A Multibacteriocin Cheese Starter System, Comprising Nisin and Lacticin 3147 in Lactococcus lactis, in Combination with Plantaricin from Lactobacillus plantarum.

Functional starter cultures demonstrating superior technological and food safety properties are advantageous to the food fermentation industry. We evaluated the efficacies of single- and double-bacteriocin-producing starters of Lactococcus lactis capable of producing the class I bacteriocins nisin A and/or lacticin 3147 in terms of starter performance. Single producers were generated by mobilizing the conjugative bacteriophage resistance plasmid pMRC01, carrying lacticin genetic determinants, or the conjugative transposon Tn5276, carrying nisin genetic determinants, to the commercial starter L. lactis CSK2775. The effect of bacteriocin coproduction was examined by superimposing pMRC01 into the newly constructed nisin transconjugant. Transconjugants were improved with regard to antimicrobial activity and bacteriophage insensitivity compared to the recipient strain, and the double producer was immune to both bacteriocins. Bacteriocin production in the starter was stable, although the recipient strain proved to be a more efficient acidifier than transconjugant derivatives. Overall, combinations of class I bacteriocins (the double producer or a combination of single producers) proved to be as effective as individual bacteriocins for controlling Listeria innocua growth in laboratory-scale cheeses. However, using the double producer in combination with the class II bacteriocin producer Lactobacillus plantarum or using the lacticin producer with the class II producer proved to be most effective for reducing bacterial load. As emergence of bacteriocin tolerance was reduced 10-fold in the presence of nisin and lacticin, we suggest that the double producer in conjunction with the class II producer could serve as a protective culture providing a food-grade, multihurdle approach to control pathogenic growth in a variety of industrial applications.IMPORTANCE We generated a suite of single- and double-bacteriocin-producing starter cultures capable of generating the class I bacteriocin lacticin 3147 or nisin or both bacteriocins simultaneously via conjugation. The transconjugants exhibited improved bacteriophage resistance and antimicrobial activity. The single producers proved to be as effective as the double-bacteriocin producer at reducing Listeria numbers in laboratory-scale cheese. However, combining the double producer or the lacticin-producing starter with a class II bacteriocin producer, Lactobacillus plantarum LMG P-26358, proved to be most effective at reducing Listeria numbers and was significantly better than a combination of the three bacteriocin-producing strains, as the double producer is not inhibited by either of the class I bacteriocins. Since the simultaneous use of lacticin and nisin should reduce the emergence of bacteriocin-tolerant derivatives, this study suggests that a protective starter system produced by bacteriocin stacking is a worthwhile multihurdle approach for food safety applications.

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.

CRISPR analysis of bacteriophage-insensitive mutants (BIMs) of industrial Streptococcus thermophilus--implications for starter design.

AIMS: An efficient approach for generation of bacteriophage-insensitive mutants (BIMs) of Streptococcus thermophilus starters was described in our laboratory [Mills et al. (2007) J Microbiol Methods70, 159-164]. The aim of this study was to analyse the phage resistance mechanism responsible for BIM formation. METHODS AND RESULTS: Three clustered regularly interspaced short palindromic repeat (CRISPR) regions have been identified in Strep. thermophilus, and Strep. thermophilus can integrate novel spacers into these loci in response to phage attack. Characterization of three sets of BIMs indicated that two sets had altered CRISPR1 and/or CRISPR3 loci. A range of BIMs of yoghurt starter CSK938 were generated with the same phage in different phage challenge experiments, and each acquired unique spacer regions ranging between one and four new spacers in CRISPR1. In addition, the BIM that acquired only one new spacer in CRISPR1 also acquired an additional spacer in CRISPR3. A fourth BIM, generated with a different phage, had two spacers deleted from CRISPR1 but acquired two spacers in CRISPR3. Analysis of the Mozzarella starter CSK939 and its associated BIMs indicated that formation of second generation BIMs does not lead to increases in spacer number but to alterations in spacer regions. BIMs of an exopolysaccharide (EPS)-producing strain that lost the ability to produce EPS did not harbour an altered CRISPR, suggesting that phage sensitivity may be related to the EPS-producing phenotype. CONCLUSIONS: Acquisition/deletion of new spacers in CRISPR loci in response to phage attack generates distinctly individual variants. It also demonstrates that other modifications may be responsible for the phage resistance of Strep. thermophilus BIMs. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolation of individual BIMs that have unique spacers towards the leader region of the CRISPR locus may be a very useful approach for rotation strategies with the same starter backbone. Upon phage infection, BIMs 'in reserve' can be slotted into the rotation scheme.

Efficient method for generation of bacteriophage insensitive mutants of Streptococcus thermophilus yoghurt and mozzarella strains.

