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Glycolysis and related reactions during cheese manufacture and ripening a

b

P. F. Fox , J. A. Lucey & T. M. Cogan a

b

Department of Food Chemistry , University College , Cork, Ireland

b

Teagasc , Moovepack, Fermoy, County Cork, Ireland Published online: 29 Sep 2009.

To cite this article: P. F. Fox , J. A. Lucey & T. M. Cogan (1990) Glycolysis and related reactions during cheese manufacture and ripening, Critical Reviews in Food Science and Nutrition, 29:4, 237-253, DOI: 10.1080/10408399009527526 To link to this article: http://dx.doi.org/10.1080/10408399009527526

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Food Science and Nutrition

Glycolysis And Related Reactions During Cheese Manufacture And Ripening

ein, although it is usually enzymatically modified to produce the desired body and texture. Coagulation of the casein is induced by 1.

P. F. Fox, J. A. Lucey, and T. M. Cogan 2.

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ABSTRACT 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 Dlactate 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.

3.

Specific enzymatic hydrolysis of the casein micelle-stabilizing protein, K-casein, through the action of selected acid proteinases, called rennets Isoelectric precipitation at ~pH 4.6, either through production of acid by lactic acid bacteria, or by the direct addition of acid or acidogen, usually gluconic acid-8lactone By heating to 80 to 90°C at pH - 5 . 2

Acid- and heat/acid-coagulated cheeses are consumed fresh (unripened), but the vast majority of rennet-coagulated cheeses are ripened (matured) after manufacture for periods ranging from 4 weeks to 2 or more years during which the characteristic flavor and texture of the individual cheese varieties develop. This review concentrates on cheeses that are ripened, although the mechanism of lactic acid production is similar in all cheeses. The manufacture of rennet-coagulated cheeses can be divided into two more-or-less distinct phases: (1) conversion of milk to curd, which is essentially complete within 24 h; and (2) ripening of the curd. Conversion of milk to curd consists of several distinct operations: 1. 2. 3. 4. 5.

Acidification Coagulation Curd syneresis/dehydration Moulding Salting

In this review we are concerned only with acidification during manufacture and the fate of lactate during the ripening process. The interested reader is referred to Fox29 for comprehensive reviews of the other operations. II. ACIDIFICATION DURING CHEESE MANUFACTURE

I. INTRODUCTION Cheese manufacture essentially involves concentrating the casein and fat in milk by a factor of 6 to 12, depending on the variety. In traditional cheese manufacture, concentration is achieved by coagulating the casein to form a gel that occludes the fat, if present. When the gel is cut or broken it contracts (synereses), expelling whey at a rate and to an extent dependent mainly on temperature, pH, agitation, protein concentration, and Ca 2+ concentration; during manufacture these parameters are manipulated to control the moisture content of the cheese. Recent developments in ultrafiltration technology also permit concentration of the total colloidal phase of milk (casein, whey proteins, fat, and colloidal salts) without coagulating the cas-

One of the primary events in the manufacture of most, if not all, cheese varieties is the fermentation of lactose to lactic acid by selected lactic acid bacteria or, in traditional cheesemaking, by the indigenous microflora. The rate and point of the process at which lactic acid is principally produced are

P. F. Fox (corresponding author), B.Sc, Ph.D.,Depaitment of Food Chemistry, University College, Cork, Ireland. J.A. Lucey and T. M. Cogan, B.Sc. D. M.Sc., Ph.D., Teagasc, Moovepack, Fermoy, County Cork, Ireland.

1990

237

Critical Reviews In characteristic of the variety; for example, in Cheddar-type cheeses, most of the acid is produced prior to molding, while in most other varieties it occurs mainly after molding. The extent of acidification prior to molding determines the method of salting that may be used, i.e., blending of salt with curd in Cheddar types or surface salting (dry or brining) for most other varieties. Regardless of the rate or point in the process at which acidifiction occurs, the pH of most rennet cheese varieties reaches ~ 5 within 5 to 12 h and certainly within 24 h.

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III. SIGNIFICANCE OF ACID DEVELOPMENT IN CHEESE Acid production affects almost all facets of cheese manufacture and hence cheese composition and quality. Among the most important consequences of pH and acid development are 1. 2. 3. 4. 5. 6. 7.

8.

9. 10. 11.

Activity of the coagulant during manufacture Retention of coagulant in cheese curd Activity and perhaps the specificity of the coagulant during ripening Activity of plasmin (indigenous milk proteinase) Curd tension Curd syneresis Solubilization of colloidal calcium and phosphate, which, among other factors, affects curd (cheese) texture, stretchability, and meltability Shelf-life — the spectrum of cheese varieties exhibits a range of shelf-life stability from low (e.g., cottage, Camembert) to high (e.g., Parmesean); pH and a,, play complementary roles in determining cheese stability (see Shaw108 and Marcos77) obviously, these factors also control the rate of ripening Prevention of the growth and/or survival of pathogens The acid taste of acid-coagulated and young rennet-coagulated cheeses Lactate serves as substrate for the production of propionic acid, acetic acid, and CO2 during ripening of Swiss-type cheeses

The significance of many of these functions will be developed in later sections.

IV. LACTOSE METABOLISM BY STARTER BACTERIA In modem cheese manufacture, acidification of milk and cheese curd is accomplished by the production of L- (mainly) and D-lactic acid from lactose through the action of specially selected cultures (starters) of lactic acid bacteria (LAB). Recent general reviews on starter cultures include those of Lawrence et al., 69 Auclair and Accolas,3 Cogan,9 Thunell et al.,113 Marshall,81 Cogan and Daly,12 Cogan and Accolas.11 238

