Investigating hydrophobicity and the effect of exopolysaccharide on aggregation properties of dairy propionibacteria isolated from Turkish homemade cheeses

Propionic acid bacteria have been used widely as starter cultures. However, their potential as probiotics has received little attention. The ability to auto- and coaggregate is a desirable property for probiotics in health-promoting foods. Therefore, in the current study, we assessed the effect of exopolysaccharides produced by dairy propionibacteria strains on the aggregative and hydrophobicity properties. All propionibacteria strains tested showed auto- and coaggregation ability with Escherichia coli ATCC 11229, but the results were strain specific and dependent on exopolysaccharides production and incubation conditions. In addition, propionibacteria strains tested were determined to be highly hydrophilic. Our results indicate that the ability to autoaggregate, together with cell-surface hydrophobicity and coaggregation abilities with an E. coli strain, can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human or animal use.     

Research on some factors influencing acid and exopolysaccharide produced by dairy propionibacterium strains isolated from traditional homemade Turkish cheeses

In this study, a total of 32 isolated strains and 5 reference strains of dairy propionibacteria were analyzed for acid and exopolysaccharide (EPS) production in skim milk and yeast extract-lactate broth (YEL) media in order to investigate the physiological background and preservative role of acid and EPS. The effects of final culture pH and optical density on acid and EPS production were also determined. On average, all strains produced more acid and reached lower final pH values in skim milk than in YEL medium. While the correlations obtained between the acid produced by propionibacterium strains and their final culture pH in skim milk medium were significant (P < 0.01), no correlations were found between optical density, final pH, and produced acid in YEL medium. Sixteen isolated and five reference strains of propionibacteria were tested further for the ability to produce propionic and acetic acids. On average, Propionibacterium freudenreichii subsp. shermanii and P. freudenreichii subsp. freudenreichii strains produced higher amounts of propionic and acetic acids than did Propionibacterium jensenii in YEL medium. The acid produced by these strains may be used as a preservative in the food industry for replacement or reduction of the increasing use of chemical additives. The EPS production by propionibacterium strains during growth in YEL medium was 72 to 168 mg/liter, while in skim milk it was 94 to 359 mg/liter. The monomer compositions of the EPSs formed by the six selected dairy propionibacteria strains were analyzed. The EPSs may have applications as food grade additives and viscosity-stabilizing agents.     

Characterization of enterococci isolated from homemade Bulgarian cheeses and katuk

A collection of 107 lactic acid bacteria (LAB) isolates was obtained from traditional Bulgarian dairy products??homemade cheeses and katuk samples, produced from heat-treated cow, goat, ewe and buffalo milk without the addition of any bacterial starter culture. The samples were collected from mountain region of Rhodope (south part of Bulgaria), Tracian valley and mountain region of Stara Planina (west part of Bulgaria). These LAB produced bacteriocin-like inhibitory substances (BLIS) and proteinases. Preliminary strain determination was performed according to their fermentation ability using API 50CHL and API 20 Strep. Most of the characterized strains (58) belong to genus Enterococcus; 21, 20 and 11 strains were identified as Lactobacillus sp., Streptococcus sp. and Lactococcus sp., respectively. Isolated enterococcal strains were characterized using phenotypic features as well as by DNA typing. The strains were identified as Enterococcus faecium (34), Enterococcus durans (22) and Enterococcus faecalis (2). The proteolytic activity varied from 0.094 to 0.455?mM/L Gly into the group of E. faecium and from 0.109 to 0.487?mM/L Gly into the group of E. durans strains, while both E. faecalis strains showed relatively high proteolytic activity. The samples obtained after 3?h of hydrolysis of ??-casein by E. faecalis B1 strain were further used for mass spectrometry analysis, and 31 peptides were identified.     

