Nonstarter lactic acid bacteria and aging temperature affect calcium lactate crystallization in cheddar cheese

The occurrence of unappetizing calcium lactate crystals in Cheddar cheese is a challenge and expense to manufacturers, and this research was designed to understand their origin. It was hypothesized that nonstarter lactic acid bacteria (NSLAB) affect calcium lactate crystallization (CLC) by producing D(-)-lactate. This study was designed to understand the effect of NSLAB growth and aging temperature on CLC. Cheeses were made from milk inoculated with Lactococcus lactis starter culture, with or without Lactobacillus curvatus or L. helveticus WSU19 adjunct cultures. Cheeses were aged at 4 or 13 degrees C for 28 d, then half of the cheeses from 4 and 13 degrees C were transferred to 13 and 4 degrees C, respectively, for the remainder of aging. The form of lactate in cheeses without adjunct culture or with L. helveticus WSU19 was predominantly L(+)-lactate (> 95%, wt/wt), and crystals were not observed within 70 d. While initial lactate in cheeses containingL. curvatus was only L(+)-lactate, the concentration of D(-)-lactate increased during aging. After 28 d, a racemic mixture of D/L-lactate was measured in cheeses containing L. curvatus; at the same time, CLC was observed. The earliest and most extensive CLC occurred on cheeses aged at 13 degrees C for 28 d then transferred to 4 degrees C. These results showed that production of D(-)-lactate by NSLAB, and aging temperature affect CLC in maturing Cheddar cheese.     

Influence of salt-to-moisture ratio on starter culture and calcium lactate crystal formation

The occurrence of l(+)-lactate crystals in hard cheeses continues to be an expense to the cheese industry. Salt tolerance of the starter culture and the salt-to-moisture ratio (S:M) in cheese dictate the final pH of cheese, which influences calcium lactate crystal (CLC) formation. This research investigates these interactions on the occurrence of CLC. A commercial starter was selected based on its sensitivity to salt, less than and greater than 4.0% S:M. Cheddar cheese was made by using either whole milk (3.25% protein, 3.85% fat) or whole milk supplemented with cream and ultrafiltered milk (4.50% protein, 5.30% fat). Calculated amounts of salt were added at milling (pH 5.40 ± 0.02) to obtain cheeses with less than 3.6% and greater than 4.5% S:M. Total and soluble calcium, total lactic acid, and pH were measured and the development of CLC was monitored in cheeses. All cheeses were vacuum packaged and gas flushed with nitrogen gas and aged at 7.2°C for 15 wk. Concentration of total lactic acid in high S:M cheeses ranged from 0.73 to 0.80 g/100 g of cheese, whereas that in low S:M cheeses ranged from 1.86 to 1.97 g/100 g of cheese at the end of 15 wk of aging because of the salt sensitivity of the starter culture. Concentrated milk cheeses with low and high S:M exhibited a 30 to 28% increase in total calcium (1,242 and 1,239 mg/100 g of cheese, respectively) compared with whole milk cheeses with low and high S:M (954 and 967 mg/100 g of cheese, respectively) throughout aging. Soluble calcium was 41 to 35% greater in low S:M cheeses (low-salt whole milk cheese and low-salt concentrated milk cheese; 496 and 524 mg/100 g of cheese, respectively) compared with high S:M cheeses (high-salt whole milk cheese and high-salt concentrated milk cheese; 351 and 387 mg/100 g of cheese, respectively). Because of the lower pH of the low S:M cheeses, CLC were observed in low S:M cheeses. However, the greatest intensity of CLC was observed in gas-flushed cheeses made with milk containing increased protein concentration because of the increased content of calcium available for CLC formation. These results show that the occurrence of CLC is dependent on cheese milk concentration and pH of the cheese, which can be influenced by S:M and cheese microflora.     

