The research described in this technical research communication examines the hypothesis that sublethal stress conditions can improve the survival of Lactococcus lactis subsp. lactis during drying and subsequent storage. After drying, the L. lactis that had adapted to acid or osmotic stresses did not differ statistically in terms of cell viability loss compared to the control samples tested (~0.38 log cycles). However, the cells that had adapted to oxidative conditions demonstrated a cell viability loss of only 0.01 log cycles. After 45 d of storage at temperatures of 4 and 25 °C, the final L. lactis sample populations were shown to be higher (112.5%) when they had been submitted to sublethal conditions of oxidative stress. When the cell samples were exposed to acid stress conditions, they exhibited a viability loss (0.82 log cycles) that was statistically different from the control sample (0.58 log cycles) after 45 d. Osmotic stress conditions did not demonstrate any influence over cell survival rates. Thus, submitting cells to oxidative stress conditions prior to storage has been shown to be a potential strategy for producing dehydrated cultures of L. lactis strains that are less sensitive to oxygen exposure.

The dairy industry uses lipase extensively for hydrolysis of milk fat. Lipase is used in the modification of the fatty acid chain length, to enhance the flavours of various chesses. Therefore finding the unlimited source of lipase is a concern of dairy industry. Due to the importance of lipase, this study was an attempt to express the lipase from Burkholderia cepacia in Lactococcus lactis. To achieve this, a gene associated with lipase transport was amplified and subcloned in inducible pNZ8148 vector, and subsequently transformed into Lc. lactis NZ9000. The enzyme assay as well as SDS-PAGE and western blotting were carried out to analysis the recombinant lipase expression. Nucleotide sequencing of the DNA insert from the clone revealed that the lipase activity corresponded to an open reading frame consisting of 1092 bp coding for a 37·5-kDa size protein. Blue colour colonies on nile blue sulphate agar and sharp band on 37·5-kD size on SDS-PAGE and western blotting results confirm the successful expression of lipase by Lc. lactis. The protein assay also showed high expression, approximately 152·2 μg/ml.h, of lipase by recombinant Lc. lactis. The results indicate that Lc. lactis has high potential to overproduce the recombinant lipase which can be used commercially for industrially purposes.

Previous studies have demonstrated that milk fermented by specific Lactococcus lactis strains significantly inhibits the activity of angiotensin I-converting enzyme (ACE). However, the relationship between the ACE inhibitor and its in vivo action has revealed discrepancies. Therefore, the aim of the present study was to investigate the antihypertensive and heart rate (HR)-lowering effect of milk fermented by specific L. lactis in a murine model. Spontaneously hypertensive male rats (271 (SD14) g) were randomised into four treatment groups that were orally administered with milk fermented by L. lactis NRRL B-50 571 or L. lactis NRRL B-50 572 at 35 or 50 mg protein/kg body weight (BW), respectively. Further, two more groups were fed with different solutions as controls: a saline solution as the negative control and Captopril™ (40 mg/kg BW), a proven ACE inhibitor, as the positive control. Blood pressure and HR were monitored by the tail-cuff method before the treatments and at 2, 4, 6 and 24 h post-oral administration. The results demonstrated that milk fermented by L. lactis NRRL B-50 571 as well as by L. lactis NRRL B-50 572 presented an important systolic and diastolic blood pressure- and HR-lowering effect. Thus, milk fermented by specific L. lactis strains may present potential benefits in the prevention and treatment of CVD associated with hypertension in humans.

Mastitis is one of the most costly diseases to the dairy farming industry. Conventional antibiotic therapy is often unsatisfactory for successful treatment of mastitis and alternative treatments are continually under investigation. We have previously demonstrated, in two separate field trials, that a probiotic culture, Lactococcus lactis DPC 3147, was comparable to antibiotic therapy to treat bovine mastitis. To understand the mode of action of this therapeutic, we looked at the detailed immune response of the host to delivery of this live strain directly into the mammary gland of six healthy dairy cows. All animals elicited signs of udder inflammation 7 h post infusion. At this time, clots were visible in the milk of all animals in the investigation. The most pronounced increase in immune gene expression was observed in Interleukin (IL)-1β and IL-8, with highest expression corresponding to peaks in somatic cell count. Infusion with a live culture of a Lc. lactis leads to a rapid and considerable innate immune response.

