Exploring the cellulolytic activity of environmental mycobacteria.

Although studies on non-tuberculous mycobacteria have increased in recent years because they cause a considerable proportion of infections, their cellulolytic system is still poorly studied. This study presents a characterization of the cellulolytic activities of environmental mycobacterial isolates derived from soil and water samples from the central region of Argentina, aimed to evaluate the conservation of the mechanism for the degradation of cellulose in this group of bacteria. The molecular and genomic identification revealed identity with Mycolicibacterium septicum. The endoglucanase and total cellulase activities were assessed both qualitatively and quantitatively and the optimal enzymatic conditions were characterized. A specific protein of around 56 kDa with cellulolytic activity was detected in a zymogram. Protein sequences possibly arising from a cellulase were identified by mass spectrometry-based shotgun proteomics. Results showed that M. septicum encodes for cellulose- and hemicellulose-related degrading enzymes, including at least an active ß-1,4 endoglucanase enzyme that could be useful to improve its survival in the environment. Given the important health issues related to mycobacteria, the results of the present study may contribute to the knowledge of their cellulolytic system, which could be important for their ability to survive in many different types of environments.

Hybrid materials based on chitosan functionalized with green synthesized copper nanoparticles: Physico-chemical and antimicrobial analysis.

Recently, the development of materials with antimicrobial properties has become a challenge under scrutiny. The incorporation of copper nanoparticles (NpCu) into a chitosan matrix appears to represent a viable strategy to contain the particles and prevent their oxidation. Regarding the physical properties, the nanocomposite films (CHCu) showed a decrease in the elongation at break (5 %) and an increase in the tensile strength of 10 % concerning chitosan films (control). They also showed solubility values lower than 5 % while the swelling diminished by 50 %, on average. The dynamical mechanical analysis (DMA) of nanocomposites revealed two thermal events located at 113° and 178 °C, which matched the glass transitions of the CH-enriched phase and nanoparticles-enriched phase, respectively. In addition, the thermogravimetric analysis (TGA) detected a greater stability of the nanocomposites. Chitosan films and the NpCu-loaded nanocomposites demonstrated excellent antibacterial capacity against Gram-negative and Gram-positive bacteria, proved through diffusion disc, zeta potential, and ATR-FTIR techniques. Additionally, the penetration of individual NpCu particles into bacterial cells and the leakage of cell content were verified by TEM. The mechanism of the antibacterial activity of the nanocomposites involved the interaction of chitosan with the bacterial outer membrane or cell wall and the diffusion of the NpCu through the cells. These materials could be applied in diverse fields of biology, medicine, or food packaging.

Fructose derived oligosaccharides prevent lipid membrane destabilization and DNA conformational alterations during vacuum-drying of Lactobacillus delbrueckii subsp. bulgaricus.

Dehydration of lactic acid bacteria for technological purposes conducts to multilevel damage of bacterial cells. The goal of this work was to determine at which molecular level fructose-oligosaccharides (FOS) and sucrose protect Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 during the vacuum-drying process. To achieve this aim, the cultivability and metabolic activity of vacuum-dried bacteria were firstly determined (plate counting and absorbance kinetics). Then, the membrane integrity and fluidity were assessed using propidium iodide and Laurdan probes (general polarization -GP-), respectively. Finally, bacterial structural alterations were determined using high throughput methods (fluorescence confocal microscopy and Raman spectroscopy coupled to Multivariate Curve Resolution analysis -MCR-). The vacuum-drying process directly affected the microorganism's cultivability and membrane integrity. Non-dehydrated cells and sugar protected bacteria (both with FOS or sucrose) presented high GP values typical from the gel state, as well as phospholipids microdomains laterally organized along the cytoplasmic membrane. On the contrary, bacteria dehydrated without protectants presented low GP values and greater water penetration, associated with membrane destabilization. Raman spectroscopy of vacuum-dried cells revealed DNA conformational changes, B-DNA conformations being associated to non-dehydrated or sugar protected bacteria, and A-DNA conformations being higher in bacteria vacuum-dried without protectants. These results support the role of FOS and sucrose as protective compounds, not only acting at the membrane organizational level but also preventing conformational alterations of intracellular structures, like DNA.

Nutritional and technological properties of a quinoa (Chenopodium quinoa Willd.) spray-dried powdered extract.

