Characterization of semipurified enterocins produced by Enterococcus faecium strains isolated from raw camel milk

Food safety has become an issue of great interest worldwide. Listeria monocytogenes is a food-borne pathogen that causes listeriosis and is difficult to control in the dairy industry. The use of lactic acid bacteria (LAB) and their antimicrobial substances against Listeria is promising in food applications. Here, we report the isolation from raw camel milk of LAB displaying antilisterial activity. Two isolates were selected for their secretion of bacteriocin(s) and identified by 16S rRNA sequencing as Enterococcus faecium S6 and R9. The produced bacteriocins were partially purified by ammonium sulfate precipitation and then biochemically characterized. Antimicrobial activity was estimated to be 6,400 and 400 AU (arbitrary units)/mL for E. faecium S6 and R9, respectively. The proteinaceous nature of the bacteriocins was confirmed via enzymatic reactions. Moreover, lipolytic and glycolytic enzymes completely inactivated the antimicrobial effect of the bacteriocins. These bacteriocins were heat-resistant and stable over a wide range of pH (2.0 to 10.0). To confirm its inactivation by lipolytic and glycolytic enzymes, the bacteriocin of E. faecium S6 was further purified by gel filtration, which suggested the existence of carbohydrate and lipid moieties. In addition, enterocin-coding genes were identified by PCR, showing DNA fragments corresponding in size to enterocins A, B, and P for E. faecium S6 and to enterocins B and P for E. faecium R9. In conclusion, these results indicate that partially purified bacteriocins from E. faecium S6 and R9 may be beneficial in controlling Listeria in the dairy industry.     

Purification of two bacteriocins produced by Enterococcus faecalis DBFIQ E24 strain isolated from raw bovine milk

Enterococcus faecalis DBFIQ E24 was formerly characterised as a producer of antibacterial and antifungal substances. This strain is vancomycin sensitive, nonhaemolytic and gelatinase negative. SDS‐PAGE revealed the presence of an active band located between 1060 and 3500 Da. After the application of a three‐step purification procedure, two antimicrobial peptides were isolated. One of them is mostly active against Bacillus cereus with a molecular mass of 1364 Da, and the other one with a molecular mass of 1686 Da is mainly active against Escherichia coli. These results confirm the technological potential of E. faecalis DBFIQ E24 strain as a GRAS food biopreservative.     

Assimilation of cholesterol and probiotic characterisation of yeast strains isolated from raw milk and fermented foods

In this study, cholesterol assimilation ratios and some probiotic characteristics of yeasts were investigated. For this purpose, yeasts were isolated from milk and foods that were naturally fermented and not containing starter culture. In vitro cholesterol assimilation properties were determined in media. The Cholesterol assimilation by yeast strains ranged between 1.36 and 73.33%. Twenty‐one yeast strains showing high assimilation percentage were selected, and their acid tolerance, bile tolerance, bile salt deconjugation activity and survival in simulated gastrointestinal conditions were investigated. Among the strains assessed, 12 of them showed probiotic characteristics.     

In vitro characterisation of probiotic properties of Enterococcus faecium and Enterococcus durans strains isolated from raw milk and traditional dairy products

In this study, some probiotic characteristics such as antimicrobial activity, vancomycin resistance, growth ability at pH, resistance to bile salts, bile salt deconjugation and hydrophobicity of 30 Enterococcus faecium and Enterococcus durans strains isolated and identified from raw milk and various dairy products were investigated. According to the study results, antimicrobial activity profiling, pH and bile salt resistance and bile salt deconjugation ability of Enterococcus strains varied depending on the species and strains and all the strains showed resistance to the tested bile salt concentrations. It was concluded that the E. faecium and E. durans strains tested showed probiotic characteristics and have the potential to be used in food production.     

