The Dairy Leuconostoc: Use in Dairy Products

The Dairy Leuconostoc: Use in Dairy Products E. R. VEDAMUTHU Quest International Sarasota, FL 34243

ABSTRACT

and other neutral C4 compounds. Diacetyl is the primary source of aroma and flavor in cultured dairy products such as cultured buttermilk, creamery butter, sour cream, dressed cottage cheese, and certain other soft, semi-soft, and hard cheeses. For utilitarian purposes, leuconostocs are primarily important for flavor generation in cultured dairy foods.

Leuconostocs are a relatively inert of bacteria found on plants that ¥am entrance into milk via plant material In the dairy environs. In milk, leuconostocs grow associatively with lactococci, produce important flavor compounds, and play a significant part in developing aroma and flavor in various cultured dairy products including certain varieties of cheeses. The role of leuconostocs in various dairy applications and the factors that govern their significant functions are discussed in this review. (Key words: Leuconostoc, dairy products, flavor) gr~up

Associative Growth and Compatibility

A:bbreviation key: QAC = quaternary ammonium compounds.

INTRODUCTION Leuconostocs comprise a relatively inert group of bacteria found mainly on vegetables an~ roots (18). Hucker and Pederson (28), in theIr extensive studies on leuconostocs, also mention that these bacteria, although found in milk and certain dairy products, have their ecological niche on plants and vegetables. ~ese bacteria probably gain entry into raw milk from pasture and dairy environs in which fresh, leafy fodder is present. Hucker and Pederson (28) state that the types of leuconostocs found in milk products are not in their true habitat, and, hence, the inactive characteristics of these strains reflect their inability to carry on indefinite growth in milk. In pure cultures, leuconostocs are inert in milk (28), but, in association with lactococci and sometimes with other lactic acid bacteria, they ferment citrate present in milk to yield diacetyl

. Leuconostocs function only associatively in rrulk (18), a relationship between leuconostocs a~d acid-producing lactococci that was recognized as early as 1919 by independent groups of researchers in the US, Denmark, and Holland (18). Foster et a1. (18) stated that, in mixed cultures containing lactococci and leuconostocs, the function of lactococci is to produce acid from lactose, and the role of leuconostocs is to ferment the citric acid of milk to volatile compounds. Further, together these bacteria perform a function that neither could carry out alone, an example of a synergistic effect. Thus, the associative growth relationship between these two groups of bacteria is symbi~ti~, and the functional relationship is synergIStIc. Factors contributing to the symbiotic growth relationship between lactococci and leuconostocs have been reported (18). Suffice it to say that lactococci, because of their relative metabolic versatility in milk, produce stimulatory substances necessary for the growth of leuconostocs. Because of the dependency of leuconostocs on lactococci to initiate good gro~th in milk, the selection of compatible ~trams of the two groups is necessary. The Importance of such selection was recognized by earlier workers. Foster et al. [(18) p. 293] summarize the early observations thus:

Received February 10, 1993. Accepted May 17, 1994. 1994 J Dairy Sci 77:2725-2737

2725

If one takes a series of lactic streptococci and a series of leuconostocs and attempts to make combinations of the different species, only a small number of the combinations can be expected to prove satisfactory. Present knowledge of the factors that influence establishment of the desired balance does

2726

VEDAMUTHU

not permit advance determination of whether a satisfactory combined culture may be expected.