乳酸杆菌的表面特性及其黏附能力的研究

乳酸杆菌是肠道益生菌,主要通过其表面的黏附因子定殖于肠道而发挥益生作用。为了研究乳酸杆菌表面性质与黏附能力之间的关系,选择五种乳酸杆菌,分别进行自聚集能力、表面疏水性及与致病菌大肠杆菌ATCC25922和枯草芽孢杆菌K的共聚能力测定。同时,利用氯化锂和高碘酸钠分别处理乳酸杆菌后再与致病菌进行共聚作用,研究乳酸杆菌表面参与黏附的活性物质。结果表明,约氏乳酸杆菌F0421和副干酪乳酸杆菌M5-L具有良好的表面性质和黏附特性。乳酸杆菌与枯草芽孢杆菌K的共聚作用较好。同时,氯化锂和高碘酸钠处理前后,乳酸杆菌对致病菌的聚集能力有所下降,表明菌株表面蛋白及多糖参与了黏附过程。      

Surface properties of Lactobacillus and Leuconostoc isolates from homemade cheeses showing auto-aggregation ability

Ten lactobacilli and one leuconostoc showing auto-aggregation ability were isolated from artisanal cheeses. Furthermore, non-aggregation strains were isolated from the same cheese sample, if existed. The analysis of factor(s) possibly involved in auto-aggregation was performed. The pretreatment of cells with proteinase K resulted in the disappearance of auto-aggregation ability. Moreover, cells also lost aggregation ability after three-times, successive washing in distilled water. Testing the ability of strain Lactobacillus paracasei subsp. paracasei BGSJ2-8 and its aggregation-deficient derivative BGSJ2-81 to co-aggregate with Listeria innocua ATCC33090, Escherichia coli ATCC25922 or Salmonella typhimurium TR251 showed that strain BGSJ2-8 co-aggregated with these strains, but derivative BGSJ2-81 was not. However, the treatment of L. paracasei subsp. paracasei BGSJ2-8 with proteinase K prior to co-aggregation tests resulted in losing co-aggregation ability. Surface properties of selected strains were analyzed by MATS (microbial adhesion to solvents) method. It was noticed that the strains with auto-aggregation ability were highly hydrophobic in comparison with aggregation-deficient ones. Comparative analyses of the surface features of strain L. paracasei subsp. paracasei BGSJ2-8 and its derivative BGSJ2-81 revealed notable difference.     

Inhibition of yeasts isolated from traditional Turkish cheeses by Lactobacillus spp.

Antifungal activity of some Lactobacillus strains was determined. Yeasts were isolated and identified as Saccharomyces cerevisiae (10 of 17) and one each of Candida pseudotropicalis, C. krusei, C. lipolytica, C. lusitaniae, C. ciferrii, Torulopsis glabrata and Rhodotorula rubra . Lactobacillus strains were found to have fairly strong antifungal activity against S. cerevisiae . Of the 19 bacteria tested, L. plantarum Lp 21 seemed to have the most inhibitory effect against all of the S. cerevisiae strains except for S. cerevisiae 13. We suggest that L. plantarum Lp 21 can be used with starter cultures for cheese 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.     

A polyphasic approach to highlight genotypic and phenotypic diversities of Lactobacillus helveticus strains isolated from dairy starter cultures and cheeses

In the present work, 67 strains of Lactobacillus helveticus isolated from whey starter cultures and cheeses were identified and grouped by genotypic and phenotypic methods. Strains were identified by sugar fermentation pattern, by cell-wall protein profile, and by probe hybridisation. Phenotypic diversity was evaluated by a chemometric model taking into account biochemical characteristics (i.e. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by RAPD-PCR, which provided stran-specific patterns and revealed the occurrence of different strains. The RAPD-PCR profiles were clustered according to their similarities: the groups obtained, together with the cell-wall protein profiling and the chemometric information, could be sometimes correlated with the type of cheese and/or dairy niches used as sources of strains. A computerised analysis of genotypic and phenotypic information could be successfully applied for rapid and reliable differentiation and characterisation of Lb. helveticus isolates occurring in different dairy products.     

