Blanca E. García-Almendárez

Biotechnological applications of peroxidases

Peroxidases are widely distributed in nature. Reduction of peroxides at the expense of electron donating substrates, make peroxidases useful in a number of biotechnological applications. Enzymes such as lignin peroxidase and manganese peroxidase, both associated with lignin degradation, may be successfully used for biopulping and biobleaching in the paper industry, and can produce oxidative breakdown of synthetic azo dyes. Oxidative polymerization of phenols and aromatic amines conducted by horseradish peroxidase (HRP) in water and water-miscible organic solvents, may lead to new types of aromatic polymers. Site directed mutagenesis of HRP has been used to improve the enantioselectivity of arylmethylsulfide oxidations. Peroxidase has a potential for soil detoxification, while HRP as well as soybean and turnip peroxidases have been applied for the bioremediation of wastewater contaminated with phenols, cresols, and chlorinated phenols. Peroxidase based biosensors have found use in analytical systems for determination of hydrogen peroxide and organic hydroperoxides, while co-immobilized with a hydrogen peroxide producing enzyme, they can be used for determination of glucose, alcohols, glutamate and choline. Peroxidase has also been used for practical analytical applications in diagnostic kits, such as quantitation of uric acid, glucose, cholesterol, lactose, and so on. Enzyme linked immunorbent assay (ELISA) tests on which peroxidase is probably the most common enzyme used for labeling an antibody, are a simple and reliable way of detecting toxins, pathogens, cancer risk in bladder and prostate, and many other analytes. Directed evolution methods, appear to be a valuable alternative to engineer new catalyst forms of plant peroxidases from different sources to overcome problems of stability and to increase thermal resistance.

Antimicrobial Edible Films and Coatings for Meat and Meat Products Preservation

Animal origin foods are widely distributed and consumed around the world due to their high nutrients availability but may also provide a suitable environment for growth of pathogenic and spoilage microorganisms. Nowadays consumers demand high quality food with an extended shelf life without chemical additives. Edible films and coatings (EFC) added with natural antimicrobials are a promising preservation technology for raw and processed meats because they provide good barrier against spoilage and pathogenic microorganisms. This review gathers updated research reported over the last ten years related to antimicrobial EFC applied to meat and meat products. In addition, the films gas barrier properties contribute to extended shelf life because physicochemical changes, such as color, texture, and moisture, may be significantly minimized. The effectiveness showed by different types of antimicrobial EFC depends on meat source, polymer used, film barrier properties, target microorganism, antimicrobial substance properties, and storage conditions. The perspective of this technology includes tailoring of coating procedures to meet industry requirements and shelf life increase of meat and meat products to ensure quality and safety without changes in sensory characteristics.

Physiological, morphological, and mannanase production studies on Aspergillus niger uam-gs1 mutants

Mutant strains from Aspergillus niger UAM-GS1 were produced by UV radiation to increase their hemicellulolytic and cellulolytic activity production. The mutant strains showing more enzymatic activity were those labelled GS1-S059 and GS1-S067. These strains also showed the largest relationship between diameter of hydrolysis zone and colony diameter. The mutant GS1-S067 showed a colony radial extension rate and a biomass growth rate [g biomass/(cm 2 h)], 1.17 times higher than that achieved by strain UAM-GS1. The high invasive capacity makes this mutant strain a promising alternative for its use in solid substrate fermentation (SSF). The morphological properties of the two mutant strains were evaluated by using scanning electron microscopy. The diameter of the sporangium of the mutant strains GS1-S059 and GS1-S067 was significantly larger ( P P

Monosaccharide composition and properties of a deglycosylated turnip peroxidase isozyme

