Technical note: Use of RFLP to characterize Lactococcus lactis strains producing exopolysaccharides

Restriction fragment length polymorphism (RFLP) is used to differentiate microorganisms by analysis of their DNA restriction patterns. A modified RFLP procedure is proposed for the rapid characterization of Lactococcus lactis strains producing exopolysaccharides (EPS). The availability of such effective cataloging system is likely to benefit research aimed at identifying lactococcal strains that produce novel EPS.     

Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama

Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.     

Lactococcal 936-species phage attachment to surface of Lactococcus lactis

The interactions of the 936-species phages sk1, jj50, and 64 with the cell surface of Lactococcus lactis LM0230 were analyzed. Cell envelopes (walls + plasma membrane), cell wall, or plasma membrane from L. lactis ssp. lactis LM0230 each inactivated the phages in vitro. However, other 936-species phages kh and P008, which do not infect strain LM0230, were not inactivated by any of the subcellular fractions. Treating cell walls or plasma membrane with the cell wall hydrolase mutanolysin eliminated inactivation of phage sk1. This suggested that intact cell wall fragments were required for inactivation. A role for plasma membrane in phage sk1 inactivation was further investigated. Boiling, washing in 2 M KCl, 8 M urea, or 0.1 M Na(2)CO(3)/pH 11, or treating the plasma membrane with proteases did not reduce adsorption or inactivation of phage. Adding lipoteichoic acid or antibodies to lipoteichoic acid did not reduce inactivation of phage in a mixture with membrane, suggesting that lipoteichoic acid was not involved. Inactivation by envelopes or cell wall correlated with ejection of DNA from the phage sk1 capsid. Although calcium is required for plaque formation, it was not required for adsorption, inactivation, or ejection of phage DNA by envelopes or cell wall. The results suggest that at least for phages sk1, jj50, and 64, adsorption and phage DNA injection into the host does not require a host membrane protein or lipoteichoic acid, and that cell wall components are sufficient for these initial steps of phage infection.     

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.     

The effect of lactose, NaCl and an aero/anaerobic environment on the tyrosine decarboxylase activity of Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis

The aim of this work was to study, under model conditions, combined effects of the concentration of lactose (0-1% w/v), NaCl (0-2% w/v) and aero/anaerobiosis on the growth and tyramine production in 3 strains of Lactococcus lactis subsp. lactis and 2 strains of L. lactis subsp. cremoris. The levels of the factors tested were chosen with respect to the conditions which can occur during the real process of natural cheese production, including the culture temperature (10 ± 1 °C). In all strains tested, tyrosine decarboxylation was most influenced by NaCl concentration; the highest production of tyramine was obtained within the culture with the highest (2% w/v) salt concentration applied. Two of the strains L. lactis subsp. lactis produced tyramine only in broth with the highest NaCl concentration tested. In the remaining 3 strains of L. lactis, tyramine was detected under all conditions applied. The tested concentration of lactose and aero/anaerobiosis had a less significant effect on tyramine decarboxylation. However, it was also found that at the same concentrations of NaCl and lactose, a higher amount of tyramine was detected under anaerobic conditions. In all strains tested, tyramine decarboxylation started during the active growth phase of the cells.     

Improved viability of bifidobacteria in fermented milk by cocultivation with Lactococcus lactis subspecies lactis