Effects of propionibacteria and yeast culture fed to steers on nutrient intake and site and extent of digestion

The effects of feeding Propionibacterium strain P169 (P169), yeast culture (XPY), and their combination on nutrient intake, site and extent of digestion, and ruminal kinetics were evaluated in a completely randomized experimental design. Ruminally and duodenally cannulated Angus × Hereford steers (n = 12) were assigned to 1 of 4 treatments in each of 2 periods: 1) control, fed a sorghum silage-based total mixed ration; 2) P169, fed the control plus P169 (6 × 10¹¹ cfu/steer per d); 3) XPY, fed the control plus XPY (56 g/steer per d); and 4) P169 + XPY, fed the control plus P169 and XPY (at 6 × 10¹¹ cfu/steer per d and 56 g/steer per d, respectively). Each period lasted 21 d; d 1 to 15 were used for diet adaptation and d 16 to 21 were used for fecal, duodenal, ruminal, and blood sample collection. Steers were individually housed and fed. Feeding XPY tended to decrease intake of organic matter, acid detergent fiber, and N, and decreased intake of neutral detergent fiber. However, feeding XPY alone tended to increase total tract digestibility of organic matter, N, neutral detergent fiber, and acid detergent fiber. Ruminal digestibility, duodenal flow, microbial N synthesis, microbial efficiency, and fluid and particulate passage rates were not affected by dietary treatments. Feeding P169 tended to decrease molar proportion of acetate, increased molar proportion of propionate (by 9.7%), and tended to decrease acetate:propionate ratio compared with control steers. No other effects of XPY or P169 on ruminal fermentation were observed. Plasma glucose and insulin concentrations were not affected by dietary treatment. Our results suggest that feeding P169 alters ruminal metabolism toward increased propionate without affecting feed intake or ruminal kinetics, whereas feeding XPY alone tended to increase total tract digestibilities of nutrients.     

植物乳杆菌表面性质及对Caco-2细胞的黏附

通过对10 株不同来源的植物乳杆菌进行自聚集能力、表面疏水性以及体外黏附Caco-2细胞能力的测定,探究植物乳杆菌表面性质与黏附能力之间的关系,利用LiCl对植物乳杆菌进行处理,探究参与植物乳杆菌对细胞黏附过程的物质。结果表明：植物乳杆菌3-4对Caco-2细胞的黏附能力最强。所选的不同植物乳杆菌之间自聚集能力、表面疏水性以及对Caco-2细胞的黏附能力存在差异性;对细胞的黏附能力与表面疏水性存在着显著的相关性（P＜0.05）,因此,自聚集能力和疏水性可以作为筛选具有高黏附细胞能力的植物乳杆菌的参考指标。同时LiCl处理前后,植物乳杆菌自聚集能力和对细胞的黏附能力均有所下降,表明菌株表面蛋白类物质及其他大分子物质均参与自聚集和黏附过程。     

Probiotic potential of Lactobacillus strains isolated from dairy products

Twenty-nine Lactobacillus strains of dairy origin were examined in vitro for their probiotic potential. Only a few strains were able to survive at pH 1 or in the presence of pepsin, while all were unaffected by pH 3, pancreatin and bile salts. Strains exhibited variable bile salt hydrolase activity. None was haemolytic. The majority of strains were resistant to vancomycin and teicoplanin, but sensitive to chloramphenicol and tetracycline. A few strains were able to adhere to Caco-2 cells. Although no bacteriocin activity was detected in vitro, strains L. casei Shirota ACA-DC 6002, L. plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were able to inhibit the adhesion of Escherichia coli and Salmonella typhimurium to Caco-2 cells. They also induced the secretion of pro- and anti-inflammatory cytokines by human peripheral blood mononuclear cells. These three strains were therefore found, in vitro, to possess desirable probiotic properties.     

Antibiotic susceptibility patterns of Enterococcus strains isolated from Turkish Tulum cheese

The aim of this study was to detect the plasmid profiles, haemolytic activity and antibiotic susceptibility patterns of 47 Enterococcus strains isolated from Turkish Tulum cheese. Enterococcus strains were found to comprise 1–9 plasmids with molecular weight from 2.0 to 47.6 kb. None of the Enterococcus strains displayed haemolytic activity. The Enterococcus strains were found mostly resistant to tetracycline (30 μg) followed by high‐level streptomycin (300 μg). The Enterococcus faecalis strains were found to be highly resistant to antibiotics than Enterococcus faecium and Enterococcus durans strains. In total, 4.3% of the strains exhibited multiple antibiotic resistance patterns.     

