Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Inhibition of seafood-borne bacteria in cooked mackerel (Rastrelliger kanagurta) fish meat by lactic acid bacteria

Antagonistic activity of lactic acid bacteria (LAB) namely Streptococcus faecalis, Pediococcus cerevisiae and Lactobacillus casei was tested against seafood-borne bacteria such as Staphylococcus aureus, Bacillus cereus, Escherichia coli, Clostridium perfringens and Listeria monocytogenes. Three lactic acid bacteria such as Streptococcus faecalis, Lactobacillus casei and Pediococcus cerevisiae were coated on cooked mackerel meat, individually and in combination against fish-borne bacteria. S. faecalis inhibited C. perfringens in individual coat by 3.7 log units as compared to control, whereas L. casei did not inhibit C. perfringens. P. cerevisiae inhibited S. aureus by 5 log units. L. casei, inhibited L. monocytogenes by 3.3 log units on the third day of storage as compared to control. On the other hand, S. aureus and B. cereus were inhibited on the third and second day by 4.9 log and 5.2 log units respectively. B. cereus, S. aureus, L. monocytogenes were the most sensitive to all three LAB. C. perfringens was the least inhibited among all the seafood-borne bacteria tried. Multiple LAB or LAB strains in combination showed much earlier inhibitory activity on seafood-borne bacteria than single LAB coat.

“Green preservatives” – combating fungi in the food industry by applying antifungal lactic acid bacteria

Fungal spoilage is the most common type of microbial spoilage in food leading to significant economical and health problems throughout the world. Fermentation by lactic acid bacteria (LAB) is one of the oldest and most economical methods of producing and preserving food. Thus, LAB can be seen as an interesting tool in the development of novel bio-preservatives for food industry. The overall objective of this study was to demonstrate, that LAB can be used as a natural way to improve the shelf-life and safety of a wide range of food products. In the first part of the thesis, 116 LAB isolates were screened for their antifungal activity against four Aspergillus and Penicillium spp. commonly found in food. Approximately 83% of them showed antifungal activity, but only 1% showed a broad range antifungal activity against all tested fungi. The second approach was to apply LAB antifungal strains in production of food products with extended shelf-life. L. reuteri R29 strain was identified as having strong antifungal activity in vitro, as well as in sourdough bread against Aspergillus niger, Fusarium culmorum and Penicillium expansum. The ability of the strain to produce bread of good quality was also determined using standard baking tests. Another strain, L. amylovorus DSM19280, was also identified as having strong antifungal activity in vitro and in vivo. The strain was used as an adjunct culture in a Cheddar cheese model system and demonstrated the inhibition of P. expansum. Significantly, its presence had no detectable negative impact on cheese quality as determined by analysis of moisture, salt, pH, and primary and secondary proteolysis. L. brevis PS1 a further strain identified during the screening as very antifungal, showed activity in vitro against common Fusarium spp. and was used in the production of a novel functional wortbased alcohol-free beverage. Challenge tests performed with F. culmorum confirmed the effectiveness of the antifungal strain in vivo. The shelf-life of the beverage was extended significantly when compared to not inoculated wort sample. A range of antifungal compounds were identified for the 4 LAB strains, namely L. reuteri ee1p, L. reuteri R29, L. brevis PS1 and L. amylovorous DSM20531. The identification of the compounds was based on liquid chromatography interfaced to the mass spectrometer and PDA detector