Traditionally, these cultures are divided into mesophilic, with optimum temperatures of ~ 30°C, and thermophilic, with optima of ~ 45°C. Mesophilic cultures always contain Streptococcus cremoris and sometimes also S. lactis, S. diacetylactis, and/or Leuconostoc spp. S. lactis and S. cremoris are responsible for acid production, while S. diacetylactis and the leuconostocs are required for flavor development (mainly diacetyl and acetate) and eye formation in Dutch-type cheese (due to CO2 production). Thermophilic cultures are usually composed of S. thermophilus and a lactobacillus — Lb. helveticus and/or Lb. lactis for Swiss cheese and Lb. bulgaricus for yogurt manufacture. However, starter cultures for some hard cheeses, e.g., Beaufort and Grana, contain only lactobacilli, i.e., Lb. helveticus or Lb. lactis. Application of modem molecular techniques, especially DNA or RNA hybridizations, to the LAB found in starter cultures has resulted in several changes in their taxonomic status (Garvie,36 Kandler and Weiss,62 Schleifer and Kilpper-Balz106). Some of these changes, for example, the transfer of 5. cremoris, S. lactis, and S. diacetylactis to a new genus, Lactococcus, and their relegation to subspecies status, the retention of S. thermophilus in the genus Streptococcus and the relegation of Lb. bulgaricus and Lb. lactis to subspecies of Lb. delbruekii give a clearer understanding of cultures since organisms in the same group generally have similar properties, of which the transport and fermentation of sugars are good examples (see Section IV.A). For simplicity, the abbreviations S. lactis, S. cremoris, and S. diacetylactis are used in this review. A. Sugar Transport and Metabolism The detailed mechanisms of sugar transport in LAB have been identified and characterized only recently. Basically, two mechanisms are involved — active transport and the phosphoenolpyruvate (PEP) — phosphotransferase system (PTS) —• both of which require energy. In active transport, the lactose is essentially taken up against a thermodynamically unfavorable concentration gradient. This results in intracellular concentrations several thousand times that in the extracellular medium. The lactose is unchanged during transport and so the system is often called the "lactose permease system". In the PEP/ PTS system (Dills et al.24), the lactose is phosphorylated by PEP during transport, entering the cell as lactose phosphate. The system is also called the "group translocation system" and has been thoroughly reviewed recently by Thompson.127 There is no concentration gradient in this case, as the form of the sugar inside and outside the cell is different. The system requires three enzymes, El, EII, and EIII, and a low-molecularweight, heat-stable protein (HPr) (Figure 1). EII and EIII are specific for the particular sugar and are associated with the cell wall and/or membrane. The enzyme El is nonspecific and is responsible for the initial transfer of energy from PEP to a histidine residue in the HPr. Lactose and lactose phosphate are hydrolyzed by P-galactosidase (p-gal) and phospho-(J-galac-

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Food Science and Nutrition

SUGAR-P

• SUGAR

PYRUVATE

tm-p

S

IN

OUT

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FIGURE 1. Phosphoenol pyruvaterphosphotransferase system for sugar metabolism. El, enzyme I; EH, enzyme II; Em, enzyme HI; HPr, heat stable phosphocarrier protein; P, phosphorylated compound. (From Dills et al., Microbiol. Rev., 44, 385, 1980. With permission.)

tosidase (P-3-gal), respectively, and the presence of these enzymes indicates the transport mechanism used. The transport and metabolism (Figure 2) of lactose in each group of bacteria involved in starter cultures are discussed in the following sections. 1. Lactococcus Fairly detailed information is available on this group of starter bacteria, in which the transport of sugars is by the PEP/PTS system; however, a low-affinity PTS system and a high-affinity permease system have been observed for galactose (Thompson126 and Park and McKay94). Lactose (4-0-3-D-galactopyranosylct-D-glucopyranoside) is phorphorylated at carbon 6 of the galactose moiety during transport. Inside the cell, lactose phosphate is hydrolyzed by P-|3-gal to glucose and galactose-6phospshate. The former is metabolized through the normal glycolytic sequence to L-lactic acid, while the latter is metabolized through several tagatose derivatives (Thompson125). Tagatose is a stereoisomer of fructose, but separate enzymes are involved in the formation of fructose-6-phosphate and tagatose6-phosphate, and of fructose-1, 6-diphosphate (FDP) and tagatose-1, 6-diphosphate (TDP). Lactose transport and the enzymes of the tagatose system are plasmid-encoded properties in these bacteria (McKay84 and Crow et al.18). The terminal reactions of both glucose and tagatose metabolism are similar, finally leading to the production of L-lactate from pyruvate in a NADH-requiring reaction, catalyzed by lactic dehydrogenase (LDH). One of the reasons for the formation of lactic acid by lactococci is the need to regenerate stoichiometric amounts of oxidized NAD to continue the fermentation. For this reason, cells contain large amounts of LDH, an allosteric enzyme that is activated by FDP and TDP (Thomas114). L-Lactate is generally the sole product of fermentation, but when these bacteria are grown on galactose or low concentrations of glucose, other products besides L-lactate are produced from pyruvate, e.g., formate, ethanol, and acetate (Thomas et al.124 and Thomas and Turner121). This is due mainly to decreases in the intracellular concen-