In order to establish cut-off values for Lactococcus lactis to six antibiotics to distinguish susceptible and intrinsically resistant strains from those having acquired resistances, the minimum inhibitory concentration (MIC) of tetracycline, erythromycin, clindamycin, streptomycin, chloramphenicol and vancomycin was determined in 93 different Lc. lactis strains using the Etest. These bacterial strains were originally isolated from dairy and animal sources in widely separated geographical locations. Cut-offs were defined on the basis of the distribution of the MICs frequency of the studied antibiotics, which in the absence of acquired determinants should approach to a normal statistical distribution. In general, the new cut-off values proposed in this study are higher than previously defined (European Commission, 2005. The EFSA Journal 223, 1–12). Based on these new values, all the strains tested were susceptible to erythromycin, chloramphenicol and vancomycin, and 79 susceptible to all six antibiotics. However, 11 strains (around 12%) were considered resistant to tetracycline (six of which had been identified after screening of a large collection of lactococci strains for tetracycline resistance) and five (5·4%) resistant to streptomycin. Of these, two fish isolates proved to be resistance to both tetracycline and streptomycin. From the tetracycline resistant strains, tet(M) and mosaic tet(L/S) genes were amplified by PCR, demonstrating they harboured acquired antibiotic resistance determinants.

Microbial diversity of the raw milk for the production of Fior di Latte di Agerola and its changes during cheesemaking were studied. Viable counts showed that at the end of curd ripening, loads of lactic acid bacteria, both mesophilic and thermophilic rods and cocci, higher than those commonly evidenced in similar cheeses produced by using natural or commercial starters, were detected. Identification of 272 isolates, supported by molecular diagnostic aids, evidenced representative cultures of a high number of bacterial taxa of interest as participating in the process, although most of the isolates belonged to Lactococcus lactis and Lactobacillus helveticus species. RAPD-PCR and REA-PFGE biotyping were performed for the isolates of the above species and it was shown that most of the strains isolated from the raw milk occurred during the whole cheesemaking process, and an active role of these strains in the fermentation was supposed. The results offer further proof of the importance of the raw milk as source of technologically interesting strains of lactic acid bacteria capable of driving the fermentation of traditional cheeses.

The thermal resistance of the lactococcal bacteriophage P008 was investigated between 55 and 80 °C. Inactivation kinetics revealed an order of reaction above 1 and could be determined by a non-1st-order regression model. Phage inactivation was influenced by the medium (milk and Ca-M17-broth). Within the investigated temperature range, milk had a protective effect on phage P008. This was reflected in the rate constant and in the activation energy. Thermal phage inactivation studies reported in literature were re-analysed using non-1st-order regression. The obtained kinetic parameters showed that phage P008 belongs to the most heat resistant lactococcal phages investigated so far.

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.

A new method for characterization of acid production by dairy starter cultures is presented. Microplates with integrated optical pH sensors are developed. Two fluorophores, a pH-sensitive and a pH-insensitive one are immobilised at the bottom of a polystyrene 96-well microtitre plate. The pH-insensitive fluorophore serves as an internal reference and makes calibration unnecessary. The sensor measures pH accurately in optically well-defined media. Particles and fluorophores contained in the bulk medium disturbed the measurements. Despite these disturbances it was possible to clearly sense differences in inoculum type and in inoculum sizes of cultures of Lactococcus lactis and of Streptococcus thermophilus at 30 and 37°C. Besides a pH-related signal there is information about other changes during milk fermentation. The cultivation results were compared with those from the established CINAC-method. From this comparison it can be concluded that the new method can be used reliably to characterize particularly a large number of strains for screening purposes but also for quality control.

Since genetically modified (GM) lactic acid bacteria (LAB) might be released in open environments for future nutritional and medical applications, the purpose of this study was to determine an upper limit for the horizontal gene transfer (HGT) in the digestive tract (DT) from Lactococcus lactis carrying heterologous genes (lux genes encoding a bacterial luciferase) to Enterococcus faecalis. Two enterococcal wide host-range conjugative model systems were used: (i) a system composed of a mobilizable plasmid containing the heterologous lux genes and a native conjugative helper plasmid; and (ii) a Tn916-lux transposon. Both systems were tested under the most transfer-prone conditions, i.e. germfree mice mono-associated with the recipient E. faecalis. No transfer was observed with the transposon system. Transfers of the mobilizable plasmid carrying heterologous genes were below 102 transconjugants per g of faeces for a single donor dose and reached between 103 and 104 transconjugants per g of faeces when continuous inoculation of the donor strain was used. Once established in mice, transconjugants persisted at low levels in the mouse DT.