Alan G. Williams

Enhancement of amino acid catabolism in Cheddar cheese using α-ketoglutarate : amino acid degradation in relation to volatile compounds and aroma character

The effectiveness of the transaminase acceptor α-ketoglutarate in enhancing amino acid catabolism and manipulating the aroma profile of Cheddar cheese has been studied. Utilisation of α-ketoglutarate, catabolism of amino acids, volatiles production, and aroma profile of the cheese were monitored after 6, 12 and 24 weeks ripening. Glutamate and GABA were considerably enhanced on addition of the transaminase acceptor while levels of phenylalanine, leucine, isoleucine, alanine, valine, methionine and threonine were reduced. Addition of α-ketoglutarate increased volatile components originating from the catabolism of branched chain and aromatic amino acids. These compounds included acetic, propanoic, 2-methylpropanoic and 3-methylbutanoic acids, 3-methylbutanol, phenylacetaldehyde and benzaldehyde. Additionally enhanced production of 3-OH-2-butanone was evident. Addition of α-ketoglutarate increased aroma intensity, creamy and fruity aromas. Effects obtained must be verified by tasting cheeses made with food grade α-ketoglutarate, but results suggest potential benefits in accelerated maturation, low fat systems and manipulation of flavour profiles.

Energy sources of non-starter lactic acid bacteria isolated from Cheddar cheese

Abstract The range of carbon sources available in cheese curd during maturation that could be used as energy and growth substrates by 60 cultures of non-starter lactic acid bacteria isolated from Cheddar cheese was determined by the detection of tetrazolium salt reduction in Biolog MT1 microplates ™ . There were marked inter-species and strain differences in the range of carbon substrates catabolized by the 11 Lactobacillus spp. and 2 Weissella spp. examined. Sugars were used widely among the NSLAB with 90, 100 and 85% of the isolates metabolizing lactose, glucose and galactose, respectively. In addition, ribose, N -acetyl-galactosamine and sialic acid, potentially derived from nucleic acid and casein deglycosylation, were catabolized by 58, 48 and 22% of the isolates, respectively. Lactic acid was also a potential substrate for 15% of the isolates but Tween 80 was not an effective substrate. Although 50% of the NSLAB removed citric acid from the growth medium it was not an independent energy source. Peptides and amino acids were also catabolized by up to 27% of the NSLAB provided that an exogenous source of α -ketoglutaric acid was present to facilitate the aminotransferase-mediated transamination degradative pathway. The MT1 microplate method facilitates the rapid screening for isolates able to establish in the cheese curd and for the detection of specific metabolic activities in isolates undergoing evaluation for use as adjunct cultures in cheesemaking trials.

Multiple protease activities in Giardia intestinalis trophozoites

Azocasein and a wide range of chromogenic and fluorogenic peptidyl substrates, representing 21 different peptide sequences, were degraded by the intracellular proteases present in lysates of trophozoites of Giardia intestinalis (syn lamblia) Portland 1 strain. Total protease activity differed considerably with the substrate with the p-nitroanilide derivatives Bz-pro-phe-arg-pNA and Bz-phe-val-arg-pNA being most rapidly hydrolysed (specific activities 4.2 +/- 0.4 and 1.1 +/- 0.6 U/mg protein, respectively). Activities were increased (16-72%) by addition of 1 mM dithiothreitol and were maximal, for the substrates monitored, in the range pH 5.5-7.0. These data and the inhibitor susceptibilities of the trophozoite proteases confirmed that the activity was predominantly due to cysteine proteases, although the presence of some serine protease, aspartic protease and aminopeptidase activity in the lysates was also indicated. The multiplicity of the protease activities was confirmed by gelatin-polyacrylamide electrophoretic analysis. Eighteen proteolytic activities with Mr values in the range 30,000 to > 211,000 were detected. These gelatinase activities were enhanced by dithiothreitol, were maximal at or close to pH 6, and were inhibited by cysteine proteinase and some serine proteinase inhibitors. Four of the proteases present in the gels exhibited activity towards fluorogenic amidomethylcoumarin peptides, but with different substrate preferences. The results show that G. intestinalis contains multiple proteases, many of which are of the cysteine type.

Variability of the species and strain phenotype composition of the non-starter lactic acid bacterial population of cheddar cheese manufactured in a commercial creamery

Abstract The non-starter lactic acid bacteria (NSLAB) present in cheddar cheese manufactured in a commercial creamery was monitored phenotypically to the strain level over a period of 12 months to examine the effects of maturity status and manufacturing practices on the composition of the population. Five Lactobacillus spp. and Leuconostoc lactis were identified among the 459 isolates selected. The predominant NSLAB, Lactobacillus paracasei and Lactobacillus brevis , were present in 59 and 31% of the cheeses examined and represented 52.7 and 25.8%, respectively, of the isolates identified. Among the NSLAB screened 71 different phenotypic profiles were identified and these included 26 biotypes of Lb. paracasei , 14 Lb. brevis , 11 Lactobacillus plantarum , 10 Lactobacillus curvatus and 7 Leuc. lactis . The average number of strains recovered from a cheese was 3.9±2.1 and ranged from 1 to 11. Although approximately 70% of the cheese samples were dominated by three or less strains the NSLAB populations were heterogeneous and the majority (61.5%) were comprised of four or more strains of one or more species. Only 30 of the biotypes were recovered from more than one population. There was no evidence for the repeated recurrence of any of the strains isolated although some of the Lb. paracasei strains were present intermittently in cheeses throughout the 12-month manufacturing period. Six Lb. brevis strains also recurred in some of the cheeses produced in a limited period during the autumn. Pronounced shifts in the species complement and strain profile occurred during maturation, while the average number of strains present in the cheese decreased with increasing maturity. Microbiological examination of the NSLAB population of cheese either produced in different vats during the same production run or manufactured in the same vat but in different production runs (vat fills) indicated that the number of strains common to paired samples from two vats or a single vat in successive production runs was only 1.7±1.4 and 1.5±1.2, respectively, and confirmed the inherent variability that exists, both within and between production runs, in the non-starter population of cheese manufactured in a commercial creamery.