trations of FDP and triose phosphates, which activate LDH and inhibit pyruvate-formate lyase, respectively. The net result is that more pyruvate is metabolized to formate, acetate, and ethanol at the expense of lactate (Fordyce et al.27). Pyruvate is also produced from citrate by the flavor-producing bacteria (Leuconostoc spp. and S. diacetylactis), but much of this pyruvate is used in the production of acetoin and diacetyl (Cogan9). Pyruvate produced from citrate could be used in lactate formation, but the stoichiometry between the NAD required in glycolysis and lactate production suggests that it is not. PEP is an intermediate of both lactose transport and lactose metabolism and it therefore plays a pivotal role in determining the balance between transport and metabolism. The regulation of sugar metabolism is very complicated and essentially relies on the allosteric enzyme, pyruvate kinase (PK), and the intracellular concentrations of various effectors, especially FDP and inorganic phosphate (Pi) (Thompson127). FDP is a positive effector, while Pi is a negative effector of PK. In growing cells, the concentration of FDP is high, while that of Pi is low, so that PK activity is maintained and pyruvate is produced. In contrast, during starvation, when the PTS is inoperative, the concentration of FDP is low, while that of Pi is high, and, as a result, the concentration of PEP increases because of the lack of PK activity. 2. Leuconostocs Little is known about the transport of lactose, or indeed of other sugars, in these bacteria. A p-galactosidase (p-gal) has been identified in Leuconostoc cremoris ATCC 8081 (Singh et al.109), implying that lactose is transported by a permease system; however, this organism was misidentified and is in fact a Pediococcus acidilactici (Felton and Niven26 and Anon1). Nevertheless, the presence of a permease system in leuconostocs is inferred from the results of Romano et al.105 who found that only facultative anaerobes that ferment sugars via glycolysis contain a PEP/PTS system. Leuconostoc spp. ferment sugar by the phosphoketolase pathway (Gunsalus and Gibbs45 DeMoss et al.23) in which lactose is hydrolyzed by 0gal to glucose and galactose. The former is metabolized to equimolar concentrations of lactate, ethanol, and CO2, while the latter is probably metabolized by the Leloir pathway to glucose-6-P, using the enzymes, galactokinase (GK), galactose-1-P uridyl transferase, and UDP-4-epimerase. Lactate and ethanol are produced owing to the need to regenerate NAD/ NADP to continue the fermentation, but, unlike the lactococci, the D isomer of lactate is formed. Co-metabolism of glucose and citrate by leuconostocs results in a switch from ethanol to acetate formation and the production of more lactate than can be accounted for in terms of the sugar used. This is due to the reduction of the pyruvate produced from both citrate and glucose to lactate. This relieves the cell from reducing acetaldehyde to ethanol to regenerate NAD/NADP and instead the precursor, acetyl-P, is metabolized to acetate and ATP, which increases the growth rate of the cells (Jordan and Cogan57). 1990

239

Critical Reviews In CKOUfM STKtnvCOCCt

UUCCHOSTOeS

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It. lUlGAtlCUS U . LACTIS LACTOSE

LACTOSE EXTERNAL ENVIRONMENT

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1 CLUCOSE

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I CALACTOSE-IP

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TAGATOSE-f-P

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1 • PHOSTHOGLYCE KATE

ACETALDEHYDE

r"

2 - PHOSPHOGLYCE KATE

PMCSPHOENOLPYWIYATE

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FIGURE 2. Probable pathways for the metabolism of lactose in mesophilic and thermophilic lactic acid bacteria.

3. Streptococcus thermophilus An extensive review of carbohydrate metabolism by 5. thermophilus has been published recently (Hutkins and Morris53). There are contradictions about the presence of P-p-gal and pgal in this organism. Some workers (Tinson et al.129 and Thomas and Crow118) found only p-gal, while others (Hemme et al.46 and Somkuti and Steinberg111) found both enzymes with much higher levels of 3-gal than P-P-gal. These apparent contradictions could be due to strain differences or the presence of P240

P-gal artifact due to hydrolysis of the substrate ONPG-P (0nitrophenylgalactose phosphate), used to assay for P-p-gal activity by phosphatase to ONPG, which is the substrate used to assay P-gal (Hickey et al.48). It should be noted that both enzymes are assayed with artificial substrates and a low activity of one does not necessarily mean that activity on the natural substrate is also low. Thus, in S. thermophilus, transport of lactose is probably by a permease with the initial step of lactose hydrolysis catalyzed by p-gal. However, only the glucose moiety

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Food Science and Nutrition is metabolized subsequently; galactose is excreted in amounts equimolar with the lactose used. Because of this, another organism capable of metabolizing galactose must be used in cheese made with 5. thermophilus to reduce the sugar levels so that organisms capable of causing defects do not grow in the cheese. In Swiss cheese, Lb. helveticus and gal + strains of Lb. lactis fulfill this function. Although most strains of S. thermophilus are gal", a few are gal + (Somkuti and Steinberg111). Gal + strains can also be selected by growing gal" strains in lactose-limited chemostats (Thomas and Crow118), but these revert to gal" status within 10 subcultures (100 generations). Wild-type gal + strains also excrete galactose when grown on lactose (Hutkins et al.54), but they will metabolize galactose in the presence of low (3.5 mM) levels of lactose (Tinson et al.129). High levels of hexokinase, aldolase, glyceraldehyde-3-P dehydrogenase, PK, and LDH and low levels of the Leloir pathway enzymes are found in cells growing on lactose, and no evidence for a tagatose pathway has been found in this organism (Thomas and Crow118). Phosphorylation of galactose by GK appears to be the ratelimiting step in the utilization of galactose (Thomas and Crow118 and Hutkins et al.54). Three pieces of information support this contention: 1. 2.

3.

GK activity in gal" strains growing on lactose, with one exception, is lower than in gal + strains55 (Hutkins et al.52) Wild-type gal + strains growing on galactose contain 10 times more GK activity than when grown on lactose (Hutkins et al.52) gal + strains selected from gal" strains in lactose-limited chemostats contained 3 to 5 times more GK activity than gal" strains grown on lactose (Thomas and Crow118).

These data suggest that lactose and/or glucose repress GK activity, and that while glucose can be readily metabolized the capacity of the organism to metabolize the equivalent amount of galactose produced from lactose is exceeded, and therefore galactose is excreted. Similar results have been observed with other organisms. Like the lactococci, S. thermophilus metabolizes sugars by the glycolytic pathway, but, unlike the former, metabolizes galactose homofermentatively (Thomas and Crow118). There is very little information on the regulation of sugar metabolism in S. thermophilus. 3-Gal is induced by lactose and repressed by glucose; the latter effect can be offset by cAMP (Somkuti and Steinberg110), high concentrations of which are found in this bacterium (Radliff et al.101). These data suggest that cAMP has a regulatory function. In contrast to the group N streptococci, the LDH of 5. thermophilus is not affected allosterically by FDP (Garvie34 and Thomas and Crow118), but PK is activated by FDP, fructose-6-P, and inhibited by Pi (Hutkins and Morris52). However, the regulatory role of PK is probably not as important when these bacteria metabolize lac-