Food preservation using antifungal lactic acid bacteria

Fungal spoilage of food and feed prevails as a major problem for the food industry. The use antifungal-producing lactic acid bacteria (LAB) may represent a safer, natural alternative to the use of chemical preservatives in foods. A large scale screen was undertaken to identify a variety of LAB with antifungal properties from plant, animal and human sources. A total of 6,720 LAB colonies were isolated and screened for antifungal activity against the indicator Penicillium expansum. 94 broad-spectrum producers were identified through 16S rRNA sequencing with the majority of the population comprising Lactobacillus plantarum isolates. Six broad-spectrum isolates were consequently characterised. Pedicococcus pentosaceous 54 displayed potent anti-mould capabilities in pear, plum and grape models and may represent an ideal candidate for use in the beverage industry. Two antifungal Lb. plantarum isolates were assessed for their technological robustness and potential as biopreservatives in refrigerated foods. Lb. plantarum 16 and 62 displayed high levels of tolerance to freeze-drying, low temperature exposure and high salt concentrations. Both lactobacilli were introduced as supplements into orange juice to retard the growth of the spoilage yeast Rhodotorula mucilaginosa. Furthermore the isolates were applied as adjuncts in yoghurt production to successfully reduce yeast growth. Lb. plantarum 16 proved to be the optimal inhibitor of yeast growth in both food matrices. To date there is limited information available describing the mechanisms behind fungal inhibition by LAB. The effects of concentrated cell-free supernatant (cCFS), derived from Lb. plantarum 16, on the growth of two food-associated moulds was assessed microscopically. cCFS completely inhibited spore, germ tube and hyphal development. A transcriptomic approach was undertaken to determine the impact of antifungal activity on Aspergillus fumigatus Af293. A variety of genes, most notably those involved in cellular metabolism, were found to have their transcription modulated in response to cCFS which is indicative of global cellular shutdown. This study provides the first insights into the molecular targets of antifungal compounds produced by LAB. The genome sequence of the steep water isolate Lb. plantarum 16 was determined. The complete genome of Lb. plantarum16 consists of a single circular chromosome of 3,044,738 base pairs with an average G+C content of 44.74 % in addition to eight plasmids. The genome represents the smallest of this species to date while harbouring the largest plasmid complement. Some features of particular interest include the presence of two prophages, an interrupted plantaricin cluster and a chromosomal and plasmid encoded polysaccharide cluster. The sequence presented here provides a suitable platform for future studies elucidating the mechanisms governing antifungal production.

Investigation of lactic acid bacteria mediated bioprotection with applications in cereal industry. Case-study: malting process

Antifungal compounds produced by Lactic acid bacteria (LAB) metabolites can be natural and reliable alternative for reducing fungal infections pre- and post-harvest with a multitude of additional advantages for cereal-base products. Toxigenic and spoilage fungi are responsible for numerous diseases and economic losses. This thesis includes an overview of the impact fungi have on aspects of the cereal food chain. The applicability of LAB in plant protection and cereal industry is discussed in detail. Specific case studies include Fusarium head blight, and the impact of fungi in the malting and baking industry. The impact of Fusarium culmorum infected raw barley on the final malt quality was part of the investigation. In vitro infected barley grains were fully characterized. The study showed that the germinative energy of infected barley grains decreased by 45% and grains accumulated 199 μg.kg-1 of deoxynivalenol (DON). Barley grains were subsequently malted and fully characterized. Fungal biomass increased during all stages of malting. Infected malt accumulated 8-times its DON concentration during malting. Infected malt grains revealed extreme structural changes due to proteolytic, (hemi)-cellulolytic and starch degrading activity of the fungi, this led to increased friability and fragmentation. Infected grains also had higher protease and β-glucanase activities, lower amylase activity, a greater proportion of free amino and soluble nitrogen, and a lower β-glucan content. Malt loss was over 27% higher in infected malt when compared to the control. The protein compositional changes and respective enzymatic activity of infected barley and respective malt were characterized using a wide range of methods. F. culmorum infected barley grains showed an increase in proteolytic activity and protein extractability. Several metabolic proteins decreased and increased at different rates during infection and malting, showing a complex F. culmorum infection interdependence. In vitro F. culmorum infected malt was used to produce lager beer to investigate changes caused by the fungi during the brewing processes and their effect on beer quality attributes. It was found, that the wort containing infected malt had a lower pH, a higher FAN, higher β-glucan and a 45% increase in the purging rate, and led to premature yeast flocculation. The beer produced with infected malt (IB) had also a significantly different amino acid profile. IB flavour characterization revealed a higher concentration of esters, fusel alcohols, fatty acids, ketones, and dimethylsulfide, and in particular, acetaldehyde, when compared to the control. IB had a greater proportion of Strecker aldehydes and Maillard products contributing to an increased beer staling character. IB resulted in a 67% darker colour with a trend to better foam stability. It was also found that 78% of the accumulated mycotoxin deoxynivalenol in the malt was transferred into beer. A LAB cell-freesupernatant (cfs), produced in wort-base substrate, was investigated for its ability to inhibit Fusarium growth during malting. Wort was a suitable substrate for LAB exhibiting antifungal activity. Lactobacillus amylovorus DSM19280 inhibited 104 spores.mL-1 for 7 days, after 120 h of fermentation, while Lactobacillus reuteri R29 inhibited 105 spores.mL-1 for 7 days, after 48 h of fermentation. Both LAB cfs had significant different organic acid profiles. Acid-base antifungal compounds were identified and, phenyllactic, hydroxy-phenyllactic, and benzoic acids were present in higher concentrations when compared to the control. A 3 °P wort substrate inoculated With L. reuteri R29 (cfs) was applied in malting and successfully inhibited Fusarium growth by 23%, and mycotoxin DON by 80%. Malt attributes resulted in highly modified grains, lower pH, higher colouration, and higher extract yield. The implementation of selected LAB producing antifungal compounds can be used successfully in the malting process to reduce mould growth and mycotoxin production.