Nonstarter lactic acid bacteria biofilms and calcium lactate crystals in Cheddar cheese

A sanitized cheese plant was swabbed for the presence of nonstarter lactic acid bacteria (NSLAB) biofilms. Swabs were analyzed to determine the sources and microorganisms responsible for contamination. In pilot plant experiments, cheese vats filled with standard cheese milk (lactose:protein = 1.47) and ultrafiltered cheese milk (lactose:protein = 1.23) were inoculated with Lactococcus lactis ssp. cremoris starter culture (8 log cfu/mL) with or without Lactobacillus curvatus or Pediococci acidilactici as adjunct cultures (2 log cfu/mL). Cheddar cheeses were aged at 7.2 or 10°C for 168 d. The raw milk silo, ultrafiltration unit, cheddaring belt, and cheese tower had NSLAB biofilms ranging from 2 to 4 log cfu/100 cm². The population of Lb. curvatus reached 8 log cfu/g, whereas P. acidilactici reached 7 log cfu/g of experimental Cheddar cheese in 14 d. Higher NSLAB counts were observed in the first 14 d of aging in cheese stored at 10°C compared with that stored at 7.2°C. However, microbial counts decreased more quickly in Cheddar cheeses aged at 10°C compared with 7.2°C after 28 d. In cheeses without specific adjunct cultures (Lb. curvatus or P. acidilactici), calcium lactate crystals were not observed within 168 d. However, crystals were observed after only 56 d in cheeses containing Lb. curvatus, which also had increased concentration of d(−)-lactic acid compared with control cheeses. Our research shows that low levels of contamination with certain NSLAB can result in calcium lactate crystals, regardless of lactose:protein ratio.     

Starter strain related effects on the biochemical and sensory properties of Cheddar cheese

A detailed investigation was undertaken to determine the effects of four single starter strains, Lactococcus lactis subsp. lactis 303, Lc. lactis subsp. cremoris HP, Lc. lactis subsp. cremoris AM2, and Lactobacillus helveticus DPC4571 on the proteolytic, lipolytic and sensory characteristics of Cheddar cheese. Cheeses produced using the highly autolytic starters 4571 and AM2 positively impacted on flavour development, whereas cheeses produced from the poorly autolytic starters 303 and HP developed off-flavours. Starter selection impacted significantly on the proteolytic and sensory characteristics of the resulting Cheddar cheeses. It appeared that the autolytic and/or lipolytic properties of starter strains also influenced lipolysis, however lipolysis appeared to be limited due to a possible lack of availability or access to suitable milk fat substrates over ripening. The impact of lipolysis on the sensory characteristics of Cheddar cheese was unclear, possibly due to minimal differences in the extent of lipolysis between the cheeses at the end of ripening. As anticipated seasonal milk supply influenced both proteolysis and lipolysis in Cheddar cheese. The contribution of non-starter lactic acid bacteria towards proteolysis and lipolysis over the first 8 months of Cheddar cheese ripening was negligible.     

The effect of some manufacturing conditions on the development of flavour in Cheddar cheese

Organoleptic assessments by the NIRD panel of Cheddar cheeses made with Streptococcus cremoris NCDO 924 or 1986, either in enclosed vats excluding nonstarter flora or in open vats, showed that high viable starter populations in curd did not give stronger-flavoured cheese, but led to the development of bitterness. Cheeses made in open vats developed typical flavour more rapidly than those made in enclosed vats. Maturation temperature was the most important factor in determining the flavour intensity; cheese ripened at 13d?C for six months had stronger flavour than corresponding ones ripened at 6d?C for nine months, irrespective of the starter or vat used.     

Isolation and characterisation of exopolysaccharide-producing Weissella and Lactobacillus and their application as adjunct cultures in Cheddar cheese

This study characterised exopolysaccharide-producing lactic acid bacteria and examined their potential for use in Cheddar cheese manufacture. Two strains were chosen for incorporation as adjunct cultures in Cheddar cheese manufacture: namely, the homopolysaccharide-producers Weissella cibaria MG1 and Lactobacillus reuteri cc2. These strains both produce dextrans with molecular masses ranging from 10⁵ to 10⁷ Da. Both strains were used in the production of miniature Cheddar cheeses that employed a conventional commercial cheese starter culture Lactococcus lactis R604. A cheese was also included that used purified dextran as an ingredient. The W. cibaria strain survived in cheese with levels increasing by 1.5 log cycles over the ripening period. All experimental cheeses (adjunct or exopolysaccharide ingredient) had higher moisture levels compared with the control cheese made using starter alone. Inclusion of the adjunct strains had no detectable negative effects on cheeses in terms of proteolysis.     