tose as it is in the lactococci. It is conceivable that regulation of PK would be important if these bacteria were growing on sugars transported by a PTS system. 4. Thermophilic lactobacilli The thermophilic lactobacilli important in starter cultures are Lb. acidophilus, Lb. helveticus, Lb. delbrueckii subsp. lactic, and Lb. delbrueckii subsp. These bacteria contain both P-gal and P-P-gal (Premi et a l . " and Hickey et al.49), implying that lactose is transported by both the PTS and permease systems, but the levels of p-gal are much greater, suggesting that the permease is the more important mechanism (Hickey et al.49). Some evidence for a glucose-specific PEP/PTS system has been found (Hickey et al.49). Only Lb. helveticus and a few strains of Lb. lactis ferment, while all strains of Lb. bulgaricus do not (Turner and Martley131 and Hickey et al.49). Like 5. thermophilus, the gal-lactobacilli, when growing on lactose, excrete galactose in amounts equimolar to the lactose used. Some strains can metabolize galactose, but only when low (4.0 mM) concentrations of lactose are present (Hickey et al.49). Further studies are required to establish the reasons for the defective metabolism of galactose. Sugar metabolism by these organisms is by glycolysis and different isomers of lactate are formed. A summary of the different mechanisms of transport and metabolism of lactose in various starter bacteria is given in Table 1. 5. Mesophilic lactobacilli and pediococci These organisms are important contaminants of many cheeses, reaching numbers as high as 108 per g of cheese and are often called the "nonstarter lactic acid bacteria" (NSLAB). The particular species involved are thought to be Lb. casei, Lb. plantarum, and Ped. pentosaceus (Thomas et al.125). Whether the NSLAB are also involved in flavor formation in cheese is unclear, but they are responsible for the isomerization of L- to D-lactate in Cheddar cheese, eventually producing a racemic mixture. The mechanism involves oxidation of L-lactate by a L-LDH, which is then reduced by D-LDH to D-lactate (Thomas and Crow117). Whether these activities are catalyzed by one or two enzymes has not been categorically determined. This reaction could also be catalyzed by a racemase, but this was ruled out in experiments that showed simultaneous loss of Dlactate formation and the NAD required as co-factor. Furthermore, racemases are rare in LAB, so far being found only in Lb. curvatus and Lb. sake (Garvie35). Lb. casei and Lb. plantarum vary in their complement of lactose-hydrolyzing enzymes, with P-P-gal predominating in most strains of the former and p-gal in the latter (Premi et al., 99 Jimeno et al., 58 and Chassy and Thompson7). However, some strains of Lb. casei possess only P-gal. Presumably, these differences are also reflected in the transport systems with the PTS system being involved in those organisms that contain P-p-gal and the permease in p-gal-containing organisms. A galactose PTS has also been identified in Lb. casei (Chassy and Thompson8). 1990

241

Critical Reviews In Table 1 Salient Features of Lactose Metabolism of the Lactic Acid Bacteria Found in Starter Cultures Transport pathway

Organisms Group N Streptococci Leuconostoc S. thermophilus Thermophilic lactobacilli

PEP/PTS ATP? ATP ATP

GLY PK GLY GLY

Cleavage enzyme P-0-GAL P-GAL 0-GAL (3-GAL

Products (mol/mol used) 4 2 4 4

Lactate Lactate + 2 ethanol + 2CO2 Lactate Lactate

Isomer of lactate L D L DL or D

Little information is available on pediococci. A 3-gal has been isolated from one strain of Pediococcus (Singh et al.109), implying that, at least in this strain, active transport of lactose occurs.

V. PRODUCTION OF LACTIC ACID IN CHEESE CURD AND FACTORS AFFECTING IT As discussed in the following sections, the rate and extent of acid production have major consequences for cheese characteristics and quality. Owing to greater curd demineralization, the consequences of acid production prior to whey drainage are more pronounced (e.g., Camembert, Cheddar) than when acid is produced mainly after molding (e.g., Gouda, Emmental). This is due mainly to the relatively small proportion of whey removed after initial drainage. Changes in the concentration of lactose and lactate are shown in Figure 3a and b. On

coagulation, the great majority of the lactic acid bacteria are occluded within the semi-rigid casein network (Czulak20) and therefore lactate is produced faster within the curd than in the surrounding whey up to the point of whey drainage. The extent of acid production prior to whey drainage is characteristic of the variety and is determined by several factors, the most important of which are discussed below. A. Preincubation of Inoculated Cheesemilk This is practiced for several varieties; for example, in Camembert manufacture, rennet is not added until the pH of the milk reaches ~6.0; under these conditions ~50% of the colloidal calcium phosphate is solubilized and is therefore removed in the whey. B. Cooking Temperature The optimum growth temperature of S. lactis and S. cremoris is ~30°C and most strains will not multiply above ~38°C; S.

B

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Note: PEP/PTS, phosphoenolpyruvate-phosphotransferase system; GLY, glycolysis; PK, phosphoketolase.

Month

o o I

FIGURE 3. Changes in the concentrations of lactose (A) and lactate (B) in milk (A) and curd (o) during the manufacture and ripening of Cheddar cheese. (From Huffman, L. M. and Kristoffersen, T., N. Z. J. Dairy Sci. Technol., 19, 151, 1984. With permission.)

