Identification and characterization of a glucan-producing enzyme from Lactobacillus hilgardii TMW 1.828 involved in granule formation of water kefir

Water kefir is a home made fermented beverage based on a sucrose solution with fruit extracts. The inoculum of such fermentations consists of macroscopic granula containing lactic and acetic acid bacteria, and yeasts, which are embedded in an exopolysaccharide (EPS) matrix. In this work, a strain of Lactobacillus hilgardii producing large amounts of the granule-forming dextran could be isolated. The glycosyltransferans (Gtf) commonly called glucansucrase responsible for the production of this dextran was purified from L. hilgardii. Characteristic enzyme kinetic data were obtained. Optimum activity was observed between pH 4.3 and 4.6 and temperatures between 40 °C and 45 °C. A Michaelis–Menten kinetic could be fit to the experimental data and a K_M of 0.0385 M was calculated. The corresponding gtf gene was identified and characterized. It encodes a 1448 amino acid protein with higher homologies to Gtfs of Lactobacillus parabuchneri, Lactobacillus sakei and Lactobacillus fermentum followed by lower homologies to Lactobacillus reuteri Gtfs. By knockout experiments the role of this gene in granule dextran production was demonstrated.     

Evaluation of culture media for enumeration of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium animalis in the presence of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus

The study compared the growth capability of probiotic (Lactobacillus acidophilus La05, Lactobacillus casei Lc01 and Bifidobacterium animalis Bb12) and non-probiotic (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus) cultures on twenty-one culture media grouped according to selectivity: non-selective agars, selective agars without antibiotics and MRS agars containing different combinations of lithium chloride, cystein, bile salts and antibiotics. Four of these media were selected for quantitative enumeration of L. acidophilus La05, L. casei Lc01, and B. animalis Bb12. The best culture media and incubation conditions for enumeration of the probiotic cultures were: B. animalis: MRS agar with dicloxacillin, 37 °C or 42 °C, anaerobiosis; L. acidophilus: MRS agar with bile salts, 37 °C or 42 °C, aerobiosis; L. casei: MRS agar with lithium chloride and sodium propionate, 37 °C or 42 °C, aerobiosis or anaerobiosis. Plating on MRS with glucose replaced by maltose, 37 °C or 42 °C, anaerobiosis, will distinguish probiotic from non-probiotic cultures. For enumeration of each probiotic in a mixed culture, the following media and incubation conditions were recommended: B. animalis: 4ABC-MRS, 42 °C, anaerobiosis, L. acidophilus: LC medium, 42 °C, aerobiosis or anaerobiosis and L. casei: LP-MRS, 42 °C, aerobiosis or anaerobiosis. In all experiments, differences in counts using pour plating or surface plating were not significant (P ≤ 0.05).     

Survival of Lactobacillus acidophilus and Bifidobacterium animalis ssp. lactis in stirred fruit yogurts

The effect of commercial fruit preparations (mango, mixed berry, passion fruit and strawberry) on the viability of probiotic bacteria, Lactobacillus acidophilus LAFTI^® L10 and Bifidobacterium animalis ssp. lactis LAFTI^® B94 in stirred yogurts during storage (35 days) at refrigerated temperature (4 °C) was evaluated. The results showed that addition of either 5 or 10 g/100 g fruit preparations had no significant (p>0.05) effect on the viability of the two probiotic strains except on L. acidophilus LAFTI L10 yogurt with 10 g/100 g passion fruit or mixed berry. After the addition of fruit preparation, 96% of the yogurts incorporated with fruit preparation did not exhibit a greater loss in the viability of probiotic bacteria compared to plain yogurt during the storage period. A correlation between the post-storage pH in yogurts and the survival of probiotic bacteria was observed. All the yogurts, however, contained the recommended levels of (10⁶-10⁷ cfu/g) probiotic bacteria at the end of 35-day shelf life.     

The substitution of a traditional starter culture in mutton fermented sausages by Lactobacillus acidophilus and Bifidobacterium animalis

Common starter cultures used in fermented mutton sausages were substituted by probiotic strains of Lactobacillus acidophilus CCDM 476 and Bifidobacterium animalis 241a. Technological properties of the traditional and the probiotic sausages were compared. The potential probiotic effect was evaluated by enumeration of bifidobacteria and lactobacilli in stool samples of 15 volunteers before and after a 14-day consumption period. The numbers of lactobacilli (10⁷ cfu/g) and bifidobacteria (10³ cfu/g) in the final product did not affect the technological properties. The use of L. acidophilus as a starter culture was found more beneficial than the use of B. animalis. Even after 60 days of storage, high counts of L. acidophilus (10⁶ cfu/g) were detected; on the other hand, the counts of B. animalis were under the detection limit. Regarding sensory properties, the probiotic products showed better texture, and, curiously, a reduction of the typical smell of mutton. The numbers of lactobacilli in stool samples increased significantly after the consumption of the probiotic sausages.     

