Biomodification of acenocoumarol by bifidobacteria.

The increased interest of consumers in probiotic foods requires a deeper knowledge on the possible interactions with drugs, because their pharmacological properties could be modified. In this context, these studies are relevant for drugs such as acenocoumarol, whose dosage must be controlled due to, among other factors, food-drug interactions. Acenocoumarol is an oral anticoagulant with a narrow therapeutic range. The aim of the present research is to evaluate, in vitro, the effect of bifidobacteria on acenocoumarol. The drug was incubated with Bifidobacterium bifidum CIDCA 5310 or Bifidobacterium adolescentis CIDCA 5317 in MRS broth at 37°C for 24 h in anaerobic conditions. The effect of incubation with sterilized spent culture supernatants (SSCS) was also evaluated. Analysis by RP-HPLC showed that both bifidobacterial strains reduced the area of the acenocoumarol peak and two new peaks were evidenced. In addition, a decrease in the intensity of the bands at 1650, 1390 and 1110/cm was observed in the FTIR spectroscopic determinations. Moreover, a new band appeared at 1720/cm. No effect on the drug was observed when incubation was performed with SSCS. The present study showed a significant change in the concentration of the anticoagulant after incubation with bifidobacteria and results are compatible with biomodification of the drug due to enzymatic activity of bifidobacteria.

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.

Development of a method based on chemometric analysis of Raman spectra for the discrimination of heterofermentative lactobacilli.

In this work, a method based on Raman spectroscopy in combination with Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) has been developed for the rapid differentiation of heterofermentative related lactobacilli. In a first approach, Lactobacillus kefir strains were discriminated from other species of heterofermentative lactobacilli: Lb. parakefir and Lb. brevis. After this first approach, PCA allowed for a clear differentiation between Lb. parakefir and Lb.brevis. For the first level of discrimination, PCA was performed on the whole spectra and also on delimited regions, defined taking into consideration the loading values. The best regions allowing a clear differentiation between Lb. kefir and non-Lb. kefir strains were found to be: the 1700-1500 cm(-1), 1500-1185 cm(-1) and 1800-400 (whole spectrum) cm(-1) Raman ranges. In order to develop a classification rule, PLS-DA was carried out on the mentioned regions. This method permitted the discrimination and classification of the strains under study in two groups: Lb. kefir and non-Lb. kefir. The model was further validated using lactobacilli strains from different culture collections or strains isolated from kefir grains previously identified using molecular methods. The second approach based on PCA was also performed on the whole spectra and on delimited regions, being the regions 1700-1500 cm(-1), 1500-1185 cm(-1) and 1185-1020 cm(-1), i.e., those allowing the clearest discrimination between Lb. parakefir and Lb. brevis. The results obtained in this work, allowed a clear discrimination within heterofermentative lactobacilli strains, proteins being the biological structures most determinant for this discrimination.

Heterogeneity of S-layer proteins from aggregating and non-aggregating Lactobacillus kefir strains.

Since the presence of S-layer protein conditioned the autoaggregation capacity of some strains of Lactobacillus kefir, S-layer proteins from aggregating and non-aggregating L. kefir strains were characterized by immunochemical reactivity, MALDI-TOF spectrometry and glycosylation analysis. Two anti-S-layer monoclonal antibodies (Mab5F8 and Mab1F8) were produced; in an indirect enzyme-linked immunosorbent assay Mab1F8 recognized S-layer proteins from all L. kefir tested while Mab5F8 recognized only S-layer proteins from aggregating strains. Periodic Acid-Schiff staining of proteins after polyacrylamide gel electrophoresis under denaturing conditions revealed that all L. kefir S-layer proteins tested were glycosylated. Growth of bacteria in the presence of the N-glycosylation inhibitor tunicamycin suggested the presence of glycosydic chains O-linked to the protein backbone. MALDI-TOF peptide map fingerprint for S-layer proteins from 12 L. kefir strains showed very similar patterns for the aggregating strains, different from those for the non-aggregating ones. No positive match with other protein spectra in MSDB Database was found. Our results revealed a high heterogeneity among S-layer proteins from different L. kefir strains but also suggested a correlation between the structure of these S-layer glycoproteins and the aggregation properties of whole bacterial cells.

Interaction between Lactobacillus kefir and Saccharomyces lipolytica isolated from kefir grains: evidence for lectin-like activity of bacterial surface proteins.

Several microbial interactions involving yeast and lactobacilli have been suggested in fermented products. Co-aggregation between Lactobacillus kefir and yeast Saccharomyces lipolytica isolated from kefir grains was studied by scanning electron microscopy and aggregation assays. Six out of twenty Lb. kefir strains were able to co-aggregate with Sacch. lipolytica CIDCA 812 and showed thermolabile non-covalently bound surface molecules involved in this interaction. Co-aggregation inhibition after Lb. kefir pre-treatment with 5 m-LiCl or 20 g SDS/l showed that bacterial S-layer proteins play an important role in this interaction. Presence of different sugar (mannose, sucrose and fructose) or yeast pre-treatment with sodium periodate inhibited co-aggregation between Lb. kefir and Sacch. lipolytica. Co-aggregating Lb. kefir strains were also able to agglutinate with human red blood cells and they lost this ability after treatment with 5 m-LiCl. These results and the capacity of purified S-layer proteins of Lb. kefir to haemagglutinate, strongly suggest that a lectin-like activity of bacterial surface proteins (S-layer) mediates the aggregation with yeast cells.