The foregoing statements were published in 1957, and even today not much is known of the intricacies of symbiotic relationships between lactococci and leuconostocs. Recent work done at Oregon State University (ytI. E. Sandine, 1994, personal communication) demonstrated that only a limited number of compatible strains of the two groups exist in culture collections. In the Oregon State University study, the investigators examined 60 Leuconostoc strains in their collection for biochemical characteristics for taxonomic identification. Twenty cultures typed as Leuconostoc mesenteroides ssp. cremoris. All 20 Leuc. mesenteroides ssp. cremoris strains were paired with 10 strains of Lactococcus lactis ssp. lactis and 10 strains of Lactococcus lactis ssp. cremoris. Only 2 of the 20 leuconostocs were compatible with both Lactococcus subspecies. The criterion for compatibility was based on a positive reaction for King's test in milk (35). Thus, renewed research is needed to screen for compatible strains of leuconostocs and for basic studies relating to factors that govern compatibility. A great deal of research pertaining to leuconostocs was done when ripened cream butter was widely made (42). With the decline of ripened cream butter as a lucrative and popular item of commerce, research on Leuconostoc strains dwindled. Many successful butter cultures containing good aroma-producing leuconostocs were lost because of neglect (41). Need for Sufficient Numbers

For functionality, associative culturing of lactococci and leuconostocs needs not only compatible strains, but also sufficient numbers of the bacteria that produce acid and aroma. To obtain maximum benefit from the associative growth, the leuconostocs must be pennitted to reach a high population before pH drops to a low level, so that there will be enough Leuconostoc cells to carry through citrate metabolism (18, 63). This aspect is closely tied to the temperature of incubation. A balanced growth of lactococci and leuconostocs occurs during incubation between 21 and 25°C. At temperatures above 25°C, the ratio is skewed toward Journal of Dairy Science Vol. 77, No.9, 1994

the metabolically more active lactococci, which grow at a faster rate at higher temperatures (between 25 and 32°C) relative to the leuconostocs (12, 63). The lack of sufficient aroma and flavor in cultured buttennilk incubated at temperatures above 25'C may in part reflect insufficient numbers of leuconostocs at the end of fennentation (61). Higher incubation temperatures are often used in the industry to obtain a rapid coagulum, which facilitates quick turnover of setting tanks and filling equipment (61). The interrelationship between growth rate and citrate metabolism by leuconostocs in milk in the presence of lactococci can be more easily followed now, since the availability of sophisticated analytical techniques such as high perfonnance liquid chromatography for diacetyl and citrate detenninations and differential counting procedures using vancomycin as the selective agent (5). Leuconostocs are inherently resistant to relatively high concentrations of vancomycin, but lactococci are inhibited. The need for a sufficiently high number of Leuconostoc bacteria to give good diacetyl flavor in cultured dairy products was recognized by early investigators. Michaelian et al. (45) found that, by building up high numbers of Leuconostoc bacteria before pH adjustment and citrate fortification, followed by an additional growth period, they could obtain high aroma and flavor in butter. Their procedure called for growing pure cultures of leuconostocs for 15 to 24 h in pasteurized milk, after which .15% citric acid was added, and the pH was adjusted to 4.0 to 4.3 with sulfuric acid. After these manipulations, the mixture was incubated at 21°C for an additional 24 h. Fabricius and Hammer (16) used a modification of this procedure to prepare a modified butter culture. They found that with the ample availability of Leuconostoc cells, more unifonn production of aroma and flavor between different lots of butter could be ensured. Further modification of the procedure by Hoecker and Hammer (27), allowing the unimpeded increase in cell numbers of leuconostocs, achieved diacetyl concentrations ranging from 4.48 to 11.84 ppm. This principle was adapted by Mather and Babel (43) to develop a practical method to obtain creamed cottage cheese with a good aroma and flavor. The principles em-

SYMPOSIUM: THE DAIRY LEUCONOSTOC

bodied in these studies by Mather and Babel led to the issue of a US patent (3). Propagation of Leuconostocs