Postprandial variations in the activity of polysaccharide-degrading enzymes of fluid- and particle-associated ruminal microbial populations

The distribution and specific activities of polysaccharide-degrading enzymes were monitored during the postprandial period in the liquid-associated bacteria (LAB), liquid-associated protozoa (LAP), and solid-associated microbes (SBFP) isolated from ruminal contents of cattle fed a high-cereal diet. Polysaccharide depolymerase activities were highest in the SBFP and the LAP populations. The postprandial variations in the specific activity of amylase were similar within the subpopulations. However, carboxymethylcellulase and xylanase activities increased in the first 5 h after feeding in the LAP, but were highest at the end of the postprandial period in the SBFP. Glycosidases involved in the fermentation of soluble carbohydrates increased significantly immediately after feeding in the liquid-associated microbes. β-d-Glucosidase and β-d-xylosidase were most active in the SBFP and were maximal 23 h after feeding. The activities of the plant cell wall polysaccharide-degrading enzymes and glycosidases in the SBFP were inversely related to ruminal pH; however, the activity of enzymes in the liquid-associated populations were highest in the immediate post-feed period when ruminal pH was lowest.

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the rumen ciliate Dasytricha ruminantium

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen isotrichid ciliate Dasytricha ruminantium were investigated. Principal metabolic products were lactic, butyric and acetic acids, H2 and CO2. Traces of propionic acid were also detected; formic acid present in the incubation supernatants was found to be a fermentation product of the bacteria closely associated with this rumen ciliate. 13C NMR spectroscopy revealed alanine as a minor product of glucose fermentation by D. ruminantium. Glucose uptake and metabolite formation rates were influenced by the headspace gas composition during the protozoal incubations. The uptake of exogenously supplied D-glucose was most rapid in the presence of O2 concentrations typical of those detected in situ (i.e. 1-3 microM). A typical ruminal gas composition (high CO2, low O2) led to increased butyrate and acetate formation compared to results obtained using O2-free N2. At a partial pressure of 66 kPa CO2 in N2, increased cytosolic flux to butyrate was observed. At low O2 concentrations (1-3 microM dissolved in the protozoal suspension) in the absence of CO2, increased acetate and CO2 formation were observed and D. ruminantium utilized lactate in the absence of extracellular glucose. The presence of both O2 and CO2 in the incubation headspaces resulted in partial inhibition of H2 production by D. ruminantium. Results suggest that at the O2 and CO2 concentrations that prevail in situ, the contribution made by D. ruminantium to the formation of ruminal volatile fatty acids is greater than previously reported, as earlier measurements were made under anaerobic conditions.

Proteinase activity in rumen ciliate protozoa

Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4.0-5.0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.

The influence of ruminal concentrations of O2 and CO2 on fermentative metabolism of the rumen entodiniomorphid ciliateEudiplodinium maggii

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen ciliateEudiplodinium maggii were investigated. The principal metabolic products were butyrate, acetate, lactate, propionate, H2, and CO2.13C NMR spectroscopy revealed glycerol to be an important, but previously unidentified, fermentation product of this organism. Glucose uptake and metabolite formation rates were influenced by the headspace composition during protozoal incubations. Glucose uptake was most rapid in the presence of low O2 in N2 (1–3 µM O2 dissolved in the protozoal suspension). Pathways located in the hydrogenosomes were O2 sensitive, and low O2 concentrations resulted in lowered acetate, H2, and CO2 formation. The presence of high CO2 (65% gaseous headspace by volume) resulted in elevated acetate and butyrate formation; fumarate and propionate were similarly found to accumulate at higher concentration than previously detected in the supernatants. Results suggest that under conditions similar to those prevailing in the rumen (i.e., high CO2),Eu. maggii produces higher levels of important ruminal volatile fatty acids, and thus its relative contribution to rumen metabolism may have been underestimated.

Induction of xylan-degrading enzymes inButyrivibrio fibrisolvens

The formation of xylanolytic enzymes byButyrivibrio fibrisolvens NCFB 2249 was induced by xylan, xylo-oligosaccharides, and xylobiose. Inhibition of RNA or protein synthesis prevented inducetion, and enzyme formation occured only when anaerobiosis was maintained. The rate of enzyme inducetion by xylan was affected by pH and inducer concentration, and highest levels of activity occurred when the initial pH and xylan concentration were pH 6.5–7 and ≥2 mg/ml respectively. The ability of the cells to respond to the inducer was reduced in slowly growing cells, although cells that were grown at dilution rates that appertain in the rumen ecosystem responded rapidly to the inducer.Butyrivibrio fibrisolvens also exhibited diauxic characteristics of carbohydrate utilization, and in consequence enzyme induction and xylanolysis were delayed until readily metabolized sugars (e. g., glucose, arabinose) had been consumed.