Discrimination of dairy industry isolates of the Lactobacillus casei group

Lactobacilli are a major part of the microflora of the gut and of many fermented dairy products, and are found in a variety of environments. Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus zeae form a closely related taxonomic group within the facultatively heterofermentative lactobacilli. The classification and nomenclature of these bacteria are controversial. In this study, relationships between these species were investigated using type strains and dairy industry isolates examined with DNA-based techniques and conventional carbohydrate use tests. Carbohydrate use patterns gave poor discrimination of some species, but DNA PCR using specific primers targeted to sequences of the 16S rRNA gene discriminated 4 types consistent with the currently recognized species. Pulsed-field agarose gel electrophoresis of chromosomal NotI restriction fragments identified 18 different band patterns from 21 independent Lactobacillus isolates and confirmed the identity of L. casei strains from 2 culture collections (CSCC 5203 and ASCC 290), both representing the type strain of L. casei. Some isolates were reclassified as L. rhamnosus, suggesting that the prevalence of L. rhamnosus as a natural component of the microflora of dairy foods and dairy environments has previously been underestimated. These methods can provide a practical basis for discrimination of the species and identification of individual industrial strains.     

Technological and probiotic role of adjunct cultures of non-starter lactobacilli in soft cheeses

The influence of two cheese-isolated Lactobacillus strains on cheese composition, acceptability and probiotic capacity was assessed. Soft cheeses with and without the addition of Lactobacillus plantarum I91 or Lactobacillus paracasei I90 were prepared. Gross composition was assessed and secondary proteolysis was described by soluble fractions and free amino acids profiles. Acceptability was determined by a panel of 98 non-trained consumers. Cheeses harboring added Lactobacillus strains were also studied in vivo to evaluate their probiotic capacity. Gross composition of the cheeses was similar for control and treated (Lactobacillus-added) cheeses. Peptidolysis increased in cheeses with added lactobacilli, which was evidenced by a higher free amino acid content. Overall, the acceptability of the cheeses was good: 65%–80% of the consumers said that they “liked very much” or “liked” the cheeses. Cheeses with L. plantarum I91 showed the highest changes in composition and proteolysis and were the most accepted ones. On the contrary, composition of cheeses with L. paracasei I90 was similar to that of the controls, but these samples were less accepted than cheeses without lactobacilli. The oral administration of cheese containing L. plantarum I91 or L. paracasei I90 proved to be safe and able to enhance the number of IgA + cells in the small intestine lamina propria of mice. The use of selected strains of NSLAB exerted a technological and probiotic role: it contributed to the standardization of cheese quality and induced benefic health effects at the gut mucosa in vivo.     

Probiotic potential of Lactobacillus strains with anti-allergic effects from kimchi for yogurt starters

Lactobacillus strains were isolated from kimchi and investigated for their potential use for probiotic yogurt starters. Two of the isolated strains have over 90% survival rate to artificial gastric acid (pH 2.5, 1% pepsin) and artificial bile acid (0.3% oxgall). These strains were identified as Lactobacillus plantarum by 16S rRNA sequencing, and named as L. plantarum SY11 and L. plantarum SY12. The known carcinogenic enzyme, β-glucuronidase was not produced by L. plantarum SY11 and L. plantarum SY12. These 2 strains were found to be resistant to several antibiotics and strongly adhered to intestinal cells. Antiallergic effect of L. plantarum SY11 and L. plantarum SY12 was demonstrated using nitric oxide (NO) and cytokine production. L. plantarum SY11 and L. plantarum SY12 were capable of significantly decreasing NO production, and reduced T helper 2-associated cytokines, cyclooxygenase-2, tumor necrosis factor-α, and inducible nitric oxide synthase compared to control. Yogurt samples produced using L. plantarum SY11 and L. plantarum SY12 did not show significant differences in microbiological, physicochemical, and sensory properties compared with yogurt sample produced using commercial strain. Therefore, these two strains could be used as probiotic yogurt starters with antiallergic effects.     

Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.     

Genotypic diversity of stress response in Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus

Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum are three closely related species which are widespread in food and non-food environments, and are important as starter bacteria or probiotics. In order to evaluate the phenotypic diversity of stress tolerance in the L. plantarum group and the ability to mount an adaptive heat shock response, the survival of exponential and stationary phase and of heat adapted exponential phase cells of six L. plantarum subsp. plantarum, one L. plantarum subsp. argentoratensis, one L. pentosus and two L. paraplantarum strains selected in a previous work upon exposure to oxidative, heat, detergent, starvation and acid stresses was compared to that of the L. plantarum WCFS1 strain. Furthermore, to evaluate the genotypic diversity in stress response genes, ten genes (encoding for chaperones DnaK, GroES and GroEL, regulators CtsR, HrcA and CcpA, ATPases/proteases ClpL, ClpP, ClpX and protease FtsH) were amplified using primers derived from the WCFS1 genome sequence and submitted to restriction with one or two endonucleases. The results were compared by univariate and multivariate statistical methods. In addition, the amplicons for hrcA and ctsR were sequenced and compared by multiple sequence alignment and polymorphism analysis. Although there was evidence of a generalized stress response in the stationary phase, with increase of oxidative, heat, and, to a lesser extent, starvation stress tolerance, and for adaptive heat stress response, with increased tolerance to heat, acid and detergent, different growth phases and adaptation patterns were found. Principal component analysis showed that while heat, acid and detergent stresses respond similarly to growth phase and adaptation, tolerance to oxidative and starvation stresses implies completely unrelated mechanisms. A dendrogram obtained using the data from multilocus restriction typing (MLRT) of stress response genes clearly separated two groups of L. plantarum strains from the other species but there was no correlation between genotypic grouping and grouping obtained on the basis of the stress response pattern, nor with the phylograms obtained from hrcA and ctsR sequences. Differences in sequence in L. plantarum strains were mostly due to single nucleotide polymorphisms with a high frequency of synonymous nucleotide changes and, while hrcA was characterized by an excess of low frequency polymorphism, very low diversity was found in ctsR sequences. Sequence alignment of hrcA allowed a correct discrimination of the strains at the species level, thus confirming the relevance of stress response genes for taxonomy.     

Study of the inhibitory activity of phenolic compounds found in olive products and their degradation by Lactobacillus plantarum strains

Lactobacillus plantarum is the main species responsible for the spontaneous fermentation of Spanish-style green olives. Olives and virgin oil provide a rich source of phenolic compounds. This study was designed to evaluate inhibitory growth activities of nine olive phenolic compounds against four L. plantarum strains isolated from different sources, and to explore the L. plantarum metabolic activities against these phenolic compounds. None of the nine compounds assayed (oleuropein, hydroxytyrosol, tyrosol, as well as vanillic, p-hydroxybenzoic, sinapic, syringic, protocatechuic and cinnamic acids) inhibited L. plantarum growth at the concentration found in olive products. Oleuropein and tyrosol concentrations higher than 100 mM were needed to inhibit L. plantarum growth. On the other hand, sinapic and syringic acid showed the highest inhibitory activity since concentrations ranging from 12.5 to 50 mM inhibited L. plantarum growth in all the strains analyzed. Among the nine compounds assayed, only oleuropein and protocatechuic acid were metabolized by L. plantarum strains grown in the presence of these compounds. Oleuropein was metabolized mainly to hydroxytyrosol, while protocatechuic acid was decarboxylated to catechol. Metabolism of oleuropein was carried out by inducible enzymes since a cell-free extract from a culture grown in the absence of oleuropein was unable to metabolize it. Independent of their isolation source, the four L. plantarum strains analysed showed similar behaviour in relation to the inhibitory activity of phenolic compounds, as well as their ability to metabolize these compounds.     