With the widespread use of commercial cultures and the development of concentrated frozen or lyophilized cultures for direct setting of vats (17), it became important to formulate suitable media and to concentrate and preserve Leuconostoc cells with high viability and functionality. Only a few publications (20, 60) deal with these aspects, and commercial procedures have remained proprietary. The most commonly used laboratory media for the propagation of leuconostocs are MRS broth or Elliker's lactic broth (I), both of which support luxuriant growth of leuconostocs. Lundstedt (40) described a wheybased medium for leuconostocs that is adaptable for mother and intermediate culture preparation in dairy plants. He found that in the citrate and whey medium, different species comprising aroma bacteria, namely, Leuc. mesenteroides ssp. cremoris. Leuconostoc mesenteroides ssp. dextranicum. and Lactococcus lactis ssp. lactis biovar diacetylactis, had distinctive growth patterns. He also reported that sterilized citrated cottage cheese whey containing a commercial preparation of pancreas extract (Procheez) was an excellent medium for propagation and long-term storage (30 d) of leuconostocs in the refrigerator (4 to 5'C). Gilliland et al. (20) described a procedure to prepare frozen concentrated cultures of Leuc. mesenteroides ssp. cremoris. Their growth studies were conducted in 4-L volumes in a 7.5-L fermentor equipped with an autoclavable pH probe and controls for pH. The medium consisted of 2% laboratory grade tryptone, .5% yeast extract, 1% glucose, and .5% sodium citrate. The medium was autoclaved at 121°C for 15 min, cooled to 2SoC, and aseptically transferred to the sterilized fermentor vessel. The temperature was controlled at 2SoC, and a 4% inoculum of a fresh broth culture of Leuconostoc mesenteroides ssp. cremoris was added. Maximum cell numbers were obtained after 23 h growth (1.4 billion viable cells/ml) when the pH was controlled at 6.5 or 7.0. Although diacetyl produced by concentrated cells previously grown at pH S.S was greater by SO% compared with cell crops grown at higher pH, Gilliland et al. (20) suggested that

2727

the increased cell numbers at higher pH would yield more total diacetyl. No differences were noted between the use of sodium or ammonium hydroxide for pH control in cell yields or for diacetyl produced by the concentrated cells. Walker and Gilliland (63) described another medium for preparing frozen concentrates of Leuc. mesenteroides ssp. cremoris: 5% peptonized milk nutrient (Sheffield Products, Norwich, NY), 2% primatone (Sheffield Products), 2% lactose, .1 % sodium citrate, and .1 % Tween 80. The medium was sterilized at 121°C for IS min, cooled to 25°C, and inoculated with S% (voVvol) of a 24-h broth culture. The pH was controlled at 6.0 with 20% sodium carbonate. The fermentation period was between 22 and 24 h. Concentrated cell slurry was frozen in liquid nitrogen. Walker and Gilliland (63) did not report the viable cell yields from the fermentations, but emphasized their flavor-yielding potential. Cell yields of leuconostocs are usually much less than those of lactococci. In composite commercial culture concentrates containing acid-producers and aroma-producing leuconostocs, the proportion of leuconostocs varied from S to 10% of the culture (17). Because of the lower viable cell numbers of the aroma bacteria per unit volume or weight of cell concentrate relative to those of the lactococci, often products made with such cultures lack full aroma and flavor. Research is needed in process development and preservation to obtain high numbers of viable cells of leuconostocs with good functionality. Need for Acidic Environment

Early studies with butter cultures containing lactococci and leuconostocs showed that acid production by lactococci was necessary before the aroma bacteria could convert citrate in milk to aromatic compounds (18). From these observations, the need for an acidic environment for the synthesis of diacetyl was realized and applied in the development of technologies for flavorful butter and creamed cottage cheese. The requirement for an acidic environment for the biosynthesis of diacetyl by aroma bacteria was explained by physiological studies conducted by Harvey and Collins (2S). They found that citrate permease, the enzyme that facilitates the uptake of citrate, was active only Journal of Dairy Science Vol. 77. No.9. 1994

