The impact of lactic acid bacteria on cheese flavor

The impact of lactic acid bacteria on cheese flavor

FEMS MicrobiologyReviews87 (1990) 131-148 Pubfishedby Elsevier i31 FEMSRE00165 Applications The impact of lactic acid bacteria on cheese flavor N o...

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FEMS MicrobiologyReviews87 (1990) 131-148 Pubfishedby Elsevier


FEMSRE00165 Applications

The impact of lactic acid bacteria on cheese flavor N o r m a n F. Olson Centerfor Dairy Research and Department of Food Science, Universityof Wisconsin. Madison, WI, U.S.A.

Key words: Cheese flavor; Cheese proteolysis; Lactococcus; Lactobacillus

1. SUMMARY Cheese flavor is a manifestation of complex interactions of volatile and non-volatile flavor-active compounds plus tactual perception. Numerous agents, including lactic acid bacteria, produce the flavor sensations. The effect of lactic acid bacteria is more dominant in cheese varieties with limited growth of secondary flora. This review describes the indirect and direct impacts of lactic acid bacteria in cheese with emphasis on carbohydrate fermentation, changes in oxidation-reduction potential, interactions with non-starter bacteria, autolysis, proteolytic and peptidolytic activities, transport of metabolites and flavor production.

2. INTRODUCTION Flavor of cheeses is expressed as a continuum of perceptions of tactual properties, volatile components and non-volatile compounds. Pronounced differences in flavor that exist between classes of cheese result primarily from metabolism of secondary flora. Variations in flavor within a cheese variety have evolved because groups of consumers in different geographical areas view their specific

Correspondence to: N.F. Olson, Center for Dairy Research, Babcock Hall, Universityof Wisconsin,Madison,Wl 53706, U.S.A.

variant as possessing the authentic flavor for that variety. The complexity can be stated simplistically by saying, "There is a cheese for every taste preference and a taste preference for every cheese". The flavor profiles of cheeses are complex and variety or type specific. Only limited information is available on characterization of flavor of most cheese varieties. None are characterized sufficiently to permit duplication of their complete flavor by mixtures of pure compounds. This has prompted use of indirect methods to enhance the intensity of cheese flavor or the rate of cheese maturation. Controlled (elevated) temperatures during cheese maturation and added enzymes and bacteria have been used most commonly. Variability in effectiveness and the tendency to produce off-flavors illustrates the delicate balance of environmental conditions and chemical, biological and physical changes that occur in cheeses during flavor synthesis. In spite of the variability in the properties of cheese, there are several constant factors in manufacturing and maturation. Milk-clotting enzymes form the coagulum (c~d) f~r ~ ' = ~ E y :2] cheeses that are matured. Lactic acid bacteria are used in these varieties, although different species have evolved for specific cheese varieties. The commonality of enzymatic milk-clotting and lactic acid bacteria should dictate similarities between cheese varieties. Lack of homogeneity is created by differences in cheese manufacturing procedures, cheese composition, pH, secondary flora, and added enzymes or induced enzymatic

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132 activities. Cheese flavor is affected by these variables by direct influence on synthesis of flavor compounds or by indirectly altering flavor perception. Lactic acid bacteria serve several functions in cheese manufacturing and maturation that directly or indirectly impact on cheese flavor. These are: (1) fermentation and depletion of fermentable sugars which control growth and composition of adventitious flora; (2) creating a low oxidationreduction potential during early stages of cheese maturation; (3) competition and synergism with adventitious bacteria during cheese manufacturing and early stages of maturation; (4) protein hydrolysis; and (5) synthesis of flavor compounds. Information to verify the last function is meager and does not completely describe the mechanisms for production of the full flavor of mature cheese or if the lactic bacteria had a major direct role in forming the flavors. Consequently, this review will integrate the indirect influences of lactic bacteria into the maturation processes of cheeses and will focus on developments during the past 5 years. Several reviews have described the flavor profiles of cheese [1,2].

3. LACTOSE FERMENTATION Fermentation of lactose in cheeses occurs rapidly during the early stages of maturation. The residual concentration of lactose depends upon metabolic activity of the lactic acid bacteria, the amount of lactose removed during cheese manufacturing and the sodium chloride concentration of cheese [3-5]. For example, Cheddar cheese of normal composition should contain < 0.1~ lactose after several weeks of maturation. However, increases in sodium chloride concentrations in the water phase of Cheddar cheese cause a linear increase in lactose levels in Cheddar because of inhibition of the lactic starter culture. Salt concentrations exceeding 5~ in moisture phase of Cheddar cheese inhibited lactose metabolism [4]. The residual sugar serves as substrate for more salt tolerant bacteria, such as lieterofermentative lactobacilli, that may form undesirable flavors and gas.

Negligible amounts of glucose and galactose accumulate in cheeses made with Lactococcus iactis ssp. ceemoris and LacWcoccus lactis ssp. lactis (Streptococcus cremoris and S. iactis ) since lactose is transported intact into the cell by the phosphoenolpyruvate-phosphotransferase system. Streptococcus salivarus ssp. thermophilus ( S. theemophilus), Lactobacillus bulgaricus and Lb. heiveticus use a permease and hydrolyse the lactose with intracellular fl-galactosidase. $. thermophilus and Lb. bulgaricus can not metabolize the released galactose and excrete it from the cells. Concentrations of 0.4-0.5~ galactose have been reported in Mozzarella cheese after 1 month of maturation [6]. This monosaccharide could serve as an energy source or substrate for flavor synthesis by adventitious bacteria in cheese.

4. OXIDATION-REDUCTION POTENTIAL Metabolism of the lactic acid bacteria during cheese manufacturing and the first few days of maturation lowers the oxidation reduction potential of Cheddar cheese to about - 200 mV [7]. The effect is short-lived since the redox potential increased to about - 50 mV during the first week of maturation of Cheddar cheese before a subsequent steady decline to almost - 300 mV [8]. The limited effect of the lactic starter culture may be related to its apparent requirement for lactose to exhibit oxidative activity. It was proposed that enzymes, such as N A D H oxidase, removed 02 during lactose metabolism. The disappearance of lactose during the first week of cheese maturation terminates the reductive effect of the starter culture. The second decrease presumably resulted from metabolism of residual sugar by non-starter lactic acid bacteria. Contrary to these observations, Galesloot reported a slow increase in redox potential during maturation of Dutch cheese [9]. Presence of Lb. easel and Lb. plantarum in this cheese did not reverse the trend. The implications of packaging materials were not discussed in these studies. Non-starter lactic acid bacteria (NSLAB) possess oxidative activities that should be operative under the conditions found in cheese [7]. Sufficient substrates, especially lactate, are present

133 to saturate the lactate oxidation system of the NSLAB. Oxidative activities on lactate and a cheese extract varied substantially between species and strains within a species. Presumably this could create variability in the redox potential of cheeses during maturation and potentially influence the flavor profile [10,11]. This premise has not been tested but lactate oxidation has been shown to function in Cheddar cheese [12].


