A large amount of cheese is lost every year due to mold contamination. Biopreservation, which is the use of biological entities (microbes) and their metabolites to suppress microbial spoilage instead of chemical preservatives has lately gained increasing interest. Lactic acid bacteria (LAB) have the potential for use in biopreservation, because they are safe to consume and naturally exist in many foods. In this study, fifteen strains of lactobacilli isolated from dairy products, vegetables, and fermented pickles were tested by agar overlay assay for their anti-mold activity. Six strains grown on MRS agar showed strong inhibitory activity against a target mold (Penicillium sp. at 105 spores/ml) isolated from the surface of Cheddar cheese. The isolates were identified by biochemical tests using API CHL50 strips. Five strains were identified as Lactobacillus plantarum, and one strain as Pediococcus pentasaceus. Well-diffusion method was used to demonstrate anti-mold activity in concentrated cell-free supernatants. Supernatants from all strains showed inhibition of the target mold (indicator). The anti-mold compound(s) produced by all the strains was heat-resistant (100o C for 15 min). Supernatants from 5 strains retained the anti-mold activity when the pH was adjusted to 6.8 ± 0.2, while one strain DC2 isolated from cheese lost its anti-mold activity at that pH. Temperature of incubation of cultures affected anti-mold activity. The optimum was 37o C. Very little or no inhibition was noted when cultures were incubated at either 10 or 55 °C. A preliminary study of applying anti-mold lactobacilli in Cheddar cheese was completed. Anti-mold LAB was added to the cheese milk as an adjunct to give 105 cfu/ml. After 1-week and 1-month ripening, mold (10~20spores) was added on to the surface, and the cheese was wrapped loosely. The appearance of the mold on cheese surface was monitored. Mold was not present on the 1-week old cheese “NB in milk” until the 6th day after the control cheese (made without strain NB) showed signs of mold. The 1-month old cheese “NB in milk ” extended the shelf life 17 days longer than the control cheese.

Lactobacillus plantarum and subspecies of Lactobacillus casei were isolated from good quality mature Cheddar cheese and characterized with respect to metabolic functions that would allow their use in cheesemaking. In this way microbiological control of the maturation process with particular emphasis on protein catabolism was achieved. The lactobacilli isolated were selected for low growth rates (and acid production) in milk, and low proteinase activity to allow for their addition in high numbers to cheesemilk together with the normal starter flora (group N streptococci). The growth and acid production of the starter bacteria were unaffected by the presence of the lactobacilli during cheese manufacture and it was found that the added lactobacilli were able to grow and function under the conditions prevalent in Cheddar cheese during maturation. It was also demonstrated that the lactobacilli could be grown in an artificial medium to high numbers under controlled conditions and could be harvested for the preparation of cell concentrates, a necessary characteristic for commercialization. The lactobacilli also metabolized citrate, a potential problem in cheese maturation associated with C02 production but this did not adversely affect the maturation process under the conditions used. Compared to the group N streptococci the non-starter lactobacilli possessed a proteinase system that had a higher temperature optimum and was less affected by heat and sodium chloride. They also possessed a more active peptidase system although both the lactobacilli and the starter organisms possessed a similar range of peptidases. Non-starter lactobacilli were added to normal cheese and cheese made with proteinase negative starter. The added organisms did not adversely affect manufacturing parameters and did not metabolize citrate or lead to the formation of biogenic amines. However protein catabolism rates, particularly with respect to peptide degradation, were increased, as was flavour development and intensity. It was observed that the body and texture of the cheeses was unaffected by the treatment. By controlling both the starter and non-starter microflora in the cheeses a practical system for favourably influencing cheese maturation was possible. The investigation has demonstrated that carefully selected and characterized non-starter lactobacilli can be incorporated into Cheddar cheese manufacture in order to influence flavour development during maturation. Moreover the organisms can be added to the vat stage of manufacture without causing problems to the manufacturing process. This approach is a simple cost effective means of improving the cost of Cheddar cheese production and provides an unique opportunity to improve and control quality of all Cheddar cheese produced.