The poor survival of probiotic bacteria in commercial yogurts may limit their potential to exert health benefits in humans. The objective was to improve the survival of bifidobacteria in fermented milk. Cocultivation with some strains of Lactococcus lactis ssp. lactis improved the survival of bifidobacteria in fermented milk during refrigerated storage. Studies on one strain, Lc. lactis ssp. lactis MCC866, showed that the concentrations of dissolved oxygen were kept lower in the cocultivated fermented milk during storage compared with monocultured Bifidobacterium longum BB536 or samples cocultured with another noneffective Lc. lactis ssp. lactis strain. Degradation of genomic DNA was suppressed in the cocultivating system with Lc. lactis ssp. lactis MCC866. Several genes that participated in protection from active oxygen species (e.g., genes coding for alkyl hydroperoxide reductase and Fe²⁺ transport system) were expressed at higher levels during refrigerated storage in Lc. lactis ssp. lactis MCC 866 compared with another noneffective Lc. lactis ssp. lactis strain. Concentration of free iron ion was also lower in supernatants of fermented milk cocultivated with B. longum BB536 and Lc. lactis ssp. lactis MCC866. These results suggest that Lc. lactis ssp. lactis MCC 866 is potentially superior in reducing oxygen damage and consequently improves the survival of bifidobacteria in the cocultivating system. This cocultivation system is of industrial interest for producing fermented milk containing viable bifidobacteria with long shelf life.     

Enhanced production of pediocin PA-1 in wild nisin- and non-nisin-producing Lactococcus lactis strains of dairy origin

In this work, heterologous production of pediocin PA-1 in Lactococcus lactis ESI 153 and ESI 515 (Nis⁺), two strains selected because of their technological properties for cheesemaking, was achieved after transformation with plasmids pMC117, pRK119 and pCNC1, which contain the complete pediocin operon under the control of the strong P32 promoter. The pediocin production of the L. lactis ESI 153 derivatives containing pRK119 or pCNC1 was higher (approximately 165%) than that achieved by the natural pediocin PA-1 producer Pediococcus acidilactici 347. In the case of the L. lactis ESI 515 derivatives, those containing pRK119 or pCNC1 showed a pediocin production level similar (95–100%) to that of P. acidilactici 347.     

Production of pediocin PA-1, and coproduction of nisin A and pediocin PA-1, by wild Lactococcus lactis strains of dairy origin

Heterologous production of pediocin PA-1 in nisin and non-nisin-producing Lactococcus lactis strains, which had been previously selected because of their technological properties for cheese making, was investigated. Plasmid pFI2160, which contains a hybrid gene (L-pedA) encoding the fusion between the lactococcin A leader and propediocin PA-1, and also the genes lcnC and lcnD, that encode the lactococcin A secretion apparatus, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis⁺). The pediocin production level of their respective transformants, L. lactis CL1 and L. lactis CL2 (Nis⁺), was approximately 600 and 400 ng mL⁻¹, respectively, which represents a 30% and a 20% of the quantity produced by the natural pediocin PA-1 producer Pediococcus acidilactici 347. Transformation of L. lactis ESI 515 with pFI2160 did not affect its ability to produce nisin. Pediocin bioassays showed the stability of pFI2160 in both heterologous hosts under selective and non-selective conditions.     

Enhanced nutty flavor formation in cheddar cheese made with a malty Lactococcus lactis adjunct culture

Nutty flavor in Cheddar cheese is desirable, and recent research demonstrated that 2- and 3-methyl butanal and 2-methyl propanal were primary sources of nutty flavors in Cheddar. Because malty strains of Lac-tococcus lactis (formerly Streptococcus lactis var. malti-genes) are characterized by the efficient production of these and other Strecker aldehydes during growth, this study investigated the influence of a malty L. lactis adjunct culture on nutty flavor development in Cheddar cheese. Cheeses made with different adjunct levels (0, 10⁴ cfu/mL, and 10⁵ cfu/mL) were ripened at 5 or 13°C and analyzed after 1 wk, 4 mo, and 8 mo by a combination of instrumental and sensory methods to characterize nutty flavor development. Cheeses ripened at 13°C developed aged flavors (brothy, sulfur, and nutty fla-vors) more rapidly than cheeses held at 5°C. Additionally, cheeses made with the adjunct culture showed more rapid and more intense nutty flavor development than control cheeses. Cheeses that had higher intensities of nutty flavors also had a higher concentration of 2/3-methyl butanal and 2-methyl propanal compared with control cheeses, which again confirmed that these compounds are a source of nutty flavor in Cheddar cheese. Results from this study provide a simple methodology for cheese manufacturers to obtain consistent nutty flavor in Cheddar cheese.     