5.1. Non.starter lactic acid bacteria Lactic acid synthesized by the lactic starter culture during cheese manufacturing may serve as a substrate for NSLAB [12]. Strains of Pediococcus pentosaceus and Lb. plantarum were capable of converting lactate to acetate via an O2-dependent mechanism. The rate and extent of conversion was directly related to the 02 transmission rates of the cheese packaging material and indirectly related to the size (surface to volume ratio) of the cheese. The metabolism can have a positive effect by maintaining a low redox potential in cheese to protect 02 sensitive flavor compounds, e.g. thiols. The impact can be detrimental if the concentrations of acetate are too high and affect flavor balance. An O2-independent mechanism of converting citrate to acetate can occur with strains of Lb. plantarum in cheese [12].

5.2. Effect on metabolism of propionibacteria Metabolism of lactic acid bacteria directiy and indirectly affects subsequent metabolism of propionibacteria in Swiss cheese. Lactose fermentation by S. thermophilus and Lb. helveticus a n d / o r Lb. bulgaricus during manufacturing and 24 h post-manufacturing yields variable concentrations of galactose and L(+) and D( -- )-lactic acids. The amounts depend upon the bacterial strain and species [13]. S. thermophilus dominates the initial stages t fermentation and depletes the lactose in Swiss cheese during the first 18 h. Its inability to metabolize galactose creates an increase in the

concentration of this sugar until galactose-fermeriting strains of lactobacilli become active. The lack of participation by the Lactobacillus species in lactose fermentation was evident even when the ratio of lactobacilli to $. thermophilus was increased from 1 : 100 to 1 : 1. The low levels of v ( - )-lactate and very high levels of L(+)lactate in cheese at the point of lactose depletion indicated the predominance of S. thermophilus in this stage of glycolysis even when numbers of lactobacilli were increased 100-fold. Different ratios of L( + )-lactate, D( -- )-lactate and galactose produced by lactic starters containing gal + or gal- lactobacilli impact on the subsequent propionibacterium metabolism [13,14]. In cheese made with a gal + Lactobacillus species, L( + )-lactate and to a limited extent D(-)-lactate was converted into equal concentrations of acetate and propionate by the propionibacteri& Cheese made with gad- Lb. bulgaricus contained galactose which was fermented by the propionibacteria with little utilization of I.(+)- and D( - )-lactate. SubstantiaUy more (1.5 × ) acetic acid was produced than propionic acid in this cheese. The concentration (w/w) of succinic acid was intermediate between propionic and acetic acid levels. Succinate can be produced by the propionibacterium enzyme carboxytransphosphorylase that catalyses CO2 fixation at the expense of propionic acid production. Succinate can also be produced from aspartate by propionibacteria. The relative use of these two mechanisms can impact on the rate and extent eye formation since CO 2 is depleted in the first situation and produced by the second mechanism. The ratios of propionic to acetic acids would also differ but reports on the relative importance of these acids on the "sweet taste" of Swiss cheese is contradictory [15]. The relative importance of the two pathways may depend upon the tendency of propionibacteria to produce succinate, the concentrations of different substrates for the propionic f©tmentation and the concentration of aspaxtate. The lower than anticipated ratio of propionate to acetate suggests that CO 2 fixation is important. However, the ratio of aspartate to glutamate ranged between 1 : 15 and 1 : 50 in water extracts of Swiss cheese whereas the ratio of these free amino acids in

Gouda cheese was about 1:12, suggesting that aspartate was metabolized in some of the Swiss cheese samples [16,17]. 5.3. Autolysis of lactic acid bacteria

Growth and autolysis of lactic acid bacteria in cheese potentially can release enzymes and cellular constituents that would serve as metabolites for other microorganisms in cheese. Lactic starter cultures reach densities of about 109 cfu/g cheese, equivalent to 1 mg dry weight/g cheese. Non. growing cells are slowly autolysed by their hydrolyric enzymes [18]. The products, including sugars and nucleic acids, that are released are not utilized by the remaining lactic bacteria but appeared to be used by NSLAB for growth [19]. Only traces of products such as acetate, L(+) and 13(-) lactate were formed by NSLAB during growth in the presence of autolysed L. lactis ssp. lactis. Strains of lactococci varied considerably in their impact on supporting growth of NSLAB species. Numbers of test strains of 1". pentosaceus varied by 10000-fold and Lb. casei by 300-fold when grown in buffer in the presence of different autolysed strains of the lactococci. L. lactis ssp. cremoris E s provided the greatest growth stimulation of the six Lactococcus strains tested. It is not clear if ~.!~c~e effects would be as substantial in cheese since 200 lactococcal cells were required for the doubling of one non-starter cell. This would not account for the populations of NSLAB in cheese. However, Thomas concluded that products from starters cells could b.~ a sisnificant carbon and energy source for NSLAB [19]. Autolytic tendencies of lactic acid bacteria appear to vary between strains [20-22]. This variability is one explanation for the differences in effects of lactic bacteria on NSLAB observedby Thomas [19]. Variability in rates of population decline has been observed also between lactic starter cultures during maturation of Gouda cheese [23]. The duration required for the population to drop from the maximum of about 109 cfu/g cheese at I week to 103 cfu/g cheese varied from two to 8 weeks. The reduction in numbers in cheese does not indicate autolysis but suggests that autolytlc rates may vary.

