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 (T_g) 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 80 days completed this rational analysis of the protective effect of FOS.Purification of FOS led to an increase of T_g 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 (8.9–12.6 h). 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 ± 0.14 log CFU/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 < 1 logarithmic unit after 80 days 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.     

Production of volatile aroma compounds by kefir starter cultures

Production of carbonyl compounds by single-strain cultures, kefir starter (Lactobacillus delbrueckii subsp. bulgaricus HP1+Lb. helveticus MP12+Lactococcus lactis subsp. lactis C15+Streptococcus thermophilus T15+Saccharomyces cerevisiae A13) and kefir grains during fermentation and storage of kefir was studied. The content of carbonyl compounds produced by kefir starter was greater than that produced by kefir grains. The maximum acetaldehyde concentration (18.3 μg g⁻¹) in kefir with starter culture was mainly due to the metabolic activity of Lb. delbrueckii subsp. bulgaricus HP1 isolated from kefir grains. The highest diacetyl production activity was recorded in the starter culture (1.87 μg g⁻¹) and the single-strain culture St. thermophilus T15 (1.62 μg g⁻¹), followed by Lb. helveticus MP12 (0.85 μg g⁻¹) and Lc. lactis subsp. lactis C15 (0.42 μg g⁻¹). The lactobacilli Lb. delbrueckii subsp. bulgaricus HP1 and Lb. helveticus MP12 produced acetone, which was not found in the cocci cultures. The presence of 2-butanone was related to the production ability of Lb. helveticus MP12. In comparison, Lc. lactis subsp. lactis C15 synthesized ethyl acetate more actively than the other single-strain cultures included in the starter. S. cerevisiae A13 produced ethanol and CO₂ in amounts (3975 μg g⁻¹; 1.80 g L⁻¹) that lent cultured kefir distinctive flavour and aroma characteristic of authentic kefir.     

Stability of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus in the presence of galacto-oligosaccharides and lactulose as determined by near infrared spectroscopy

The ability of two types of galacto-oligosaccharides (GOS) and lactulose to protect Lactobacillus delbrueckii subsp. bulgaricus upon freeze-drying was evaluated on the basis of their capacity to form glassy structures, using near infrared (NIR) spectroscopy.After freeze-drying in the presence of protective agents, microorganisms were stored at 4 °C at different relative humidities (RH) (from 11 to 80%) up to 21 days. The loss of viability was determined by plate count and referred to viability before freeze-drying. For each storage condition, glass transition temperatures (T_g) were determined by differential scanning calorimetry (DSC). In parallel, near infrared spectra registered in the 900–1700 nm region provided information about viability and RHs that could be correlated with the results obtained by plate counts and DSC.The comparison of NIR spectra of freeze-dried samples stored at the same RH but differing in the time of storage allowed the identification of spectral features related with viability.The spectral differences observed on samples stored in the same conditions (same time of storage and same protective agent) but differing in RHs were ascribed to the formation of amorphous and rubbery states.Based on the spectral information, a multivariate based classification model (PLS-DA) was defined to determine whether samples are in a rubbery or an amorphous state. As the physical state of starters is related with their shelf-life, this model provided support for the use of NIR spectroscopy as a quick method to assess the proper storage of starters.     

Conjugated linoleic acid production by immobilized cells of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus acidophilus

Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) and L. acidophilus (CCRC14079), immobilized with chitosan and polyacrylamide, were tested for CLA production. A 10-ml aliquot of L. delbrueckii ssp. bulgaricus cell suspension (3.59 × 10⁷ CFU/ml) was adsorbed to 0.5 g chitosan and polyacrylamide, mixed with 0.2 ml linoleic acid (0.9 g/ml), and incubated at 37 °C for 24 h at pH 5, 6, 7, and 8 for CLA production. CLA levels, produced by immobilized cells of L. delbrueckii ssp. bulgaricus and L. acidophilus with increasing cell counts to 1.08 and 1.28 × 10¹⁰ CFU/ml, respectively, at optimal reaction pHs were evaluated. More CLA was formed at pH 8 of chitosan and pH 7 of polyacrylamide-immobilized L. delbrueckii ssp. bulgaricus cell treatments. Increase in cell count resulted in higher CLA production. The adsorption of L. delbrueckii ssp. bulgaricus cells onto polyacrylamide at pH 7 showed significant improvement in total CLA level. Results demonstrated a potential for enhancing CLA production through immobilization.     