Changes in microbiological, biochemical and physico-chemical properties of Nham inoculated with different inoculum levels of Lactobacillus curvatus

The effect of inoculum level of Lactobacillus curvatus on Nham fermentation was studied. Nham inoculated with L. curvatus at 10⁴ (LC104) and 10⁶ cfu/g (LC106) exhibited a higher rate of fermentation than naturally fermented Nham (control) as indicated by greater rate of pH drop, lactic acid production, and changes in protein compositions. Based on the pH, the fermentation was completed within 72, 48 and 36 h for control, LC104, and LC106, respectively. Higher extent of proteolysis and lipolysis were observed during fermentation of Nham inoculated with starter bacteria (P<0.05). Due to higher acid production rate and extent, texture development of inoculated Nham was more rapid. Inoculated Nham exhibited higher TPA hardness and adhesiveness but lower fracturability than naturally fermented Nham (P<0.05). In terms of acceptability, control and LC104 had higher ratings on the overall liking than LC106 (P<0.05). However, LC104 was better accepted than control in terms of flavour, sourness, saltiness, and texture. Unusual smell was detected only in Nham inoculated at 10⁶ cfu/g. Based on physico-chemical properties and consumer acceptability, L. curvatus is a potential starter for Nham fermentation. However, inoculation of L. curvatus at high level may cause off-flavour in the product.     

Protective action of Lactobacillus curvatus CRL705 on vacuum-packaged raw beef. Effect on sensory and structural characteristics

Lactobacillus curvatus CRL705 was examined for its effectiveness as protective culture in the biopreservation of vacuum-packaged fresh beef stored during 60 days at 2 °C. For this purpose, L. curvatus CRL705, producer of lactocin 705 and lactocin AL705, was inoculated on the meat surface (10⁶ cfu g⁻¹). This microorganism became the dominating population throughout the storage period controlling the growth of Brochothrix thermosphacta and spoilage lactic acid bacteria naturally present on the meat. When the microstructural characteristics of the meat were evaluated using light microscopy, beef samples inoculated with the bioprotective culture showed a 10 days delay for the appearance of tissue degradation signs. Sensory analysis demonstrated that beef samples treated with L. curvatus CRL705 only developed an “acid” off-flavor after 60 days of refrigerated storage, and no undesirable off-odors were found. Therefore, inoculation with this bacteriocinogenic strain would provide an additional hurdle to improve storage life of refrigerated vacuum-packaged beef without affecting its sensory and structural characteristics.     

Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains

The biological and technological characteristics of kefiran as well as its importance in grain integrity led us to analyze the microbial kefir grain consortium with focus on Lactobacillus kefiranofaciens. The presence of L. kefiranofaciens in the nine kefir grains studied was demonstrated by denaturing gradient gel electrophoresis. By culture dependent methods applying a methodology focused on the search of this species, 22 isolates with typical morphology were obtained and identified applying a combination of SDS-PAGE of whole cell proteins, (GTG)₅-PCR and sequence analysis of the housekeeping gene encoding the α-subunit of bacterial phenylalanyl-tRNA synthase (pheS). This polyphasic approach allowed the reliable identification of 11 L. kefiranofaciens, 5 Lactobacillus paracasei, 4 Lactobacillus kefiri and 2 Lactobacillus parakefiri isolates. Isolated L. kefiranofaciens strains produced polysaccharide in strain-dependent concentrations and EPS produced by them also differed in the degree of polymerization. The isolation and accurate identification of L. kefiranofaciens is relevant taking into account the important role of this microorganism in the grain ecosystem as well as its potential application as starter in food fermentations.     

