Production of volatile sulfur compounds in cheese is associated with desirable flavors. The direct source of these compounds has been assumed to arise from the metabolism of methionine and cysteine. However, the methionine concentration in cheese rises above the amount found in casein during aging, suggesting that alternative sulfur sources are present in milk. This led us to hypothesize that lactococci may acquire sulfur from the inorganic sulfur pool of milk, in addition to methionine and cysteine, to generate volatile sulfur compounds during cheese ripening. A turbidimetric method to determine total sulfate content in milk samples was developed. The average sulfate content of milk was determined to be ~49 mg/L ± 2.0 mg/L. The limit of detection of the test was ~2.5 mg/L in Tris buffer and ~10 mg/L in milk. Skim milk samples had significantly higher total sulfate content as compared to whole milk samples. Transport of sulfate by three strains of Lactococcus sp. was studied after we determined that milk had small, but measurable amounts of inorganic sulfate. A decrease in the environmental pH increased sulfate transport. The maximum transport occurred during exponential cellular growth phase. All strains tested had the ability to transport much more sulfate than is native in milk. The last phase of study was to determine the metabolic fate of sulfate. Incorporation of radio-labeled sulfate into cellular protein was studied by two-dimensional gel-electrophoresis of crude cellular lysate followed by auto-radiography. Production of volatile sulfur compounds from inorganic sulfur was determined with analysis of the head space gas with gas chromatography and scintillation counting. The incorporation of radio-labeled sulfur from sulfate was not detected in proteins on two-dimensional gels. Detectable volatile sulfur compounds were found only in the case of gas chromatographic analysis of ML3 head space. However, radio-labeled volatile sulfur was detected in the head space of all the three strains with scintillation counting. This study defined that lactococci can fix inorganic sulfur into volatile sulfur compounds in small amounts.

Modern, large scale Cheddar cheese manufacture is dependent on reliable acid production by Lactococcus lactis subspecies cremoris and subspecies lactis starter cultures. Any inhibition of acid production may affect cheese quality, disrupt production schedules and reduce profitability. The presence of antibiotic residues in manufacturing milk resulting from the treatment of mastitis in lactating cattle is a potential source of starter culture inhibition. Therefore, a range of antibiotic concentrations was assessed for measurable inhibitory effects on acid production and compared to the minimum detectable concentrations by approved screening test procedures. Antibiotics were selected from formulations approved for use on lactating cattle for the treatment of mastitis. Novobiocin, lincomycin, oleandomycin and oxytetracyline HCl, all non-b-lactam antibiotics, inhibited acid production of one or more L. lactis strains at antibiotic concentrations below the detectable limit of standard screening procedures. / Depending on the antibiotic, either or both the Bacillus stearothermophilus (var. calidolactis) disk assay and/or the Delvo SP assay were ineffective at detecting the antibiotics at concentrations required to inhibit the starter strains. Consequently, antibiotic residues below the detectable limits of these testing procedures could cause significant starter culture inhibition, disrupting cheese making schedules. Another potential source of starter culture inhibition is related to raw milk quality and the practice of refrigerated storage prior to processing. Previous studies differed as to whether the growth of psychrotrophic organisms would have a detrimental impact on subsequent acid production by starter bacteria employed in cheese manufacture. In this study, no inhibition of acid production by a commercial L. lactis subsp. cremoris strain was evident when grown in milk that had undergone short term temperature abuse. Antimicrobial systems native to bovine milk may also have an adverse impact on starter culture performance. The present study assessed the inhibitory effect of an activated lactoperoxidase system (LPS) on a range of L. lactis cultures. All of the strains were significantly inhibited when grown on reconstituted skim milk in the presence of an active LPS. Inhibition of acid production by strains grown on glucose was also observed, leading to further investigations to describe the inhibitory process. A non-phosphoenolpyruvate phosphotransferase (PEP/PTS) dependent glucose transport system, first observed in 1980 in one L. lactis subsp. lactis strain, was hypothesised as a link in strain variations in LPS sensitivity. However, the LPS sensitive L. lactis subsp. cremoris strains tested did not take up glucose in a PEP depleted state, most likely due to their inability to utilise arginine as an ATP generating energy source. The questions remain unanswered whether cremoris strains possess this glucose transport mechanism and whether it could contribute to strain variations in LPS sensitivity. / In a subsequent investigation, galactose phosphotransferase system (PTS) deficient L. lactis strain ATCC 7962 demonstrated log phase growth inhibition when grown on galactose in the presence of the model LPS. Previously reported LPS mediated effects on the glycolytic enzyme hexokinase do not appear to explain this result. The present study confirmed strain variability in sensitivity to the model LPS among both Lactococcus lactis subspecies lactis and subspecies cremoris strains. Further, the observation that dithiothreitol significantly alleviated the inhibition of a highly sensitive cremoris strain, implicated the involvement of sulphydryl groups as the target of the transient inhibitory factors. Data collected excluded the possibility that portions of the metabolic pathways involved in fructose and galactose metabolism are sensitive to the LPS in cells possessing PEP/PTS capability. This study also identified potential directions of further work to elucidate the mechanism(s) of LPS inhibition.