Effect of acidification on the activity of probiotics in yoghurt during cold storage

The viability of Lactobacillus acidophilus LAFTI^® L10, Bifidobacterium lactis LAFTI^® B94, and L. paracasei LAFTI^® L26 and their proteolytic activities were assessed in yoghurt at different termination pH of 4.45, 4.50, 4.55, and 4.60 in the presence of L. delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342 during 28 days of storage at 4 °C. All strains achieved the recommended level of 6.00 log cfu g⁻¹ of the product with L. acidophilus LAFTI^® L10 and L. paracasei LAFTI^® L26 exceeding the number to 8.00 and 7.00 log cfu g⁻¹, respectively. Lactobacilli strains showed a good cellular stability maintaining constant concentration throughout storage period regardless of termination pH. On the other hand, the cell counts of B. lactis LAFTI^® B94 decreased by one log cycle at the end of storage. The presence of probiotic organisms enhanced proteolysis significantly in comparison with the control batch containing L. delbrueckii ssp. bulgaricus Lb1466 and S. thermophilus St1342 only. The proteolytic activity varied due to termination pH, but also appeared to be strain related. The increased proteolysis improved survival of L. delbrueckii ssp. bulgaricus Lb1466 during storage resulting in lowering of pH and production of higher levels of organic acids, which might have caused the low cell counts for B. lactis LAFTI^® B94.     

Shelf life of alginate beads containing lactobacilli and bifidobacteria: characterisation of microspheres containing Lactobacillus delbrueckii subsp. bulgaricus

Capsules, containing different microorganisms (Lactobacillus plantarum, L. casei, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, L. reuteri, L. rhamnosus, Bifidobacterium breve and B. animalis subsp. lactis), were produced through a simple extrusion protocol and dipped in a CaCl₂ solution (0.5% or 8%). Beads were characterised, to assess the encapsulation yield (EY) and the shelf life at 4 °C (defined as the time to retain inside a cell count of 6 log CFU g^?1); then, L. delbrueckii subsp. bulgaricus was used as target for system optimisation, through the study of the kinetic of cell release, as a function of the concentration of CaCl₂ solution, agitation (150 rpm) and recycling. The EY was 50% or higher, and the shelf life was at least 30 days. Concerning beads containing L. delbrueckii subsp. bulgaricus, the release of cells from capsules followed a biphasic (diauxic) trend and the microsphere could be used successfully for two times, as only after third reuse, the amount of release cells decreased.     

Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR

This study describes a procedure that allows specific detection and enumeration of viable bacteria in four species of lactic acid bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus casei subsp. casei and Lactobacillus acidophilus) and of Bifidobacterium lactis, mixed in fermented milk products. The procedure is based on the combined use of propidium monoazide (PMA), able to distinguish between viable and irreversibly damaged cells, with species-specific quantitative real-time PCR (RTi-PCR). Loss of viability of the species in a fermented milk through storage at 4 °C was similarly (P < 0.05) detected by PMA–RTi-PCR and selective plate counts. Furthermore, comparison of results obtained by both methods showed a Pearson linear correlation of 0.995. The enumeration of viable bacteria by PMA–RTi-PCR could be performed in 3 h, whereas enumeration by selective plate counts required three days. The procedure developed is a fast method for the identification, enumeration and discrimination of viability of lactic acid bacteria and bifidobacteria mixed in fermented milk products.     

Probiotic stability of yoghurts containing Jerusalem artichoke inulins during refrigerated storage

Experimentally prepared Jerusalem artichoke inulins (JAI) were compared with two commercial chicory root inulins for their prebiotic potentials in media broth model and growth-sustaining ability in non-fat yoghurts. Experimental yoghurts were made with 12% reconstituted skim milk (RSM) supplemented with 4% inulin powders, inoculated with mixed cultures of Lactobacillus casei LC-01, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (1:0.5:0.5 based on supplier’s recommendation) and incubated overnight at 37 °C. Non-supplemented yoghurt was prepared from 16% RSM and used as control. The survival and acidifying activity of lactic and probiotic cultures in all yoghurts were investigated on weekly intervals during the shelf life of 28 days at 4 °C. Incorporation of JAI resulted in improved viability of LC-01, maintaining >7.0 log CFU/g during cold storage but did not affect the viability of yoghurt bacteria in comparison with the control.     