A survey of lipolytic and glycolytic end-products in commercial Cheddar enzyme-modified cheese

The concentrations of L- and D-lactic acid and free fatty acids, C4:0 to C18:3, were quantified in a range of commercial enzyme-modified Cheddar cheeses. Lactic acid in Cheddar enzyme-modified cheeses varied markedly depending on the manufacturer. Differences in the ratio of L- to D-lactic acid indicate that cheeses of different age were used in their manufacture or contained varying levels of nonstarter lactic acid bacteria. The level of lipolysis in enzyme-modified cheese was higher than in natural Cheddar cheese; butyrate was the predominant free fatty acid. The addition of exogenous acetate, lactate, and butyrate was also indicated in some enzyme-modified cheeses and may be used to confer a specific flavor characteristic or reduce the pH of the product. Propionate was also found in some enzyme-modified cheese products and most likely originated from Swiss-type cheese used in their manufacture. Propionate is not normally associated with natural Cheddar cheese flavor; however, it may be important in the flavor and aroma of Cheddar enzyme-modified cheese. Levels of lipolysis and glycolysis appear to highly controlled as interbatch variability was generally low. Overall, the production of enzyme-modified Cheddar cheese involves manipulation of the end-products of glycolysis (lactate, propionate, and acetate) and lipolysis to generate products for specific applications.     

Effect of starter cultures on the physico‐chemical and biochemical features in ewe cheese made with extracts from flowers of Cynara cardunculus L

The effect of starter cultures on the physico‐chemical and biochemical features of Los Pedroches cheese made by using Cynara Cardunculus L extracts as vegetable coagulant was assessed. Specimens of cheese containing no starter (vats A and C) and others to which a lactic acid starter was added (vats B and D) were stored either under typical farmhouse ripening conditions (vats A and B) or in a controlled chamber at 14°C and 80% relative humidity (vats C and D) for 90 days. The addition of starter cultures and the ripening conditions where found to have no appreciable effect on the moisture, fat, protein, ash, NaCl, Ca and P contents of the cheese, nor on its water activity; however, it resulted in increased lactic acid contents and lower pH values relative to control specimens throughout ripening. The protein breakdown of the cheeses was assessed in terms of soluble nitrogen, non‐protein nitrogen, aminoacid nitrogen, ammonia nitrogen, soluble tyrosine and tryptophan, and urea‐PAGE. Proteolysis was generally more marked and rapid in cheese containing the lactic starter, the NPN, NH₃ ‐N and soluble Tyr and Trp contents of which were significantly higher than those of the cheese obtained with no starter. The PAGE technique revealed no appreciable differences between the four cheese batches studied; however, it showed the cheese to be more resistant to β‐CN hydrolysis than to α_s‐CN hydrolysis. In general, the flavour and aroma scores were higher in batches obtained with starter cultures and ripened under controlled conditions. © 1999 Society of Chemical Industry     

The design of a three strain starter system for Cheddar cheese manufacture exploiting bacteriocin-induced starter lysis

A three strain starter system was developed for increasing the level of starter cell lysis during cheese manufacture and ripening. The composition of this starter combination includes a bacteriocin (lactococcin A, B and M) producer which causes the lysis of a second strain (sensitive to bacteriocin activity) during cheese manufacture, and a third strain resistant to bacteriocin activity. The latter strain plays an essential role in ensuring acid production during cheese manufacture. Cheeses manufactured at pilot-scale (450 L vats), with the three strain starter combination were assessed for levels of the intracellular enzyme, lactate dehydrogenase, released into the cheese matrix during ripening. Experimental cheeses, manufactured with the bacteriocin-producing adjunct, exhibited higher levels of free amino acids and greater release of intracellular LDH than control cheeses manufactured in its absence. Cheese was subject to sensory analysis which revealed that experimental cheese showed a decrease in bitterness over cheeses manufactured without the bacteriocin-producing adjunct. Thus, this three strain system offers manufacturers a reliable starter system exhibiting increased lysis with concomitant improvements in cheese flavour.     