A neutral peroxidase isozyme (TP) purified from turnip (Brassica napus L. var. purple top white globe) was partially deglycosylated, using chemical and enzymatic treatment. A 32% carbohydrate removal was achieved by exposing TP to a mixture of PNGase F, O-glycosidase, NANase, GALase III and HEXase I, while m-periodate treatment removed about 88% of TP carbohydrate moiety. The glycoprotein fraction of the TP contained a relatively high mannose and fucose content (37 and 31%, w/w, respectively), 16% (w/w) galactose, and 15% (w/w) GlcNAc. Thus, the carbohydrate moiety was classified as a hybrid type. Partially deglycosylated TP had reduced activity (by 50-85%), was more susceptible to proteolysis, and showed a slight decrease in thermostability compared to the native enzyme. Circular dichroism studies strongly suggested that although the carbohydrate moiety of TP did not influence the conformation of the polypeptide backbone, its presence considerably enhanced protein conformational stability toward heat. Removal of oligosaccharide chains from TP caused a decrease in K(m) and V(max) for hydrogen peroxide. Native and chemically deglycosylated TP were similarly immunodetected by rabbit polyclonal antibodies raised against TP. The results suggest that the carbohydrate moiety of TP is important for peroxidase activity and stability.

Microencapsulation, Chemical Characterization, and Antimicrobial Activity of Mexican (Lippia graveolens H.B.K.) and European (Origanum vulgare L.) Oregano Essential Oils

The effect of solvent polarity (methanol and pentane) on the chemical composition of hydrodistilled essential oils (EOs) of Lippia graveolens H.B.K. (MXO) and Origanum vulgare L. (EUO) was studied by GC-MS. Composition of modified starch microencapsulated EOs was conducted by headspace-solid-phase microextraction (HS-SPME). The antimicrobial activity of free and microencapsulated EOs was evaluated. They were tested against Salmonella sp., Brochothrix thermosphacta, Pseudomonas fragi, Lactobacillus plantarum, and Micrococcus luteus. Thymol and carvacrol were among the main components of EOs and their free and microencapsulated inhibitory activity was tested against M. luteus, showing an additive combined effect. Chemical composition of EOs varied according to the solvent used for GC analysis and to volatile fraction as evaluated by HS-SPME. Thymol (both solvents) was the main component in essential oil of MXO, while carvacrol was the main component of the volatile fraction. EUO showed α-pinene (methanol) and γ-terpinene (pentane) as major constituents, the latter being the main component of the volatile fraction. EOs showed good stability after 3 months storage at 4°C, where antimicrobial activity of microencapsulated EOs remained the same, while free EOs decreased 41% (MXO) and 67% (EUO) from initial activity. Microencapsulation retains most antimicrobial activity and improves stability of EOs from oregano.

Cloning, Heterologous Expression and Properties of a Recombinant Active Turnip Peroxidase

Turnip (Brassica napus) roots peroxidase isoforms have been used in diagnostic kits and can also efficiently polymerize phenolic compounds from wastewaters. Heterologous expression of a turnip acidic peroxidase (BnPA) was investigated to increase availability of this widely used enzyme. The mature BnPA was ligated into the pET28a(+) vector and used to transform Escherichia coli Rosetta 2. Recombinant BnPA peroxidase was overexpressed and accumulated in inclusion bodies from which it was purified to homogeneity by immobilized metal affinity chromatography under denaturing conditions. Peroxidase activity was observed after a refolding process under oxidative conditions. The yield of pure recombinant BnPA was 29 mg L(-1) of culture with a specific activity of 981 ± 20 ABTS units mg(-1) at optimal conditions (pH 6, 45 °C). Recombinant BnPA showed similar kinetic properties compared to native turnip peroxidase, and its secondary structure evaluated by circular dichroism comprised 20% α-helix, 32% β-sheet and 48% random structure. Recombinant BnPA showed high yield and good kinetic properties which are key steps for future structure-function studies and biotechnological applications.

Improvement of Covalent Immobilization Procedure of β-Galactosidase from Kluyveromyces lactis for Galactooligosaccharides Production: Modelling and Kinetic Study

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by β‐galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane‐polyvinyl alcohol (POS‐PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL−1 initial lactose concentration and 6 U mL−1 enzyme concentration, obtaining 25.46 ± 0.01 gL−1 yield of trisaccharides. Although below the HPLC‐IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL−1 of trisaccharides and 13.79 ± 0.21 gL−1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady‐state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved.