Production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis) with bacteriocinogenic strains of Lactobacillus plantarum and Lactobacillus curvatus

Lactobacillus plantarum 423, producer of bacteriocin 423, Lactobacillus curvatus DF38, producer of curvacin DF38, and a bacteriocin-negative mutant of L. plantarum 423 (423m) were evaluated as starter cultures in the production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis). Growth of L. plantarum 423 and L. curvatus DF38 was best supported in Blesbok salami, as revealed by the highest growth rate during sweating, cold smoking and maturation, and final cell numbers after 70 days (1 × 10⁸ and 5 × 10⁷ cfu/g, respectively). Growth of Listeria innocua was the best suppressed in Blesbok salami fermented with L. plantarum 423 and L. curvatus DF38. Growth of L. innocua in horse salami was best suppressed when fermented with L. curvatus DF38. The final pH of salami fermented with L. plantarum 423 and L. plantarum 423m was slightly lower (4.4) compared to the pH of salami produced with L. curvatus DF38 (pH 4.7). No significant differences (P > 0.05) were recorded by a trained sensory taste panel amongst the three starter cultures regarding colour and venison like aroma. Horse, Blesbok and Springbok salami were rated significantly higher (P ? 0.05) in salami flavour than mutton salami, which was rated the lowest for this attribute. Blesbok salami was rated the highest for sour meat aroma, while beef salami was rated the lowest. Springbok salami was rated the highest in terms of oily mouth feel. Beef salami had the most compact structure and horse salami the softest structure of all meat types fermented. In general, salami produced with L. plantarum 423 yielded the best sour meat aroma, colour, texture, venison like flavour, sour meat flavour and oily mouthfeel and is considered superior to the L. plantarum mutant (strain 423m) and L. curvatus DF38.     

Identification of lactic acid bacteria involved in the spoilage of pasteurized “foie gras” products

The spoiling microflora of a re-packaged French “foie gras” product was studied. A total of 54 isolates, originating from two different factories, were identified using phenotypical and molecular methods (partial 16S rDNA sequencing). Weissella viridescens was the main species detected in the products from factory 1 (64% of the isolates). These products had a low lactic acid concentration and were considered as non-spoiled. The microflora of factory 2 was dominated mainly by the genus Lactobacillus (95% of the isolates), and the high lactic acid concentration of these products was linked with a strong spoilage. Among the 30 Lactobacillus strains, three species were predominant: Lactobacillus sakei (nine isolates), Lactobacillus coryniformis (eight isolates) and Lactobacillus paraplantarum (five isolates). Challenge tests were performed to confirm the involvement of the Lactobacillus strains in the spoilage of the product. Sterile “foie gras” samples were inoculated with 14 LAB strains from the collection. The most acidifying strains belonged to the species L. sakei, Lactobacillus plantarum and L. paraplantarum. This confirmed the role of the strains from the Lactobacillus genus as the main spoilers of “foie gras” products and will be useful to design new quality protocols and extend the shelf-life of these products.     

Molecular Cloning and Sequence Analysis of Bile Salt Hydrolase Gene (bsh) from Lactobacillus plantarum MBUL90 Strain of Human Origin

This study provided genetic information on a bile salt hydrolase (bsh) of a Lactobacillus plantarum strain of Indian origin, MBUL90. L. plantarum strains were screened by PCR for the determination of the bsh locus in their genome using specific primers. None of the lactobacilli strains produced the expected size of amplicon (～ 1.0 kb) except L. plantarum strains, which proved the specificity of the primers. The bsh amplicon of L. plantarum MBUL90 was cloned into pDrive vector, and nucleotide sequences were determined. Sequence analysis of bsh genes revealed a high level of similarity within the species of L. plantarum as well as with other species of Lactobacillus. The resulting nucleotide sequence of an ORF of 975 bp encoded a predicted protein of 324 amino acids representing a theoretical molecular mass of 37 kDa with a pI of 4.92. The protein deduced from the complete ORF had high similarity with other Bsh proteins, and four highly conserved amino acid motifs (YFGRNXD, NEXGLXXAGLNF, VXVLTNNPXF, and SXSRFVRXAF) were located around the active site. Genetic data presented in this paper provide a sound foundation for better understanding the genetic diversity of bsh in Lactobacillus genus and may provide a new genetic marker for phylogenetic study.     