Growth and metabolic behaviour of Lactococcus lactis subsp. lactis IPLA 947 in anaerobic lactose-limited chemostat cultures

Lactococcus lactis subsp. lactis IPLA 947 is a starter strain isolated from a Spanish farmhouse acid-coagulated cheese. To use this strain as starter culture in Afuegal Pitu cheese manufacture, anaerobic lactose-limited chemostat cultures were used to optimise the active biomass production in a simple medium (BRFS). Growth, lactose consumption and metabolite production were measured at different pH values (5.5, 6.0 and 6.5) and dilution rates (0.06 to 0.96 h^–1). The influence of both variables on the quoted parameters was described by means of mathematical models. These provided a satisfactory representation of the experimental data. The bacterial response surface showed a significant increase in the biomass when the pH increased and the dilution rate decreased. As dilution rate increased, biomass yield increased but only at the lowest pH. The lactose consumption and the ratio of metabolic end products were also influenced by both variables. As dilution rate decreased, the residual lactose decreased and increased amounts of metabolites other than lactate (acetate, formate, ethanol) were produced. The minimum lactose consumption was obtained at pH 5.5 and 0.96 h^–1 dilution rate. A significant rise of acetate and formate levels occurred as pH increased. A maximum lactate level, along with an acetate, formate and ethanol production close to the minimum, was predicted by mathematical modelling at 0.5173 h^–1 dilution rate and pH 6.054.

In vitro antimicrobial effect and in vivo preventive and therapeutic effects of partially purified lantibiotic lacticin NK34 against infection by Staphylococcus species isolated from bovine mastitis

Lantibiotics are small (<5 kDa), polycyclic peptides produced by gram-positive bacteria; they are also known as gram-positive bacteriocins. The high antimicrobial activity of lacticins and the continuing appearance of antibiotic-resistant bacteria in recent years have resulted in a renewed interest in lantibiotics. A partially purified form of lacticin NK34 (a Lactococcus lactis product isolated from the Korean fermented fish jeotgal) was tested to determine its antimicrobial effects against Staphylococcus aureus (n = 20) and coagulase-negative Staphylococcus (CNS, n = 20) strains isolated from the raw milk of cows with subclinical bovine mastitis in the present study. The spot-on-lawn assay was used to identify the 2 strains from each group with the greatest lacticin NK34 susceptibility, and the minimal lethal dose (MLD) was measured in ICR (imprinting control region) mice. The preventive and therapeutic effects of lacticin NK34 on the mouse infection model were determined for the first time. Lacticin NK34 demonstrated antimicrobial effects in 14 of 20 (70%) S. aureus indicator strains and in 18 of 20 (90%) CNS strains. Staphylococcus aureus 69 and S. simulans 55 demonstrated the greatest susceptibility to lacticin NK34 in the spot-on-lawn assay. The S. aureus 69 MLD was measured at 1.53 × 10⁹ cfu/mouse, whereas the S. simulans 55 MLD was 3.59 × 10⁹ cfu/mouse. Mice infected experimentally with S. aureus 69 MLD or S. simulans 55 MLD were treated with lacticin NK34. Treated mice demonstrated an 80% survival rate (48 of 60 mice) compared with a survival rate of 7.5% (3 of 40 mice) in control mice treated with distilled water. These data suggest that lacticin NK34 might be useful in the control of bovine mastitis and systemic bacterial infection.     