Antihypertensive activity of casein-enriched milk fermented by Lactobacillus helveticus

Milk proteins contain peptidic angiotensin I-converting enzyme (ACE) inhibitors, which can be released by proteolysis during milk fermentation by some strains of Lactobacillus helveticus. Reconstituted milk media containing skim milk powder (12%), skim milk powder (10%) with added sodium caseinate (5%) or whey protein isolate (5%) were fermented by L. helveticus strains R211 and R389, and further tested for bacterial growth, proteolysis (free NH₃ groups) and ACE-inhibitory activity. The antihypertensive activity in spontaneously hypertensive rats (SHR) was also investigated with caseinate-enriched milk unfermented (UFM) and fermented by the two strains of L. helveticus. Caseinate-enriched milk fermented by both strains showed higher proteolysis and ACE-inhibitory activity, indicating that ACE-inhibitory peptides are probably released from caseins during milk fermentation. Significant decreases in mean arterial blood pressure in SHR rats were measured following oral administration of UFM milk at doses of 1.0 and 2.5 g kg⁻¹ of body weight, and milk fermented by R211 or R389 strains at doses of 0.5, 1.0 and 2.5 g kg⁻¹ of body weight. The antihypertensive activity of UFM could be explained by the release of ACE-inhibitory peptides from caseins during the digestion process.     

Potential probiotic characteristics of Lactobacillus and Enterococcus strains isolated from traditional dadih fermented milk against pathogen intestinal colonization

Traditional fermented buffalo milk in Indonesia (dadih) has been believed to have a beneficial impact on human health, which could be related to the properties of the lactic acid bacteria (LAB) involved in its fermentation process. In previous studies, it was discovered that strains of dadih lactic isolates possessed some beneficial properties in vitro. In the present study, the adhesion capacity of specific LAB isolates from dadih to intestinal mucus was analyzed. Further, the ability to inhibit model human pathogens and displace them from mucus was assessed. The adhesion of tested LAB strains was strain-dependent and varied from 1.4 to 9.8%. The most adhesive Lactobacillus plantarum strain was IS-10506, with 9.8% adhesion. The competition assay between dadih LAB isolates and pathogens showed that a 2-h preincubation with L. plantarum at 37 degrees C significantly reduced pathogen adhesion to mucus. All tested LAB strains displaced and inhibited pathogen adhesion, but the results were strain-specific and dependent on time and pathogen strains. In general, L. plantarum IS-10506 showed the best ability against pathogen adhesion.     

花鲈鱼肠道中产细菌素粪肠球菌的筛选和抑菌效果研究

为筛选出产细菌素的乳酸菌,应用于低温物流水产品保鲜中以提高水产品的品质。从花鲈鱼肠道中分离出18株乳酸菌,以大肠杆菌为指示菌,采用牛津杯琼脂扩散法检测菌株的抑菌谱大小,并经过有机酸排除、H2O2排除、蛋白酶水解检测试验,最终筛选得到1株产细菌素的菌株。经形态学、生理生化分析以及16S r DNA序列同源性分析,鉴定该菌株为Enterococcus faecalis。该菌株可以抑制水产品中的一些腐败菌,比如希瓦氏菌和假单胞菌;而对杀鲑气单胞菌的抑菌作用最强。因此,该菌株可作为一种潜在的生物保鲜剂应用在水产品保鲜中,以延长水产品的货架期。     

Antibacterial potential of Enterococcus faecium strains isolated from ewes’ milk and cheese

The study was conducted to evaluate the antibacterial activity of three bacteriocin-producing Enterococcus faecium strains (TW15, TW20 and TW22) isolated from ewes’ milk and cheese sampled in the Patagonian region of Argentina. The strains were tested against spoilage and pathogens microorganisms showing antimicrobial activity towards 4 strains of Listeria monocytogenes, one strain of Listeria innocua and 2 strains of Staphylococcus aureus. E. faecium TW15, E. faecium TW20 and E. faecium TW22 were sensitive to vancomycin. Furthermore, investigation of virulence factors revealed the absence of the genes encoding them. The bacteriocin-like substances (BLISs) produced by the 3 strains were thermostable, pH resistant and can be expressed even in the presence of NaCl (3.0 g/100 g). Moreover, they prove to have a bactericidal mode of action. Results from physicochemical and biochemical characteristics of BLIS produced by these E. faecium strains make them potential candidates to aid in preservation of foods.     

Proteolytic activity among psychrotrophic bacteria isolated from refrigerated raw milk

Psychrotrophic bacteria were isolated from refrigerated raw milk from a processing plant in Southern Brazil. Psychrotrophic counts were between 4.9 and 7.8 log cfu/mL, and 5.3 to 7.2 log cfu/mL, for samples collected at the truck and the milk storage silo, respectively. Among the bacterial isolates, 90% were Gram-negative. Most strains presented low proteolytic activity, but strains of Burkholderia cepacia, Klebsiella oxytoca and Aeromonas sp. showed higher than 20 U/mL on azocasein as substrate. Crude proteases from selected strains were resistant to conventional heat treatments and caused coagulation of UHT milk after 5 days storage at room temperature.     

