Screening of lactic acid bacteria for antimicrobial properties from mayonnaise-based products and raw materials

A collection of lactic acid bacteria isolated from both mayonnaise-based products and raw materials used to manufacture them was tested for antimicrobial activity. Out of 144 strains (97 lactobacilli, 23 lactococci and 24 enterococci) only three supernatants of Enterococcus spp. strains (EN3, EN14 and EN15) exhibited activity against lactobacilli and lactococci. The supernatant of the strain EN3 exclusively inhibited the growth of Bacillus cereus and Listeria monocytogenes. Enterococcus strains EN3, EN14 and EN15 produced thermostable bacteriocins, which had antibacterial activity.     

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.     

Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants

Antimicrobial behavior of lactic acid bacteria (LAB) has been explored since many years to assess their ability to produce bacteriocin, a natural preservative, to increase the shelf life of food. This study aims to characterize bacteriocin producing strains of lactic acid bacteria isolated from acidic to slightly acidic raw vegetables including tomato, bell pepper and green chili and to investigate their potential to inhibit food related bacteria. Among twenty nine LAB screened for antimicrobial activity, three exhibited antagonism against closely related bacterial isolates which was influenced by varying temperature and pH. They were identified up to strain level as Lactococcus lactis subsp. lactis TI-4, L. lactis subsp. lactis CE-2 and L. lactis subsp. lactis PI-2 based on 16S rRNA gene sequence. Their spectrum of inhibition was observed against food associated strains of Bacillus subtilis and Staphylococcus aureus. Moreover, L. lactis subsp. lactis PI-2 selected on the basis of higher antimicrobial activity was further evaluated for bacteriocin production which was detected as nisin A and nisin Z. These findings suggest the possible use of L. lactis strains of vegetable origin as protective cultures in slightly acidic as well as slightly alkaline food by the bio-preservative action of bacteriocins.     

Assessing the ability of nisin A and derivatives thereof to inhibit gram-negative bacteria from the genus Thermus

Nisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains.     

Probiotic potential and biochemical and technological properties of Lactococcus lactis ssp. lactis strains isolated from raw milk and kefir grains

Lactococcus lactis ssp. lactis is one of the most important starter bacteria used in dairy technology and it is of great economic importance because of its use in the production of dairy products, including cheese, butter, cream, and fermented milks. Numerous studies have evaluated the biochemical and probiotic properties of lactococci; however, limited studies on the probiotic characteristics of lactococci were conducted using strains originating from raw milk and dairy products. Characterizing the probiotic properties of strains isolated from raw milk and fermented milk products is important in terms of selecting starter culture strains for the production of functional dairy products. In this study, biochemical properties (including antibiotic sensitivity, lipolytic activity, amino acid decarboxylation, antioxidant activity) and probiotic properties (including antimicrobial activity, growth in the presence of bile salts, bile salts deconjugation, and hydrophobicity) of 14 Lactococcus lactis strains isolated from raw milk and kefir grains were investigated. Strains originating from kefir grains had better characteristics in terms of antimicrobial activity and bile salt deconjugation, whereas strains from raw milk had better hydrophobicity and antioxidant activity characteristics. None of the strains were able to grow in the presence of bile salt and did not show amino acid decarboxylation or lipolytic activities. Biochemical and probiotic properties of L. lactis strains varied depending on the strain and some of these strains could be used as functional cultures depending on their properties. However, these strains did not possess all of the properties required to meet the definition of a probiotic.     

Milk fermentation by functional mixed culture producing nisin Z and exopolysaccharides in a fresh cheese model

A nisin Z-producing strain, Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 and two nisin-sensitive cultures, Lactobacillus rhamnosus RW-9595 M producing exopolysaccharide (EPS), and Lc. lactis subsp. cremoris for acidification, were tested in pure and mixed cultures during milk fermentation. The mixed culture of the three strains showed a higher acidifying capacity at 34°C and 38°C, even though populations of Lc. cremoris were largely reduced compared with pure cultures. Bacteriocin production was 3.1–4.6-fold higher in mixed cultures than for pure cultures of Lc. diacetylactis UL719. These data can be explained by commensalism behavior relying on high proteolytic activity of Lc. cremoris and autolysis and nisin Z-induced lysis. In mixed culture, EPS production was 3-fold lower than for Lb. rhamnosus RW-9595 M pure culture. Our data showed that this strain combination, with nisin-producing and sensitive strains, can be used in mixed cultures for manufacture of fresh cheese with improved functional properties.     