Cooperation between wild lactococcal strains for cheese aroma formation

Several wild lactococcal strains were tested for their ability to produce aroma compounds during growth in milk. Strains were incubated alone and in combination with Lactococcus lactis IFPL730, which is characterized by showing α-keto acid decarboxylase activity. Volatile compounds from incubated milks were analyzed by means of solid-phase microextraction (SPME) and evaluated by gas chromatography–mass spectrometry (GC–MS). Incubated milks were also sniffed for sensory analysis to describe aroma attributes. The combination of L. lactis IFPL326 that showed the highest branched chain aminotransferase activity with IFPL730 contributed to the highest formation of leucine-derived volatile compounds, such as 3-methylbutanal, 3-methyl-1-butanol and 2-hydroxy-4-methyl pentanoic acid methyl ester. In addition, the milk incubated with this combination of strains was awarded, by the test panellists, the highest scores for “ripened cheese” attribute and aroma intensity. The results indicate that combination of L. lactis strains harbouring complementary catabolic routes can contribute to improved cheese aroma formation, the combined cultures with L. lactis IFPL730 resulting in higher volatile compound formation than isolate strains.     

Arginine metabolism in sugar deprived Lactococcus lactis enhances survival and cellular activity, while supporting flavour production

Flavour development in cheese is affected by the integrity of Lactococcus lactis cells. Disintegrated cells enhance for instance the enzymatic degradation of casein to free amino acids, while integer cells are needed to produce specific flavour compounds from amino acids. The impact of the cellular activity of these integer cells on flavour production remains to be elucidated. In this study we investigated whether lactose-deprived L. lactis cells that use arginine as an alternative energy source can extend cellular activity and produce more specific flavours. In cheese experiments we demonstrated that arginine metabolising cells survived about 3 times longer than non-arginine metabolising cells, which suggests prolonged cellular activity. Cellular activity and flavour production of L. lactis was further studied in vitro to enable controlled arginine supplementation. Comparable with the results found in cheese, the survival rates of in vitro incubated cells improved when arginine was metabolised. Furthermore, elongated cellular activity was reflected in 3-4-fold increased activity of flavour generating enzymes. The observed prolonged cellular activity resulted in about 2-fold higher concentrations of typical Gouda cheese flavours. These findings provide new leads for composing starter cultures that will produce specific flavour compounds.     

Antimicrobial activity of pediocin PA-1 against Oenococcus oeni and other wine bacteria

Pediocin PA-1 is an antimicrobial peptide produced by lactic acid bacteria (LAB) that has been sufficiently well characterised to be used in food industry as a biopreservative. Sulphur dioxide is the traditional antimicrobial agent used during the winemaking process to control bacterial growth and wine spoilage. In this study, we describe the effect of pediocin PA-1 alone and in combination with sulphur dioxide and ethanol on the growth of a collection of 53 oenological LAB, 18 acetic acid bacteria and 16 yeast strains; in addition, production of pediocin PA-1 by Pediococcus acidilactici J347-29 in presence of ethanol and grape must is also reported. Inhibitory concentrations (IC) and minimal bactericide concentrations of pediocin PA-1 were determined against LAB, and revealed a bacteriostatic effect. Oenococcus oeni resulted more sensitive to pediocin PA-1 (IC₅₀ = 19 ng/ml) than the other LAB species (IC₅₀ = 312 ng/ml). Cooperative inhibitory effects of pediocin PA-1 and either sulphur dioxide or ethanol were observed on LAB growth. Moreover, the pediocin PA-1 producing P. acidilactici strain J347-29 was able to grow and produce the bacteriocin in presence of ethanol (up to 4% ethanol in the fermentation broth) and grape must (up to 80%), which indicated that pediocin PA-1 can be considered as a potential biopreservative in winemaking.     