Bacteriophage infection of Streptococcus thermophilus is becoming increasingly problematic in many industry fermentations such as yoghurt and mozzarella manufacture. This study describes the development of an efficient and rapid 3-step approach for the generation of bacteriophage insensitive mutants (BIMs) of these starter strains. The method initially involves infection of a culture in solid media at a multiplicity of infection (M.O.I.) of 10 which is then incubated in milk overnight. BIMs are then isolated following successive rounds (20-25) of growth in 10% reconstituted skimmed milk (RSM) in the presence of high phage titres. The method selects for BIMs which can grow efficiently in milk. Using this approach BIMs of two industrial strains were generated, whose starter performance was comparable to the parent starters in terms of performance in milk. Genomic fingerprinting used to validate the identity of each BIM, revealed a number of restriction fragment length polymorphisms (RFLPs) in two of the resultant BIMs. This method provides a simple and reliable method for generation of BIMs of industrial starters which does not require any specialised equipment and should be widely applicable.

Cardiovascular abnormalities in patients with a carcinoid syndrome.

BACKGROUND: Heart failure is an important reason for morbidity and mortality in patients with carcinoid. Carcinoid heart disease is caused by increased levels of circulating serotonin. Because carcinoids also produce catecholamines, we evaluated cardiovascular manifestations of autonomic dysfunction in patients with a carcinoid syndrome. METHODS: Twenty patients with a midgut carcinoid, who had a carcinoid syndrome with a median duration of 72 months, and markedly elevated urinary 5-hydroxyindoleacetic acid (5-HIAA) excretion were studied. RESULTS: Ten patients had no symptoms of heart failure, i.e. New York Heart Association (NYHA) functional class I, 6 had class II, and 4 class III heart failure. Transthoracic echocardiography (TTE) showed right-sided valvular abnormalities in 13 of 19 evaluable patients (mild n=8, severe n=5). Fourteen of the 20 patients (70%) had an elevated concentration of plasma N-terminal atrial natriuretic peptide (N-ANP), which correlated with NYHA class, TTE abnormalities, and increased urinary metanephrine excretion. Heart rate variability (HRV) parameters, in particular those associated with increased sympathetic activity (low frequency power, p=0.002 versus healthy individuals), were impaired but were independent of NYHA class and TTE findings and correlated with urinary metanephrine excretion (r=-0.49, p<0.05). CONCLUSION: In these 20 carcinoid patients with substantial secretory activity of the tumour, overt cardiac morphological changes were present in a minority of patients. However, N-ANP values and HRV profile were markedly abnormal, and related to enhanced urinary excretion of catecholamine and metabolites, suggesting autonomic derangement. These abnormalities possibly herald the development of more severe cardiac dysfunction and may be indicative of the need for preventive drug treatment.

Food-grade controlled lysis of Lactococcus lactis for accelerated cheese ripening.

An attractive approach to accelerate cheese ripening is to induce lysis of Lactococcus lactis starter strains for facilitated release of intracellular enzymes involvement in flavor formation. Controlled expression of the lytic genes lytA and lytH, which encode the lysin and the holin proteins of the lactococcal bacteriophage phi US3, respectively, was accomplished by application of a food-grade nisin-inducible expression system. Simultaneous production of lysin and holin is essential to obtain efficient lysis and concomitant release of intracellular enzymes as exemplified by complete release of the debittering intracellular aminopeptidase N. Production of holin alone leads to partial lysis of the host cells, whereas production of lysin alone does not cause significant lysis. Model cheese experiments in which the inducible holinlysin overproducing strain was used showed a fourfold increase in release of L-Lactate dehydrogenase activity into the curd relative to the control strain and the holin-overproducing strain, demonstrating the suitability of the system for cheese applications.

Proteolytic enzyme activity in lactococci grown in different pretreated milk media.

Lactococcus lactis ssp. lactis MG1363, harbouring plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, was used to study the effect of two different treatments of the growth medium milk on the activity levels of PrtP and the intracellular localized aminopeptidase PepN and X-prolyl-dipeptidyl aminopeptidase PepXP. All three proteolytic enzymes showed lower activity levels in cells grown in high heat-treated milk as compared to cells grown in non-heat-treated milk. Highest activity levels of the three studied enzymes were found in cells grown in milk heat treated for 30 min at 63 degrees C. Using cells of strain L. lactis ssp. lactis MG1363, harbouring plasmid pNZ544, which encodes reporter gene gusA under control of the prtP promoter, it was demonstrated that the regulation of PrtP takes place at the transcription initiation level. After separation of the pH 4.6 soluble fraction of high heat-treated milk with reverse phase HPLC, it was found that the hydrophilic small peptide fraction of the milk was responsible for this regulation. Amino acid analysis of this fraction confirmed that this fraction consisted of peptides only. Ultrafiltration of milk, which increases the dry matter of the milk specifically through increase of its protein content, only significantly affected the levels of PrtP and PepN in cells of strain MG1363. Highest activity was found during growth in unconcentrated milk, and the lowest level was found during growth in four times concentrated retentate. Using cells of strain MG1363(pNZ544), it was demonstrated that also in this case regulation of PrtP takes place at the level of transcription initiation. Approximately 40% of the decrease in activity of PrtP and PepN could be explained by the presence of higher amounts of purified whey proteins and higher amounts of dry mass. This suggests the presence of another factor, concentrated by ultrafiltration, which controls the production different proteolytic enzymes in concentrated retentate.