The efficacy of nisin can drastically vary when produced in situ in model cheeses

Nisin, a bacteriocin produced by strains of Lactococcus lactis, has a broad inhibitory effect against Gram-positive bacteria. This study investigated the efficacy of nisin Z against Lactobacillus sakei when produced by a nisin-producing strain L. lactis in model cheeses manufactured with ultrafiltrated milk. These cheeses, containing 0, 4 or 10% of gelatin in their dry matter, were inoculated with both strains. Measurement of Lb. sakei loss of viability was an indirect indicator of nisin in situ efficacy. After 24 h, the loss of viability of Lb. sakei was from 0.73 ± 0.14 to 3.30 ± 0.60 log₁₀ cfu g⁻¹ in the cheeses with 0 and 10% of gelatin, respectively, indicating a better in situ efficacy of nisin when gelatin was incorporated. However, the concentration of nisin produced by Lactococcus was similar (3.5 μg g⁻¹) in all model cheeses when measured using an enzyme-linked immune sorbent assay (ELISA). The growth of Lactococcus was slightly improved when gelatin was incorporated, leading to a higher lactate concentration, which is one of the factors explaining the increased nisin efficacy. These results reinforced previous observations that prediction of nisin efficacy in complex food systems remains difficult.     

The effects on vegetative cells and spores of three bacteriocins from lactic acid bacteria

The sensitivities of vegetative cells of strains ofListeria, Clostridium, Staphylococcus, Lactococcus, Lactobacillus, MicrococcusandPediococcus, and of spores ofClostridiumandBacillusto three broad spectrum bacteriocins (nisin A, nisin Z and pediocin) from lactic acid bacteria were determined by a critical dilution micro-assay. The minimal inhibitory concentrations (MIC) of partially purified bacteriocins, prepared by a pH-dependent adsorption/desorption process, were determined and expressed in arbitrary units ml⁻¹and in μ g ml⁻¹of pure bacteriocin. The MICs of bacteriocins varied considerably between species and even between strains of the same species, as clearly shown for nine strains ofListeria monocytogenes. When bacteriocin activity was expressed in μ g ml⁻¹, pediocin was more effective againstListeria monocytogenesthan nisin A or nisin Z. The latter bacteriocins, in concentrations between 23 and 69 μ g ml⁻¹, prevented outgrowth ofClostridiumandBacillusspores for at least 10 days. Although pediocin at 17 μ g ml⁻¹prevented outgrowth ofB. stearothermophilusandC. butyricumspores for up to 7 days, it apparently activated the germination ofB. subtilisspores.     

Characterization of bacteriocins from two Lactococcus lactis subsp. lactis isolates

In this study, bacteriocins from two Lactococcus lactis subsp. lactis isolates from raw milk samples in Turkey designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after alpha-amylase treatment suggested that they form aggregates with carbohydrates. Molecular masses of the purified bacteriocins were determined by SDS-PAGE. PCR amplification was carried out with specific primers for the detection of their structural genes. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Examination of plasmid contents of the isolates and the results of plasmid curing and conjugation experiments showed that in L. lactis subsp. lactis OC1 strain the 39.7-kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity, whereas the 16.0-kb plasmid is responsible for lacticin 481 production and lactose fermentation in L. lactis subsp. lactis OC2 strain.     

Angiotensin-converting enzyme inhibitory activity of milk fermented by wild and industrial Lactococcus lactis strains

Angiotensin I-converting enzyme inhibitory (ACEI) activity was evaluated and compared in <3 KDa water-soluble extracts (WSE) isolated from milk fermented by wild and commercial starter culture Lactococcus lactis strains after 48 h of incubation. The highest ACEI activities were found in WSE from milk inoculated with wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures. On the other hand, the lowest ACEI activities were found in WSE from milk inoculated with wild strains isolated from vegetables. Moreover, the IC₅₀ values (concentration that inhibits 50% activity) of WSE from artisanal dairy products were the lowest, indicating that these fractions were the most effective in inhibiting 50% of ACE activity. In fact, a strain isolated from artisanal cheese presented the lowest IC₅₀ (13 μg/mL). Thus, it appears that wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures showed good potential for the production of fermented dairy products with ACEI properties.     

Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria

The present work compares, under the same stated experimental conditions, the antimicrobial activity of crude and purified enterocin L50, pediocin PA-1, nisin A and lactocin S, produced by lactic acid bacteria (LAB) isolated from Spanish dry-fermented sausages. The bacteriocins were purified to homogeneity by ammonium sulphate precipitation, gel filtration (for lactocin S), and cation-exchange, hydrophobic-interaction, and reverse-phase-chromatography; high yields of pure bacteriocins were obtained. Minimal inhibitory concentration (MIC) of pure enterocin L50, pediocin PA-1, nisin A and lactocin S was determined against a broad spectrum of Gram-positive bacteria, including spoilage and foodborne pathogenic bacteria. The purified bacteriocins showed a broad antimicrobial spectrum similar to that exerted by crude bacteriocins. Enterocin L50 and pediocin PA-1 were very active againstListeria monocytogenes, which was quite resistant to nisin A and lactocin S. Enterocin L50 also displayed antimicrobial activity againstStaphylococcus aureus,Clostridium perfringensandClostridium botulinum. However, these pathogens were weakly inhibited, or not at all, by the other pure bacteriocins.     

Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui,a Brazilian fermented,salted and dried meat product

A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0–10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui.     

Peptidase and proteinase activity ofLactococcus lactis,Lactobacillus casei andLactobacillus plantarum

The proteolytic system of several non-commercial strains of lactococci and lactobacilli that were isolated directly from traditional-Spanish, semi-hard, goats' milk cheese was studied. The aminopeptidase, X-prolyldipeptidyl aminopeptidase, dipeptidase and proteinase activity of these new strains was measured for the cytoplasmic, cell-wall/membrane and spontaneously released fractions. The aminopeptidase activity was exclusively intracellular and higher forLactobacillus casei subsp.casei than forLactococcus lactis subsp.lactis. Lactobacillus plantarum showed higher dipeptidase activity thanL. casei. The highest level of proteinase activity was recorded for the cell-wallmembrane fraction ofLactococcus lactis subsp.lactis IFPL 359, and was higher on β-casein than on α_s-casein for all the strains studied. These results suggest some different contribution of these strains to the proteolysis of cheese during ripening and they seem to complement each other when used together in the starter culture.     

Proteolytic activities,peptide utilization and oligopeptide transport systems of wild Lactococcus lactis strains

The increasing demand for tasty dairy products has raised the interest for strains of lactic acid bacteria with novel properties. In this study we have explored the proteolytic system of 24 wild Lactococcus lactis strains isolated previously from Spanish raw milk cheeses, to select proteolytic strains with high peptidase activity and efficient peptide transport capability. Large variations in overall proteolysis, proteolytic activities, as well as in peptide utilization, were recorded among strains. Peptide utilization correlated well with the presence of oligopeptide transport systems (Opp and Dpp) in wild L. lactis strains. Eighteen of the 24 wild L. lactis strains possessed both Opp and Dpp systems, what supports the theory that the presence of both oligopeptide transport systems could confer an advantage to the strain. Differences in the nucleotide sequence of genes coding for the oligopeptide binding proteins were shown by means of restriction endonuclease analyses. Based on the characteristics determined in this work, L. lactis strains ESI197, M21 and P21 seem to be promising candidates for use as components of starter cultures.     

Consequences of the development of nisin-resistant Listeria monocytogenes in fermented dairy products

Wild Listeria isolates representing serovars found in artisanal cheeses commercialized in Asturias (northern Spain) were assessed for their susceptibility to several bacteriocins. Pediocin PA-1 was the most active bacteriocin followed by enterocin AS-48, nisin, and plantaricin C. However, some Listeria monocytogenes and Listeria innocua strains were already highly resistant to PA-1. Among the wild L. monocytogenes populations, the frequency of development of nisin resistance ranged from 10(-6) up to 10(-3), depending on the strain. Highly stable mutants with increased nisin resistance (two- to fourfold) were isolated and tested for potential cross-resistance to lysozyme, EDTA, and various NaCl concentrations and pH values. All mutants were cross-resistant to lysozyme but sensitive to EDTA. In contrast, no clear correlation could be established between nisin resistance and an altered susceptibility to NaCl or pH changes. Nisin-resistant variants were able to survive and even to multiply in milk fermented by a nisin-producing Lactococcus, but the growth of the wild-type strain was inhibited. The different phenotypes evaluated in this study are indicative of the unpredictability of the consequences of the development of nisin resistance in a dairy environment. This resistance should be considered when making a risk assessment of the long-term use of nisin to control L. monocytogenes.     