Characterization of Lactobacillus helveticus strains isolated from cheeses by distribution studies of insertion sequences

A collection of 38 Lactobacillus helveticus strains, isolated from a number of different artisan Italian cheeses, and 4 reference strains were studied with respect to the presence of insertion sequences and their distribution and abundance. The mobile genetic element ISLh1, that contains one open reading frame coding for a putative transposase of the IS982 family, was used for DNA fingerprinting, together with IS1201 and ISL2, previously isolated from L. helveticus. The number of insertion sequences per strain and the size of DNA restriction fragments containing them, was variable and allowed the discrimination at the strain-level. The genomic distribution of the three unrelated insertion sequences showed significant correlations and allowed the differentiation of the strains also with regard to the specific ecological niche of origin of the isolates. Consequently, insertion sequences comparison may be useful in determining the history of a group of strains known to be related because of identity and offers a further parameter for evaluating the population polymorphism in L. helveticus.     

Determination of biofilm production by Candida tropicalis isolated from hospitalized patients and its relation to cellular surface hydrophobicity,plastic adherence and filamentation ability

Candida tropicalis is an emerging virulent species. The aim of this study is to determine the biofilm‐forming ability of 29 strains of C. tropicalis isolated from inpatients, and to examine its relation with other virulence factors such as cellular surface hydrophobicity (CSH), immediate (15 min, IA) and late (24 h, LA) plastic adherence and filamentation ability. The study was performed in parallel using two incubation temperatures – 37 and 22 °C – to determine the effect of growth temperature variations on these pathogenic attributes of C. tropicalis. Biofilm formation (BF) was measured by optical density (OD) and by XTT reduction (XTT); Slime index (SI), which includes growth as a correction factor in BF, was calculated in both methods. All strains were hydrophobic and adherent – at 15 min and 24 h – at both temperatures, with higher values for 22 °C; the adhered basal yeast layer appears to be necessary to achieve subsequent development of biofilm. Filamentation ability varied from 76.2% of strains at 37 °C to 26.6% at 22 °C. All C. tropicalis strains were biofilm producers, with similar results obtained using OD determination and XTT measurement to evaluation methods; SI is useful when good growth is not presented. BF at 37 °C was similar at 24 h and 96 h incubation; conversely, at 22 °C, the highest number of biofilm‐producing strains was detected at 96 h. CSH is an important pathogenic factor which is involved in adherence, is influenced by the filamentation of yeast, and plays a critical role in BF. Copyright © 2013 John Wiley & Sons, Ltd.     

Safety assessment of dairy microorganisms: aerobic coryneform bacteria isolated from the surface of smear-ripened cheeses

The group of "coryneform bacteria" belongs to the class of Actinobacteria including a diverse and heterogeneous collection of bacteria of various genera. Most of them are known as environmental residents and/or commensal flora of humans and they are isolated frequently in clinical studies. Actinobacteria include also several aerobic species, present at the surface of smear-ripened cheeses for decades and used as ripening culture in the dairy industry. Their clinical significance is controversial because an easy combination of phenotypic and molecular methods to characterize Actinobacteria at the species level is still lacking. A bibliographical survey was conducted to assess the safety status of Actinobacteria species used as starter culture in fermented dairy foods, according to their technological interest. Aerobic coryneform bacteria isolated from smear-ripened cheeses are most commonly recovered from soil, the environment or food. To date, no clinical infection or food toxi-infection related to smear cheese coryneform bacteria ingestion has been reported. From a taxonomic viewpoint, dairy species are distant from the reference species associated with known pathologies. From a physiological viewpoint, cheese smear coryneform bacteria appear to be related to particular ecological niches: they are all oxidative species, and most are psychrotrophic and unable to grow at 37 degrees C whereas medically relevant coryneform bacteria are facultative anaerobes and grow at 35-37 degrees C. Consequently, technological strains must be selected according to taxonomic criteria (nonpathogenic species) and ecological criteria.     