Lactococcus is an economically important group in lactic acid bacteria (LAB) that are often used in the dairy industry as starters for cheese production. Good starter strains should possess the ability to grow, ferment milk sugar, and produce desirable flavor compounds during cheese making. Therefore, it is essential to understand the physiology of these starters during cheese processing in order to obtain high-quality cheese products. Cheese manufacturing compromises several stress factors that affect the growth of starter lactococci. Among these stressed environmental parameters, sugar starvation is the most important one to overcome to obtain energy for cellular processes. It is known that degradation of arginine produces energy. In this study, we investigated arginine utilization by Lactococcus lactis ssp. lactis strain ML3 via the arginine deiminase (ADI) pathway to see its influence on cellular physiology after exhaustion of a primary energy source. During the cell growth in a carbohydrate-limited environment, we observed that metabolic pathways switched between lactose utilization arginine degradation. The statistical model described in this study suggested lactose and arginine were co-metabolized during cell growth. These results initially showed arginine was a good candidate of secondary energy source after exhaustion of primary energy source (lactose). To confirm these observations, cell counts, cellular ATP levels, ADI enzyme activities, and total protein expression were compared in arginine-positive L. lactis ssp. lactis ML3 and arginine-negative L. lactis ssp. cremoris Sl grown in medium containing 0.2% lactose and 2% arginine. Results showed ATP levels remained high in strain ML3, in which a transition stage of protein expression pattern was also observed. This physiological evidence highlights the important roles of arginine degradation in starved ML3, perhaps by producing extra ATP and modulating external pH. The genes involved in the ADI pathway of strain ML3 were cloned, sequenced, and characterized. Genes involved in this pathway formed a unique multi-operon cluster structure that we termed MOC. It was organized as arcA, arcBD1, arcC1C2, and arcTD2. The influence of different environmental parameters including pH, various amino acids, and phosphate (organic and inorganic) on the expression of the ADI MOC was tested. No single factor regulated the entire MOC simultaneously. It is concluded that the unique structure of the MOC appears to allow the ADI pathway to occur in discrete sections in response to fluctuated external conditions, such as sugar starvation and low environmental pH.