Enhancement of functional characteristics of mixed lactic culture producing nisin z and exopolysaccharides during continuous prefermentation of milk with immobilized cells

Antagonistic phenomena between strains often occur in mixed cultures containing a bacteriocinogenic strain. A nisin Z producer (Lactococcus lactis ssp. lactis biovar. diacetylactis UL719) and 2 nisin-sensitive strains for acidification (Lactococcus lactis ssp. cremoris ATCC19257) and exopolysaccharide (EPS) production (Lactobacillus rhamnosus RW-9595M) were immobilized separately in gel beads and used to continuously preferment milk at different temperatures, with pH controlled at 6.0 by fresh milk addition. The process showed high volumetric productivity, with an increase from 8.0 to 12.5 L of prefermented milk per liter of reactor volume and hour as the temperature was increased from 27 to 35°C. Lactococcus lactis ssp. lactis biovar. diacetylactis UL719 counts in prefermented and fermented (22-h batch fermentation) milks were stable during 3 wk of continuous fermentation (8.1 ± 0.1 and 8.9 ± 0.2 log cfu/mL, respectively). The L. lactis ssp. cremoris population (estimated with real-time quantitative PCR) decreased rapidly during the first week of continuous culture to approximately 4.5 log cfu/mL and remained constant afterward. Lactobacillus rhamnosus counts in prefermented and fermented milks significantly increased with prefermentation time, with no temperature effect. Nisin Z reached high titers in fermented milks (from 177 to 363 IU/mL), with EPS concentration in the range from 43 to 178 mg/L. Immobilization and continuous culture led to important physiological changes, with Lb. rhamnosus becoming much more tolerant to nisin Z, and Lb. rhamnosus and L. lactis ssp. lactis biovar. diacetylactis UL719 exhibiting large increases in milk acidification capacity. Our data showed that continuous milk prefermentation with immobilized cells can stimulate the acidification activity of low-acidifying strains and produce fermented milks with improved and controlled functional properties.     

High-level coproduction of the bacteriocins nisin A and lactococcin A by Lactococcus lactis

In this study, a two-plasmid system for enhanced and consistent biosynthesis of the model lactococcal bacteriocin lactococcin A in non-producing Lactococcus lactis hosts was developed. The system comprised a plasmid carrying the genes lcnA and lciA under the control of the nisin-inducible nisA promoter, and a second plasmid harbouring the lcnC and lcnD genes. The introduction of both plasmids into two strains containing the nisRK genes required for nisin-controlled expression, Lc. lactis FI5876 (a nisin A-producer strain) and FI7847, resulted in production of extracellular lactococcin A at a higher level than that for the parental strain, Lc. lactis WM4. In addition, transformation of the nisin-producing host with both plasmids led to a high-level production of both lactococcal bacteriocins, which may provide a means to exploit their complementary properties in cheese ripening.     

Diversity Analysis,Batch Fermentation and Characterization of Nisin in Identified Strains of Lactococcus lactis spp. lactis

Thirty-one strains of Lactococcus lactis spp. lactis were identified out of 89 isolates of lactic acid bacteria (LAB) from dairy and nondairy sources. Of the 31 strains, 24 (46.1%) were obtained from dairy and seven (18.9%) from non-dairy sources. The cluster analysis of rep-PCR showed that (GTG)₅-PCR followed by ERIC-PCR exhibited more discriminating potential than BOX-PCR. The obtained banding patterns characterized the polymorphism among strains. The strain level polymorphism was also obtained by the combined cluster analysis of (GTG)_5, ERIC and BOX-PCR which exhibited a level of heterogeneity among strains but not with the sources of isolation. Among 31 strains, 17 strains were able to produce zones of inhibition against Lactobacillus acidophilus NCDC 015 and therefore considered as nisin producers. Nisin production by strains was further confirmed by PCR amplification of nisA/Z of 174 bp size. The nisin activity and cell growth observed to be higher in pH controlled batch fermentation than in uncontrolled fermentation. The nisin activity, cell concentration, and acidity were high in immobilized cell system than free cell batch fermentation. The hot acid and chloroform extraction method was found to be the efficient way for the partial purification of nisin from fermented broth.