242

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Food Science and Nutrition cremoris is the more heat sensitive. In some starter systems, a blend of "fast" and "slow" strains is used to control starter population, since an excessively high number of starter cells in cheese curd is believed to result in bitterness, at least in the case of Cheddar (Lowrie et al.73). In this context, a fast strain is one that multiplies and produces acid at ~38°C, while slow strains do not multiply at that temperature but continue to produce acid at a slower rate due to uncoupling of growth from acid production (Turner and Thomas133). Cooking temperature has a major effect on acid development, which can be illustrated by comparing Cheshire, Gouda, Cheddar, and Emmental. Although milk for these cheeses is inoculated with different levels and types of starter, cooking temperature is probably the dominant factor in determining the rate of acid development. Curd for Cheshire cheese is normally cooked at ~32°C and hence acid production continues at its maximum rate during "cooking"; syneresis is achieved mainly through rapid acidification, which leads to extensive demineralization of the curd, with major consequences for cheese texture, as discussed in Section VII.B. Curd for Gouda is cooked to 34 to 36°C and hence the pH would be expected to decrease rapidly; however, the rate of acidification in Gouda is regulated by removing part of the whey and replacing it by water, which reduces the lactose content of the curd (see Section VI.E). Cheddar cheese curd, which is normally drained ~pH 6.1, is cooked to temperatures (~39°C) above the upper limit of mesophilic starters. Starter population and curd pH are controlled by matching cooking temperature to the thermal sensitivity of the starter strains used. Emmental is an example of a cheese in which the cooking temperature (52 to 55°C) is so high that it prevents growth of the thermophilic starter used. Very little acid is produced in the vat and syneresis is induced mainly through a high cooking temperature. Acidification occurs only when the curd cools during pressing (see Section Vin). The curd is highly mineralized with consequences for cheese texture (see Section VII.B). C. Phage, Antibiotics, and Other Inhibitors These agents retard or prevent starter growth and acid production in cheese. They will not be discussed further here.

VI. RETENTION OF LACTOSE IN CHEESE CURD With the notable exception of the English cheese varieties (Cheddar, Cheshire, other territorials, Stilton), most of the lactic acid is produced after molding of the curd. The concentration of lactose and lactate in Cheddar curd at milling is usually 0.8 to 1.0% and —0.6 to 0.8%, respectively. The remaining lactose is usually metabolized to lactate during the early stages of ripening, causing a further, relatively small, decrease in pH (as discussed in Section VIII). At low pH or high NaCl concentrations cheese curd or fresh

cheese may contain more lactose than the starter bacteria can metabolize. Under these circumstances, lactose may be utilized by NSLAB with the risk of gas production and product spoilage. Therefore, the level of residual lactose has a significant effect on cheese quality. The principal factors that influence the equilibrium of lactose between curd and whey are discussed below. A. Acid Development During the early stages of cheesemaking there appears to be no significant difference in pH between the.curd and whey. Lactic acid is produced from lactose within curd particles, but is readily transferred to the whey, mainly by synthesis. However, during the later stages of cheesemaking, curd pH tends to be slightly lower than that of the whey due to recent acid production inside the curd particles, although the curd has a greatly increased buffering capacity, especially below pH 5.5 (Dolby25 and Czulak et al.21). Therefore, curd pH is thought to be a more reliable indicator of recent acid production than whey pH. The faster acid rate of development inside the curd particles than in the whey results in the concentration of lactose in the curd decreasing more rapidly than in the surrounding whey. It has been suggested (Dolby,25 Czulak et al., 21 and Czulak20) that some lactose diffuses from the whey into the curd to replace that fermented. Concurrently, but in the opposite direction, lactic acid diffuses from the curd into the whey. The above authors suggested that the rate of diffusion of lactose from the whey into the curd is faster than that of lactic acid from the curd into the whey. Thus, the longer the curd remains in contact with the whey, the greater the amount of lactose that diffuses into the curd. However, Lewis72 considered that diffusion of lactose from whey into the curd particles is unlikely because 1. 2. 3.

Too little lactose is metabolized before draining to create a significant concentration gradient The diffusion coefficient for lactose is small The outflow of whey from the curd would prevent the diffusion of lactose into the curd, especially during the early stages of syneresis

B. Moisture Content of The Curd Since lactose is present in the aqueous phase of cheese curd, a decrease in the water content of curd is paralleled by a decrease in lactose. Conversely, curds with a high moisture content are likely to contain a high concentration of lactose, which can, eventually, be converted to lactic acid. A rennet coagulum undergoes considerable syneresis when cut or broken, and this is the main factor responsible for the transfer of solutes (lactose, lactic acid, etc.) from the aqueous phase of curd to the whey. The physical and biochemical aspects of syneresis were reviewed by Walstra et al.13S and Pearce and MacKinlay.97 The rate and extent of syneresis depend on 1990

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the composition of the cheesemilk, especially the fat, protein, and Ca contents, and on certain cheesemaking parameters, especially the size of curd particles, cooking temperature, rate of agitation, and the rate and extent of acid production. With cheeses cooked to a high temperature, e.g., Swiss (~52°C), the curd particles are dehydrated rapidly and become tough and rubbery, whereas with low-cook cheeses, e.g., Cheshire (32°C), the extent of dehydration is less and is due mainly to acid production. If the rate of heating is excessive, a condition similar to case hardening of the curd particles can occur, i.e., the surfaces of the particles become dehydrated and less porous, hindering free diffusion of whey. Further syneresis of the curd occurs on salt addition and on pressing. C. The Initial Lactose Content of The Cheesemilk Changes in the lactose content of cheesemilk may lead to concomitant changes in the residual lactose in the curd (Lawrence and Gilles66). The lactose content of milk decreases throughout lactation and this decrease is very marked in regions with seasonal lactation patterns, e.g., Ireland (Phelan et al.98) or New Zealand (Lawrence and Gilles66). Small interbreed variations in lactose levels have been reported (Johnson56) and marked decreases occur during mastitic infection. Reduced concentrations of lactose in milk are paralleled by a tendency for the pH of commercial New Zealand Cheddar cheese to increase (Lawrence and Gilles66). D. Alteration of The Lactose Content of Cheese Curd Intentional alteration of the lactose content of curd to produce low-lactose (LL) or high-lactose (HL) cheeses (Dolby25 and Huffman and Kristoffersen51) should help to clarify the functions and importance of lactose in cheesemaking. Cheddar cheeses made from lactose-supplemented milk had a higher residual lactose, lower pH, and lower moisture contents at 14 d, and the mature cheeses were of inferior quality to the controls (Dolby25). Huffman and Kristoffersen51 varied the lactose content of Cheddar cheese by dissolving lactose in the whey or by replacing some of the whey with buffer; most of the added lactose was lost in the whey. Relatively more lactate than lactose or moisture was expressed from the curd during cheddaring, which the authors suggested was due to the association of lactate with the calcium-phosphate-casein complex and that was released slowly (along with calcium) on acid development during cheddaring, in contrast to lactose and moisture, which were expressed mainly before draining. In all these treatments —98% of the total lactose (including lactose fermented to lactate) was removed in the whey. Alteration of the whey lactose had no detectable effect on the rate of acid development, the amount of lactate produced, the distribution of lactate between curd and whey, or the pH and moisture content of the fresh cheese. All the cheeses were of good to excellent quality, in contrast to the HL cheeses produced by Dolby.25 Breene et al.4 con-

