M. Elisabeth Sharpe

The effect of the microbial flora on the flavour and free fatty acid composition of cheddar cheese

Comparisons were made of the flavour, free fatty acids and bacterial flora of commercial cheese made at different factories and experimental cheese made under aseptic conditions: (i) with δ-gluconic acid lactone instead of starter, (ii) with starter only, (iii) with starter and added floras derived from the curd of the commercial cheeses (reference flora cheeses). Comparison of the bacterial flora of commercial and reference flora cheeses showed that replication of organisms was better with some reference floras than with others. In all the cheeses the lactobacilli increased in numbers during maturation, whilst other groups of organisms died out. The amount of acetic acid present was influenced by the starter and by the lactobacilli. Single-strain starters produced some acetic acid, most of which was lost in the whey; commercial starters produced considerably more, due to the presence in them of Streptococcus diacetilactis . Later in maturation lactobacilli increased the acetic acid content, a greater increase being observed with homo-than with heterofermentative strains. The initial levels of butyric and higher fatty acids in the milk varied with source of the milk and with the season, summer milk having higher levels than winter milk. During cheese-making a slight increase of these acids occurred in every cheese made with starter and a further small increase occurred during ripening. However, there was no increase in the content of these acids in the cheese made with δ-gluconic acid lactone, indicating that lactic acid bacteria were weakly hydrolysing the milk fat. Flavour trials showed that Cheddar flavour was present not only in the reference flora and commercial cheese, but also in the cheese made with starter only. Different starters produced different intensities of flavour; one strain produced an intense fruity off-flavour. Cheeses made with δ-gluconic acid lactone were devoid of cheese flavour.

The incidence of bacteria in cheese milk and Cheddar cheese and their association with flavour

The numbers and types of non-starter lactic acid bacteria, lipolytic bacteria and group D streptococci in cheese milks and corresponding Cheddar cheeses have been studied and their relationship to cheese flavour discussed. Different milks, heat-treatments and starters were used, and their association with flavour investigated. The pH and fat, salt and moisture contents of the cheeses were also determined. The non-starter lactic acid flora consisted mainly of Lactobacillus casei, L. plantarum, L. brevis, L. buchnerii, Pediococcus spp. and Leuconostoc spp. Lactobacillus casei was nearly always present in the milks and cheeses, predominating in most of the cheeses, particularly in those made from milk which received the most severe of the three heat-treatments tested (160°F for 17 sec). Leuconostocs were not found in any of the cheeses. Other species sometimes occurred in approximately equal numbers to L. casei , particularly in cheeses manufactured from the milder heattreated milks. Cheeses made from milk receiving the highest heat-treatment contained fewest bacteria and scored lowest for flavour. The average flavour scores increased as the number of species contributing to the non-starter lactic acid flora at the time of tasting increased. The lipolytic organisms capable of hydrolysing butterfat consisted mainly of Gram-positive cocci, predominantly Staphylococcus saprophyticus . The group D streptococci occurred infrequently, Streptococcus faecium being the most commonly occurring species. A definite correlation was found to exist between the starters used and the flavour of the cheeses. The pH of the cheeses was associated with the flavour, but the fat, moisture and salt contents of the cheeses showed no definite effect.

The contribution of starter streptococci to flavour development in Cheddar cheese

The number of starter bacteria in Cheddar cheese was increased approximately 2 or 8 times by supplementing the normal starter inoculum with starter-cell suspensions which had been incubated with lysozyme in the absence of salt. Lysozyme-treated cells were also introduced into chemically acidified cheese in an attempt to achieve ripening in the absence of a normal starter culture. The added starters did not interfere with normal cheese-making by producing acid. The lysozyme-treated starter cells were lysed when the curd was salted and lysis was detected by the release of cell-free DNA and an intracellular marker enzyme (dipeptidase) into the cheese matrix. Free amino acid concentrations in maturing cheeses were increased up to 3 times compared with control cheeses. The intensity of Cheddar flavour was not increased in starter cheeses by the presence of additional lysozyme-treated starter and no Cheddar flavour developed in chemically acidified cheese containing the lysozyme-treated cells. It is concluded that intracellular starter enzymes play no direct part in flavour formation, but produce breakdown products from which Cheddar flavour compounds may be formed by other unknown mechanisms.

699. Lactobacilli in Cheddar cheese: 1. The use of selective media for isolation and of serological typing for identification

A comparison of three different media, yeast dextrose agar, tomato dextrose agar and acetate agar (which is selective for lactobacilli) showed that the acetate medium was satisfactory for the isolation and counting of lactobacilli from Cheddar cheese during ripening. Lactobacilli were isolated on this medium in the early stages of ripening when the large numbers of starter streptococci growing on the other media were completely suppressed. Lactobacilli isolated were identified biochemically and serologically. Serological typing was found to be a useful method of identifying large numbers of isolates and of assessing the incidence and distribution of particular types in the cheese. We wish to thank Dr A. T. R. Mattick for his interest and helpful advice in this work; Dr L. A. Mabbitt for preparation of the cheese samples; Miss H. R. Chapman for making the experimental cheese; and Mrs P. Jarrett for technical assistance.