Use of high pressure treatment to attenuate starter bacteria for use as adjuncts for Cheddar cheese manufacture

Attenuated starter bacteria cannot produce acid during cheese manufacture, but contain enzymes that contribute to cheese ripening. The aim of this study was to investigate attenuation of starter bacteria using high pressure treatment, for use in combination with a primary starter for Cheddar cheese manufacture, and to determine the effect of such adjunct cultures on secondary proteolysis during ripening. Lactococcus lactis ssp. cremoris HP and L. lactis ssp. cremoris 303 were attenuated by pressure treatment at 200 MPa for 20 min at 20 °C. Cheddar cheese was manufactured using untreated cultures of both these starter strains, either alone or in combination with their high pressure-treated equivalents. High pressure-treated starters did not produce acid during cheese manufacture and starter counts in cheeses manufactured using high pressure-treated starter did not differ from those of the controls. Higher levels of cell lysis were apparent in cheese manufactured using high pressure-treated strains than in the controls after 26 d of ripening. Small differences were observed in the peptide profiles of cheeses, analysed by reversed-phase HPLC; cheeses manufactured using high pressure-treated starters also had slightly higher levels of amino acids than the relevant controls. Overall, addition of high pressure-treated starter bacteria as a secondary starter culture accelerated secondary proteolysis in Cheddar cheese.

Industrial relevance

Attenuated starters provide extra pool of enzymes, which can influence cheese ripening, without affecting the cheese making schedule. This paper presents an alternative method for attenuation of starter bacteria using high pressure treatment and their subsequent use to accelerate secondary proteolysis in Cheddar cheese during ripening.     

Survival of foot-and-mouth disease virus in cheese.

Persistence of foot-and-mouth disease virus during the manufacture of Cheddar, Mozzarella, Camembert cheese prepared from milk of cows experimentally infected with the virus was studied. Cheese samples were made on a laboratory scale with commercial lactic acid starter cultures and the microbial protease MARZYME as a coagulant. Milk was heated at different temperatures for different intervals before it was made into cheese. Food-and-mouth disease virus survived the acidic conditions of Cheddar and Camembert cheese processing but not that of Mozzarella. Foot-and-mouth disease virus survived processing but not curing for 30 days in Cheddar cheese preparaed from heated milk. However, the virus survived curing for 60 days but not for 120 days in cheese (pH 5) prepared from unheated milk. Foot-and-mouth disease virus survived in Camembert cheese (pH 5) for 21 days at 2 C but not for 35 days.     

Effect of Lactobacillus helveticus and Propionibacterium freudenrichii ssp. shermanii combinations on propensity for split defect in Swiss cheese

One of the least controlled defects in Swiss cheese is development of splits that appear during refrigerated storage after cheese is removed from the warm room. Such fissures, or cracks, in the body of the cheese can be as short as 1 cm, or long enough to span a 90-kg block. A 2 x 2 x 2 factorial experiment was used to determine the effect of different Lactobacillus helveticus/Propionibacterium freudenreichii ssp. shermanii starter culture combinations on the occurrence of split defect in Swiss cheese. Eights vats of cheese were made in summer and eight in winter. Each 90-kg block of cheese was cut into twenty-four 4-kg blocks and graded based on the presence of splits. Only small variations were found in the composition of cheeses made during the same season. There were no correlations between moisture, pH, fat, protein, calcium, lactose contents, D/L lactate ratio, or protein degradation that could be used to predict splits after 90 d of storage. However, cheese made in the summer had 2% higher moisture content and a greater prevalence of splits. There was a sixfold increase in amount of downgraded cheese between the best and worst culture combinations used during cheese manufacture. After 90-d storage, 14 to 90% of cheese had splits in the summer, and 1 to 6% in the winter. Split formation increased with time from 60 to 120 d of storage and extent of split formation was influenced by both the lactobacilli and propionibacteria cultures used.     