Graduation date: 1995

Graduation date: 1994

Graduation date: 1994

Carnocyclin A (CclA) is a remarkably stable, potent bacteriocin produced by Carnobacterium maltaromaticum UAL307. Elucidation of the amino acid and genetic sequences revealed that CclA is a circular bacteriocin. Preliminary structural studies (dynamic light scattering, NMR, circular dichroism, stereochemical analysis) indicated that CclA is monomeric and alpha-helical in aqueous conditions and composed of L-residues. The 3D structure of [13C,15N]CclA was solved by NMR, revealing a compact arrangement of four helices. To examine the structure of the precursor peptide (pCclA) several fusion proteins were constructed and overexpressed; however, pCclA could not be isolated. To investigate the requirements for cyclization, several internally hexahistidine-tagged (His6) pCclA mutants were constructed. Expression conditions are underway. PisI was heterologously expressed and confirmed to impart protection against piscicolin 126 (PisA). Labeled and unlabeled PisA and PisI were purified following overexpression as maltose-binding protein fusions (MalE-fusions) and Factor Xa cleavage. NMR studies indicated that PisI and PisA do not physically interact. The 3D structure of PisI was solved by NMR, confirming that the four-helix bundle is a conserved motif for the immunity proteins of type IIa bacteriocins. The putative receptor proteins for these bacteriocins were cloned and overexpressed as His6-fusion proteins. Experiments are underway to optimize the expression and purification of these membrane proteins. The peptidase domain of the ABC-transporter protein (CbnTP) for carnobacteriocin B2 (CbnB2) was overexpressed as a His6-fusion protein. Active protease could not be purified from inclusion bodies, but was obtained as soluble protein following low-temperature overexpression. The CbnB2 precursor pCbnB2 (and a truncated derivative pCbnB2-RP) was purified following overexpression as a MalE-fusion and Factor Xa cleavage. pCbnB2 was incubated with CbnTP and MALDI-TOF and activity testing confirmed that CbnTP cleaved the leader peptide from pCbnB2. Five CysSer CbnTP mutants were constructed. Crystallographic studies of CbnTP are underway. Six bacteriocins (nisin, gallidermin, lacticin 3147, CclA, PisA, enterocin 710C) were tested against Gram-negative bacteria (E. coli DH5, Pseudomonas aeruginosa ATCC 14207, Salmonella typhimurium ATCC 23564) in the absence and presence of EDTA. PisA and lacticin 3147 exhibited minimal activity, whereas the other bacteriocins killed at least one strain, in the presence of EDTA.

The work presented in this thesis is concerned with the effect of several antibiotics and quaternary ammonium sanitizers upon growth of lactic acid bacteria. Section I reports the purification of beta-lactamase from Lactococcus cremoris PR-108, by ion exchange chromatography, using the chromogenic substrate pyridine-2-azo-p-dimethylaniline (PADAC) as the enzymatic indicator. Section II reports a study of the influence of antibiotics on lactococcal growth, where the effects of incubation time, culture dilution and the use of seeded and spread agar plate techniques are investigated. These studies were extended, in section III, to include investigations of the effect of quaternary ammonium base sanitizer (Ster-bac) on lactic starters. In addition, this section describes an reverse phase high performance liquid chromatography assay for the detection of quaternary ammonium compounds in milk. / Graduation date: 1991

Immunomodulatory effects of lactic acid bacteria vary with strain and may vary with growth phase and medium. The ability of different lactobacilli strains (Lactobacillus helveticus R0052, L. rhamnosus R0011, L. rhamnosus GG) at different growth phases to modulate macrophage and intestinal epithelial cell cytokine production following a pro-inflammatory challenge was examined. Modulation of cytokine production by human macrophage cell lines (U-937) and intestinal epithelial cells (HT-29) induced by Tumor Necrosis Factor α was assayed by ELISA for interleukin-8 (IL-8). Granulocyte-macrophage colony stimulating factor (GM-CSF) production was assayed by ELISA in the HT-29 cell line. Strain-dependent differences were observed in the ability of viable bacteria and spent de Mann-Rogosa- Sharpe (MRS) broths from log versus stationary growth phase in HT-29 and U-937 cells. Overall, variation in the immunomodulatory activity of these lactic acid bacteria and spent broths reflects not only strain variation but potentially also differences in growth phase and substrate. / UOIT