244

cluded that a high lactose content in curds did not cause acid defects in cheese when the moisture content and the buffering capacity were controlled. E. Curd Washing or Addition of Water During the manufacture of some cheese varieties, e.g., Edam and Gouda, a portion of the whey is removed and replaced with warm water. The stage at which water is added, the temperature of the water, and the extent of washing are varied according to the cheese variety. This operation helps to control the final cheese pH by raising the cooking temperature and by extracting some lactate and lactose from the curd. The stage at which water is added is critical for proper pH control because efficient removal of lactose and lactate mainly occurs while curd particles are still undergoing significant syneresis and before the formation of a "tight skin" on these particles. Normally, washed cheeses have a low acid content and a high pH. Addition of water increases the final pH of Colby cheese by 0.1 to 0.2 units and makes the cheese texture more plastic and pliable (Lawrence and Gilles65). van den Berg and de Vries135 found that normal-sized Gouda curd particles in contact with washwater for 25 min lost 90% of their lactose content. Owing to the removal of whey and its replacement by water, the amount of lactose available for diffusion from whey into the curd (if rediffusion of lactose from the whey into the curd occurs) is reduced; indeed, the normal lactose gradient may even be reversed so that diffusion of lactose from curd into the whey is possible. F. Ultrafiltration (UF) UF is now widely used in commercial cheesemaking, and a discussion on its effects on acid production and lactose utilization is warranted. UF has three main applications in cheese manufacture: 1.

2.

3.

Precheese production, i.e., production of a concentrate with a composition somewhat similar to that of the final cheese, e.g., UF Cast Feta. Normally, the milk is concentrated at least fivefold, there is little or no drainage of whey and most of the whey proteins are retained in the curd. A modification of this procedure involves concentrating milk up to fivefold, but drainage of some whey occurs, e.g., structured UF Feta. Standardization of the protein content of cheesemilk with UF retentate is now fairly common practice, particularly for the manufacture of Camembert cheese. Preparation of cheese base by ultrafiltration, fermenting and evaporating whole milk; no rennet is used. Cheese base is a paste with approximately the same composition and pH as Cheddar, but without the characteristic texture and flavor (Glover40); it can replace up to 30% of the young cheese normally used in the production of processed cheese (Lawrence64).

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Food Science and Nutrition The highly concentrated retentates used in some UF cheeses have a high buffering capacity due to the high concentrations of protein and mineral salts and therefore it may be difficult to attain the desired pH for optimum cheese quality (Mocquot,83 Glover,40 and Kosikowski63). The distribution of calcium between the aqueous and micellar phases in milk is very pH dependent. In normal milk at pH 6.6, —30% of the calcium is soluble, but at pH 5.4 the situation is reversed, i.e., ~67% of the calcium is soluble (Glover40) and permeates prepared from acidified milk have a high calcium content (Brule et al. 6 ). Highly active starters must be used in UF retentates since large amounts of lactic acid are required to produce the desired pH changes (Lawrence64). Acid and bitter flavors can arise from excessive lactose in the precheese; therefore, the membranes used should not retain lactose. The lactose and mineral contents and the buffering capacity of the retentate may be reduced by 1. 2. 3.

Preacidification of the cheesemilk Addition of NaCl during UF (to exchange sodium for bound calcium) Diafiltration (Glover40)

Since ultrafiltration involves modification of traditional cheesemaking, UF cheese may not have the characteristic texture and/or flavor of the traditionally made cheese. For example, diafiltration after preacidification is essential for development of the stretching properties in UF Mozzarella cheese (Covacevich13) and for control of the lactose content. G. Prehydrolysis of Lactose (3-Galactosidase (lactase) hydrolyses lactose to galactose and glucose. Gilliland et al.39 suggested that glucose is preferred as a substrate over lactose by starter bacteria and this may explain why a faster rate of acid production was observed during Cheddar cheesemaking with Iactose-hydrolyzed milk (Thompson and Brower128 and Marschke and Dulley78). Flavor development was also accelerated and maturation times reduced for these cheeses. However, several workers (Marschke et al.79 and Grieve et al.42) have demonstrated that the increased proteolysis, and hence the accelerated ripening, in lactosehydrolyzed cheeses is due to contaminating proteinases in the lactase preparations used.

VII. CONSEQUENCES OF ACID PRODUCTION Proper acid production is recognized as the most important factor determining cheese quality; some of the consequences are discussed in the following sections. A. Buffering Capacity Changes The pH of cheese curd is determined not only by the amount