2728

VEDAMUTHU

below pH. 6.0. Based on this observation, the associative functional relationship between lactococci and leuconostocs may be envisioned as follows. Leuconostocs are relatively inert in pure cultures in milk, but they possess the enzymes necessary for the metabolism of citrate to diacetyl. Lactococci can actively ferment the lactose in milk, but they lack the mechanisms to use citrate. Although the leuconostocs contain the enzymes necessary for citrate metabolism, the intake of citrate is active only below pH 6.0. The normal pH of milk is between 6.4 to 6.6, and, to initiate citrate uptake by leuconostocs inoculated into milk, sufficient acid production by lactococci is needed to depress the pH below 6.0. The interrelationship of pH and diacetyl accumulation in a milk system containing both lactococci and leuconostocs, however. is quite different from milk containing leuconostocs alone. In a pure aroma culture system, optimal pH is between 4.1 and 4.4. In a mixed culture fermentation involving lactococci and leuconostocs, Lundstedt and Corbin (41) found that 86% of the milk citrate was used up below pH 5.2. Michaelian et al. (45) reported in 1933 that, for good diacetyl production by leuconostocs in pure cultures, the pH should be adjusted to 4.0 to 4.3. Additional studies by Fabricius and Hammer (16) and Hoecker and Hammer (27) showed that, for high diacetyl production by pure cultures of leuconostocs, an acidic environment was needed. Mather and Babel (43) conducted extensive studies using a pure Leuc. mesenteroides ssp. cremoris strain for the development of a highly aromatic creaming mixture for cottage cheese and found that, after sufficient cell numbers were attained, downward adjustment of pH to 4.24 was necessary. Cogan (9) found the pH optimum for citrate metabolism by leuconostocs was 5.4. Citrate Concentration

Hammer (18) established in the 19205 that the precursor for the flavor compounds produced by lactic cultures was citric acid in milk. Citrate concentration in milk exhibits a wide fluctuation, which in many cases is seasonal. On average, milk contains about .2% citrate. Fortification of milk or cream with citrate boosts the flavor of cultured products. Journal of Dairy Science Vol. 77, No.9, 1994

The Code of Federal Regulations (7) allows the addition of .15% citrate. Addition of citrate not only provides more substrate, but also compensates for variations in flavor from fluctuations in the native citrate content of milk. The importance of citric acid or citrate addition found application in the development of aromatic butter cultures (43) and cottage cheese dressing (3). In some of these procedures, citric acid played the dual role of providing additional precursor for flavor compounds and of adjusting pH to the desired acidic range. In some instances, during the curd washing step for cottage cheese, citric acid is used to acidify the chilled wash water (15), thereby introducing some substrate for conversion to flavor compounds by flavor bacteria added to the cream dressing. Fortification with citrate also helps in stabilizing the level of diacetyl in cultured products. This aspect is discussed in a later section. Production and Destruction of DIRcatyl by Leuconostocs

Diacetyl is synthesized from citric acid. Citrate permease, an inducible enzyme active below pH 6.0, transports citrate into the cell. Citrate is first cleaved by an inducible enzyme, citritase. into acetic and oxaloacetic acids. The metabolism of citrate by leuconostocs is discussed in detail by Collins (10) and Kempler and McKay (34). The inducible nature of citrate permease and citritase among Leuconostoc strains requires that, in the production of cell concentrates of these bacteria for application in generating flavor compounds, the propagation media contain citrate as an ingredient. In the absence of citrate, cell crops obtained from fermentations failed to produce any diacetyl (19). In dairy fermentations, concentration of diacetyl neither accumulates indefinitely nor remains unchanged. The reason for the disappearance of diacetyl was explained by Hammer et al. (23), who found that leuconostocs reduce diacetyl to acetoin and 2,3-butanediol. The quantitative depletion of added diacetyl by a pure culture of Leuc. mesenteroides ssp. dextranicum was demonstrated by Elliker (14). Seitz et al. (56) showed that the destruction of diacetyl was an irreversible phenomenon mediated by the enzyme diacetyl reductase. The reduction of diacetyl to acetoin and further