Propionic acid production by cofermentation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough

Cooperative metabolism of lactobacilli in silage fermentation converts lactate to propionate. This study aimed to determine whether propionate production by Lactobacillus buchneri and Lactobacillus diolivorans can be applied for bread preservation. Propionate formation was observed in cofermentation with L. buchneri and L. diolivorans in modified MRS broth as well as sourdough with low, medium and high ash contents. 48 mM of propionate was formed in sourdough with medium ash content, but only 9 and 28 mM propionate were formed in sourdoughs prepared from white wheat flour or whole wheat flour, respectively. Acetate levels were comparable in all three sourdoughs and ranged from 160 to 175 mM. Sourdough fermented with L. buchneri and L. diolivorans was used in breadmaking and its effect on fungal spoilage was compared to traditional sourdough or propionate addition to straight doughs. Bread slices were inoculated with Aspergillus clavatus, Cladosporium spp., Mortierella spp. or Penicillium roquefortii. The use of 20% experimental sourdough inhibited growth of three of the four moulds for more than 12 days. The use of 10% experimental sourdough deferred growth of two moulds by one day. Bread from traditional sourdough with added acetate had less effect in inhibiting mould growth.     

Isolation and partial characterization of the surface protein of Lactobacillus acidophilus strains

A surface protein layer was found to be present in some Lactobacillus acidophilus strains. it could be visualized with electron microscopy and extracted with sodium dodecyl sulfate or guanidine hydrochloride. The protein was resistant to several proteolytic enzymes, but sensitive to pronase. Amino acid composition analysis revealed about 30% hydrophobic amino acids. Hydrophobicity study with a hexadecane partition assay demonstrated that the cells were hydrophobic and removal of the surface layer with pronase destroyed the hydrophobic nature of the cells. Several growth conditions studies were found to have little effect on synthesis of the surface protein.     

Isolation and characterization of acid-sensitive Lactobacillus plantarum with application as starter culture for Nham production

The aim of this study was to derive new starter culture variants that are unable to grow below pH 4.6, the desirable pH of the Thai fermented pork sausage, Nham, specified by Thailand Food Standard, and apply them in Nham fermentation. Several acid-sensitive mutants of one of the commercial Nham starter cultures, Lactobacillus plantarum BCC 9546, were isolated as spontaneous neomycin-resistant mutants. The growth of three representative mutants was characterized in MRS broth, which revealed that their cell numbers and acid production were lower than that of the wild-type. The H⁺-ATPase activities of the three mutants were found significantly lower than that of the wild-type under either neutral or acidic conditions. Consequently, internal pH values of the mutants appeared to be lower, especially in acidic environment (pH 5). The most acid-sensitive mutant was applied in experimental Nham production and the pH of Nham fermented with the mutant had significantly higher pH at the end of fermentation (3 days) and after an additional 4 days of storage at 30 °C. These results indicate that the use of acid-sensitive L. plantarum as starter culture can reduce the severity of post-acidification and increase the shelf life of Nham at ambient temperature.     

Identification of seven species of the Lactobacillus acidophilus group by FT-IR spectroscopy

Fourier transform infrared (FT-IR) spectra of 102 strains of the seven species of the Lactobacillus acidophilus group were collected and investigated for their potential use in classification and identification on species level. The database built contains more than 370 spectra. Various procedures of pre-processing and classification methods have been compared with respect to their predictive ability. The most encouraging results were achieved with linear discriminant analysis (LDA) of the absorbance values of normalized spectra at selected wavenumbers. The rate of correct species assignment in cross-validation (Jackknife procedure with one spectrum left out for model building) were 95%, 95%, 69%, 100%, 88%, 100%, and 91% for L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, L. helveticus, and L. johnsonii, respectively. Very distinct grouping was found for L. gallinarum and L. helveticus, the most difficult differentiation in LDA was between the pairs L. crispatus/L. amylovorus and L. gasseri/L. johnsonii.     

Impact of inulin and okara on Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 viability in a fermented soy product and probiotic survival under in vitro simulated gastrointestinal conditions

The effect of inulin and/or okara flour on Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 viability in a fermented soy product (FSP) and on probiotic survival under in vitro simulated gastrointestinal conditions were investigated throughout 28 days of storage at 4 °C. Employing a 2² design, four FSP trials were produced from soymilk fermented with ABT-4 culture (La-5, Bb-12, and Streptococcus thermophilus): FSP (control); FSP-I (with inulin, 3 g/100 mL of soymilk); FSP-O (with okara, 5 g/100 mL); FSP-IO (with inulin + okara, ratio 3:5 g/100 mL). Probiotic viabilities ranged from 8 to 9 log cfu/g during the 28 days of storage, and inulin and/or okara flour did not affect the viability of La-5 and Bb-12. Bb-12 resistance to the artificial gastrointestinal juices was higher than for La-5, since the Bb-12 and La-5 populations decreased approximately 0.6 log cfu/g and 3.8 log cfu/g, respectively, throughout storage period. Even though the protective effect of inulin and/or okara flour on probiotic microorganisms was not significant, when compared to a fresh culture, the FSP matrix improved Bb-12 survival on day 1 of storage and may be considered a good vehicle for Bb-12 and could play an important role in probiotic protection against gastrointestinal juices.     