Description of the bacteriocins produced by two strains of Enterococcus faecium isolated from Italian goat milk

In this study two strains of Enterococcus faecium, M241 and M249, isolated from goat milk, were studied for their capability to produce antibacterial compounds. It was determined that the bacteriocins produced by both strains were active towards Listeria monocytogenes and Clostridium butyricum, and they did not have any activity with respect to other species of lactic acid bacteria. Enterocins A and B were targeted by polymerase chain reaction (PCR) and sequenced, after cloning, in both strains. The bacteriocins contained in the cell free supernatants were stable when subjected to treatments at high and low temperatures or with lipase, catalase and alpha-amylase. Whereas, the activity was lost when proteinases were used. Lastly, a co-culture experiment with L. monocytogenes in skimmed milk was performed. In the presence of the E. faecium strains, the pathogen showed a delay in the growth of about 6h and it reached a maximum counts of about 10(6) colony forming unit, two orders of magnitude low with respect to the control. This result suggests the possibility to use the strains studied as starter cultures to enhance food safety of dairy products.     

分离自传统发酵乳制品中具有优良发酵特性的保加利亚乳杆菌的筛选

以分离自传统发酵乳制品中的11株德氏乳杆菌保加利亚亚种(Lactobacillus delbeueckii subsp.bulgaricus)为研究对象,通过全脂乳单菌发酵,对其发酵乳的发酵时间、pH值、滴定酸度、乙醛和双乙酰含量等指标进行分析,最终筛选出1株具有优良发酵特性的菌株IMAU20775。该菌株发酵时间较短,后酸化能力弱,产乙醛能力较好,产双乙酰能力较稳定,适于进一步作为发酵剂菌种的筛选和应用研究。     

Antihypertensive effect of peptides obtained from Enterococcus faecalis-fermented milk in rats

Previous studies have demonstrated that milk fermented with Enterococcus faecalis decreases the systolic blood pressure (SBP) and the diastolic blood pressure (DBP) of spontaneously hypertensive rats. In this study, we evaluated the antihypertensive activity of the following peptide sequences: LHLPLP, LHLPLPL, LVYPFPGPIPNSLPQNIPP, VLGPVRGPFP, and VRGPFPIIV. These peptides isolated from E. faecalis-fermented milk showed in vitro angiotensin I-converting enzyme-inhibitory activity. Because the most potent angiotensin I-converting enzyme-inhibitory sequences were LHLPLP and LVYPFPGPIPNSLPQ-NIPP, we administered different doses of these peptides to spontaneously hypertensive rats. High doses of the remaining sequences were also administered to these animals. Water served as a negative control and captopril as a positive control. All products were administered orally. The SBP and DBP were measured before administration and also at 2, 4, 6, 8, and 24 h after administration. Before administration of the different products, spontaneously hypertensive rats showed SBP and DBP values of 218 ± 2.5 and 157 ± 5.9 mmHg, respectively (n = 30). The sequences LHLPLP, LVYPF-PGPIPNSLPQNIPP, VLGPVRGPFP, and VRGPFPIIV caused clear and significant decreases in SBP, DBP, or both in the animals. In particular, the antihypertensive effect could be clearly established when 2 or 3 mg/kg of LHLPLP was administered. These 2 doses of LHLPLP showed similar antihypertensive properties. Four hours after administration of captopril or the highest doses of the different peptides, the decreases in the SBP and the DBP (mmHg) were as follows: captopril (SBP = 52 ± 5.8, DBP = 38.8 ± 3.8), 3 mg/kg of LHLPLP (SBP = 25.3 ± 8.2, DBP = 29.5 ± 7.6), 6 mg/kg of LVYPFPGPIP-NSLPQNIPP (SBP = 14.9 ± 3.7, DBP = 8.7 ± 4.4), 10 mg/kg of LHLPLPL (SBP = 7.7 ± 4.1, DBP = 9.4 ± 3.1), 10 mg/kg of VLGPVRGPFP (SBP = 16.2 ± 5.8, DBP = 21.64 ± 3.2), and 10 mg/kg of VRGPFPIIV (SBP = 16.05 ± 2.74, DBP = 9.19 ± 3.49). The results obtained suggest that the sequences LHLPLP, LVYPFPGPIPNSLPQ-NIPP, VLGPVRGPFP, and VRGPFPIIV could be responsible, at least in part, for the antihypertensive properties described for E. faecalis-fermented milk.     