Effect of Lactococcus garvieae, Lactococcus lactis and Enterococcus faecalis on the behaviour of Staphylococcus aureus in microfiltered milk

The effect of four strains of Lactococcus garvieae, three strains of Lactococcus lactis and one strain of Enterococcus faecalis on Staphylococcus aureus SA15 growth in microfiltered milk was evaluated. Lactococcus and Enterococcus strains were co-cultured with S. aureus in microfiltered milk and in medium buffered at pH 6.8. All Lactococcus and Enterococcus strains were able to inhibit S. aureus growth after 6h of incubation. Inhibition by L. lactis and E. faecalis strains could be partially attributed to the decrease in pH below 6.0 as it has been observed in medium buffered at pH 6.8. L. garvieae strains were the most effective to inhibit S. aureus growth without acidification. Inhibition of S. aureus could not be attributed neither to production of lactate, acetate or nor to antistaphylococcal substance. Amino acids competition was not involved in the inhibition by L. garvieae as addition of valine, isoleucine, threonine, methionine and phenylalanine did not suppress the inhibition of S. aureus.     

Genetic characterization of the CcpA-dependent, cellobiose-specific PTS system comprising CelB, PtcB and PtcA that transports lactose in Lactococcus lactis IL1403

Lactose metabolism is one of the most important areas of research on Lactic Acid Bacteria (LAB). In rapidly acidifying industrial Lactococcus lactis strains, lactose is transported by a lactose-specific phosphotransferase system (PTS) encoded by a plasmid. However, an alternative lactose catabolic pathway was evidenced in the plasmid-cured, and thus initially lactose-negative L. lactis IL1403. We showed that in this strain the chromosomally-encoded cellobiose-specific PTS system comprising the celB, ptcB and ptcA genes is also able to transport lactose. By expression studies in the wild type IL1403 strain and IBB550, its ccpA-deficient derivative, we demonstrated that celB, ptcB and ptcA are tightly regulated by the general catabolite repression system, whereas celB additionally requires the presence of cellobiose to be fully induced. The comparison of expression levels of sugar catabolic genes indicated that the efficiency of CcpA-mediated catabolic repression depends on conservation of the cre sequence, and that in the case of perfect matching with the cre consensus, CcpA still drives a strong repression even under non-repressing conditions.     

Validation of a model for growth of Lactococcus lactis and Listeria innocua in a structured gel system: effect of monopotassium phosphate

The effect of monopotassium phosphate (KH(2)PO(4)) on the chemical environment and on growth of Listeria innocua and Lactococcus lactis in coculture were investigated in a liquid and in a gelled microbiological medium at 12 degrees C and an initial pH of 6.2. As expected, addition of KH(2)PO(4) to both the liquid and gelled media resulted in an increase in buffering capacity. This effect on buffering capacity changed the profiles of lactic acid dissociation and pH evolution. At all gelatin concentrations studied, addition of KH(2)PO(4) increased the growth rate and the stationary cell concentration of L. lactis. In addition, the growth rate of L. innocua slightly increased but, in contrast, the stationary cell concentration remained unchanged. A new class of predictive models developed previously in our research team to quantify the effect of food model gel structure on microbial growth [Antwi, M., Bernaerts, K., Van Impe, J. F., Geeraerd, A. H., 2007. Modelling the combined effect of food model system and lactic acid on L. innocua and L. lactis growth in mono- and coculture. International Journal of Food Microbiology 120, 71-84] was applied. Our analysis indicate that KH(2)PO(4) influenced the parameters of the chemical and microbiological subprocesses of the model. Nonetheless, the growth model satisfactorily predicted the stationary cell concentration when (i) the undissociated lactic acid concentrations at which L. innocua and L. lactis growth cease were chosen as previously reported, and (ii) all other parameters of the chemical and microbiological subprocesses were computed for each medium. This confirms that the undissociated lactic acid concentrations at which growth ceases is a unique property of a bacterium and does not, within our case study, depend on growth medium. The study indicates that microbial growth depends on the interplay between the individual food components which affect the physicochemical properties of the food, such as the buffering capacity. Towards future research, it can be concluded that mathematical models which embody the effect of buffering capacity are needed for accurate predictions of microbial growth in food systems.