid, the caffeic acid-3-O-α-d-glucopyranoside displayed 3-fold higher water solubility, 1.66-fold higher antilipid peroxidation effect, 15% stronger inhibition of colon cancer cell growth, and 11.5-fold higher browning resistance. These results indicate that this caffeic acid-3-O-α-d-glucopyranoside may be a suitable functional component of food and pharmaceutical products.

Growth inhibitory properties of lactose fatty acid esters.

Sugar esters are biodegradable, nonionic surfactants which have microbial inhibitory properties. The influence of the fatty acid chain length on the microbial inhibitory properties of lactose esters was investigated in this study. Specifically, lactose monooctanoate (LMO), lactose monodecanoate (LMD), lactose monolaurate (LML) and lactose monomyristate (LMM) were synthesized and dissolved in both dimethyl sulfoxide (DMSO) and ethanol. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined in growth media. LML was the most effective ester, exhibiting MIC values of <0.05 to <5 mg/ml for each Gram-positive bacteria tested (Bacillus cereus, Mycobacterium KMS, Streptococcus suis, Listeria monocytogenes, Enterococcus faecalis, and Streptococcus mutans) and MBC values of <3 to <5 mg/ml for B. cereus, M. KMS, S. suis, and L. monocytogenes. LMD showed MIC and MBC values of <1 to <5 mg/ml for B. cereus, M. KMS, S. suis, L. monocytogenes, and E. faecalis, with greater inhibition when dissolved in ethanol. LMM showed MIC and MBC values of <1 to <5 mg/ml for B. cereus, M. KMS, and S. suis. LMO was the least effective showing a MBC value of <5 mg/ml for only B. cereus, though MIC values for S. suis and L. monocytogenes were observed when dissolved in DMSO. B. cereus and S. suis were the most susceptible to the lactose esters tested, while S. mutans and E. faecalis were the most resilient and no esters were effective on Escherichia coli O157:H7. This research showed that lactose esters esterified with decanoic and lauric acids exhibited greater microbial inhibitory properties than lactose esters of octanoate and myristate against Gram-positive bacteria.

Functional characterization of purified pear protease and its proteolytic activities with casein and myofibrillar proteins.

This study was performed to characterize pear protease proteolytic activity and investigate the use of pear protease as a meat tenderizer. Pear protease was purified and stabilized by 5% dextrin during lyophilization (dry) or concentration (liquid). Pear protease was further characterized with respect to pH, thermodynamics, and enzyme kinetics. Pear protease was stable at a pH range of 5-8 with an optimum pH of 6.5. From Arrhenius plots, liquid protease showed higher temperature dependency (23.49 kJ/mol) than dry protease (18.62 kJ/mol) due to its higher activation energy. The kcat/Km, catalytic efficiency of enzyme, was similar with 2.9 and 2.7 µM/min with dry and liquid proteases. Pear protease was evaluated for its proteolytic activities with casein and beef myofibrillar proteins by individually and combination with fig and kiwifruit proteases. These result indicated that pear and kiwifruit proteases could be complementary to be a desirable product for meat tenderization.

Determining the Effects of High Intensity Ultrasound on the Reduction of Microbes in Milk and Orange Juice Using Response Surface Methodology.

This study investigated the effects of high intensity ultrasound (temperature, amplitude, and time) on the inactivation of indigenous bacteria in pasteurized milk, Bacillus atrophaeus spores inoculated into sterile milk, and Saccharomyces cerevisiae inoculated into sterile orange juice using response surface methodology. The variables investigated were sonication temperature (range from 0 to 84°C), amplitude (range from 0 to 216 µm), and time (range from 0.17 to 5 min) on the response, log microbe reduction. Data were analyzed by statistical analysis system software and three models were developed, each for bacteria, spore, and yeast reduction. Regression analysis identified sonication temperature and amplitude to be significant variables on microbe reduction. Optimization of the inactivation of microbes was found to be at 84.8°C, 216 µm amplitude, and 5.8 min. In addition, the predicted log reductions of microbes at common processing conditions (72°C for 20 sec) using 216 µm amplitude were computed. The experimental responses for bacteria, spore, and yeast reductions fell within the predicted levels, confirming the accuracy of the models.

Investigating the effectiveness of St John's wort herb as an antimicrobial agent against mycobacteria.

A persistent need exists for effective treatment agents for mycobacterial infections. This research investigated the effectiveness of the Hypericum perforatum herb (commonly known as St John's wort; SJW) in its growth inhibition of mycobacteria. A SJW extract was effective at inhibiting five nonpathogenic Mycobacterium isolates and Bacillus subtilis, but not Escherichia coli. Quantitative studies of concentration sensitivity to the SJW extract were performed with minimal bactericidal concentrations (MBC) ranging from 0.33 to 2.66 mg extract/mL. The SJW compounds hyperforin (Hfn), hypericin (Hpn), and pseudohypericin (Phn) were quantified in the extract using HPLC. The SJW extract solution of 133 mg extract/mL used in this study contained 2.3 mg Hfn/mL, 0.8 mg Hpn/mL, and 2.1 mg Phn/mL. Purified Hfn, Hpn, and Phn were tested for inhibitory activity against Mycobacterium JLS (M. JLS) at similar concentrations used in the crude extract. While Hfn was inhibitory at 46 µg/mL, none of the purified SJW constituents were bactericidal at concentrations corresponding to SJW treatments. Scanning electron microscopy (SEM) analysis of SJW-treated M. JLS cells showed changes in cell surface morphology.