Antioxidant activity of Lactobacillus plantarum strains isolated from traditional Chinese fermented foods

Eleven Lactobacillus plantarum strains isolated from traditional Chinese fermented foods were investigated for their in vitro scavenging activity against hydroxyl and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals, and their resistance to hydrogen peroxide. L. plantarum C88 at a dose of 10(10) CFU/ml showed the highest hydroxyl radical and DPPH scavenging activities, with inhibition rates of 44.31% and 53.05%, respectively. Resistance of intact cells to hydrogen peroxide was also found in all strains. L. plantarum C88 was the most resistant strain against hydrogen peroxide. When L. plantarum C88 was administered to senescent mice suffering oxidative stress induced by d-galactose, the serum superoxide dismutase activity, the glutathione peroxidase activity and the total antioxidant capacity in liver increased significantly, while the level of malondialdehyde in liver decreased significantly. L. plantarum C88 isolated from traditional Chinese fermented dairy tofu could be considered as a potential antioxidant to be applied in functional foods.     

Assessment of multifunctional activity of bioactive peptides derived from fermented milk by specific Lactobacillus plantarum strains

Milk-derived bioactive peptides with a single activity (e.g., antioxidant, immunomodulatory, or antimicrobial) have been previously well documented; however, few studies describe multifunctional bioactive peptides, which may be preferred over single-activity peptides, as they can simultaneously trigger, modulate, or inhibit multiple physiological pathways. Hence, the aim of this study was to assess the anti-inflammatory, antihemolytic, antioxidant, antimutagenic, and antimicrobial activities of crude extracts (CE) and peptide fractions (<3 and 3–10 kDa) obtained from fermented milks with specific Lactobacillus plantarum strains. Overall, CE showed higher activity than both peptide fractions (<3 and 3–10 kDa) in most of the activities assessed. Furthermore, activity of <3 kDa was generally higher, or at least equal, to the 3 to 10 kDa peptide fractions. In particular, L. plantarum 55 crude extract or their fractions showed the higher anti-inflammatory (723.68–1,759.43 μg/mL of diclofenac sodium equivalents), antihemolytic (36.65–74.45% of inhibition), and antioxidant activity [282.8–362.3 µmol of Trolox (Sigma-Aldrich, St. Louis, MO) equivalents]. These results provide valuable evidence of multifunctional role of peptides derived of fermented milk by the action of specific L. plantarum strains. Thus, they may be considered for the development of biotechnological products to be used to reduce the risk of disease or to enhance a certain physiological function.     

The lipolytic and proteolytic activity of bacteria isolated from blended raw milk

Psychrotrophic bacteria have been isolated from supplies of blended raw milk over a period of 14 months. The isolates were partially classified and their ability to degrade milk fat and protein (at 6C) examined. Pseudomonads were the predominant bacterial type and on average 43 per cent of these isolates were of the fluorescent type. These latter isolates together with isolates classified as Flavobacterium and Alcaligenes had associated with them the highest levels of lipase and protease activity.     

Identification and functional characterization of Lactobacillus strains isolated from milk and Gioddu,a traditional Sardinian fermented milk

Raw milk from Sardinian cows and sheep, and sheep's milk yoghurt (Gioddu), are food products that are poorly characterized in terms of the presence of mesophilic Lactobacillus strains, and may be good sources from which to isolate novel bacterial strains. Sixteen Lactobacillus strains were identified by genotypic and phenotypic methods from samples of sheep's milk (n=7), fermented sheep's milk (n=7) and one sample of cows’ milk. These strains were further characterized for the presence of functional traits useful for probiotic applications, such as resistance to simulated gastric acid, resistance to bile salts, and adhesion of two different cell lines (Caco-2 and MIM/PPK). Moreover, selected aspects correlated with safety (antimicrobial susceptibility and presence of plasmids) and ecological roles (ability to produce biofilms and bacteriocin-like substances) were tested. All but 1 of the 16 strains were unambiguously allotted to three species, L. paracasei (5 strains), L. plantarum (8 strains), and L. reuteri (2 strains). Many of the strains exhibited characteristics suggesting that they would survive in the gastrointestinal tract and also had the capability to attach to various cells. Greater variability was observed for the other traits analyzed. These data suggest that Sardinian artisan dairy products are interesting sources for the isolation of bacterial strains with characteristic and differentiated functional traits.     