2729

SYMPOSIUM: THE DAIRY LEUCONOSTOC

reduction to 2,3-butanediol were coupled to concomitant oxidation of NADH2. The reductive reactions thus playa physiological role in regenerating the cofactor. In stabilizing diacetyl concentrations in dairy products, a good strategy is to provide alternate routes for regenerating oxidized cofactor, thus sparing the reduction of the diketone. Pack et al. (49) investigated the fate of diacetyl synthesized from native milk citrate by a commercial mixed-strain starter containing only Leuconostoc spp. as flavor producers, when incubated at 21 and 32°C. At both temperatures, diacetyl concentration graphed over time showed a bell-shaped curve. At 21°C, diacetyl accumulation started later than at 32°C, but the maximum diketone concentration was twice as great at 21°C. Once the maximal dicarbonyl was reached, diketone concentration declined rapidly over time. Parallel citrate assays showed that the buildup of diacetyl in the milk culture occurred when the depletion of the precursor was rapid. The decline in diacetyl concentration started when the citrate concentration fell below a critical threshold [(49, Figure 1)]. From these studies of kinetics, Pack et al. (49) proposed that diacetyl synthesis and its destruction (reduction to acetoin) occur in tandem, but, in early stages when the citrate level is high, the synthetic phase is more prominent and accumulates diketone. As the precursor is used, and its concentration falls below the critical point, the reductive phase becomes dominant. Based on these observations, the commercial practice of fortifying the dairy mix with citrate is useful and valid. Addition of citrate provides more precursor, which helps to obtain a higher diacetyl concentration. More substrate also aids in delaying the onset of declining phase, which allows processors to develop more acidity in cultured buttermilk to meet market demands (60). Fortification with precursor also provides a safety margin in preventing flavor loss when large volumes of fermented product have to be cooled. Cooling cultured products to temperatures below 7"C arrests the destruction of diacetyl by retarding diacetyl reductase activity. It is also reported that citrate has a slightly repressive effect on diacetyl reductase (29). Recent work at Oregon State University ~. E. Sandine, 1994, personal communication) showed that the addition of .1 to .15% sodium

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Hours Figure I. Diacetyl production and destruction in milk by a mixed-strain starter (A) containing leuconostocs (0, 21'C; ., 32'C) contrasted with a mixed culture (B) containing Lactococcus lactis ssp. lactis biovar diacetylactis (.6., 21'C; &, 32'C). From Pack et al. (49).

citrate to cultured buttermilk or sour cream when the coagulum is stirred enhanced diacetyl concentrations in these products. Importance of Cooling

Using the data on the kinetics of diacetyl synthesis and reduction by flavor bacteria in milk cultures, Pack et al. (49) explored practical means by which a relatively high concentration of diacetyl could be achieved and circumvent the loss of accumulated diketone. The first series of experiments conducted in milk examined the effect of rapid cooling on diacetyl concentrations in aliquots collected at various stages of culturing at 21°C over 96 h. Pack et al. (49) found that immediate cooling of the culture at peak diacetyl concentration in the culture (after 12 h of culture) not only stabilized the flavor over a period of 4 d but also resulted in an increase in the diacetyl concentration. Cooling of the culture at the early stages of incubation (6 h of incubation) did not result in any great increase in diketone concentration. Pack et al. (49) suggested that, Journal of Dairy Science Vol. 77, No.9, 1994

2730

7

VEDAMUTHU

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Figure 2. Effect of cooling and cold storage (2'C) on Jiacetyl accumulation in milk cultured with a mixed-strain starter containing leuconostocs. Samples were cooled at the indicated intervals during incubation at 2I'e. From Pack el al. (49).

after 6 h of incubation. acid development was insufficient to depress the pH sufficiently for optimal citrate permease activity. and. hence. not much accumulation of diacetyl occurred. Cooling after 24 h at the end of reductive phase failed to reverse the flavor loss (Figure 2). In another experiment, Pack et al. (49) found that cooling of cultures halfway through the destructive phase resulted in the immediate stabilization of flavor and increased the diacetyl concentration during cold storage. The increase in the diketone concentration in the cold was attributed to chemical or enzymatic conversion of diacetyl precursor(s) to dicarbonyl (49). Under practical conditions, the time intervals at 21·C to attain a desirable aroma may vary, and, hence, cooling of the product should be adjusted accordingly. According to Foster et al. (18) in cultured buttermilk, too much acid development results in a harsh, acid flavor and loss of diacetyl. They recommended that an acid range of .8 to .9% yields the optimal physical and organoleptic properties, at which point the product should be rapidly cooled. Aeration

