Direct enzymatic hydrolysis of solid wheat straw with endo-xylanases: Effect of the temperature on the hemicellulose release and the product profile modulation.

Prebiotics are non-digestible compounds that promote intestinal microbiota growth and/or activity. Xylooligosaccharides (XOS) are new prebiotics derived from the hemicellulose fraction of lignocellulosic materials. Challenges in using those materials as sources for prebiotic compounds lie in the hemicellulose extraction efficiency and the safety of those ingredients. In this sense, this work aims to optimize hemicellulose extraction and XOS production through direct enzymatic hydrolysis of alkali pre-treated wheat straw without undesired byproducts. By increasing the temperature of the enzymatic step from 40 °C to 65 °C we achieved an improvement in the extraction yield from 55 % to 80 %. Products with different degrees of polymerization were also noticed: while XOS ≤ X6 where the main products at 40 °C, a mixture of long arabinoxylan derived polymers (ADPo) and XOS ≤ X6 was obtained at 65 °C, irrespective of the extraction yield. Thus, a modulatory effect of temperature on the product profile is suggested here. Among the XOS ≤ X6 produced, X2-X3 were the main products, and X4 was the minor one. At the end of the hydrolysis, 146.7 mg XOS per gram of pre-treated wheat straw were obtained.

Microfluidics potential for developing food-grade microstructures through emulsification processes and their application.

The food sector continues to face challenges in developing techniques to increase the bioavailability of bioactive chemicals. Utilising microstructures capable of encapsulating diverse compounds has been proposed as a technological solution for their transport both in food and into the gastrointestinal tract. The present review discusses the primary elements that influence the emulsification process in microfluidic systems to form different microstructures for food applications. In microfluidic systems, reactions occur within small reaction channels (1-1000 µm), using small amounts of samples and reactants, ca. 102-103 times less than conventional assays. This geometry provides several advantages for emulsion and encapsulating structure production, like less waste generation, lower cost and gentle assays. Also, from a food application perspective, it allows the decrease in particle dispersion, resulting in a highly repeatable and efficient synthesis method that also improves the palatability of the food products into which the encapsulates are incorporated. However, it also entails some particular requirements. It is important to obtain a low Reynolds number (Re < approx. 250) for greater precision in droplet formation. Also, microfluidics requires fluid viscosity typically between 0.3 and 1400 mPa s at 20 °C. So, it is a challenge to find food-grade fluids that can operate at the micro-scale of these systems. Microfluidic systems can be used to synthesise different food-grade microstructures: microemulsions, solid lipid microparticles, microgels, or self-assembled structures like liposomes, niosomes, or polymersomes. Besides, microfluidics is particularly useful for accurately encapsulating bacterial cells to control their delivery and release on the action site. However, despite the significant advancement in these systems' development over the past several years, developing and implementing these systems on an industrial scale remains challenging for the food industry.

Development and sensory test of a dairy product with ACE inhibitory and antioxidant peptides produced at a pilot plant scale.

A scale-up process was carried out to obtain potent bioactive peptides from whey protein through a simple hydrolysis process. The scale-up was satisfactory, with results similar to those obtained at lab scale: a fraction of peptides < 1 kDa with ACE inhibitory activity of 18.44 ± 2.47 µg/mL, a DPPH value of 69.40 ± 0.44%, and an ORAC value of 3.37 ± 0.03 µmol TE/mg protein. The peptide sequences responsible for the ACE inhibitory activity were also similar to those identified at lab scale: PM, LL, LF, HFKG and PT. The hydrolysate was used as a functional ingredient in a low-fat yoghurt. The consumer sensory taste panel found no significant difference (p > 0.05) between the bitterness of the control and the functional yoghurt, and about 50% of consumers would buy it. The hydrolysate maintained its bioactivities for 4 months at -20 °C (after thawing and pasteurisation), and for 1 week in yoghurt at 4 °C.

Microencapsulation of Lactobacillus plantarum in W/O emulsions of okara oil and block-copolymers of poly(acrylic acid) and pluronic using microfluidic devices.