of lactic acid produced, but also by the buffering capacity of the curd that is determined primarily by the casein and partly by soluble inorganic phosphate and citrate. According to Dolby,23 curd (young cheese) has a relatively low buffering capacity in the pH range 6.8 to 5.5, but buffering increases sharply in the range 5.5 to 4.5. Due to the high buffering capacity of the curd below pH 5.5, even extensive acid production after salting may have only a small effect on the final pH of cheese. The greater buffering capacity 5.1), to "mealy" (pH 5 % S/M. Dutch-type cheese contains up to ~ 1.4% lactose at pressing, but this decreases to < 0 . 1 % after pressing and to undetectable levels after brining (Raadsveld100). The levels of lactate in Camembert, Swiss, and Cheddar have been reported to be 1.0, 1.4, and 0.5%, respectively (Karahadian and Lindsay61 and Turner et al.,131 Turner and Thomas134). The fate of lactic acid in cheese during ripening has also received little attention until recently. Turner and Thomas,134 Thomas and Pearce,119 and Tinson et al.130 showed that experimental and commercial Cheddar cheeses contain considerable concentrations of D-lactate, which could be formed from residual lactose by lactobacilli (see Garvie35) or by racemization of L-lactate (see Section FV.A.5). Except in cases where the postmilling activity of the starter is suppressed (e.g., by S/M >6%, as discussed above), racemization is likely to be the principal mechanism (Thomas and Crow117). These authors showed that pediococci are most likely responsible for racemization. All 27 pediococci isolated from Cheddar cheese and P. pentosaceus NDCO 1220 were capable of converting L-lactate to D-lactate, eventually giving a racemic mixture, while only 5 of 16 Lactobacillus isolates were capable of racemizing L-lactate, at much slower rates and to a lesser extent than the pediococci. Racemization of L-lactate by both pediococci and lactobacilli was pH dependent (optima: 4 to 5) and was retarded by NaCl concentrations > 2 % or >6% for pedicocci and lactobacilli, respectively. Racemization of lactate in a Cheddar cheese inoculated with pediococci was complete in — 19 d, while this required ~ 3 months in a control cheese with much lower levels of NALAB, especially pediococci (Thomas and Crow117). Both lactobacilli and pediococci possess L( + )-LDH and D(-)-LDH, both of which are NAD """-dependent. The proposed pathway for lactate racemization is discussed in Section IV.A.5. Racemization of L-lactate appears to occur in several cheese varieties (Thomas and Crow117); most of the mature varieties studied contained a more or less racemic mixture. The relatively low proportion of D-lactate in Gouda cheese may be due to the short ripening time for this cheese. The level of L- or D-lactate in Camembert is very low due to the metabolism of lactate by the mold, as discussed below. Metabolism of lactate by the mold in blue cheese might also be expected, but this does not appear to occur. The racemization of L-lactate is probably not significant from the flavor viewpoint, but D-lactate may have undesirable nutritional consequences in infants. Calcium D-lactate is less soluble than Ca-L-lactate and may crystallize in cheese, causing undesirable white specks, especially on cut surfaces (Pearce et al. 96 Severn et al., 107 and Dybing et al.25a). Oxidation of lactate can also occur in cheese. Thomas et al.123 showed that pediococci produced 1 mol of acetate and 1

248

mol of CO2 and consumed 1 mol of O 2 per mole of lactate utilized (Thomas et al.123). The pH optimum was 5 to 6 and depended on the lactate concentration. The concentration of lactate in cheese far exceeded that required for optimal oxidation, and lactate was not oxidized until all sugars had been exhausted. The lactate oxidative system remained active in 6month-old cheese. The oxidative activity of suspensions of starter, and NSLAB isolated from cheese, with lactose, lactate, citrate, free amino acids and peptides was studied by Thomas.115 Starter bacteria oxidized lactose only; L. casei oxidized only citrate, while L. plantarum, L. brevis, and P. pentosaceus oxidized lactose, peptides, and L- and D-lactate, but not citrate. The oxidation of lactate to acetate in cheese obviously depends on the NSLAB population and on the availability of O2, which is determined by the size of the block and the oxygen permeability of the packaging material (Thomas116). Acetate, which may also be produced by starter bacteria from lactose (Thomas et al.122) or citrate (see Section LX) or from amino acids by starter bacteria and lactobacilli (Nakae and Elliott87), is usually present at fairly high concentrations in Cheddar cheese and is considered to contribute to cheese flavor, although high concentrations may cause off-flavors (see Aston and Dulley2). Thus, the oxidation of lactate to acetate must make some contribution to Cheddar cheese flavor. Presumably, the oxidation of L-lactate to acetate is more or less common to all hard and semihard cheese varieties. Of all cheese varieties, the metabolism of lactate is probably most extensive in the surface mold-ripened varieties, e.g., Camembert, Brie, and Carre de l'Est. The concentration of lactic acid in these cheeses at 1 d is ~ 1.0%, produced mainly or exclusively by the mesophilic starter, and hence, presumably, is L-lactate. Secondary organisms quickly colonize and dominate the surface of these cheeses — first Geotrichum candidum and yeasts, followed by Penicillium caseicolum, and, in traditional manufacture, by Brevibacterium linens. G. candidum and P. caseicolum rapidly metabolize lactate to CO2 and H2O, causing an increase in pH. Deacidification occurs initially at the surface, resulting in a pH gradient from the surface to the center and causing lactate to diffuse outward (Figure 7). When the lactate has been exhausted, P. caseicolum metabolizes proteins, producing NH 3 , which diffuses inward, further increasing the pH. The concentration of calcium phosphate at the surface exceeds its solubility at the increased pH and precipitates as a layer of Ca3(PO4)2 on the surface, thereby causing a calcium phosphate gradient within the cheese; reduction of the calcium phosphate concentration in the interior helps to soften the body of the cheese. The elevated pH stimulates the action of plasmin, which, together with residual coagulant, is responsible for proteolysis in this cheese rather than proteinases secreted by the surface microorganisms, which, although very potent, diffuse into the cheese to only a very limited extent, although the products of their action on the

Volume 29, Issue 4

Food Science and Nutrition

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Brie and Camembert. B. linens does not grow < pH 5.8 and does not colonize the cheese surface until the pH has been raised (Lenoir,71 Karahadian and Lindsay,61 and Noomen89). The fermentation of lactose in Swiss-type cheeses has been described comprehensively by Turner et al.132 (Figure 8). Typically, Emmental cheese contains —1.7% lactose 30 min after molding, which is rapidly metabolized by S. thermophilus to undetectable levels within 1 d with the production of up to 1.2% L-lactate. As discussed in Section IV.A.C, only the glucose moiety of lactose is metabolized by S. thermophilus and consequently galactose accumulates to a maximum of —0.7% at —10 h, when the Iactobacilli begin to multiply. These metabolize galactose to a mixture of D- and L-lactate, reaching —0.35 and 0.7%, respectively, at 14 d, by which time the galactose had been metabolized completely. The concentration of L-lactate did not change between days 1 and 14. L-Lactate is preferentially metabolized over the D-isomer by propionibacteria (Crow17), and on transfer to the warm room, L-lactate is metabolized rapidly to propionate, acetate, and CO2 to reach 0.2% at day 35. The concentration of D-lactate continued to increase to —0.4% during the early days in the warm room, before being metabolized by propionibacteria. Increasing the level of Lactobacillus 10-fold accelerated sugar metabolism and caused a higher concentration of both D- and L-lactate, but suppressed the growth of propionibacteria and delayed the production of propionate and acetate (Turner et al.132). In the absence of Iactobacilli or with gal~ Iactobacilli, no D-lactate was formed, while galactose accumulated to 0.7%. In contrast to the control cheese in which propionibacteria did not multiply until transfer to the warm room, the number of these organisms increased 100-fold between days 1 to 14. The levels of propionate and acetate in these cheeses increased more rapidly and to higher concentrations than in control cheeses, with the rapid disappearance of both L-lactate and galactose. In Swiss-type cheese, the propionibacteria metabolize lactate to propionate, acetate, and CO2:

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surface proteins may diffuse into the body of the cheese. The combined action of increased pH, loss of calcium (necessary for the integrity of the protein network), and proteolysis are necessary for the very considerable softening of the body of

2CH3CH2COOH + CH3COOH + CO2 The CO2 generated is responsible for eye development, a characteristic feature of these varieties. As indicated above, Llactate is preferentially metabolized by propionibacteria; hence the ratio of L- to D-lactate, and therefore the proportion of Iactobacilli in the starter, probably influence the production of CO2 and volatile acids. Production of lactate in Romano cheese was monitored by Mora et al.86 As with other varieties, the L-isomer predominated initially, reaching a maximum of — 1.9% at 1 d (Deiana et 1990

249

Critical Reviews In -,10 (D + V4h P + 4h

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FIGURE 8. Lactose and lactate metabolism in Swiss-type cheese manufactured with an inoculum of 0.005% L. helvelicus strain 5001. Lactose (o), glucose (A), galactose ( ^ ) , D-lactate (•), L-lactate (•), acetate (V), propionate (V), and propionibacterium count (•). Arrowed positions P + '/2 h, P + 4 h, and P + 10 h indicate sampling times V2, 4, and 10 h after the commencement of pumping. (From Turner, K. W., Morris, H. A., and Martley, F. G., N. Z. J. Dairy Sci. Technol.. 18, 117, 1983. With permission.)

al.22). The concentration began to decrease at 10 d and had reached 0.2 to 0.6% at 150 to 240 d. Some of the decrease was accounted for by racemization to D-lactate, which reached a maximum at ~ 90 d (up to 0.6% in some batches) and then declined somewhat. In some batches, acetate reached very high levels (1.2%) at ~ 30 d, but decreased to 2*0.2% at 90 d. The agents responsible for acetate metabolism were not identified, but yeasts (Debaryomyces hansenii) may have been involved. The significance of the primary lactose fermentation to Llactate is well recognized. However, little significance has been attached to the subsequent changes in lactose, and lactate metabolism has received relatively little attention and that only recently. It should be obvious from the foregoing that these changes are of major proportions: in quantitative terms, the metabolism of lactose and lactate is probably the principal metabolic event in most cheese varieties. 9. Citrate Metabolism The relatively low concentration of citrate in milk (~8 mAf) belies the importance of its metabolism in many cheeses made using mesophilic cultures (for reviews see Cogan9 and Cogan and Daly12). Citrate is not metabolized by S. lactis or S. cremoris, but is metabolized by S. lactis subsp. diacetylactis and Leuconostoc spp. with the production of diacetyl and CO2- It is not metabolized by S. thermophilus or by thermophilic lac250

tobacilli (Hickey et al.48), but several species of mesophilic lactobacilli metabolize citrate with the production of diacetyl and formate (Fryer31); the presence of lactose influences the amount of formate formed. Citrate is not used as an energy source by 5. lactis subsp. diacetylactis or Leuconostoc spp., but is metabolized very rapidly in the presence of fermentable carbohydrate by the pathway outlined in Figure 9. Due to CO2 production, citrate metabolism is responsible for the characteristic eyes of Dutch-type cheese, and for the undesirable openness and floating curd in Cheddar and cottage cheese, respectively. Due mainly to the formation of diacetyl, citrate metabolism is very significant in aroma/ flavor formation in cottage cheese, Quarg, and many fermented milks. Diacetyl also contributes to the flavor of Dutch-type cheeses and possibly of Cheddar cheese (Manning,75-76 McGugan,83 and Aston and Dulley2). Acetate produced from citrate may also contribute to cheese flavor. Approximately 90% of the citrate in milk is soluble and most of this is lost in the whey; however, the concentration of citrate in the aqueous phase of cheese is ~ 3 times that in whey (Fryer et al. 32 ), presumably reflecting the concentration of colloidal citrate. Cheddar cheese contains 0.2 to 0.5% (w/w) citrate. These workers showed, using cheese with a controlled microflora, that in cheese made using S. cremoris only, citrate remained constant at 0.2% up to 3 months, but decreased to

Volume 29, Issue 4

Food Science and Nutrition ACETATE

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Acetoin Diacetyl + reductase reductase 2.3BUTYLENEGLYCOL DIACETYL ^ . ACETOIN pH 5.4), but apart from such high values, pH appeared to exercise little consistent effect on quality. According to Lelievre and Gilles,70 pH had no consistent effect on the quality of New Zealand Cheddar cheese within the range 4.9 to 5.2, and within the limits prescribed by Gilles and Lawrence,38 composition was not a good predictor of cheese quality.

REFERENCES 1. Anon., American type culture collection, Catalog of Strains, 15th ed. 1982, 162. 2. Aston, J . W. and Dulley, J . R., Aust. J. Dairy TechnoL, 37, 59, 1982. 3. Aodair, J . and Accolas, J . P., Irish J. Food Sci. TechnoL. 7, 27, 1983. 4. Breene, W . M., Price, W. V., and Ernstrom, C. A., / . Dairy Sci., 41, 840, 1964. 5. Breene, W. M., Price, W. V., and Ernstrom, C. A., J. Dairy Sci., 47, 1173, 1964.

1990

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