Four strains of L lactis ssp. cremoris exhibited temperature-induced lysis when heated to 3840°C, whereas two other strains continued to grow [24]. The temperature insensitive strains have been identified as producing bitterness in cheese but the temperature-sensitive strains have not been identified with that defect. Thermoinducible mutants of L lactis ssp. lactis C2 that are highly autolytic at temperatures used during cheesemaking have been isolated and characterized [24]. This technology is being used to construct L lactis ssp. cremoris strains that release flavor-enhancing enzymes to accelerate cheese maturation.

6. LIPOLYTIC ACTIVITY Lipolytic and esterolytic activities of lactic acid bacteria appear to be limited and probably do not contribute substantially to cheese flavor. Lipases and esterases from microorganisms and glottal tissues of selected animal species are used to make cheese varieties requiring a perceptible flavor from free fatty acids [25]. It is possible that lipolytic activities of lactic acid bacteria yield levels of free fatty acids that contribute to the background flavor of many cheese varieties. Lipase and esterase activities have been detected in cell-free extracts of numerous Lacwcoccus and Lactobacillus species [26]. A preference for liberation of shorter-chain fatty acids has been observed for lactococci [26,27] and lactobaOlli [28]. In contrast, Yu [29] reported that Ion8 chain fatty acids (C16:0 and C18:1) accumulated in greatest quantities when lipases from Lb. casei were incubated with milkfat. Intracellular and extracellular lipases have been reported for lactococci and lactobacilli but the experimental conditions to differentiate the locations have not been defined as carefully as those used in research on proteinases and peptidases. Weak lipolysis has been observed with lactococci on tributyrin in agar plates, which suggests extracellular enzyraes or release of enzymes upon cell autolysis [26]. However, most studies on lipase and esterase characterization have utilized enzyraes in cell-free extracts. Yu [29] reported the

135 action of extraeeilular (cell-suspension) and intracellulax (cell-free extract) enzymes of Lb. casei on milldat. The intraeellular llpase liberated greater amounts of fatty acids except for Cs:o and Clo:o which appeared to be released preferentially by the extraceHular enzyme. An intraeellular location would require autolysis of the bacteria which raises questions about the effectiveness in cheese if autolysis is somewhat limited [30]. Also, diffusion of the substrate, milkfat, is negligible in cheese and diffusion of lipases would be slow. As indicated above, however, reasonable diffusion of lipases must have occurred in agar. It is noteworthy that lipolytic activity against milkfat was similar at 5 and 37°C, since cheese is matured at the lower temperature [31]. 7. PROTEOLYTIC ACTIVITIES 7.1. !.nCtiC acid bacteria proteinases

The proteinase system of lactic bacteria has been fairly well characterized and has been associated with rates of cheese maturation [32,33-35]. Most of the proteolytic activity oi lactococci is localized in or on the cell wall [36-38]. The cell wail-associated proteinases are stabilized by Ca 2+ and there is no reason that this stabilization should not persist in cheese, since Ca 2+ would not be limiting. Although some variability in specificity towards caseins has been reported, t-casein seems to be more susceptible. All 23 strains of L. lactis ssp. cremoris tested in one study were able to degrade ~-casein but none exhibited activity towards as-casein [39]. Other L. lactis ssp. cremoris strains, AM1 and $Kll, hydrolysed as1-, to- and ~-caseins [40]. Lb. helveticus attacked as" and /~-caseins and Lb. bulgaricus degraded all the major caseins but #-casein was most susceptible [41,42]. Hugenholtz et al. [36] described and partially characterized three cell wall-associated proteinases, identified as A, B and C. They suggested that proteinase B is responsible for bitterness in cheese since proteinase A was found in all strains tested. Proteinase B was present in L. lactis ssp. cremoris Wg2, a bitterness-producing strain, whereas proteinase C but not B was observed in a

non-bitter strain, L. lactis ssp. cremoris F. A previous survey of /.. lactis ssp. cremoris strains also indicated three types of proteolytic activities based on their pH and temperature dependence [43]. These observations support the active role of lactococcal strains in producing bitter peptides during cheese maturation. It does not discount the role of lactococcal peptidases in eliminating bitter peptides. The identification and characterization of cell-wall associated proteinases is under investigation. It is likely that the number of distinguishable proteinases may change based upon elucidation of their specificity and gene linkage. Intracellular proteinases have been characterized but the reports seem contradictory [26]. Recently, intraceHular activity of L lactis ssp. lactis NCDO 763 (prt + variant) was found to constitute only 0.8-4.3~ of total proteolytic activity [38]. The relatively low activity compared to cell wall-associated proteinase activity has been reported for other strains of iactococci [43,44]. Muset et al. [38] reported that intracellular proteinase activity was not affected by the growth medium (reconstituted skim milk medium versus M17 medium), whereas extracellnlar proteinase activity was 7-fold greater in cells grown in the presence of casein. Also intracellular activity was similar in prt- and prt + variants even though the former were deficient in cell wall-associated proteinases. Purified intracellular proteinase slowly hydrolysed soluble fi-casein; two major HPLC peaks were tentatively identified as peptides corresponding to 1-41 and 1-46 residues. In contrast, cell wall-associated proteinases from this Lactococcus strain cleaved peptides from the C-terminm of/3-casein [46]. The contribution of intraccllular laotococcul protvinases to cheese maturation has not been clarified but would be seem to be sli[ht. The low activity compared to cell wall-associated enzymes and the delayed release of the enzymes because of the apparent prolonged integrity of lactococcal cells in cheese. The starter bacteria attack the polypeptides generated by the milk-clotting enzymes and by cell-wall proteinases and cause slow degradation of ~-casein. Starter bacteria have been shown to be responsible for increased concentration of small peptides and free amino acids in

136 cheese. It has been suggested that the peptidases of lactic streptococci are capable of complete hydrolysis of the chymosin-generated peptides from casein but only about 3~ of the total N of 6month.old Cheddar cheese was present as free amino acids [32,33]. The mechanism by which starter bacteria degrade and utilize peptides in cheese has not been elucidated and has contradictory aspects. There is agreement that cell-wall associated proteinases participate in proteolysis and maturation of cheese [32,34,40]. The role of cell-wall-bound peptidases is uncertain. Cell or membrane bound peptidases have been reported [35,47] but their existence has been questioned [32]. 7.2. Lactic acid bacteria peptidases