Detection of changes in the cellular composition of Salmonella enterica serovar Typhimurium in the presence of antimicrobial compound(s) of Lactobacillus strains using Fourier transform infrared spectroscopy

It was previously established that Lactobacillus fermentum ACA-DC 179, Lactobacillus plantarum ACA-DC 287 and Lactobacillus plantarum ACA-DC 2350 exhibit antimicrobial activity against Salmonella enterica serovar Typhimurium. In order to further investigate the killing effect of these microorganisms against Salmonella cells, we employed Fourier transform infrared spectroscopy (FT-IR). Salmonella cells were incubated with different concentrated lactobacilli supernatants and their FT-IR spectra were recorded. The second derivative transformation of the original spectra revealed changes in spectral regions corresponding to absorptions of major cellular constituents (e.g. cell wall, cell membrane, and proteins of the cell) among the Salmonella cells treated with the supernatants and those treated with the control samples. Principal component analysis of the second derivative transformed spectra showed that the yet unidentified antimicrobial compound(s) produced by the lactobacilli tested clearly interfered with the fatty acids of the cell membrane, as well as the polysaccharides of the cell wall in Salmonella cells, pointing towards a dual killing mode. Our study shed light for the first time in the anti-Salmonella activity of the particular Lactobacillus strains.     

Biochemical,antimicrobial and molecular characterization of a noncytotoxic bacteriocin produced by Lactobacillus plantarum ST71KS

Lactobacillus (Lb.) plantarum ST71KS was isolated from homemade goat feta cheese and identified using biochemical and molecular biology techniques. As shown by Tricine-SDS-PAGE, this lactic acid bacterium produces a bacteriocin (ST71KS) with an estimated molecular weight of 5.0 kDa. Bacteriocin ST71KS was not affected by the presence of α-amylase, catalase and remained stable in a wide range of pH and after treatment with Triton X-100, Triton X-114, Tween 20, Tween 80, NaCl, SDS, urea and EDTA. This bacteriocin also remained active after being heated at 100 °C for 2 h and even after 20 min at 121 °C; however, it was inactivated by proteolitic enzymes. Production of bacteriocin ST71KS reached 6400 AU/mL during stationary growth phase of Lb. plantarum cultivated in MRS at 30 °C and 37 °C. Bacteriocin ST71KS displayed a bactericidal effect against Listeria monocytogenes strains 603 and 607 and did not adsorb to the producer cells. Lb. plantarum ST71KS harbors two bacteriocin genes with homology to plantaricin S and pediocin PA-1. These characteristics indicate that bacteriocin ST71KS is a class IIa bacteriocin. The peptide presented no toxic effect when tested in vitro with kidney Vero cells, indicating safe technological application to control L. monocytogenes in foods.     

Effect of exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii subsp. bulgaricus strains to bacteriophage and nisin sensitivity of the bacteria

Lactobacillus strains used in this study were isolated from village-type yogurt and raw milk. The isolates were identified as Lactobacillus delbrueckii subsp. bulgaricus by 16 s rDNA sequence analysis and API 50 CHL identification systems. The exopolysaccharide (EPS) production of the strains growth in skim milk were investigated. In addition sensitivity and insensitivity of these strains against domestic bacteriophages and nisin were examined. It was deduced that those strains which had relatively high EPS-producing capacity were insensitive against phages and nisin. Linear relationships were determined between EPS production of the bacteria and bacteriophage and nisin insensitivity of the bacteria.There was a negative correlation between EPS production quantity and phage and nisin sensitivity of the bacteria. Of all the strains, L. delbrueckii subsp bulgaricus B3 produced the highest EPS quantity, and it was insensitive against phages and nisin. Based on these results, it is suggested that L. delbrueckii subsp bulgaricus B3 can be used with the starter culture in dairy industry for stable and high-quality yogurt production.     

Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses

Ninety-eight Lactobacillus plantarum strains isolated from Italian and Argentinean cheeses were evaluated for probiotic potential. After a preliminary subtractive screening based on the presence of msa and bsh genes, 27 strains were characterized. In general, the selected strains showed high resistance to lysozyme, good adaptation to simulated gastric juice, and a moderate to low bile tolerance. The capacity to agglutinate yeast cells in a mannose-specific manner, as well as the cell surface hydrophobicity was found to be variable among strains. Very high β-galactosidase activity was shown by a considerable number of the tested strains, whereas variable prebiotic utilization ability was observed. Only tetracycline resistance was observed in two highly resistant strains which harbored the tetM gene, whereas none of the strains showed β-glucuronidase activity or was capable of inhibiting pathogens. Three strains (Lp790, Lp813, and Lp998) were tested by in vivo trials. A considerable heterogeneity was found among a number of L. plantarum strains screened in this study, leading to the design of multiple cultures to cooperatively link strains showing the widest range of useful traits. Among the selected strains, Lp790, Lp813, and Lp998 showed the best probiotic potential and would be promising candidates for inclusion as starter cultures for the manufacture of probiotic fermented foods.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

Coculture with specific bacteria enhances survival of Lactobacillus plantarum NC8, an autoinducer-regulated bacteriocin producer,in olive fermentations

Bacteriocin production in Lactobacillus plantarum NC8 is activated by coculture with specific bacteriocin production-inducing bacterial strains. The system is further regulated by a three-component regulatory system involving a specific autoinducer peptide (PLNC8IF). We have used L. plantarum NC8 as a starter culture in Spanish-style green olive fermentations and examined the influence of coculturing in its survival. We found that L. plantarum NC8 greatly enhanced its growth and survival in the olive fermentations when coinoculated with two specific bacteriocin-production inducing strains, i.e. Enterococcus faecium 6T1a-20 and Pediococcus pentosaceus FBB63, when compared to singly-inoculated fermentations. In addition, a constitutive bacteriocin-producer NC8-derivative strain was used as a control in the olive fermentations and showed also better viability than the parental NC8 strain. Our results suggest the involvement of bacteriocin production in the viability enhancement found in both cases. We postulate that the presence of specific bacteria is recognized by L. plantarum NC8 as an environmental stimulus to switch a specific adaptive response on, most probably involving bacteriocin production. The design of novel bacteriocin-producing starter cultures for food fermentations should consider their constitutive versus regulated character.     

Selection of Lactobacillus plantarum strains to use as starters in fermented table olives: Oleuropeinase activity and phage sensitivity

Fermented table olives (Olea europaea L.) are largely diffused in the Mediterranean area. Olives are picked at different stages of maturity and after harvesting, processed to eliminate the characteristic bitterness caused by the presence of the oleuropein glucoside and to become suitable for human consumption. The spontaneous fermentation of table olives mainly depends on lactic acid bacteria (LAB), and in particular on Lactobacillus plantarum which plays an important role in the degradation of oleuropein. The hydrolysis of oleuropein is attributed to the β-glucosidase and esterase activities of the indigenous LAB microflora. This study investigated the potential of L. plantarum strains isolated from dairy products and olives to be used as starters for fermented table olives. Forty-nine strains were typed by RAPD-PCR and investigated for the presence of the β-glucosidase (bglH) gene. The full sequence of the bglH gene was carried out. All the 49 L. plantarum strains were also tested for phage resistance. A total of six strains were selected on the basis of genotypic polymorphism, bglH gene sequence analysis, and phage resistance profile. These strains were further characterized to assess the acidifying capability, the growth at different temperatures, the tolerance to different NaCl concentrations, and the oleuropeinolytic activity. Although further characterizations are required, especially concerning the influence on sensory properties, L. plantarum proved to have the potential to be used as a debittering and fermentative agent in starter culture for fermented table olives.