Isolation and characterisation of antifungal compounds from lactic acid bacteria and their application in wheat and gluten-free bread

As part of the “free-from” trend, biopreservation for bread products has increasingly become important to prevent spoilage since artificial preservatives are more and more rejected by consumers. A literature review conducted as part of this thesis revealed that the evaluation of more suitable antifungal strains of lactic acid bacteria (LAB) is important. Moreover, increasing the knowledge about the origin of the antifungal effect is fundamental for further enhancement of biopreservation. This thesis addresses the investigation of Lactobacillus amylovorus DSM19280, Lb. brevis R2: and Lb. reuteri R29 for biopreservation using in vitro trials and in situ sourdough fermentations of quinoa, rice and wheat flours as biopreservatives in breads. Their contribution to quality and shelf life extension on bread was compared and related to their metabolic activity and substrate features. Moreover, the quantity of antifungal carboxylic acids produced during sourdough fermentation was analysed. Overall a specific profile of antifungal compounds was found in the sourdough samples which were strain and substrate dependently different. The best preservative effect in quinoa sourdough and wheat sourdough bread was achieved when Lb. amylovorus DSM19280 fermented sourdough was used. However, the concentration of the antifungal compounds found in these biopreservatives were much lower when compared with Lb. reuteri R29 as the highest producer. Nevertheless, the artificial application of the highest concentration of these antifungal compounds in chemically acidified wheat sourdough bread succeeded in a longer shelf life than achieved only by acidifying the dough. This evidences their partial contribution to the antifungal activity and their synergy. Additionally, a HRGC/MS method for the identification and quantification of the antifungal active compounds cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) was successfully developed by using stable isotope dilutions assays with the deuterated counterparts. It was observed that the concentrations of cyclo(Leu-Pro), cyclo(Pro-Pro), and cyclo(Phe-Pro) increased only moderately in MRS-broth and wort fermentation by the activity of the selected microorganism, whereas the concentration of cyclo(Met-Pro) stayed unchanged.

Identification of lactic acid bacteria strains modulating incretin hormone secretion and gene expression in enteroendocrine cells

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones released from intestinal enteroendocrine (EE) cells and have well-established glucose-lowering actions. Lactic acid bacteria (LAB) colonise the human intestine, but it is unknown whether LAB and EE cells interact. Acute co-culture of LAB with EE cells showed that certain LAB strains elicit GLP-1 and GIP secretion (13-194-fold) and upregulate their gene expression. LAB-induced incretin hormone secretion did not appear to involve nutrient mechanisms, nor was there any evidence of cytolysis. Instead PCR array studies implicated signalling agents of the toll-like receptor system, e.g. adaptor protein MyD88 was decreased 23-fold and cell surface antigen CD14 was increased 17-fold. Mechanistic studies found that blockade of MyD88 triggered significant GLP-1 secretion. Furthermore, blocking of CD14 completely attenuated LAB-induced secretion. A recent clinical trial clearly shows that LAB have potential for alleviating type 2 diabetes, and further characterisation of this bioactivity is warranted.