The release of intracellular dipeptidase from starter streptococci during Cheddar cheese ripening

Intracellular dipeptidase of Streptococcus cremoris NCDO 924 has been investigated as a marker enzyme for detecting the release of starter enzymes into Cheddar cheese during maturation. Dipeptidases were produced extra- and intracellularly by Str. cremoris in broth cultures, but the intracellular enzyme was distinguishable by its relatively high activity against L-alanylglycine at low temperatures. The presence of dipeptidase activity of intracellular origin was demonstrated in extracts of Cheddar cheese freed from whole starter cells, opaque material of high molecular weight and free amino acids by a combination of centrifugation and gel filtration on Sephadex G200. The enzyme was not detected in cheeses made without starter (δ-gluconic acid lactone cheeses). In fresh curd with a high viable count of starter the dipeptidase activity was low and mostly extracellular. As the count declined the proportion of intracellular enzyme activity increased, reaching a maximum when 90% of the starter cells had died. The total dipeptidase subsequently decreased until the level remained 30% higher than that of the original curd, and changed little on further ripening up to 120 d. There remained a significant proportion of extracellular dipeptidase activity throughout the maturation period studied.

The effect of non-starter bacteria on the chemical composition and the flavour of Cheddar cheese

Differences in flavour scores and in the concentrations of free fatty acids, methyl ketones and H 2 S were measured in Cheddar cheeses containing various groups of non-starter bacteria or starter streptococci alone, made under controlled bacteriological conditions by the aseptic vat technique. The non-starter bacteria were made up of lactobacilli, leuconostocs, pediococci, micrococci and Gram-negative rods isolated in commercial creameries from raw milk or fresh cheese curd. These were added to the experimental cheese as single groups or as complete floras (reference floras). Several bacterial groups influenced the measured concentrations of the flavour compounds, but flavour differences were not correlated with these chemical differences. Only cheese containing a curd-derived whole reference flora or cheese made in open vats in the N.I.R.D. Experimental Dairy had significantly better flavour than starter-only cheese, but this improvement was not attributable to any particular group of bacteria.

Amino acid nutrition of some commercial cheese starters in relation to their growth in peptone-supplemented whey media

Some strains of commercial cheese starters were obtained in higher yield after batch culture in whey-based media supplemented with soy peptones than in those supplemented with papain digested skim-milk. The soy peptones contained a greater proportion of small peptides than did the skim-milk digest. Also the average size of peptides was lower (3·4–3·9 residues) in soy preparations than in the milk digest (6·8). Small peptides isolated from the soy peptones were utilized by growing starters more completely than the equivalent peptides isolated from the papain digested milk. These observations may account for the difference in cell yields with soy and milk-digest peptones.

Neutral volatiles in cheddar cheese made aseptically with and without starter culture

Analyses of the neutral volatile components from 3 Cheddar cheese are presented. Two were made aseptically in an aseptic vat, with and without starter culture; the third was made with starter culture in an open vat. Gas–liquid chromatography and mass spectrometry detected the same volatiles in starterless cheese having little or no Cheddar flavour as in cheese made with starter and having a characteristic Cheddar flavour. Methyl disulphide and dimethyl sulphide were the only compounds consistently detected in higher concentrations in the cheese made with starter than in the cheese made without starter. However, using a total trapping technique, it was found that the combination of components recovered from the effluents of the chromatographic columns did not have the cheese-like aroma of the distillate vapours that were injected. Reasons for this are discussed.

733. Lactobacilli in Cheddar cheese: III. The source of lactobacilli in cheese

Lactobacilli occur in large numbers in cheese (1,2,3,4) and may be responsible for or influence the formation of flavour. It is therefore desirable to find the source of the lactobacilli and the route by which they enter the cheese, so that the cheese flora may be controlled

Effect of proteolytic raw milk psychrotrophs on Cheddar cheese-making with stored milk

The effect of proteolytic, psychrotrophic strains of Pseudomonas fluorescens, Ps. putida and Acinetobacter spp. on cheese-making with stored milk has been investigated. Ps. fluorescens and Ps. putida growing for 72 h in raw milk at 7·5 °C to levels of approx. 10 7 colony-forming units/ml caused a low degree of β - and к -casein breakdown detectable by gel electrophoresis, but this was insufficient to affect N losses in whey or cheese yields. Variations in cheese-making times with pasteurized milks were not attributable to the counts of psychrotrophs in the corresponding raw milks. The water-soluble and TCA-soluble N fractions of maturing cheeses were unaffected by psychrotroph counts in raw milks, but small differences in levels of casein fractions of cheeses made from milks stored for 72 h were detected by quantitative polyacrylamide gel electrophoresis. The incidence of casein breakdown in raw milk and subsequently in cheese were not necessarily related. None of the cheeses developed off flavour related to excessive protein breakdown but many became lipolytically rancid, despite the selection of strains with low lipolytic activity on a diagnostic medium. It is concluded that the numbers of psychrotrophic bacteria likely to occur in stored raw milk under commercial conditions are unlikely to cause significant changes in the yields or quality of Cheddar cheese through their proteolytic activity.