Effect of lactose monolaurate on pathogenic and nonpathogenic bacteria.

The antimicrobial activities of sucrose monolaurate and a novel ester, lactose monolaurate (LML), were tested. Gram-positive bacteria were more susceptible than Gram-negative bacteria to both esters. The minimal bactericidal concentrations of LML were 5 to 9.5 mM for Listeria monocytogenes isolates and 0.2 to 2 mM for Mycobacterium isolates.

Translocation of green fluorescent protein by comparative analysis with multiple signal peptides.

Type I and II secretory pathways are used for the translocation of recombinant proteins from the cytoplasm of Escherichia coli. The purpose of this study was to evaluate four signal peptides (HlyA, TorA, GeneIII, and PelB), representing the most common secretion pathways in E. coli, for their ability to target green fluorescent protein (GFP) for membrane translocation. Signal peptide-GFP genetic fusions were designed in accordance with BioFusion standards (BBF RFC 10, BBF RFC 23). The HlyA signal peptide targeted GFP for secretion to the extracellular media via the type I secretory pathway, whereas TAT-dependent signal peptide TorA and Sec-dependent signal peptide GeneIII exported GFP to the periplasm. The PelB signal peptide was inefficient in translocating GFP. The use of biological technical standards simplified the design and construction of functional signal peptide-recombinant protein genetic devices for type I and II secretion in E. coli. The utility of the standardized parts model is further illustrated as constructed biological parts are available for direct application to other studies on recombinant protein translocation.

Solid-phase capture of pathogenic bacteria by using gangliosides and detection with real-time PCR.

We developed a method for concentrating pathogens from samples without enrichment. Immobilized gangliosides concentrated bacteria for detection with real-time PCR. A sensitivity of approximately 4 CFU/ml (3 h) in samples without competing microflora was achieved. Samples with competing microflora had a sensitivity of 40,000 CFU/ml. The variance was less than one cycle.

Characterization of interactions between Escherichia coli molecular chaperones and immobilized caseins.

The molecular chaperones were affinity purified with immobilized alpha-casein (45mg protein/g beads) and beta-casein columns (30 mg protein/g beads) from two heat-induced E. coli strains, NM522 and BL21. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. The eluates from affinity columns were analyzed by SDS-PAGE and Western analysis. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES in eluates. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The beta-casein column showed a higher chaperone binding capacity than the alpha-casein column. A higher concentration of chaperones was purified from strain BL21 than strain NM522 which may have been due to the lack of lon protease in the BL21 strain.

Comparison of milk produced by cows cloned by nuclear transfer with milk from non-cloned cows.

Cloning technologies, including embryo splitting and nuclear transfer, were introduced into dairy cattle breeding in the early 1980s. With the recent worldwide attention on the cloning of sheep ("Dolly") and cows ("Gene"), the potential food safety concerns for food products derived from cloned animals needs to be addressed. There has been no study of the composition of milk produced by cloned cows. In this preliminary study, we evaluated the composition of milk from 15 lactating non-embryonic cell cloned cows and six non-cloned lactating cows over a single season. The cloned cows came from five unique genetic lines and three distinct breeds. Milk samples were analyzed for total solids, fat, fatty acid profile, lactose, protein and compared to non-cloned and literature values. Gross chemical composition of milk from cloned cows was similar to that of the non-cloned cows and literature values. Our results lead us to conclude that there are no obvious differences in milk composition produced from cloned cows compared to non-cloned cows.

Affinity purification and characterization of the Escherichia coli molecular chaperones.

The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well-documented affinity for proteins lacking tertiary structure. The molecular chaperones were induced from lon(-) Escherichia coli mutants, affinity purified with an immobilized beta-casein column, and assayed for refolding activity with thermally and chemically denatured carbonic anhydrase B (CAB). Chaperones were induced with three treatments: heat shock at 39 degrees C, heat shock 42 degrees C, and alcohol shock with 3% ethanol (v/v). Lysates were applied to an immobilized beta-casein (30 mg/g beads) column. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water or 1 mM Mg-ATP. The cold water and Mg-ATP eluates were analyzed by SDS-PAGE. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES. The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP. Refolding denatured CAB in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured CAB and a 68% recovery of chemically denatured CAB. The use of affinity matrices for the chaperone purification which are effective as in vitro folding aids will be presented.