Effect of Nanoemulsified and Microencapsulated Mexican Oregano (Lippia graveolens Kunth) Essential Oil Coatings on Quality of Fresh Pork Meat

Fresh meat is a highly perishable food. This work aimed to evaluate the influence of Mexican oregano (Lippia graveolens Kunth) incorporated into active coatings (ACs) spread on fresh pork meat as free (FEO), nanoemulsified (NEO), and microencapsulated (MEO) essential oil (EO), on its microbiological, physicochemical and sensory properties during 15 d at 4 ± 1 °C. Thymol and γ-terpinene were identified in the EO. In vitro effect of 2.85 mg EO/cm2 was tested against Brochothrix thermosphacta, Micrococcus luteus, Lactobacillus plantarum, Pseudomonas fragi, and Salmonella Infantis. FEO antioxidant capacity (DPPH assay) was significantly higher than that of thymol, NEO and MEO (93.53%, 89.92%, 77.79%, and 78.50% inhibition, respectively), and similar to BHA (96.03%) and gallic acid (95.57%). FEO, NEO, and MEO ACs on meat caused growth inhibition of lactic acid bacteria (5 log population reduction) and Pseudomonas spp. (4 log reduction), whereas ≤1.5 log population reduction was observed for B. thermosphacta and Salmonella Infantis. Meat microbiota was more efficiently controlled by MEO than by FEO or NEO. ACs delayed lipid and oxymyoglobin oxidation of fresh pork meat. After 15 d of cold storage meat added with EO coatings was desirable for panelists, whereas untreated (UT) samples were undesirable. Active coatings are a significant alternative method for fresh meat preservation.

Physical, Structural, Barrier, and Antifungal Characterization of Chitosan–Zein Edible Films with Added Essential Oils

Edible films (EFs) have gained great interest due to their ability to keep foods safe, maintaining their physical and organoleptic properties for a longer time. The aim of this work was to develop EFs based on a chitosan–zein mixture with three different essential oils (EOs) added: anise, orange, and cinnamon, and to characterize them to establish the relationship between their structural and physical properties. The addition of an EO into an EF significantly affected (p < 0.05) the a* (redness/greenness) and b* (yellowness/blueness) values of the film surface. The EFs presented a refractive index between 1.35 and 1.55, and thus are classified as transparent. The physical properties of EFs with an added EO were improved, and films that incorporated the anise EO showed significantly lower water vapor permeability (1.2 ± 0.1 g mm h−1 m−2 kPa−1) and high hardness (104.3 ± 3.22 MPa). EFs with an added EO were able to inhibit the growth of Penicillium sp. and Rhizopus sp. to a larger extent than without an EO. Films’ structural changes were the result of chemical interactions among amino acid side chains from zein, glucosamine from chitosan, and cinnamaldehyde, anethole, or limonene from the EOs as detected by a Raman analysis. The incorporation of an EO in the EFs’ formulation could represent an alternative use as coatings to enhance the shelf life of food products.

Isolation and characterization of bacteriocinogenic lactic bacteria from M‐Tuba and Tepache, two traditional fermented beverages in México

Mexican Tuba (M-Tuba) and Tepache are Mexican fermented beverages prepared mainly with pineapple pulp and coconut palm, respectively. At present, reports on the microbiota and nutritional effects of both beverages are lacking. The purpose of this study was to determine whether M-Tuba and Tepache contain cultivable lactic acid bacteria (LAB) capable of producing bacteriocins. Tepache and M-Tuba contain mesophilic aerobic bacteria, LAB, and yeast. Bacillus subtilis, Listeria monocytogenes, Listeria innocua, Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, and Salmonella spp, were the microorganisms most susceptible to metabolites produced by bacterial isolates. M-Tuba and Tepache contain bacteria that harbor genes coding for nisin and enterocin, but not pediocin. The presence of Lactococcus lactis and E. faecium in M-Tuba and Tepache, was identified by 16S rDNA. These bacteria produced bacteriocins of ∼3.5 kDa and 4.0–4.5 kDa, respectively. Partial purified bacteriocins showed inhibitory effect against Micrococcus luteus, L. monocytogenes, L. innocua, Str. agalactiae, S. aureus, Bacillus cereus, B. subtilis, E. faecalis, and K. pneumoniae. We characterized, for the first time, cultivable microbiota of M-Tuba and Tepache, and specifically, identified candidate lactic bacteria (LAB) present in these beverages that were capable of synthesizing antimicrobial peptides, which collectively could provide food preservative functions.