Viscosity of model yogurt systems enriched with barley β-glucan as influenced by starter cultures

The effect of barley β-glucan (BG) on the growth of two yogurt starter cultures (SCs) each consisting of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus was investigated. Model yogurt systems were prepared with or without the incorporation of barley BG and lactose to mimic natural levels in milk and allowed to ferment for 8 h at 42 °C. The fermentation efficacy of the SCs was monitored through pH measurements. Samples were collected and analyzed for pH and viscosity at 0, 2, 4, and 8 h. Viscosity was determined using a rotational viscometer at shear rates of 1.29–129 s⁻¹ and 20 °C. The pH measurements indicated no adverse effect on the growth of SCs as the acidification process was consistent with normal pH development, but viscosity measurements suggested that BG was depolymerized by SCs when lactose became a limiting nutrient during fermentation.     

Incorporation of selected nutraceuticals and probiotic bacteria into a fermented milk

Yoghurts were produced from a base milk containing three important nutraceuticals, namely ω-3-fatty acids, isoflavones and phytosterols. The cultures employed to make the yoghurts were single probiotic strains of Lactobacillus gasseri or Bifidobacterium infantis and, to achieve a short production time, a two-stage fermentation procedure was used with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus providing the rapid acidification. Yoghurts containing counts of >1.0×10⁸ cfu mL⁻¹ of the individual probiotics and high counts of the traditional species from yoghurt were awarded overall scores for sensory acceptability >4.0 out of 5.0; the nutraceuticals appeared to have no adverse effect on flavour. Storage trials at 5 °C showed that the viability of the probiotic cultures was retained over 15 days.     

Identification of cultivable lactic acid bacteria isolated from Algerian raw goat's milk and evaluation of their technological properties

One hundred and fifty-eight strains of lactic acid bacteria isolated from Algerian raw goat's milk were identified and technologically characterized. Five genera were found: Lactobacillus (50.63%), Lactococcus (25.94%), Streptococcus (14.56%), Leuconostoc (7.59%) and Pediococcus (1.26%). The predominant species were Lactococcus lactis (32 strains), Streptococcus thermophilus (23 strains), Lactobacillus bulgaricus (19 strains), Lb. helveticus (16 strains) and Lb. plantarum (14 strains).Approximately 39% of the lactic acid bacteria isolated produced more than 0.6% lactic acid (w/v) after 18 h of incubation, and belonged to the Lactococcus and Lactobacillus genera. The highest proteolytic activity was approximately 3 mg tyrosine l⁻¹ for mesophilic strains and nearly 5 mg tyrosine l⁻¹ for thermophilic lactobacilli after 72 h. High aromatic activity (more than 0.8 mg diacetyl l⁻¹ after 16 h) was detected in 14% of the strains.Nine strains were used to make dairy products (a yoghurt-like product and Edam-type cheese) on a pilot scale in the laboratory. The best-liked organoleptic characteristics were noted in a yoghurt produced with a mixed culture made up of S. thermophilus (strain 16TMC+) and Lb. helveticus (strain 20TMC) and in a cheese made with a starter composed of Lc. lactis subsp. lactis (strain 10MCM) and L. lactis subsp. lactis (V.P. +) (strain 19MCM).     

Survival of Lactobacillus delbrueckii UFV H2b20 in ice cream produced with different fat levels and after submission to stress acid and bile salts

The survival of Lactobacillus delbrueckii UFV H2b20 in three ice cream formulations (low fat, fat free and high fat) was evaluated after the processing and storage at ?16 °C. The survival of L. delbrueckii UFV H2b20 was not significantly affected (P > 0.05) in three ice cream formulations after processing. The same result was observed during storage for 40 days at ?16 °C. Cells of L. delbrueckii UFV H2b20 incorporated in three ice cream formulation survived when exposed to acid stress and bile salts. The results demonstrate that L. delbrueckii UFV H2b20 has potential for being used in ice cream and capacity to resist acid stress and to grow in the presence of bile salts. This demonstrates that reduction of fat in ice cream does not compromise the viability of L. delbrueckii UFV H2B20.     