Okara oil is a by-product remaining from defatting okara, the solid residue generated after extracting the aqueous fraction of grounded soybeans in the elaboration of soy beverages. The goal of this work was to encapsulate the probiotic Lactobacillus plantarum CIDCA 83114 into W/O emulsions composed of a block-copolymer constituted of pluronic® and acrylic acid (PPP12) and okara oil, prepared in microfluidic devices. For comparative purposes, alginate was also included as a second dispersed phase. Lactobacillus plantarum CIDCA 83114 was suspended in PPP12 or alginate giving rise to dispersed phases with different compositions, named I, II, III and IV. Controls were prepared by suspending microorganisms in water as dispersed phase. 6-carboxyfluorescein was added as bacterial marker in all the emulsions. The presence of green dyed bacteria in the dispersed phases, inside the droplets of the emulsions and the absence of fluorescence outside them, confirmed the complete encapsulation of bacteria in the dispersed phases. After being prepared, emulsions were freeze-dried. The exposure to gastric conditions did not lead to significant differences among the emulsions containing polymers. However, in all cases bacterial counts were significantly lower than those of the control. After exposing emulsions to the simulated intestinal environment, bacterial counts in assays I, II and III (emulsions composed of only one dispersed phase or of two dispersed phases with bacteria resuspended in the PPP12 one) were significantly greater than those of the control (p < 0.05) and no detectable microorganisms were observed for assay IV (emulsions composed of two dispersed phases with bacteria resuspended in the alginate one). In particular, bacterial cultivability in emulsions corresponding to assay I (only PPP12 as dispersed phase) exposed to the intestinal environment was 8.22 ± 0.02 log CFU/mL (2 log CFU higher than the values obtained after gastric digestion). These results support the role of PPP12 as an adequate co-polymer to protect probiotics from the gastric environment, enabling their release in the gut, with great potential for food or nutraceutical applications.

Optimisation of bovine ß-lactoglobulin hydrolysis using cardosins from dried flowers of Cynara cardunculus.

Bovine whey protein was hydrolysed using cardosins A and B purified from dried flowers of Cynara cardunculus by combining diafiltration, anion-exchange chromatography and ultrafiltration. The proteolysis experiments were performed using different whey protein concentrations and enzyme/substrate (E/S) ratios. Complete hydrolysis of the main whey proteins, ß-Lactoglobulin (ß-Lg) and α-lactalbumin (α-La), was achieved after 4 h, at E/S ratios of 1/150 U/mg, regardless the initial protein concentration. In previous reports, the authors suggested that cardosins could not hydrolyse ß-lactoblogulin. However, our promising results open up new possibilities to further explore the action of cardosins on whey proteins for the production of bioactive peptides.

Effectiveness of proteolytic enzymes to remove gluten residues and feasibility of incorporating them into cleaning products for industrial purposes.

The development of protocols for efficient gluten elimination is one of the most critical aspects of any allergen management strategy in the industry. The suitability of different proteolytic enzymes to be included in a cleaning formulation that allows the effective elimination of gluten residues was studied. Alcalase (ALC), neutrase (NEUT) and flavourzyme (FLAV) were selected from in silico analysis. The presence of 1% (v/v) of linear alkylbenzene sulphonate (LAS), a common anionic detergent, improved the gluten solubility, which may favour its elimination. Chromatographic analysis showed that the three enzymes studied were able to hydrolyse gluten in the presence of LAS. The highest percentage of short peptides (< 5 kDa) was achieved with ALC, what increases the probability of reducing the gluten antigenicity. Besides, in the presence of ALC and detergent LAS have detected the lowest levels of gluten with ELISA kits. So, effective amounts of ALC and LAS were added to a cleaning formulation, where its proteolytic activity was maintained above 90% after 37 days at 4 °C and 25 °C (under dark). Preliminary validation of the effectiveness enzymatic cleaning formulation to hydrolyse gluten was performed in a ready-to-eat/frozen food company, in which previous episodes of cross-contamination with gluten have been detected. The gluten content decreased to values below 0.125 µg/100 cm2 when the cleaning formulation was tested on different surfaces with different cleaning protocols, demonstrating the high suitability of the enzymatic cleaning formulation developed.

One-step chromatographic method to purify α-lactalbumin from whey for nanotube synthesis purposes.