Evidence of a relationship between autolysis of starter bacteria and lipolysis in cheddar cheese during ripening

Cell viability, autolysis and lipolysis were studied in Cheddar cheese made using Lactococcus lactis subsp. cremoris AM2 or Lactococcus lactis subsp. cremoris HP. Cheddar cheese was made in triplicate over a 3 month period and ripened for 238 days at 8 degrees C. Cell viability in cheese was lower for AM2 (a non-bitter strain) than for strain HP (a bitter strain). Autolysis, monitored by the level of the intracellular marker enzyme, lactate dehydrogenase (EC 1.1.1.27) in cheese 'juice' extracted by hydraulic pressure, was much greater in the cheese made using AM2 than that made with HP. Lipolysis was determined by the increase during ripening of individual free fatty acids (FFA) from butyric (C4:0) to linolenic acid (C18:3) measured using a high performance liquid chromatographic technique. Levels of individual FFA from butyric (C4:0) to linolenic (C18:3) acids increased significantly (P<0.05) during ripening in cheeses made with either starter culture. Palmitic (C16:0) and oleic (C18:1) acids were the most abundant FFA throughout ripening in all cheeses. Levels of caprylic (C8:0), myristic (C14:0), palmitic (C16:0) and stearic (C18:0) acids were significantly higher (P<0.05) in cheeses manufactured with Lc. lactis subsp. cremoris AM2 than in cheeses manufactured with Lc. lactis subsp. cremoris HP. Differences in levels of lipolysis between strains was not due to differences in the specific lipolytic or esterolytic activities in cell free extracts of the strains as measured by activity on triolein (lipase) and p-nitrophenylbutyrate (esterase) substrates. Therefore, evidence is provided for a relationship between the extent of starter cell autolysis and the level of lipolysis during Cheddar cheese ripening.     

Glycolysis and related reactions during cheese manufacture and ripening

Fermentation of lactose to lactic acid by lactic acid bacteria is an essential primary reaction in the manufacture of all cheese varieties. The reduced pH of cheese curd, which reaches 4.5 to 5.2, depending on the variety, affects at least the following characteristics of curd and cheese: syneresis (and hence cheese composition), retention of calcium (which affects cheese texture), retention and activity of coagulant (which influences the extent and type of proteolysis during ripening), the growth of contaminating bacteria. Most (98%) of the lactose in milk is removed in the whey during cheesemaking, either as lactose or lactic acid. The residual lactose in cheese curd is metabolized during the early stages of ripening. During ripening lactic acid is also altered, mainly through the action of nonstarter bacteria. The principal changes are (1) conversion of L-lactate to D-lactate such that a racemic mixture exists in most cheeses at the end of ripening; (2) in Swiss-type cheeses, L-lactate is metabolized to propionate, acetate, and CO2, which are responsible for eye formation and contribute to typical flavor; (3) in surface mold, and probably in surface bacterially ripened cheese, lactate is metabolized to CO2 and H2O, which contributes to the increase in pH characteristic of such cheeses and that is responsible for textural changes, (4) in Cheddar and Dutch-type cheeses, some lactate may be oxidized to acetate by Pediococci. Cheese contains a low level of citrate, metabolism of which by Streptococcus diacetylactis leads to the production of diacetyl, which contributes to the flavor and is responsible for the limited eye formation characteristic of such cheeses.     

Quality and ripening changes of Ras cheese made by direct acidification

Trials were carried out to produce Ras cheese of good quality without the use of starter. Cheese was made from pasteurized cow's milk acidified with lactic acid or citric acid to pH 5.8 alone or coupled with mixing the curd with glucono δ lactone (4.5 g/kg curd). Control cheese was made from milk ripened with a starter culture of S. lactis. Resultant cheeses showed poor body and texture, weak flavour intensity and low levels of soluble nitrogen compounds and free volatile fatty acids. Incorporation into the cheese curd of mixtures containing Fromase 100 (fungal protease) and Piccantase B (fungal lipase) or Fromase 100 and Capalase K (animal lipase) enhanced flavour intensity, improved body characteristics and accelerated the formation of both soluble nitrogen compounds and free volatile fatty acids. The organoleptic properties of the experimental cheeses with added enzymes were comparable to those of the control cheese.     

