E. Elizabeth Tymczyszyn

Galacto-oligosaccharides as protective molecules in the preservation of Lactobacillus delbrueckii subsp. bulgaricus.

In this work, the protective capacity of galacto-oligosaccharides in the preservation of Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 was evaluated. Lactobacillus bulgaricus was freeze-dried or dried over silica gel in the presence of three commercial products containing galacto-oligosaccharides. The freeze-dried samples were stored at 5 and 25°C for different periods of time. After desiccation, freeze-drying or storage, samples were rehydrated and bacterial plate counts were determined. According to the results obtained, all galacto-oligosaccharides assays demonstrated to be highly efficient in the preservation of L. bulgaricus. The higher content of galacto-oligosaccharides in the commercial products was correlated with their higher protective capacity. Galacto-oligosaccharides are widely known by their prebiotic properties. However, their role as protective molecules have not been reported nor properly explored up to now. In this work the protective capacity of galacto-oligosaccharides in the preservation of L. bulgaricus, a strain particularly sensitive to any preservation process, was demonstrated. The novel role of galacto-oligosaccharides as protective molecules opens up several perspectives in regard to their applications. The supplementation of probiotics with galacto-oligosaccharides allows the production of self-protected synbiotic products, galacto-oligosaccharides exerting both a prebiotic and protecting effect.

Critical water activity for the preservation of Lactobacillus bulgaricus by vacuum drying.

Lactobacillus delbrueckii subsp. bulgaricus was dried under vacuum at different temperatures and its preservation evaluated analyzing the evolution of three parameters throughout the process: lag time, percentage of membrane damage and zeta potential. Microorganisms were dehydrated at 30, 45 and 70 degrees C in a vacuum centrifuge for different times. The aw achieved for each time of drying was correlated with the cell recovery at all the temperatures assayed. The recovery of microorganisms was evaluated by means of: a) kinetics of growth in milk after drying, as a measure of the global damage; b) quantification of the membrane damage using the fluorescent dyes SYTO 9 and PI; c) determination of changes in the superficial charges (zeta potential) as measured of the increase in the hydrophobic residues exposed in the bacterial surface after dehydration. These changes correlate well with the bacterial damage occurred during the dehydration process. The Pages equation allowed fitting of aw and time of drying, thus making possible the determination of the appropriate dehydration conditions (time-temperature ratios) for which no cell damage occurs. The evaluation of three parameters (lag time, percentage of membrane damage and zeta potential) allowed us to conclude that at the lowest temperature of dehydration, the first target of damage is the cell membrane. However, this damage is not decisive for the bacterial recovery after rehydration, as are the increase in the lag time and the changes in the zeta potential, as was observed for L. bulgaricus dehydrated at 45 and 70 degrees C for larger times.

Effect of physical properties on the stability of Lactobacillus bulgaricus in a freeze-dried galacto-oligosaccharides matrix

The ability of galacto-oligosaccharides (GOS) to protect Lactobacillus delbrueckii subsp. bulgaricus upon freeze drying was analyzed on the basis of their capacity to form glassy structures. Glass transition temperatures (T(g)) of a GOS matrix at various relative humidities (RH) were determined by DSC. Survival of L. bulgaricus in a glassy GOS matrix was investigated after freezing, freeze drying, equilibration at different RHs and storage at different temperatures. At 32 °C, a drastic viability loss was observed. At 20 °C, the survival was affected by the water content, having the samples stored at lower RHs, the highest survival percentages. At 4°C, no decay in the cells count was observed after 45 days of storage. The correlation between molecular mobility [as measured by Proton nuclear magnetic resonance (¹H NMR)] and loss of viability explained the efficiency of GOS as cryoprotectants. The preservation of microorganisms was improved at low molecular mobility and this condition was obtained at low water contents and low storage temperatures. These results are important in the developing of new functional foods containing pre and probiotics.

Use of whey permeate containing in situ synthesised galacto-oligosaccharides for the growth and preservation of Lactobacillus plantarum

Galacto-oligosaccharides (GOS) are prebiotics that have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. GOS are commonly produced from lactose in an enzymatic reaction catalysed by β-galactosidase, named transglycosylation. Lactose is the main constituent of whey permeate (WP), normally wasted output from the cheese industry. Therefore, the main goal of this work was to optimise the synthesis of GOS in WP using β-galatosidase from Aspergillus oryzaea. WP and whey permeate enzymatically treated (WP-GOS) were used as culture media of Lactobacillus plantarum 299v. Lb. plantarum 299v attained the stationary phase in approximately 16 h, reaching 3·6 and 4·1×108 CFU/ml in WP and WP-GOS, respectively. The in situ synthesised GOS were not consumed during growth. No significant differences were observed in the growth kinetics of microorganisms in both media. After fermentation, microorganisms were dehydrated by freeze-drying and spray-drying and stored. The recovery of microorganisms after fermentation, dehydration and storage at 4 °C for at least 120 d was above 108 CFU/g. These studies demonstrated that WP is an appropriate substrate for the synthesis of GOS and the obtained product is also adequate as culture medium of Lb. plantarum 299v. The coexistence of GOS and dehydrated viable probiotic microorganisms, prepared using an effluent as raw material, represents the main achievement of this work, with potential impact in the development of functional foods.