Characteristics and functions of lactic acid bacteria peptidases were reviewed recently by Thomas and Pritchard [32], Kamaly and Marth [26] and Laan et al. [48]. Most of the previous reports have dealt with exopeptidases e.g. aminopeptidases, iminopeptidases, dipeptidases, tripeptidases and arylamidases, with six recent papers describing endopeptidases [49-54]. Two endopeptidases characterized by Yah et al. [50] are intriguing since they hydrolysed the as1-CN(f123) peptide which is released from asl-casein during early stages of cheese maturation. One endopeptidase (LEP 1) exhibited high affinity towards the Glu-Asn bond in as1-CN(f1-23 ) and aslCN(fgl-100). Other peptide bonds were hydrolysed in hormonal peptides that were tested which suggests a specificity which also characterized LEP-II. The LEP-I was not active against larger proteins such as as-casein, ~-casein, K-casein, a-laetalbumin and ~O-lactoglobulin. The LEP-I hydrolysed asl-CN(fl-23 ) into asl-CN(f1-18 ) and asl-CN(f19-23). However, these two peptides were not major components in Gouda-type cheese during maturation [55]. It was suggested that these two peptides were further hydrolysed by other endopeptidases and exopeptidases. In contrast, LEP-II cleaved aslCN(fl-9) and asl-CN(fl-13 ) from asl-CN(f123); these two peptides have been identified in Gouda cheese during maturation [55].

The LEP-II is similar to LEP-I in its substrate specificity but is able to hydrolyse pepfides that are slightly larger than those cleaved by LEP-I. The largest peptide susceptible to LEP-II action is the oxidized B-chain of insulin (Mw 3390) which was not attacked by LEP-I. As indicated previously, LEP-II exhibited a broader peptide bond specificity than LEP-I. Both LEP-I and II were recovered from cell extracts produced from dried cells that were sonicated and the suspension centrifuged at 35 000 × g. Yan et al. [50] indicated that most of the LEP-I was identified with the cell wall fraction but data were not shown. Kolstad and Law [54] observed that endopeptidase and aminopeptidase activities were not detected in cell wall fractions but were detected in intracellular preparations of L. lactis ssp. cremoris and L lactis ssp. lactis. It is logical for these endopeptidases to be located on the cell periphery unless they function in degrading signal peptides. Exopeptidases of lactic bacteria have been characterized to a greater extent than endopeptidases although their cellular location and their role in peptide transport has not been resolved. A variety of exopeptidases have been identified in lactic bacteria. A broad spectrum of peptidases in lactococci and lactobacilli commonly found in cheese suggest that peptides can be cleaved rather extensively and at a number of points in the peptide chain. The large number of proline residues in cheese requires peptidas¢ activities capable 9f cleaving bonds on both sides of this residue. Aminopeptidase P, proline iminopeptidase and iminodipeptidase have been observed in L. lactis ssp. cremoris, L~ lactis ssp. lactis and L lactis ssp. lactis ear. diacetylactis [32]. Prolyldipeptidylaminopeptidase has been purified from Lactococcus species, S. thermophilus and Lactobacillus species [56-58]. The enzymes were recovered from cell extracts so it is not clear whether they were intracellular or cell-wall associated. Either locality should be effective since lactic bacteria are capable of transporting up to pentapeptides into the cell although some species of L. lactis ssp. cremoris do not possess a dipeptide transport system [59]. The location of peptidases, especially aminopeptidases, in cells is controversial. Kolstad and Law [54] detected two peptidases in cell walls of

137 lactococci; one attacked only trileucine whereas the other hydrolysed a number of di- and tripeptides. Purified cell membranes possessed one peptidase with a very narrow specificity. Three to four intracellular peptidases, including aminopeptidase activity, were detected in different lactococci. The intracellular di- and tripeptidases hydrolysed a narrower range of di- and tripeptides as compared to the cell wall-associated peptidases. Of interest was the loss of cell wall-associated peptidases when grown in M17 broth as compared to growth in milk. However, no differences were detected between zymograms of intracellular extracts of cells grown in these two media. An intracellular aminopeptidase capable of cleaving bonds adjacent to hydrophobic amino acid residues, except proline, was characterized by Neviani et al. [60]. The enzyme was not a metalloenzyme unlike most aminopeptidases from lactococci and lactobacilli. The presence of this aminopeptidase plus X-prolyl dipeptidyl aminopeptidases should provide cells with the capability of utilizing peptides, containing high amounts of hydrophobic amino acids, that are released by cell wall proteinases and endopeptidases. A small amount, equal to 15~ of total cell activity, of the aminopeptidase was thought to be exocellular and associated with the cell wall.

Exterkate [53] proposed a cell wall-membrane interface as the location for alanyL leucyL prolyl and lysyl aminopeptidases. Glutamate aminopeptidase and membrane-associated lysyl aminopeptidase were detected also intracellularly. Exterkate and De Veer [51] described an interesting aminopeptidase complex which they claimed was located outside the cell membrane. The enzyme cluster was composed of an L-a-glutamyl aminopeptidase (glu-AP) and a low and high temperature phenylalanyl aminopeptidases (phe-AP). Coupled reactions by the cluster were demonstrated with the substrate, N-glutaryl-L-phenylalanine-4-nitroanilide. Gintaryl residues were released initially by glu-AP and the intermediate phe-pNA was hydrolysed by the phe-AP. A coupled system t h a t is localized on or in a cell has obvious advantages in minimizing diffusion of reaction intermediates. An extensive analysis of substrate specificities of aminopeptidases from L lactis ssp. lactis a n d L lactis ssp. c r e m o m strains produced three groups of similar characteristics [61]. The enzymatic activities of cell-free extracts against 54 substrates were used for this cluster analysis. The authors summarized the relative peptidase activities for each of the clusters as shown in Table 1. The differences in relative activities amongst the

Table 1 Groupingof strainsof lactococciby clusteranalysisof peptidaseactivities (A)


ML-3, SK-1,SKo2,SK-3,527; L. lactis ssp. cremoris C-13

L. lactis ssp. cremoris

R-l, E-S,K











L. lactis ssp. lactis

(1) Dipeptidasespecificfor hydrophobicaminoacid-X (2) Dipeptidasclike aminopeptidaseB (3) Prolinediaminopeptidase specificfor Pro-X (4) Aminopeptidese-Pspecific for X-Pro (5) Oencmlaminopeptidase with widespecificity (6) Tripeptidasespecific for X-X-X Taken from [61]with permission.