Production of traditional Greek yoghurt using Lactobacillus strains with probiotic potential as starter adjuncts

Lactobacillus plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were examined for their potential application as adjuncts in the production of traditional Greek set-type yoghurt. Both strains displayed low milk acidification activity, while no inhibition was observed towards or from the yoghurt starters used. Yoghurt produced with L. paracasei subsp. tolerans ACA-DC 4037 exhibited the best sensory properties, with a rich traditional smooth taste, and the strain was selected for further trials. Yoghurt produced with this strain as an adjunct had good physicochemical properties. After 2 weeks of refrigerated storage, microbial loads (>7.0 log cfu g⁻¹) were in accordance with international recommendations and guidelines for probiotic and starter cultures in milk products. Increasing the microbial load further, using concentrated and encapsulated inocula (10–11 log cfu g⁻¹), gave yoghurt with long fermentation times and poor organoleptic properties.     

Identification and technological properties of lactic acid bacteria isolated from raw goat milk of four Algerian races

The characteristics of 725 lactic acid bacteria isolated from raw goat milk of four Algerian races were studied. They were phenotypically classified as Lactobacillus (31.6%), Lactococcus (28.4%), Leuconostoc (22.2%), Streptococcus (13.7%) and Pediococcus (4.1%). No major differences were observed as to the distribution of genus isolates in the milk of three races (Makatia, Makatia-Chamia and Kabyle). However, the number of isolates of both Leuconostoc and Lactococcus predominated in Arabia milk while the samples collected from the other races mostly contained lactobacilli isolates.The phenotypic and biochemical analyses gave a diversity of species (28 presumptive species). The most abundant species were Streptococcus thermophilus (46 isolates), Lactobacillus helveticus (45 isolates), Lactobacillus plantarum (30 isolates), Lactobacillus delbruecki subsp. bulgaricus (26 isolates), Lactobacillus brevis (25 isolates) and Lactococcus lactis subsp. lactis (25 isolates).The technological properties of the isolates were as follows: 38.6% of isolates showed a fast acidifying rate (more than 60°D during 18 h of incubation); 25.9% of isolates had a high proteolytic capacity (more than 6 ppm leucine/72 h) and 14.1% of isolates had a high diacetyl production (more than 0.8 ppm diacetyl/16 h).Nine isolates were selected to prepare yoghurt and cheese. Sensory tests performed by a trained panel revealed a pleasant yoghurt prepared with mixed culture St. thermophilus 16 TMC+L. helveticus 20TMC. Cheese prepared with isolates L. lactis subsp. lactis biovar diacetylactis 19 MCA and L. lactis subsp. lactis 10 MCM had a very good sensory quality and a pleasant taste.     

Inactivation of Salmonella typhimurium and Lactobacillus plantarum by UV-C light in flour powder

This research evaluates the potential use of ultraviolet C light (UV-C) as a decontamination method for powdered foods, particularly of refined flour. This technology's lethal effectiveness was evaluated on Salmonella enterica subsp. Enterica serotype Typhimurium and Lactobacillus plantarum in wheat flour, and in laboratory liquid media of different a_w and turbidities to evaluate the action mechanisms of UV-C light in powdered products. Initial results showed a large variability of lethality in flour, obtaining between 0.2 and 3.0 log₁₀ cycles of inactivation. Results obtained in laboratory media and SEM analysis of contaminated flour indicated that the variability was due to a shadow effect on the efficacy of UV-C light and not due to the low water a_w of the flour or starch content. Based on these conclusions, a 2-m vertical tunnel with twelve 480 W UV-C lamps was designed to treat flour by forming a continuous cloud of dust (0.05–2.4 kg/h). Inactivation levels of 4.0 to 1.7 log₁₀ cycles of the population of L. plantarum in flour were achieved at flow rates of 0.2 and 2.4 kg/h respectively, with a maximum residence time of 4 s.Industrial relevanceThis investigation demonstrated the lethal efficacy of the application of UV–C light to inactivate microorganisms, both pathogenic and spoilage, present in flour. 4-log10 cycles of inactivation of both Salmonella Typhimurium and Lactobacillus plantarum were inactivated with UV-C treatments. A UV-C facility was built up which enabled to treat flour in continuous conditions creating a cloud of dust with treatments of 4 s and lethalities of 4-log10 reductions.     