Determination of amorphous/rubbery states in freeze-dried prebiotic sugars using a combined approach of near-infrared spectroscopy and multivariate analysis

Galacto-oligosaccharides (GOS) and lactulose are well-recognized prebiotics widely used in functional food and pharmaceutical products, but there is still a lack of knowledge regarding their physical-chemical properties. In this study, a physical-chemical approach on two GOS of different composition (GOS Cup Oligo H-70® and GOS Biotempo) and lactulose was assessed. Mid infrared and Raman spectra of the freeze-dried sugars allowed their structural characterization in the amorphous state, lactulose, showing the main spectral differences. Freeze-dried sugars were then equilibrated at 4°C at relative humidity (RH) ranging from 11% to 80%. Near-infrared reflectance spectra were registered in each condition in the 900- to 1700-nm region. A principal component analysis (PCA) was performed on the three sugars equilibrated at different RH. In all the three sugars, the groups observed explained more than 95% of the variance and were related with the RH of the samples. According to the loading plots of PC1, the main differences related with RH were observed in the 1380- to 1500-nm region. As the amorphous states are very sensitive to changes in temperature and moisture content, and the moisture content is related with the parameter T-Tg (T: storage temperature; Tg: vitreous transition temperature), an effort was made to determine this parameter directly from the NIR spectra. To this aim, a partial least square model (PLS) was defined. Tg values obtained by differential scanning calorimetry (DSC) were used to calculate the T-Tg values of reference. The model was validated with an independent set of data. The mean of predicted values fitted nicely T-Tg obtained from DSC (correlation=0.966; R2=0.934), thus supporting the use of the PLS model to investigate unknown samples. The stability of amorphous sugars in foods and pharmaceuticals is of practical and economical importance because it affects different quality attributes of foods, including texture, aroma retention and shelf life. Therefore, predicting T-Tg, a parameter that is independent on the sugar investigated, directly from their NIR spectra is of utmost importance to determine the shelf life of food and food-related products and up to our knowledge has never been determined hereto.

Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Spray-Drying

In this work maltodextrins were added to commercial galacto-oligosaccharides (GOS) in a 1:1 ratio and their thermophysical characteristics were analyzed. GOS:MD solutions were then used as matrices during spray-drying of Lactobacillus plantarum CIDCA 83114. The obtained powders were equilibrated at different relative humidities (RH) and stored at 5 and 20°C for 12 weeks, or at 30°C for 6 weeks. The Tgs of GOS:MD matrices were about 20–30°C higher than those of GOS at RH within 11 and 52%. A linear relation between the spin-spin relaxation time (T2) and T-Tg parameter was observed for GOS:MD matrices equilibrated at 11, 22, 33, and 44% RH at 5, 20, and 30°C. Spray-drying of L. plantarum CIDCA 83114 in GOS:MD matrices allowed the recovery of 93% microorganisms. In contrast, only 64% microorganisms were recovered when no GOS were included in the dehydration medium. Survival of L. plantarum CIDCA 83114 during storage showed the best performance for bacteria stored at 5°C. In a further step, the slopes of the linear regressions provided information about the rate of microbial inactivation for each storage condition (k values). This information can be useful to calculate the shelf-life of spray-dried starters stored at different temperatures and RH. Using GOS:MD matrices as a dehydration medium enhanced the recovery of L. plantarum CIDCA 83114 after spray-drying. This strategy allowed for the first time the spray-drying stabilization of a potentially probiotic strain in the presence of GOS.

Effect of acclimation medium on cell viability, membrane integrity and ability to consume malic acid in synthetic wine by oenological Lactobacillus plantarum strains

The aim of this work was to evaluate the effect of acclimation on the viability, membrane integrity and the ability to consume malic acid of three oenological strains of Lactobacillus plantarum.

Use of Raman spectroscopy and chemometrics for the quantification of metal ions attached to Lactobacillus kefir

Aims:  To set‐up an experimental and analytical methodology to evaluate the feasibility of developing simple, accurate and quantitative models based on Raman spectroscopy and multivariate analysis for the quantification of metal ions adsorbed to the bacterial surface of Lactobacillus kefir.

Applications of Infrared and Raman Spectroscopies to Probiotic Investigation

In this review, we overview the most important contributions of vibrational spectroscopy based techniques in the study of probiotics and lactic acid bacteria. First, we briefly introduce the fundamentals of these techniques, together with the main multivariate analytical tools used for spectral interpretation. Then, four main groups of applications are reported: (a) bacterial taxonomy (Subsection 4.1); (b) bacterial preservation (Subsection 4.2); (c) monitoring processes involving lactic acid bacteria and probiotics (Subsection 4.3); (d) imaging-based applications (Subsection 4.4). A final conclusion, underlying the potentialities of these techniques, is presented.

Galacto-oligosaccharides and lactulose as protectants against desiccation of Lactobacillus delbrueckii subsp. bulcaricus

Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 was dehydrated on desiccators containing silica gel in the presence of 20% w/w of two types of galacto‐oligosaccharides (GOS Biotempo and GOS Cup Oligo H‐70®) and lactulose, until no changes in water desorption were detected. After rehydration, bacterial growth was monitored at 37°C by determining: (a) the absorbance at 600 nm and (b) the near infrared spectra (NIR). Principal component analysis (PCA) was then performed on the NIR spectra of samples dehydrated in all conditions. A multiparametric flow cytometry assay was carried out using carboxyfluorescein diacetate and propidium iodide probes to determine the relative composition of damaged, viable, and dead bacteria throughout the growth kinetics. The absorbance at 600 nm and the position of the second derivative band at ∼1370 nm were plotted against the time of incubation. The efficiency of the protectants was GOS Biotempo > GOS Cup Oligo H‐70® > lactulose. The better protectant capacity of GOS Biotempo was explained on the basis of the lower contribution of damaged cells immediately after rehydration (t = 0). PCA showed three groups along PC1, corresponding to the lag, exponential and stationary phases of growth, which explained 99% of the total variance. Along PC2, two groups were observed, corresponding to damaged or viable cells. The results obtained support the use of NIR to monitor the recovery of desiccated microorganisms in real time and without the need of chemical reagents. The use of GOS and lactulose as protectants in dehydration/rehydration processes was also supported.