(c) MI-14,

L. lactis ssp. c r ~











three groups raise intriguing questions about the impact of this diversity on cheese flavor. This has yet to be tested in detail. Other characteristics of the strains such as acid producing activities, proteolytic activities and rate of autolysis would also have to taken into consideration in comparison of strains in cheese manufacturing trials. Lactobacilli also exhibit a wide range of peptid ~ activities [62-64]. Amh'Jopeptidase activity of cell-free extracts of Lb. helveticus CNRZ 303 was 16-18-fold greater than Lb. lactis CNRZ 250 or Lb. bulgaricus CNRZ 369 [62]. Dipeptidase and caseinolytic activities did not vary as greatly betwe~n the strains. Three aminopeptidases, designated as API, APII and APIII, were isolated and characterized from Lb. bulgaricus CNRZ 397 [64]. APII was localized in the cell wall and appeared to be associated more clearly with the peptidoglycan than with the proteinases on the cell surface. It was able to hydrolys¢ a wide range of aminoacyl-fl-naphthylamides and amino-acyl-pNAs. Mutants devoid of APII exhibited a greater X-proline-dipeptidyl-AP activity when grown in milk. Sinc~ APII deficient mutants grew in milk, other peptidases with overlapping activities must carry out the necessary hydrolysis of peptides. Several peptidases with broad specificities were isolated from Lb. casei NCDO 151 [65]. One pepridase, exhibiting the broadest substrate specificity, hydrolysed various ~i- and tri-peptides and carbobenzoxypcptides. Dipeptides with proline as the C-terminus were not hydrolysed but dipeptides with N-terminal proline were cleaved. Peptidase activity with the broad specificity was also detected in two strains of Lb. plantarum. Growth of Lb. casei in skim milk induced two dipeptidases and possibly a cell-bound p'rotvinase. Cell wall-associated dipeptidases have been reported in two strains of /_, lactis ssp. cremoris [66,67]. These enzymes from the two strali~s differed in molecular weights, specificities and metal ion dependencies. Turn-over numbers on certain dipeptides differed by 500-600-fold between the strains. The L, lactis ssp. cremoris Wg2 peptidase was inhibited by 1 mm Co2+ but the L, lactis ssp. cremoris H61 enzyme activity was enhanced. The dipeptidase from L lactis ssp.cremoris Wg2 that was inhibited by EDTA exhibited an activity peak

at 0.1 mM Co2+ and was reactlvated by Mn 2+ above 0.15 raM. Both enzymes exhibited broad substrate specificity. A majority of peptidases are metalloenzyrnes and are inactivated by metal chelators such as EDTA. Kim and Olson [68] suggested that citrate and other carboxylic acids in the serum phase of cheese may affect peptidase activities. They observed a 20-25-fold reduction in fungal peptidase activities in a citrate-phosphate buffer at pH 5.6 as compared to a phosphate buffer. The leucineaminopeptidase activities of the fungal proteinase-peptidase preparations were reduced by 97~ in the presence of citrate equivalent to concentrations that are in cheese serum, before maturation commences. This observation has not been tested in cheese but the effect would depend upon citrate levels, competition for citrate between cations in cheese serum and those associated with enzymes and the rate and extent of metabolism of citrate in cheese. Indirect evidence for the inhibitory effects of citrate on proteolysis of cheese was suggested in several studies in which citrate fermenting bacteria were incorporated into the lactic culture. Nakanishi and Tokida [69] reported that levels of free amino acid nitrogen were lower in cheese made with L. lactis ssp. lactis and L lactis ssp. cremoris as compared to a BD-typ¢ culture which contains citrate fermenters. Qvist et ai. [70] also observed faster citrate utilization in Havarti cheese made with citrate fermenters and this effect was also related to some degree with amino acid nitrogen levels. However, differences in peptidas¢ activities between the species in the cheese cultures may be the determining factor. 7.3. Transport of peptides and amino acida

Transport of peptides and amino acids is energy-dependent [71]. The proton motive force during the transport depends upon ATP-hydrolysis by a proton-translocating ATPase or on end product efflux. Added energy sources such as lactose increased uptake of peptides by L. !actis ssp. cremoris. Stationary phase cultures of Lb. easel energized with glucose assimilated certain amino acids very rapidly in a phosphate buffer [72]. The critical role of an energy source was evident by a

139 90~g reduction in transport activity 5 min after depletion of the glucose. Non-energized (no glucose) cells exhibited slow transport of leucine and phenylalanine even though the proton motive force was reasonably high. The inabifity of an imposed proton motive force to drive the transport of glutamine, glutamate or asparagine in de-energized cells and the inhibition of transport in glucose-energized cells by arsenate suggests that phosphate-bond energy is needed for uptake of these amino acids [72]. The need for energy-driven transport of amino acids in lactic streptococci and lactobacilli common to cheese raises questions about the ability of these bacteria to metabolize these components in a cheese environment. Thomas and Pritchard [32] questioned the role of membrane-bound peptidases in hydrolysis of peptides in cheese and suggested that intracellular enzymes were involved. The efficacy of intracellular peptidases would require transport of peptides into the cell or release of intracellular enzymes. The latter may not occur to a great extent since electron micrographs of 5-month-old Cheddar cheese showed lactococcal cells that were reasonably intact [30]. Most cells exhibited one to two fissures but release of cell contents appeared to be fairly limited. Transport of peptides into cells is, of course, an alternative to transport of amino acids. Lactococci possess separat,e transport systems for amino acids, di-, and oligopeptides [47,73]. Understandably, mechanisms for uptake of peptides have not been clearly defined. Uptake of lysyMeucine has been linked to both a phosphate-bond high energy intermediate or ATP or to energy coupling mediated by proton-motive force [59]. In relation to conditions in maturing cheese, de-energized cells of lactococci were unable to transport glutamic acid but displayed a slow influx of leucyMeucine and alanyl-glutamic acid [74]. This secondary transport system is driven by the chemical gradient of dipeptide that is maintained by the intracellular peptidase action. These researchers proposed that this mechanism which is not linked to energy from high-energy phosphate bonds or proton-motive force might play a role during cheese maturation when energy sources are depleted. Limited passive transport of amino acids has been reported by