Quality attributes of yogurt with Lactobacillus casei and various prebiotics

The objective was to determine the effect of chain length of inulins on the characteristics of fat-free plain yogurt manufactured with Lactobacillus casei. Probiotic fat-free plain yogurts were manufactured using Streptococcus thermophilus, Lactobacillus bulgaricus and L. casei. The treatments were inulins of short (P95), medium (GR) and long (HP) chain lengths. The inulins were incorporated at a concentration of 1.5 g/100 g yogurt mix. Inulins of various chain lengths did not affect viscosity, L*, a*, b* and appearance of yogurts manufactured with L. casei. Yogurt with HP had less syneresis compared to the control, while yogurt with P95 had syneresis comparable to the control. Yogurt with P95 had a significantly lower pH than the control, while the pH of the yogurts with other treatments was not different from the control. Flavor scores of the control were comparable to yogurt with P95. The flavor scores for yogurts with P95 were significantly higher than for yogurts with HP. The yogurts with HP had better body and texture compared to the control and P95. Chain length of prebiotics affected some characteristics of the yogurts.     

The application of flow cytometry to the characterisation of a probiotic strain Lactobacillus reuteri DPC16 and the evaluation of sugar preservatives for its lyophilization

In this study, we applied flow cytometric (FCM) analyses to characterize the resistance of a probiotic strain Lactobacillus reuteri DPC16 (DPC16) against diverse stresses. Two fluorescent probes, propidium iodide (PI) and carboxylfluorescein diacetate (cFDA), were combined to the FCM method to reveal multiple cellular statuses of DPC16. The FCM results confirmed that the DPC16 strain had probiotic potential in respect of acid tolerance and bile acid resistance, whereby more than 60% of DPC16 bacteria remained intact after 1 h exposure to pH 2.0, and over half of DPC16 bacteria were unaffected by the presence of bile salts at a concentration of 0.2 g/100 mL for 1 h even without nutrient supply. In addition, the comparison among a number of lyo-preservatives for the DPC16 strain confirmed that lactose was able to maintain over 60% viable DPC16 bacteria after the lyophilization and subsequent storage period, outperforming all the other selected sugars. To conclude, the superior stress resistance of the novel DPC16 strain was confirmed by the FCM analyses in this study. The FCM technique also proved to be readily incorporated into probiotics research, and capable of providing insightful information.     

The effect of probiotics (Lactobacillus rhamnosus HN001, Lactobacillus paracasei LPC-37, and Lactobacillus acidophilus NCFM) on the availability of minerals from Dutch-type cheese

The use of probiotic cultures in the production of Dutch-type cheeses did not lead to significant changes in their chemical composition but it lowered their acidity. The availability of calcium and magnesium analyzed by in vitro enzymatic hydrolysis was 19 and 35%, respectively; the availability of phosphorus was significantly higher, at >90%. The use of probiotic cultures significantly increased the availability of calcium (~2.5%), phosphorus (~6%), and magnesium (~18%). The in vitro method supports accurate determination of the effect of the Lactobacillus spp. cultures on the availability of mineral compounds ingested with Dutch-type cheese.     

Acid tolerant mutants of Bifidobacterium animalis subsp. lactis with improved stability in fruit juice

The potential of UV mutagenesis combined with a specific selection step to generate more acid-resistant Bifidobacterium animalis subsp. lactis Bb-12 strains with improved viability in low pH food matrices was investigated in this study. A total of 144 Bb-12 UV-mutants were initially characterised. After prolonged storage in apple juice (pH 3.5) and various pheno- and genotypic tests (acid and bile tolerance, substrate utilisation, antibiotic susceptibility, aerotolerance, RAPD) two mutants (2.20 and 2.56) were chosen for further studies including cell surface morphology, stability of various traits after repeated inoculations, and performance in a fermenter and during down-stream processing. 2.20 and 2.56 showed over two Log-values better viability in pH 3.5 juice compared to Bb-12. Alterations in cell surface structures of 2.20 and 2.56 were detected with AFM, whereas other studied traits remained unchanged.In conclusion, UV mutagenesis and subsequent incubation in acidic medium enabled improving the stability of B. animalis subsp. lactis in low pH juice. Acid tolerance testing (HCl, pH 2.5, 2 h) results did not predict long-term stability of the strains in acidic food matrix (apple juice pH 3.5).