The relevance of an appropriate nutrition requires innovation in the design of food ingredients. The goal of this work was to obtain a powdered extract of quinoa by using spray-drying. To this aim, quinoa flour was suspended in water to obtain a soluble fraction mainly composed of proteins, starch, fiber, lipids, antioxidants and minerals. The spray-drying conditions of this quinoa soluble fraction were set-up in terms of inlet temperatures (150, 160, 170 and 180 °C) and feed flow (4.5, 7.5, 10.5 mL/min). The obtained powders were characterized by determining the proximate composition, antioxidant activity, microstructure, fatty acids' profile, and starch and proteins' structures. A correlation among the drying parameters and the chemical and functional attributes of the powders was addressed using principal component analysis. From a technological viewpoint the use of moderate feed flows (7.5 mL/min) and high inlet temperatures (180 °C) was the best combination to obtain high powder yields (85% d.b.), low aw (0.047 ± 0.005) and high solids content (0.956 ± 0.005). The drying temperature positively affected the structure of starch, improving swelling and favoring moderate agglomeration which increases the encapsulation properties of quinoa. These results support the use of spray-drying as a suitable method to obtain powdered extracts of quinoa without affecting the nutritional value, thus supporting their use as functional ingredients in the formulation of ready-to-eat foods.

Synthesis of fructo-oligosaccharides using grape must and sucrose as raw materials.

Grape must market has been rising and there is an increasing interest to use it as a "natural" replacement for traditional sugars. Food or beverages with prebiotic compounds, including fructo-oligosaccharides (FOS), emerge as an alternative for the new health style trend. The aim of this work was to investigate whether the combination of grape must with sucrose was a suitable raw material for the synthesis of FOS. This way, a prebiotic syrup containing fructose and FOS, potentially useful for the formulation of foods and beverages, could be obtained. The main process consisted of three stages, namely conditioning of grape must (oxidation of the initial glucose concentration, stage 1), synthesis of FOS [incorporation of 20, 30 and 55% (w/w) sucrose, and 3.5% v/v Viscozyme L - 4.2 U/mg-, stage 2], and conditioning of the final product (oxidation of the glucose generated during the synthesis, stage 3). At stage 1, glucose concentration decreased from 222.8 mg/mL to 47.2 mg/mL, representing a decay of about 80% regarding the initial concentration of glucose. At stage 2, incorporating 20% (w/w) sucrose was not enough to impulse FOS synthesis. In turn, although 30 and 55% (w/w) sucrose produced very similar concentrations of total FOS (DP3 + DP4), 55% (w/w) sucrose led to higher glucose generation and less DP4 formation. Hence, 30% (w/w) sucrose was the condition selected for the synthesis and further conditioning of the obtained product (stage 3). In these conditions, the final product consisted of more than 30% of short chain FOS (19% and 13% of DP3 and DP4, respectively), 55% fructose and less than 11% of glucose and sucrose. Considering that fructose has approximately double sweetening power than glucose, the obtained syrup has a bigger sweetening power in comparison with the original grape must, also providing the prebiotic benefits of FOS.

Flour from mature Prosopis nigra pods as suitable substrate for the synthesis of prebiotic fructo-oligosaccharides and stabilization of dehydrated Lactobacillus delbrueckii subsp. bulgaricus.

Prosopis nigra, a sucrose-rich crop, was used to enzymatically synthesize fructo-oligosaccharides (FOS). The obtained products were used as stabilizing matrices during freeze-drying and storage of Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333. The centesimal composition of P. nigra flour was firstly determined. FOS were synthesized using Viscozyme L as biocatalyst. The progress of the enzymatic reaction was monitored by HPLC and compared with a reaction carried out using equivalent concentrations of pure sucrose as substrate (control). Then, P. nigra containing or not the obtained FOS (P. nigra + FOS or P. nigra) were used as matrices for freeze-drying and storage of L. delbrueckii subsp. bulgaricus CIDCA 333. P. nigra flour was rich in simple sugars (sucrose and fructose), total dietary fiber, and polyphenols. The main products of synthesis were FOS with degrees of polymerization (DP) within 3 and 5, and these results were comparable with those of the controls. DP3 was the first product obtained, attaining the maximal production after 1.29 hours of synthesis. The maximal production of total FOS (DP3 + DP4 + DP5) was achieved after 2.57 hours, indicating that larger FOS (DP4, DP5) were produced from DP3. Glucose was obtained as secondary product, but with significantly lower Vmax and Kf (maximal velocity for the production and constant for the formation) than DP3. Both P. nigra + FOS or P. nigra matrices stabilized the highly sensitive L. delbrueckii subsp. bulgaricus CIDCA 333 strain during freeze-drying and storage for up to 140 days at 4 °C, and were significantly better protectants than the controls of sucrose (p <0.05). The concomitant presence of prebiotics (FOS), antioxidants (polypyhenols) and lactic acid bacteria in the matrices provides a smart strategy to increase the value of this underutilized regional crop, turning it in an interesting ingredient potentially useful in the food industry.