Rice et al. [73]. Passive diffusion rates calculated for amino acids exiting from L. lactis membrane vesicles indicated that the rate of diffusion is directly related to the hydrophobicity of the amino acid [75]. The mechanisms by which lactic bacteria hydrolyse polypeptides in cheese and transport the lower molecular weight peptides and amino acids into the cells has not been thoroughly clarified. The role of energy-dfven versus passive transport during the extended period of cheese maturation needs to be resolved. If energy is required, potential sources should be identified. Lactose persists in some et,eese varieties for one to two weeks and any galactose produced during cheese manufacturing will persist slightly longer. The fermentable sugars would be absent or at extremely low levels in cheese when extensive pepfidolysis occurs and when amino acids and peptides would be transported. Lack of energy source would obviate transport unless the cells contained sufficient high energy phosphate compounds. Lactic acid bacteria accumulate phosphoenoipyruvate (PEP) when sugars have been depleted from growth media but the persistence of this compounds in resting cells is not known. It has also not been demonstrated under conditions in cheese, whether ATP generated from PEP would be available for amino acid transport or for other competing energy demands. It is known that the proton motive force of batch or chemostat-grown L lactis ssp. cremoris cells drops to zero within 60-90 rain after the carbohydrate supply is exhausted [59]. 8. CHEESE PROTEIN DEGRADATION 8.1. Proteolysis

Proteolysis in cheese during maturation is a sequential process involving added milk-clotting enzymes, milk proteinases, lactic acid bacteria and adventitious or added microorganisms [76]. Initial proteolysis in most cheeses, especially those salted before pressing or fusing into their final forms, results from the action of milk-clotting enzymes. Limited but specific cleavage of the Phe23-Pheq4 bond or the Phe24-Va125 bond of asf.Casein occurs in the early stages of maturation of several cheese

140 varieties [77,78]. Substantial softening of cheese has been attributed to the hydrolysis of this single bond [79,80]. This softening enhances dispersibility during mastication which undoubtedly enhances the perception of flavors and may enhance the release of aromas. The initial specific hydrolysis of as 1-casein produces a polypeptide, asl-I (¢sl-CN(f24/25-199)) in all cheeses made with milk-clotting enzymes that have been assayed by polyacrylamide gel eleetrophoresis [81,82]. Chymosin pro;l~3,~.e¢l five additional peptides from purified asl-casein in solution: asl-CN(f24/25-169), asl-CN(f24/25149), asl-CN(f24/25-150 ), as1-CN(f29/33-199 ) and asl-CN(f56-179 ) [83,84]. Peptides have been recovered from Cheddar cheese with electrophoretic mobilities and molecular weights similar to asl-CN(f29/33-199 ) and to two non-characterized peptides, -'-lesignated as asl-VII and asl-VIlI, that are produced by chymosin action on asl-casein [82]. The asl-CN(f24/25-199 ) peptide can be degraded at variable rates during cheese maturation. Creamer [85] observed a substantial decrease in concentrations of this peptide over 14 weeks of maturation of Cheddar cheese but less change in Gouda cheese. A rapid increase in the concentration of this peptide was observed during the first 8 weeks of matfiring Cheddar cheese by Basch et al. [86]. An equally rapid disappearance occurred over the succeeding 10-12 weeks. Hydrolysis of ~-casein is slower than asl-casein in most varieties of cheese [82]. Cheeses in which /~-casein degradation is faster than in Cheddar include Gouda cheese because of the higher pH values of the cheese during maturation and the lower sodium chloride content in the cheese interior, Camembert cheese because of high pH values during maturation and Emmental cheese because of heat inactivation of chymosin [76,82,85]. All of these factors reduce the proteolytic activity of chymosin and increase the relative activity of the milk proteinase, plasmin. Also, the bonds in /~casein in solution that are readily hydrolysed by chymosin are not attacked in cheese presumably because of intermolecular hydrophobic interactions between the C-termini of fl.casein molecules that contain the ehymosin-sensitive bonds.

The principal hydrolysis products of//-casein in cheeses are 7-caseins [82]. These result primarily from the action of plasmin but an acid milk proteinase may contribute to a minor extent [87]. The rates of disappearance of casein has been modelled using non-linear regression analysis of SDS-polyacrylamide gel eleetrophoresis patterns of Cheddar cheese during maturation [86]. Calculated "half-fives" for disappearance of asl-casein and p-casein were 2 weeks and 37 weeks, respectively. It is interesting that the rate constant for disappearance of asl-casein is the only one that approaches the value for diffusion in solution. Values for degradation of other proteins are at least 10-fold lower. The role of laetococci in hydrolysis of casein in cheese is minor compared to the milk-clotting enzymes [76,17]. Whereas most laetococci possess proteinases capable of attacking ~-casein, only 20-30% of the total hydrolysis of casein could be attributed to lactococcal proteinases during maturation of Gouda cheese [17]. Also, there were no differences in levels of a s- and t-casein during maturation of aseptic cheeses or aseptic rennet-free cheeses made with different lactucoccal strains, even though the proteolytic systems of these strains were different. These estimates are based upon aseptic rennet-free and aseptic starter-free cheeses which do not exactly duplicate the traditional cheese manufacturing procedures. Some differences in pH values and moisture contents were evident between experimental groups, but these differences should not have obviated the trends observed in the studies [89]. O'Keeffe et al. [90] also observed that the n~lk-clotting enzyme played the dominant role in casein hydrolysis in Cheddar cheese and that lactic starter produced lower molecular weight peptides and amino acids. Streptococcus thermophilus is not likely to contribute to casein hydrolysis in cheese, since its proteolytic activity appears to be lower than lactococci [32]. High temperature lactobacilli are more proteolytic but direct evidence for their role in casein hydrolysis is limited. This can be inferred from studies in which "heat-shocked" Lb. helveticus CNRZ 303 was added as a starter adjunct to a low-fat cheese [91]. The added cells did


not significantly increase concentrations of noncasein nitrogen but significantly increased phosphotungstic acid soluble nitrogen (PTA-N). 8.2. Peptidolysis