Enrichment of bioactive isoflavones in soymilk fermented with β-glucosidase-producing lactic acid bacteria

This study was undertaken to investigate the possible application of β-glucosidase-producing lactic acid bacteria as a functional starter cultures to obtain the bioactive isoflavones, genistein and daidzein, in fermented soymilk. Four strains – Lactobacillus plantarum KFRI 00144, Lactobacillus delbrueckii subsp. lactis KFRI 01181, Bifidobacterium breve K-101 and Bifidobacterium thermophilum KFRI 00748 – among the 31 lactic acid bacteria tested for β-glucosidase activity using ρ-nitrophenyl-β-d-glucopyranoside as the substrate were selected. Acid development, viable populations, and quantification of isoflavones using HPLC were performed at 0, 24, and 48 h of incubation at 37 °C. The significant bioconversion (P < 0.001) of the glucoside isoflavones into their bioactive aglycones in soymilk fermented with four β-glucosidase-producing strains, with an average 7.1-fold increase of aglycones (daidzein + genistein) was observed. There appeared to be correlations between the level of growth and β-glucosidase activity of each strain, and the hydrolysis of conjugated isoflavones in soymilk fermentation. Lactobacillus sp. were able to readily proliferate in soymilk than Bifidobacterium sp. (P < 0.05) and therefore completed more rapidly the hydrolysis of glucoside isoflavones.The present study indicates that four β-glucosidase-producing lactic acid bacteria have great potential for the enrichment of bioactive isoflavones in soymilk fermentation.     

Purification and characterization of the 3-phosphoglycerate kinase from the thermophile Lactobacillus delbrueckii subsp. lactis

The 3-phosphoglycerate kinase (PGK) of the thermophile Lactobacillus delbrueckii subsp. lactis was purified to homogeneity and found to be a monomeric enzyme with a molecular weight of 45 kDa. PGK is a Michaelis–Menten type enzyme with a K_m=0.7 mm for ATP and a K_m=2.6 mm for 3-phosphoglycerate. pH tolerance of PGK was found to be oriented towards acidic conditions with an optimum at pH 6.8 and a plateau of 90% of the total activity between pH 6.0 and 7.4. The optimum temperature for PGK activity is 45°C, which is consistent with the value expected for a thermophilic bacterium with an optimal growth temperature of 45°C.     

Influence of relative humidity on carbon dioxide sorption in wheat gluten films

The influence of relative humidity (RH) on carbon dioxide sorption at 10⁵ Pa in wheat gluten films (WGF) was investigated in the range of 0–96% RH at 25 °C. The amount of water sorbed by these protein based films reached up to 60% of the dry weight at 96% RH. Carbon dioxide sorption increased with water content, ranging from 2.8 × 10⁻⁴ to 1.9 × 10⁻² mol Pa⁻¹ m⁻³ respectively at 0% and 96% RH. This behavior was tentatively explained on the grounds that sorbed water enhanced carbon dioxide accessibility to protein active sorption sites. The dependence of both the solubility and the diffusivity coefficients of CO₂ and O₂ on WGF water content explains the very good permselectivity of these films observed at high RH values (22 at 93% RH and 25 °C).     

Effect of high pressure homogenization on lactic acid bacteria phages and probiotic bacteria phages

To investigate the effect of high pressure homogenization on virus inactivation, phages specific for Lactobacillus delbrueckii, Lactobacillus helveticus, Streptococcus thermophilus, Lactococcus lactis, Lactobacillus paracasei and Lactobacillus plantarum were studied. The influence of pressure, number of passes, suspension medium and phage concentration were studied at 25 °C. Reductions in viability were proportional to pressure and number of passes, though the inactivation extent was phage-dependent. At 100 MPa, some bacteriophages were completely inactivated (6 log₁₀ reduction) after 3 or 5 passes, while others remained infective after 8 passes. For all phages, treatment at 60 MPa was insufficient for complete inactivation, even after 8 passes. No clear influence of suspending medium was observed. Inactivation seems to depend on phage concentration; the higher the initial load, the bigger the reduction achieved. Although these results showed that several phages studied are resistant to high-pressure homogenization, this strategy could be combined with others to control their presence in raw milk.