Physico-chemical and structural properties of crystalline inulin explain the stability of Lactobacillus plantarum during spray-drying and storage.

The stabilizing capacity of crystalline inulin during spray-drying and storage of Lactobacillus plantarum CIDCA 83114 was assessed. In a first step, the physical properties of the matrices were investigated, using amorphous inulin as control. Melting and glass transition temperatures, water sorption isotherms, water activity, and infrared spectra were determined. Microorganisms were spray-dried at a pilot scale in both amorphous and crystalline matrices. After that, scanning electronic and confocal microsopies provided a full landscape about the interactions between microorganisms and crystals, and also the bacterial location within the amorphous matrices. The technological properties of the dehydrated microorganisms (culturability and acidification capacity) during storage at different water activities were also evaluated. Both amorphous and crystalline inulins were adequate matrices to stabilize microorganisms. However, crystalline inulin was more stable than amorphous one, especially when the storage temperature was close to the glass transition temperature, resulting in a better matrix to protect microorganisms during pilot spray-drying and storage. Furthermore, no accumulation of insoluble inulin was observed after resuspending the dehydrated microorganisms in crystalline inulin matrices, which appears as a clear technological advantage with regard to the amorphous one. Considering the prebiotic character of inulin and the probiotic properties of L. plantarum CIDCA 83114, this work developed an integrated approach, both from a fundamental and from an applied viewpoint, supporting the incorporation of such ingredients in the formulation of food products.

Infrared spectroscopy as an alternative methodology to evaluate the effect of structural features on the physical-chemical properties of inulins.

Two types of inulins of different composition were investigated in the glassy and in the crystalline states, at relative humidities within 11 and 97%. The melting and glass transition temperatures (Tm, Tg), and their crystallinity indexes (CI) were determined by modulated differential-scanning calorimetry (MDSC) and wide-angle X-ray scattering (WAXS), respectively. In parallel assays, Fourier transform-infrared spectroscopy (FTIR) coupled to principal component analysis (PCA) enabled a physical-chemical and structural characterization of samples, explaining 90% of the total variance. Finally, partial least square (PLS) models were defined to determine Tg, Tm, and CI directly from the FTIR spectra, using the MDSC and WAXS results as reference methods. In all cases, the mean of predicted values fitted very well those of the reference methods (R2 > 0.961), thus supporting the use of the PLS models to investigate unknown samples. The robustness of the models underlines the usefulness of FTIR to easily determine physical-chemical parameters, otherwise requiring complex preparation of samples and prolonged times of analysis.

Malt sprout, an underused beer by-product with promising potential for the growth and dehydration of lactobacilli strains.

Malt sprout (MS), a by-product of the malt industry obtained by removing rootlets and sprouts from the seed of germinated barley (Hordeum vulgare L.), was used as culture, dehydration and storage medium of three strains of lactobacilli: Lactobacillus salivarius CM-CIDCA 1231B and CM-CIDCA 1232Y and Lactobacillus plantarum CIDCA 83114. The three strains were grown in MS and MS supplemented with 20% w/v fructo-oligosaccharides (MS FOS). Bacterial growth was determined by registering the decrease of pH and by plate counting. Comparable results with those of microorganisms grown in MRS (controls) were observed in terms of lag times, ΔpH and acidification rates. Furthermore, during fermentation, a significant increase of DP6 (FOS with degree of polymerization 6) was observed at expenses of inulin and DP7, probably indicating their hydrolysis. A concomitant decrease of DP3, sucrose and monosaccharides was also observed, as result of their bacterial consumption during growth. The presence of FOS in the fermented media protected microorganisms during freeze-drying and storage, as no decrease of culturability was observed after 60 days at 4 °C (> 108 CFU/mL). Using MS appears as an innovative strategy for the production of lactobacilli at large scale, supporting their use for the elaboration of functional foods containing prebiotics and probiotics.

Effect of sucrose concentration on the composition of enzymatically synthesized short-chain fructo-oligosaccharides as determined by FTIR and multivariate analysis.

Fructo-oligosaccharides (FOS) are mixtures of oligosaccharides composed of fructose and glucose units. As their composition is determined by the synthesis conditions, the goals of this work were: (a) to engineer FOS of different composition by adjusting the sucrose concentration used as initial substrate; (b) to define partial least square (PLS) based-models to quantify all the sugars present in the reaction medium directly from the FTIR spectra. The yield of each reaction was calculated as the percentage of initial sucrose converted to each oligosaccharide, as monitored by HPLC. In parallel, the reactions were followed by FTIR. Six different PLS models aiming to determine the concentration of each carbohydrate present in the reaction medium were calibrated and independently validated. The means of predicted values fitted well to those obtained by HPLC. Determining FOS composition directly from the FTIR spectra represents a useful tool to monitor enzymatic synthesis, with strong impact at both an academic and an industrial level.