There is gcueral agreement that the lactic acid bacteria contribute to the incro~se in low molecular weight peptides and amino acid concentrations during cheese maturation. Evidence for the hydrolysis by a Lactococcus species of a peptide generated by chymosin in cheese was presented by Kaminogawa et al. [55]. The first peptide, aslCN(fl-23), generated by chymosin during cheese maturation was further hydrolysed by a partially purified cell-free extract of L lactJs ssp. cremorJs H61 to generate seven principal peptides. Three of the peptides were similar to peptides isolated from Gouda cheese during maturation. Other studies have also illustrated the role of lactic starter cultures in generating small peptides and amino acids. Very tittle amino-acid N was generated in aseptic, starter-free Gouda cheese [17,89]. This effect can be inferred also from experiments in which the chymosin levels were reduced by one-half in aseptic cheese. Definite reductions occurred in levels of nitrogen soluble in 0.15% CaCI 2 (equivalent to non-casein N) with the lower amounts of chymosin but no differences were observed in amino-acid N. Kleter [92] compared levels of proteolysis in aseptic cheeses made with L. lactis ssp. cremoris E8 with cheeses acidified with 81ucono-&lactone. Concentrations of non-casein nitrogen were slightly lower (20%) in the cheese without lactic starter culture but amino-acid N was virtually non-detectable in this cheese. The concentration of free proline in Swiss cheese was directly related to the amount of Lb. bulgaricus added in the lactic starter culture [93]. Strains of lactic acid bacteria vary in their peptidolytic activities in cheese. Levels of amino acid N in aseptic Gouda cheese matured for 6 months differed by two-fold between the two most divergent strains [17]. In contrast, levels of nitrogen soluble in 0.15% CaCl 2 (non-casein N) varied by only 20% between cheeses made with the different strains. The non-casein nitrogen levels in

aseptic Gouda cheese made with L. lactis ssp. cremoris E8 were similar to those in cheese made with L laclis ~sp. cremons Wg2 but levels of amino acid N were ~ignificantly higher in the former cheese during maturation [94]. The N soluble in 0.48 N TCA in Swiss cheeses made with five different strains of lactobacilli in the lactic starter culture varied by two-fold [95]. Cheddar cheese made with L. lactis ssp. cremoris strains contained lower levels of 12% TCA soluble-N as compared to cheese made with L lactis ssp. cremoris strains [96]. It was suggested that the effect resulted from a greater proportion of prtmutants or from lower number of cells in the L. lactis ssp. cremoris starters. The effects of prt- mutants of Lactococcus species in lactic starter cultures is another index of the effects these bacteria on degradation of polypeptides in cheese [32]. The prt- mutants are deficient in cell-wall proteinase but possess peptide transport systems and peptidases similar to parent strains. They have been advocated because of improved yields of cheese and reduced bitterness in cheese. However, elimination of prt + mutants appears to reduce rates of cheese maturation because of lower rates of hydrolysis of the cheese polypeptides that is thought to be a rate limiting step [97-99]. Proteolysis was compared in Gouda cheeses made with a 1.0% inoculum of a control starter (80% prt- and 20% prt + variants), a 0.6% inoculum of prt + variants or an 8.0% inoculum of prt- variants. Noncasein-N concentrations were similar in all cheeses during maturation. Levels of amino-acid N in cheese made with the prtvariants were about one-half the levels detected in the other cheeses after 3 and 6 months of maturation. Amino-acid N concentrations in the control cheeses were 10% lower than that in cheeses made -a~th the prt + variants, indicating that as little as 20% of the culture needed to be prt + to attain the same degree of proteolysis and peptidolysis as 100% prt+ variants. Thermal- and freeze-shocked cells of selected Lb. heiveticus strains increased protcolysis of cheese when added as an adjunct to the traditional lactic culture [91,100-102].The degree of proteolysis and peptidolysis in cheese varied between strains and species of the tested bacteria. Bartels

142 et al. [100] observed little increase in PTA-soluble N during cheese maturation when two stains of Lb. bulgaricus or a strain of S. thermophilus was used. A strain of Lb. heiveticus produced the highest level of PTA-soluble N but two other strains yielded lesser amounts during cheese maturation. Ardo et al. [91] found no increase in non-casein nitrogen during maturation of a low-fat cheese when heat-shocked Lb. helveticus CNRZ 303 was added as a starter adjunct but PTA-soluble N was about 2.5-fold higher as compared to control cheeses.

9. CHEESE FLAVOR Correlating cheese flavor intensity with specific or groups of compounds has not been completely successful although some reports indicate moderately good estimation. Barlow et al. [103] tested the correlation between Australian Cheddar cheese flavor (combined intensity and quality evaluation) and 21 analytical measurements on 220 samples from three manufacturing operations over two seasons. The best-fitting subset of variables to explain Cheddar cheese flavor at 12 months was ethanol, In of propan-l-ol + ethanol, In of butanone + ethanol, H2S, In of propan-2-ol + ethanol (direct headspace measurement), and water-soluble nitrogen. The coefficient of variation (R 2) was 0.794 for these factors; inclusion of L-lactic acid increased the coefficient to 0.796. Prediction of cheese flavor quality at 12 months of maturation :vas best attained by a combination of sensory analysis of the cheese at 3 months plus analysis of H2S and water-soluble N. Manning [104] related English Cheddar cheese flavor intensity and quality to concentrations of 2-pentanone, methanethiol and methanol in headspace samples of cheese. The relationship between methanethiol and cheese flavor intensity has been questioned by several r e ~ c h e r s including the recent study by Barlow et ai [103]. However, a trend between flavor intensity and methanethiol levels was observed in one of the Australian cheese manufacturing sources but R 2 for this relationship was 0.39 for all samples [103]. Correlating flavor intensity