Physicochemical, microbiological and sensory profiles of fermented milk containing probiotic strains isolated from kefir

A two-strain starter culture containing Lactobacillus plantarum CIDCA 83114, a potential probiotic strain isolated from kefir grains, and Streptococcus thermophilus CIDCA 321 was tested for the preparation of a fermented milk product. Kluyveromyces marxianus CIDCA 8154, a yeast with immunomodulatory properties was included to formulate a three-strain starter culture. Supernatants of enterohaemorragic Escherichia coli, shiga-toxin-producing strain, along with a two-strain or a three-strain starter culture were included in the medium of Vero-cell surface cultures. The results demonstrated that these combinations of microorganisms antagonize the cytopathic action of shiga toxins. The cell concentration of Lb. plantarum did not decrease during fermentation, indicating that the viability of this strain was not affected by low pH, nor did the number of viable bacteria change during 21 days of storage in either fermented products. The number of viable yeasts increases during fermentation and storage. Trained assessors analyzed the general acceptability of fresh fermented milks and considered both acceptable. The milk fermented with the two-strain starter culture was considered acceptable after two week of storage, while the product fermented with the three-strain starter culture remained acceptable for less than one week. The main changes in sensory attributes detected by the trained panel were in sour taste, milky taste and also in fermented attributes. The correlation between different sensory attributes and acceptability indicated that the panel was positively influenced by milky attributes (taste, odour, and flavour) as well as the intensity of flavour. In conclusion, the two-strain starter culture would be the more promising alternative for inclusion of that potential probiotic lactobacillus in a fermented milk product.     

Nisin and lacticin 481 coproduction by Lactococcus lactis strains isolated from raw ewes’ milk

Bacteriocin-producing lactococci were isolated from raw ewes’ milk samples obtained from 5 different Protected Designation of Origin Zamorano cheese manufacturers. Thirteen isolates with antimicrobial activity against Lactococcus lactis HP were selected. Eleven were identified by a PCR technique as L. lactis ssp. lactis and 2 were identified as L. lactis ssp. cremoris. They were grouped under 4 different pulsed-field gel electrophoresis patterns. The presence of structural genes of both nisin and lacticin 481 was detected in 10 L. lactis ssp. lactis isolates belonging to 2 different pulsed-field gel electrophoresis patterns. Coproduction of nisin and lacticin 481 was confirmed after semipurification by using selective indicators. The production of 2 bacteriocins by the same strain is an uncommon property, with relevance in food safety. Nisin and lacticin 481 L. lactis-producing strains might be used as adjunct cultures to the commercial starter in the manufacture of dairy products.     

Antimicrobial resistance and virulence of Enterococcus faecalis isolated from retail food

Although enterococci are considered opportunistic nosocomial pathogens, their contribution to foodborne illnesses via dissemination through retail food remains undefined. In this study, prevalence and association of antimicrobial resistance and virulence factors of 80 Enterococcus faecalis isolates from retail food items were investigated. The highest rates of resistance were observed for lincomycin (73 of 80 isolates, 91%) and bacitracin (57 of 80 isolates, 71%), and lower rates of resistance (< or =40%) were found for chloramphenicol, ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, nitrofurantoin, penicillin, and tylosin. Overall resistance to antimicrobials was low for most isolates tested. Of the virulence factors tested, the majority of isolates were positive for ccf (78 of 80 isolates, 98%), efaAfs (77 of 80, 96%), and cpd (74 of 80, 93%). Isolates also commonly contained cob (72 of 80 isolates, 90%) and gelE (68 of 80, 85%). Very few isolates contained cylMBA (12 of 80 isolates [15%] for cylM and 9 of 80 isolates [11%] for both cylB and cylA) and efaAfm (2 of 80 isolates, 3%). Positive statistical associations (significance level of 0.05) were found between agg and tetracycline resistance, cylM and erythromycin resistance, and gelE and efaAfs and lincomycin resistance. The presence of the cylB and cylA alleles also was positively correlated with bacitracin and tetracycline resistance. Negative correlations were observed between many of the virulence attributes and resistance to ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, and tylosin. These data suggest that both positive and negative associations exist between antimicrobial resistance genes and virulence factors in E. faecalis isolates from foods commonly purchased from grocery stores.