A one-step anion-exchange chromatography method (NaCl gradient elution on a DEAE Sepharose™ Fast Flow gel column) was developed to purify α-lactalbumin (α-LA) from whey protein isolate. α-LA nearly 100% pure (based on the total protein content) was obtained with a yield of about 39%. Besides pure α-LA, which was the main objective of this work, highly pure ß-lactoglobulin was also obtained with a yield of about 59%. The high purity of the obtained α-LA samples allowed its use to synthesise protein nanotubes with excellent gelation properties for their use as food thickeners and bioactive carriers. The samples' purity degree obtained (based on the total protein content) was critical in the formation of proper nanotubes instead of random aggregates, which produced opaque and weak gels, less useful for food applications.

Design of whey protein nanostructures for incorporation and release of nutraceutical compounds in food.

Whey proteins are widely used as nutritional and functional ingredients in formulated foods because they are relatively inexpensive, generally recognized as safe (GRAS) ingredient, and possess important biological, physical, and chemical functionalities. Denaturation and aggregation behavior of these proteins is of particular relevance toward manufacture of novel nanostructures with a number of potential uses. When these processes are properly engineered and controlled, whey proteins may be formed into nanohydrogels, nanofibrils, or nanotubes and be used as carrier of bioactive compounds. This review intends to discuss the latest understandings of nanoscale phenomena of whey protein denaturation and aggregation that may contribute for the design of protein nanostructures. Whey protein aggregation and gelation pathways under different processing and environmental conditions such as microwave heating, high voltage, and moderate electrical fields, high pressure, temperature, pH, and ionic strength were critically assessed. Moreover, several potential applications of nanohydrogels, nanofibrils, and nanotubes for controlled release of nutraceutical compounds (e.g. probiotics, vitamins, antioxidants, and peptides) were also included. Controlling the size of protein networks at nanoscale through application of different processing and environmental conditions can open perspectives for development of nanostructures with new or improved functionalities for incorporation and release of nutraceuticals in food matrices.

Pediocin SA-1: A selective bacteriocin for controlling Listeria monocytogenes in maize silages.

In this study, we assessed the potential as silage additive of a bacteriocin produced by Pediococcus acidilactici Northern Regional Research Laboratory (NRRL) B-5627 (pediocin SA-1). Maize was inoculated either with a bacterial starter alone (I) or in combination with the bacteriocin (IP), and untreated silage served as control. We monitored the products of fermentation (ethanol, and lactic and acetic acids), the microbial population, and the presence of the indicator strain Listeria monocytogenes Colección Española de Cultivos Tipo (CECT) 4032 (1×10(5) cfu/g) after 1, 2, 5, 8, 16, and 30d of ensiling. Our results indicated antilisterial activity of the bacteriocin, anticipating the disappearance of L. monocytogenes in IP compared with I and control silages. The PCR-denaturing gradient gel electrophoresis analysis revealed the addition of the bacteriocin did not affect the bacterial communities of the spontaneous fermentation, and the inoculant-containing bacteria (Lactobacillus plantarum, Lactobacillus buchneri, and Enterococcus faecium) were found in addition to the bacterial communities of untreated maize silages in I and IP silages. Both treatments increased the concentration of antimicrobial compounds (acetic acid, ethanol, and 1,2-propanodiol) and led to lower residual sugar contents compared with the control, which would provide enhanced aerobic stability. The fact that the identified species L. plantarum, L. buchneri, and E. faecium produce some of these inhibitory compounds, together with their persistence throughout the 30d of fermentation, suggest these bacteria could actively participate in the ensiling process. According to these results, pediocin SA-1 could be used as an additive to control the presence of L. monocytogenes in maize silages selectively, while improving their fermentative quality and eventually their aerobic stability.

Temperature- and pH-sensitive nanohydrogels of poly(N-Isopropylacrylamide) for food packaging applications: modelling the swelling-collapse behaviour.