Role of mono- and oligosaccharides from FOS as stabilizing agents during freeze-drying and storage of Lactobacillus delbrueckii subsp. bulgaricus.

The aim of this work was to assess the role of mono- and oligosaccharides present in fructo-oligosaccharides (FOS) mixtures as protective agents during freeze-drying and storage of Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333. Different FOS mixtures were enzymatically obtained from sucrose and further purified by removing the monosaccharides produced as secondary products. Their glass transition temperatures (Tg) were determined at 11, 22 and 33% relative humidity (RH). Bacterial cultures were freeze-dried in the presence of 20% w/v solutions of the studied FOS. Their protective effect during freeze-drying was assessed by bacterial plate counting, and by determining the lag time from growth kinetics and the uptake of propidium iodide (PI). Plate counting during bacterial storage at 4°C, and 11, 22 and 33% RH for 80days completed this rational analysis of the protective effect of FOS. Purification of FOS led to an increase of Tg in all the conditions assayed. Microorganisms freeze-dried in the presence of non-purified FOS were those with the shortest lag times. Bacteria freeze-dried with pure or commercial FOS (92% of total FOS) showed larger lag times (8.9-12.6h). The cultivability of microorganisms freeze-dried with non-purified FOS and with sucrose was not significantly different from that of bacteria before freeze-drying (8.74±0.14logCFU/mL). Pure or commercial FOS were less efficient in protecting bacteria during freeze-drying. All the protectants prevented membrane damage. The cultivability of bacteria freeze-dried with FOS decayed <1logarithmicunit after 80days of storage at 11% RH. When storing at 22 and 33% RH, pure and commercial FOS were those that best protected bacteria, and FOS containing monosaccharides were less efficient. The effect of FOS on bacterial protection is the result of a balance between monosaccharides, sucrose and larger FOS in the mixtures: the smallest sugars are more efficient in protecting lipid membranes, and the larger ones favor the formation of vitreous states.

Edible methylcellulose-based films containing fructo-oligosaccharides as vehicles for lactic acid bacteria.

The goal of this work was to investigate the physicochemical properties of methylcellulose (MC) based films as stabilizers of two strains of lactobacilli: Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 and Lactobacillus plantarum CIDCA 83114. The incorporation of 3% w/v fructo-oligosaccharides (FOS) into the MC film formulation improved the viability of L. delbrueckii subsp. bulgaricus CIDCA 333 after film preparation. L. plantarum CIDCA 83114 was intrinsically more resistant as no viability loss was observed upon preparation of the films in the absence of FOS. Scanning electronic microscopy images also showed a good incorporation of microorganisms without affecting the homogeneity of the films. FTIR spectroscopy provided structural information about the bacteria-loaded films. Water sorption isotherms showed an impervious behavior at low aw but on exceeding 0.7 of aw the film started to dissolve and form syrup, causing a drastic drop of bacterial viability (log N/N0≤-5). Dynamic mechanical analysis (DMA) demonstrated that the incorporation of microorganisms into the MC films had no effect on vitreous transition temperatures. FOS incorporated into the MC films had a plasticizing effect. Microorganism-loaded films were stored at relative humidities (RH) ranging from 11 to 75%. Both strains could be stored at 11% RH for 90days. At 33 and 44% RH L. delbrueckii subsp. bulgaricus CIDCA 333 could be stored up to 15days and L. plantarum CIDCA 83114 up to 45days. At 75% RH only L. plantarum CIDCA 83114 could be equilibrated (log N/N0: -2.05±0.25), but CFU/g films were undetectable after 15days of storage. The results obtained in this work support the use of MC films containing FOS as a good strategy to immobilize lactic acid bacteria, with potential applications in the development of functional foods.

Characterization of cellulolytic activities of environmental bacterial consortia from an Argentinian native forest.

Cellulolytic activities of three bacterial consortia derived from a forest soil sample from Chaco region, Argentina, were characterized. The phylogenetic analysis of consortia revealed two main highly supported groups including Achromobacter and Pseudomonas genera. All three consortia presented cellulolytic activity. The carboxymethylcellulase (CMCase) and total cellulase activities were studied both quantitatively and qualitatively and optimal enzymatic conditions were characterized and compared among the three consortia. Thermal and pH stability were analyzed. Based on its cellulolytic activity, one consortium was selected for further characterization by zymography. We detected a specific protein of 55 kDa with CMCase activity. In this study, we have shown that these consortia encode for cellulolytic enzymes. These enzymes could be useful for lignocellulosic biomass degradation into simple components and for different industrial applications.