L’utilisation de bactéries lactiques (BL), telle que Lactococcus lactis (LL), comme vecteur de transfert d’ADN, constitue une stratégie prometteuse dans la mesure où elles sont considérées sans risque pour la santé. Des souches sauvages (wt) ou recombinantes de LL ont été décrites comme capables de transférer un plasmide dans des cellules épithéliales in vitro et in vivo. Cependant, les mécanismes d'action grâce auxquels certaines souches de LL ont la capacité de transférer de l’ADN plasmidique sont toujours inconnus. C’est pourquoi, nous avons décidé de construire une nouvelle souche recombinante de LL exprimant l’internaline mutée (mlnlA,) à partir de la souche pathogène Listeria monocytogenes, de manière à comprendre par quel procédé l’ADN est transféré à des cellules eucaryotes. Nous avons détecté l’expression de mInIA par FACS et montré que la souche LLmInIA était plus invasive que la souche sauvage wt après co-incubation avec des cellules épithéliales intestinales (IECs) non confluentes ou polarisées. La microscopie confocale confirme ces propriétés d’invasivité de la souche LL-mLnLA capable de transférer plus efficacement le vecteur d’expression eucaryote codant pour l’allergène de la β-lactoglobuline, pValac :BLG, in vitro dans des IECs et dans des cellules dendritiques (DCs). La souche LL-mInIA a aussi la capacité de transférer le vecteur pValac:BLG à des DCs à travers une monocouche de IECs différenciées. Des essais in vivo montrent que des bactéries invasives du genre Lactococcus ont tendance à augmenter l’expression de BLG chez la souris. De plus, il est montré qu’une souche non invasive de LL, ou la souche invasive LL-mInIA, stimulent la sécrétion de la cytokine pro-inflammatoire IL-12 dans des DCs, et que, in vivo, après des essais d’immunisation oraux ou intra nasaux, la souche LL non invasive oriente la réponse immunitaire plutôt vers le type 1, alors que la souche LL invasive génère une réponse de type 2 chez des animaux immunisés. Tous ces résultats apportent un nouvel éclairage sur le mécanisme d’assimilation des lactocoques en tant que vecteurs de transfert de molécules actives. / The use of Lactic Acid Bacteria (LAB), such as Lactococcus lactis (LL), as DNA delivery vehicles represents an interesting strategy as they are regarded as safe. Wild type (wt) LL or recombinant invasive LL, were able to trigger DNA expression by epithelial cells both in vitro and in vivo. However, important information about how LL can transfer DNA plasmids is still missing. Therefore, we decided to construct a new recombinant invasive LL strain expressing mutated Internalin A (mInlA) from the pathogen Listeria monocytogenes to understand the manner by which the DNA is transferred to mammalian cells. mInlA expression was detected by FACS analysis and LL-mInlA strain showed to be more invasive than the wt strain after co-incubation assays with non-confluent or polarized intestinal epithelial cells (IECs). Confocal microscopy confirmed the invasive status of LL-mInlA which demonstrated to deliver more efficiently the eukaryotic expression vector coding the allergen β-lactoglobulin, pValac:BLG, in vitro to IECs and to dendritic cells (DCs). LL-mInlA was also capable to transfer pValac:BLG to DCs across a monolayer of differentiated IECs. In vivo, invasive lactococci tended to increase the number of mice expressing BLG. Moreover, noninvasive or invasive LL-mInlA stimulated the secretion of the pro-inflammatory cytokine IL-12 in DCs and, in vivo, after oral or intranasal immunization trials, non-invasive LL polarized the immune response more in the type 1 direction while invasive LL generated a Th2-type response in immunized animals. All these data gives new insights on the mechanism of lactococci uptake for delivery of therapeutics.

The use of Lactic Acid Bacteria (LAB), such as Lactococcus lactis (LL), as DNA delivery vehicles represents an interesting strategy as they are regarded as safe. Wild type (wt) LL or recombinant invasive LL, were able to trigger DNA expression by epithelial cells both in vitro and in vivo. However, important information about how LL can transfer DNA plasmids is still missing. Therefore, we decided to construct a new recombinant invasive LL strain expressing mutated Internalin A (mInlA) from the pathogen Listeria monocytogenes to understand the manner by which the DNA is transferred to mammalian cells. mInlA expression was detected by FACS analysis and LL-mInlA strain showed to be more invasive than the wt strain after co-incubation assays with non-confluent or polarized intestinal epithelial cells (IECs). Confocal microscopy confirmed the invasive status of LL-mInlA which demonstrated to deliver more efficiently the eukaryotic expression vector coding the allergen β-lactoglobulin, pValac:BLG, in vitro to IECs and to dendritic cells (DCs). LL-mInlA was also capable to transfer pValac:BLG to DCs across a monolayer of differentiated IECs. In vivo, invasive lactococci tended to increase the number of mice expressing BLG. Moreover, noninvasive or invasive LL-mInlA stimulated the secretion of the pro-inflammatory cytokine IL-12 in DCs and, in vivo, after oral or intranasal immunization trials, non-invasive LL polarized the immune response more in the type 1 direction while invasive LL generated a Th2-type response in immunized animals. All these data gives new insights on the mechanism of lactococci uptake for delivery of therapeutics.