15株乳酸菌的表面性质及其黏附能力

为了初步探讨乳酸菌的黏附机制,对15株乳酸菌的表面疏水性、自凝聚能力以及体外黏附Caco-2细胞的能力进行了测定,筛选出高黏附性乳酸菌菌株。采用化学试剂和酶处理嗜酸乳杆菌KLDS 1.0901细胞壁表面成分,再经过cFDA-SE荧光标记后,测定其对Caco-2细胞黏附能力的变化,分析影响黏附的主要黏附素。结果表明:嗜酸乳杆菌KLDS 1.0901对Caco-2细胞的黏附率最高,为12.73%;不同乳酸菌之间的表面疏水性、自凝聚能力以及对Caco-2细胞的黏附能力存在差异;经高碘酸钠、苯酚、胃蛋白酶、胰蛋白酶,尤其是氯化锂和热处理,能显著降低KLDS 1.0901的黏附性(p<0.05)。表明KLDS 1.0901对Caco-2细胞的黏附可能是以表层蛋白为主的多种黏附素共同作用的结果。     

高粘附性戊糖片球菌的筛选、标记及其表面疏水与自凝聚性特征

通过常规乳酸菌分离技术结合形态观察、生理生化试验,以及16S r DNA同源性分析等方法,从臭豆腐发酵卤水中分离获得7株戊糖片球菌分离株,并通过随机扩增多态性DNA技术构建了不同菌株的特征指纹图谱;以Caco-2细胞和固定化肠黏液蛋白质作为体外模型,研究了菌株的粘附能力,并探讨了菌株粘附能力与基因型以及表面疏水性、自凝聚能力等表型特征的相关性。结果显示,发酵卤水中的戊糖片球菌存在高度分子多样性,7个分离株中存在6种不同的指纹图谱模式,其中A5型菌株(F28-8和Y27-4)对Caco-2细胞和肠黏液蛋白质的粘附性最强,并显示了高度的疏水性(﹥90%)和较强的自凝聚能力(﹥25%)。相关性分析表明,戊糖片球菌表面疏水率和自凝聚率与Caco-2细胞粘附率测定结果呈显著正相关(r=0.900和0.792,P﹤0.05),但是与肠黏液蛋白质粘附率测定结果相关性不显著(r=0.426和0.700,P﹥0.05)。研究成果为建立高粘附性戊糖片球菌快速筛选方法及其体内定植和分布研究提供了依据。     

Relationship of hydrophobicity and solubility with some functional properties of cowpea (Vigna unguiculata) protein isolate

The relationship of hydrophobicity and solubility with some functional properties of cowpea protein isolate was determined. Cowpea protein isolate was prepared by alkali extraction followed by precipitation at pH 4.5. The precipitated proteins were then neutralized to pH 7. Heating of the protein isolate to 100°C for 10 min followed by cooling to room temperature resulted in a significant (P ≦ 0.05) decrease in aromatic hydrophobicity (ARH) when compared to the native protein isolate. The inclusion of sodium dodecyl sulfate (SDS) during heating gave a significant (P ≦ 0-05) 1.7-fold increase while inclusion of mercaptoethanol (ME) gave a significant (P ≦ 0.05) 2.5-fold increase in ARH of the cooled protein solution. Protein solubility (PS), foam expansion (FE) and emulsification activity index (EAI) of the isolate generally increased significantly (P ≦ 0.05) upon heating or treatment with urea or SDS or a combination of SDS and ME. Backward stepwise multiple regression was used to obtain equations for predicting emulsifying and foaming properties of the protein isolate from solubility and hydrophobicity parameters. PS, ARH and aliphatic hydrophobicity (ALH) were important in predicting foam stability and emulsion stability, while PS and ALH were important for predicting FE. ALH alone was important for predicting EAI.