Temperature-sensitive poly(N-isopropylacrylamide) (PNIPA) nanohydrogels were synthesized by nanoemulsion polymerization in water-in-oil systems. Several cross-linking degrees and the incorporation of acrylic acid as comonomer at different concentrations were tested to produce nanohydrogels with a wide range of properties. The physicochemical properties of PNIPA nanohydrogels, and their relationship with the swelling-collapse behaviour, were studied to evaluate the suitability of PNIPA nanoparticles as smart delivery systems (for active packaging). The swelling-collapse transition was analyzed by the change in the optical properties of PNIPA nanohydrogels using ultraviolet-visible spectroscopy. The thermodynamic parameters associated with the nanohydrogels collapse were calculated using a mathematical approach based on the van't Hoff analysis, assuming a two-state equilibrium (swollen to collapsed). A mathematical model is proposed to predict both the thermally induced collapse, and the collapse induced by the simultaneous action of two factors (temperature and pH, or temperature and organic solvent concentration). Finally, van't Hoff analysis was compared with differential scanning calorimetry. The results obtained allow us to solve the problem of determining the molecular weight of the structural repeating unit in cross-linked NIPA polymers, which, as we show, can be estimated from the ratio of the molar heat capacity (obtained from the van't Hoff analysis) to the specific heat capacity (obtained from calorimetric measurements).

Effects of feeding of two potentially probiotic preparations from lactic acid bacteria on the performance and faecal microflora of broiler chickens.

The aim of this study was to evaluate the potential of two probiotic preparations, containing live lactic acid bacteria (Lactococcus lactis CECT 539 and Lactobacillus casei CECT 4043) and their products of fermentation (organic acids and bacteriocins), as a replacement for antibiotics in stimulating health and growth of broiler chickens. The effects of the supplementation of both preparations (with proven probiotic effect in weaned piglets) and an antibiotic (avilamycin) on body weight gain (BWG), feed intake (FI), feed consumption efficiency (FCE), relative intestinal weight, and intestinal microbiota counts were studied in 1-day posthatch chickens. The experiments were conducted with medium-growth Sasso X44 chickens housed in cages and with nutritional stressed Ross 308 broiler distributed in pens. Consumption of the different diets did not affect significantly the final coliform counts in Sasso X44 chickens. However, counts of lactic acid bacteria and mesophilic microorganisms were higher in the animals receiving the two probiotic preparations (P < 0.05). In the second experiment, although no differences in BWG were observed between treatments, Ross 308 broilers receiving the probiotic Lactobacillus preparation exhibited the lowest FCE values and were considered the most efficient at converting feed into live weight.

Modelling the biphasic growth and product formation by Enterococcus faecium CECT 410 in realkalized fed-batch fermentations in whey.

The influence of initial pH on growth and nutrient (total sugars, nitrogen, and phosphorous) consumption by Enterococcus faecium CECT 410 was studied during batch cultures in whey. With these data, two realkalized fed-batch fermentations were developed using different feeding substrates. The shift from homolactic to mixed acid fermentation, the biphasic kinetics observed for cell growth and nitrogen consumption and the increase in the concentrations of biomass and products (lactic acid, acetic acid, ethanol, and butane-2,3-diol) were the most noteworthy observations of these cultures. Modelling the fed-batch growth of Ent. faecium with the Logistic and bi-Logistic models was not satisfactory. However, biomass production was best mathematically described with the use of a double Monod model, which was expressed in terms of biomass, product accumulation, and nitrogen utilization. Product formation was successfully modelled with a modified form of the Luedeking and Piret model developed in this study.

Intramuscular fatty acid composition of "Galician Mountain" foals breed. Effect of sex, slaughtered age and livestock production system.

The effects of sex, slaughtered age (9 vs. 12 months) and livestock production system (freedom extensive system vs. semi extensive system) of "Galician Mountain" foals breed on the fatty acid composition were studied. The sex and slaughtered age of the animals had no statistical significance in the intramuscular fatty acids. Furthermore, the livestock production system showed differences in the fatty acid profiles from the Longissimus dorsi. The feeding system showed significant differences in PUFA content (P<0.001) higher in freedom extensive production system that semi extensive system, whereas MUFA content was significantly (P<0.001) higher in semi extensive system. This major PUFA content in freedom extensive production showed a significant (r=0.70, P<0.01) correlation with C18:3n-3 content and can be attributed to their eaten only pasture until slaughtered. The higher MUFA contents (P<0.001) observed for semi extensive production system foals were very significant (r=0.98, p<0.01) correlated with C18:1cis-9 content and less significant (r=0.81, P<0.01) correlated with C16:1cis-9 content. In addition, the data reinforced the evidence that foals from extensive production system on wood pasture have a higher nutritional quality (mainly due to the higher levels of n-3 PUFA) when compared to concentrate-fed foals, as a result of the beneficial effects of grass on meat fatty acid profiles.