The behavior of selected microorganisms during the manufacture of high moisture Jack cheeses from ultrafiltered milk.

Whole milk was pasteurized and concentrated two times by ultrafiltration. Starter cultures, Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis, were propagated in either reconstituted skim milk, two times UF retentate, or UF permeate, or a direct vat system was used for the starter culture. The cheese milk was simultaneously inoculated with starter culture and Pseudomonas fragi 4973, Staphylococcus aureus 196E, and Salmonella typhimurium var. Hillfarm. Control whole milk, UF control milk, inoculated whole milk, and inoculated UF milk were made into Monterey Jack cheese using traditional procedures. The process of cheese manufacture was followed by determination of pH, titratable acidity, and microbial population levels. The cheeses were stored for 6 mo and analyzed every month for percentage solids and microbial population levels. Generally, numbers of contaminant microbes increased at a similar rate during manufacture in all cheeses. During the 6-mo ripening period, bacterial starter culture population levels remained high, psychrotrophs declined slowly, Staphylococcus levels remained stable, and Salmonella populations decreased. No Staphylococcus enterotoxin was detected by reverse passive latex agglutination assay.     

Effect of wild strains of Lactococcus lactis on the volatile profile and the sensory characteristics of ewes' raw milk cheese

The production of volatile compounds by wild strains of Lactococcus lactis used as starter cultures and their effect on the sensory characteristics of ewes' raw milk cheese were investigated. Sixteen vats of cheese were manufactured and ripened for 120 d in two experiments, each of them duplicated. In the first experiment, milk was inoculated with different ratios of four wild Lactococcus lactis strains, two producing and two not producing branched-chain volatile compounds, and in the second experiment with different ratios of a commercial starter culture and the two strains producing branched-chain volatile compounds. Cheese pH, proteolysis, and aminopeptidase activity increased when the strains producing branched-chain volatile compounds were inoculated at a higher rate. Fifty volatile compounds were identified in cheeses using a purge and trap system coupled to a gas chromatography-mass spectrometry apparatus. The relative abundances of 30 volatile compounds (8 alcohols, 5 aldehydes, 3 ketones, 12 esters, 1 sulfur compound, and 1 benzenic compound) were influenced by starter culture composition. 2-Methylpropanol, 3-methylbutanol, isobutyl acetate, isoamyl acetate, ethyl butyrate, isobutyl butyrate, and isoamyl butyrate were always more abundant in the cheeses made with a higher level of L. lactis strains producing branched-chain volatile compounds. Flavor intensity was enhanced by a high level of L. lactis strains producing branched-chain volatile compounds in the first experiment, in which four wild L. lactis strains were used as starter culture, but not in the second experiment, in which a combination of two wild L. lactis strains and the commercial starter culture were used. Flavor quality, as judged by trained panelists, was impaired in both experiments by a high level of L. lactis strains producing branched-chain volatile compounds.     

Effect of curd washing on cheese proteolysis,texture, volatile compounds,and sensory grading in full fat Cheddar cheese

Curd was washed to varying degrees during Cheddar cheese manufacture, by partial replacement of whey with water at the early stages of cooking, to give target levels of lactose plus lactic acid in cheese moisture of 5.3 (control), 4.5, 4.3 and 3.9% (w/w). The cheeses were matured at 8 °C for 270 days. While curd washing had little effect on composition or the mean levels of proteolysis (as measured by pH 4.6 soluble nitrogen and levels of free amino acids), it led to cheeses that were, overall, firmer and less brittle. Curd washing resulted in cheeses having lower levels of some volatile compounds, and being less acid, more buttery, sweeter, saltier and creamier than non-washed cheeses that had more 'sweaty', pungent and farmyard-like sensory notes. The results suggest that curd washing during Cheddar manufacture may be used as a means of creating variants with distinctive flavour profiles.