and quality with selected compositional parameters has obvious shortcomings. Compounds that exhibit a good relationship may not contribute directly to flavor but simply increase in concentration during maturation. Key flavor compounds may not be detected because of sampling and analytical limitations or they may exist at extremely low concentrations. The difficulty in defining flavor of cheeses, especially Cheddar cheese, by analyses of specific compounds prompted researchers to fractionatc cheese and trace the partitioning of flavor [105,106]. McGugan et al. [105] found the most intense flavor in the water-soluble extract of aged Cheddar cheese. The intensity was significantly higher than that of an extract from mild Cheddar cheese. In contrast, the water-insoluble proteinaceous fraction and the milkfat fractions from mild and aged cheeses did not differ substantially in flavor intensity. The water-insoluble fraction from aged cheese possessed a slightly higher intensity than that from mild cheese. The researchers attributed that effect to differences in texture since the more highly degraded aged cheese would disperse more readily in the mouth. Ashton and Creamer [106] observed similar flavor partitioning of New Zealand Cheddar cheese into the various fractions. The water-soluble fraction (WSF) exhibited the dominant flavor intensity. In recomblned samples containing the water-soluble fraction from aged cheese, there were no intensity differences whether the samples contained fat from aged or mild cheeses or deodorized fat. The source of fat affected flavor intensity slightly in recombined samples containing the water soluble fraction from mild cheese. The WSF was further fractionated by Aston and Creamer [106] on Sephadex (]-15 into six su_h°£ractions. Sub-fraction 3 (SF3), which constituted 42~ of the WSF solids, exhibited greatest flavor intensity but the level was slightly lower than the intensity of WSF. The SF3 and SF2 contained the greatest concentrations of free amino acids but the flavor intensity of SF2 was significantly lower. The SF3 contained substantially less glutamic acid and valine, but substantially more methionine, isoleucine and leucine than SF2. It is noteworthy that a mixture of amino acids that was

143 compounded to simulate WSF had a significantly lower flavor intensity than WSF and SF3 which suggests that other compounds in addition to amino acids contributed to the flavor of WSF. Understandably, there are diverse opinions about the relationship between levels of small peptides and amino acids and flavor intensity of cheese. In a study on the use of lactobaciUi to accelerate maturation of Cheddar cheese, Puchade et al. [107] noted that amino acid concentrations were higher in experimental cheeses than the control especially during the first four months of maturation. The differences were less at 7 and 9 months. Flavor intensity defined as ripening index was greatest in cheese in which a strain of Lb. casei was added but total free amino concentrations were no greater in this cheese during the first 7 months of maturation than in others made with different Lactobacillus strains. Apparent differences between lactococcal species on flavor intensity of Cheddar cheese was reported by Amantea et al. [96]. Cheeses made with L lactis ssp. cremoris possessed lower flavor intensities and lower amino N contents than cheeses made with L iactis ssp. lactis. Higher concentrations of amino N were correlated with flavor intensity of Gouda cheese made with prt + and p r t - variants of lactococci [98]. The relationship between amino N and cheese flavor was also observed in Gouda cheeses made under aseptic conditions. Cheese made without lactic starter culture exhibited virtually no increase in amino N during 6 months of maturation and was devoid of flavor [92]. The oxidation-reduction potential of this cheese undoubtedly was abnormally high. Aseptic Gouda cheese made with lactococeal strains, that did not produce bitterness in cheese, exhibited considerably greater flavor intensity than cheeses made with strains that produced bitterness [10~]. Cheese made with the non-bitter inducing strains also contained more amino N. Enhanced flavor development was obtained in Cheddar cheese to which a lac- p r t - mutant was added as an adjunct to the lactic starter culture [109]. The Lactococcus population was increased 100-fold which almost tripled the rate of FFAsoluble N production but only reduced maturation

time at 8°C by 20~. These authors indicated that increasing the ripening temperature from 8 to 15°C was more effective in accelerating maturation than increasing the number of lac- p r t lactococci. Kamaly et al. [110] also observed enhanced maturation rates of cheese containing add e d p r t - lactococcal mutants. Using heat-shocked or freeze-shocked strains of Lb. helveticus as a lactic starter culture adjunct has enhanced maturation of Gouda cheese and a low-fat semihard Swedish cheese [91-102]. In all cases the enhancement seemed to be associated with greater peptidase activities and higher concentrations of amino N. Since it is unlikely that the flavor of cheeses such as Cheddar and Gouda results from the flavor of peptides and amino acids, further modification of amino acids seems essential. Catabolism of amino acids by lactic acid bacteria is possible but limited energy sources in cheese seemingly .minimize transport of these substrates. Slow passive diffusion of peptides has been observed. Non-enzymic reactions have been suggested as sources of sulfur compounds in Cheddar cheese [34]. Recent reports by Hammond and co-workers on reactions between dicarbonyls and amino acids appear to have direct implications for the flavor development of Swiss cheese but probably apply to several other varieties. Grifflth and Hammond [111] demonstrated that a wide variety of compounds were produced by reactions between dicarbonyls and amino acids. A number of these compounds were similar to those detected in cultures of Lb. bulgaricus and Lb. helveticus and in aromatic extracts from Swiss, Cheddar and Paxmesan cheeses [112,113]. Dicarbonyls used in the above reactions include glyoxal, methylglyoxal, dihydroxyacetone and acetoin. The first three and diacetyl were found in Swiss, Cheddar and Mozzarella cheeses and in cultures of Lb. bulgaricus, Lb. casei, S. thermophilus and Propionibacterium shermanii [114]. It is likely that these compounds would exist in many. varieties of cheese containing these bacterial species. The amino acids participating in the reactions produced flavors ranging from rose-like to cheesy depending upon the particular amino acid


a n d the dicarbonyl. T h e conditions for the reactions in these studies simulated cheese except for t h e presence o f oxygen. Griffith a n d H a m m o n d [111] p o i n t e d out that m e c h a n i s m for f o r m i n g s o m e o f the c o m p o u n d s required oxygen w h i c h w o u l d minimize their f o r m a t i o n in cheese except for the surfaces o f s o m e varieties. Also, o t h e r flavors might b e generated u n d e r reducing conditions.

10. C O N C L U S I O N S It is impossible to attribute direct i m p a c t s o f lactic acid bacteria o n cheese flavor. I n f e r e n c e s can b e m a d e b a s e d u p o n their effect o n the oxidat i o n - r e d u c t i o n potential o f cheese, their synergism with non-starter bacteria and other microorganisms, a n d their role in facilitating flavor-prod u c i n g reactions. Successful evaluation o f the m e c h a n i s m s o f flavor d e v e l o p m e n t in cheese will necessitate (1) careful analysis o f key flavor c o m p o u n d s t h a t exist in ng-g -1 quantities, (2) systematic evaluation o f proteolysis a n d peptidolysis in cheese, (3) t h o r o u g h analysis o f e n z y m e systems o f key lactic acid bacteria a n d (4) evaluation o f divergent strains in cheese to relate e n z y m a t i c activities to flavor a n d proteolysis profiles.

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