Lactic acid, which induces pH depression, leads to ATP efflux from muscle to extracellular space: it was reported that CFTR was involved in this process. However, the mechanism by which lactic acid activated CFTR and brought about the ATP release is still unknown. This study was performed to investigate (1) what channels may be involved or even conduct ATP release, and (2) how lactic acid activated CFTR. Expression of the possible channels that may conduct ATP release in L6 cells was investigated using RT-PCR: ClC-2, ClC-3, ClC-7, CACC, VDAC, connexin 40, connexin 43 and pannexin 3 were expressed in L6. Incubation of cultured L6 cells with lactic acid (10 mM) increased the extracellular ATP from 0.6 ± 0.06 to 1.1 ± 0.09 nM (P ? 0.05), indicating that lactic acid stimulated ATP efflux in vitro. The non-specific chloride channel inhibitor, DIDS, failed to abolish the lactic-acid-induced ATP release, suggesting that DIDS-sensitive chloride channels were not involved in the ATP efflux. Among the non-specific inhibitors of connexin channels, gadolinium inhibited acidosis-induced ATP efflux, but carbenoxolone failed to inhibit it, and so the role of connexins remains uncertain. The specific inhibitor of CFTR, CFTRinh-172, and the non-specific open-channel blocker of CFTR, glibenclamide, both abolished the acidosis-induced ATP release, but another specific inhibitor of CFTR, GlyH-101, which blocks CFTR from the external side, failed to abolish the ATP release, suggesting that acidosis-induced ATP is dependent on CFTR-activation, but does not involve ATP moving through the CFTR chloride channel. We hypothesize that, at low pH, the Na+/H+ exchanger (NHX) extruded H+ out of the cell and the resulting intracellular Na+ was transported out by Ca2+/Na+ exchanger (NCX); the localized increase in Ca2+ activated adenyl cyclase (AC), thus elevating intracellular cAMP; cAMP-activated-PKA then phosphorylated CFTR, which regulated an ATP release channel. KT-5720, an inhibitor of PKA, abolished the acidosis-induced ATP release, and forskolin, an agent that elevates cAMP, stimulated it, suggesting that the cAMP/PKA pathway was involved. The specific inhibitor of NCX, SN-6 and KB-R7943, both abolished the acidosis-induced ATP release, supporting a role for NCX in mediating this process. However, amiloride, the non-specific inhibitor of NHX failed to abolish ATP efflux. The whole cell Cl- currents were studied in L6 cells: lactic acid increased the whole cell currents from 2.33 ± 0.10 to 3.54 ± 0.34 nA (P ? 0.05), and this lactic-acid-induced increase in Cl- current could be inhibited by CFTRinh-172, suggesting that the CFTR Cl- channel was opened at low pH. Moreover, forskolin increased whole cell Cl- currents, which supported a role for the cAMP/PKA pathway in the lactic-acid-induced increase in CFTR current. These data confirm that CFTR is involved in the lactic-acid-induced ATP release from L6 cells. The roles of the NCX and cAMP/PKA pathway in activating CFTR at low pH are supported, but further studies are required to determine whether the NHX is involved in CFTR activation and whether connexins participate in ATP release. / published_or_final_version / Physiology / Master / Master of Philosophy

Successfully inducing malolactic fermentation in the production of grape wines can be challenging, especially in wines after finishing alcoholic fermentation with limited energy sources, low pH values and high ethanol concentrations. In this thesis, the kinetics of several chemicals of enological relevance were studied in a white wine (Chardonnay) and a red wine (Cab Franc) vinified by traditional, consecutive alcoholic (AF) and malolactic fermentations (MLF), and simultaneous AF/MLF, where bacteria were co-inoculated with yeast. The Chardonnay must was adjusted to four pH values (3.20, 3.35, 3.50 or 3.65), the cab Franc was kept as original pH value (3.56) and the concentrations of sugars, organic acids as well as acetaldehyde were followed throughout the fermentations. For Chardonnay the degradation of glucose and fructose was slower at the lowest must pH value (3.20) and independent from the time of bacterial inoculation. In all cases, malolactic conversion was faster after yeast-bacterial co-inoculation and was completed in simultaneous treatments at pH values of 3.35-3.65, and consecutive treatments at pH 3.50 and 3.65. No statistically significant difference was observed among the final acetic acid concentration, in all inoculation and pH treatments. For Cab Franc, it confirmed that co-inoculation shortened the fermentation periods while having minor effects on other parameters. Overall, simultaneous AF/MLF allowed for greatly reduced fermentation times, while the must pH remained a strong factor for fermentation success and determined the final concentration of various wine components. The time of inoculation influenced formation and degradation kinetics of organic acids and acetaldehyde significantly.