In cultured buttermilk manufacture, gentle agitation during cooling of the product provides a means to break the coagulum. to facilitate uniform cooling by moving the product, and also to incorporate some air into the buttermilk. Incorporation of air by agitation in Journal of Dairy Science Vol. 77. No.9. 1994

actual commercial production yields greater flavor. Pette (51) reported that oxygen was effective in stimulating diacetyl formation during vigorous fermentation of citrate by mixed cultures. Similar observations were reported by other investigators (13.42). The mechanism for greater accumulation of diacetyl by aeration was eloquently explained by Collins (10). Bruhn and Collins (6) described the characteristics of a NADH oxidase from Lact. lactis ssp. lactis biovar diacetylactis DRC-I. This enzyme is the type described as NADH:H20 oxidase by Condon (I 1). The presence of this enzyme in Leuc. mesenteroides was reported by Koike et al. (36). This enzyme catalyzes the oxidation of NADH2 directly by molecular oxygen with the formation of oxidized cofactor and water, and its elaboration is stimulated by aeration (6. 11). Collins (10) proposed three mechanisms by which NADH oxidase would boost diacetyl concentration in cultures. All of these mechanisms relate to the steps in carbohydrate metabolism that involve the regeneration of cofactor for recycling. In carbohydrate fermentation, pyruvate is a key intermediate. In sugar fermentation, pyruvate is reduced to lactic acid by hydrogen donated by reduced nicotinamide cofactor, thereby regenerating NAD. This reaction is coupled to glyceraldehyde-3-phosphate dehydrogenase reaction. If an alternate cofactor regenerating reaction is operative. pyruvate accumulates. Accumulated pyruvate would then be converted to neutral C4 compounds as a detoxification mechanism. In citrate fermenting lactic acid bacteria, the excess pyruvate comes from citrate degradation. The operation of NADH oxidase, which is stimulated by aeration, would thus spare more pyruvate (from sugar breakdown plus that derived from citrate) for conversion to diacetyl. Additionally, the alternate NADH oxidizing route would obviate the need for diacetyl to be reduced to acetoin and 2,3-butanediol. The third postulate offered by Collins (9) relates to the formation of acetyl-coenzyme A, during which lipoic acid is reduced. The regeneration of oxidized lipoic acid is coupled to the reduction of NAD to NADH 2. The NADH-oxidase could also play a part in recycling NAD required for the oxidation of reduced lipoic acid, thus facilitating greater availability of acetylcoenzyme A. In the diacetyl biosynthesis

2731

SYMPOSIUM: THE DAIRY LEUCONOSTOC

scheme proposed by Speckman and Collins (59) for flavor bacteria, the diketone is fonned as a result of condensation of active acetaldehyde thiamine pyrophosphate complex and acetyl-coenzyme A. Recent evidence, however, shows that diacetyl biosynthesis in flavor bacteria involves oxidative decarboxylation of aacetolactate (30), which was originalIy proposed by Seitz et al. (57). In the conversion of a-acetolactate to diacetyl, involvement of oxygen is postulated. The synthesis and accumulation of diacetyl are thus regulated by the oxidative-reductive power of the biosystem. The validity of the foregoing observations was recently confinned by Bassit et al. (4), who studied diacetyl production in skim milk saturated with different levels of oxygen. The organism used in these experiments was Lact. lactis ssp. lactis biovar diacetylactis. In their study, Bassit et al. (4) also detennined the celIular levels of various enzymes involved in diacetyl synthesis from celIs harvested from broth saturated with corresponding levels of oxygen. They found that, at 30'C and 0% saturation with oxygen, little or no diacetyl was produced over 25 h. At 100% saturation, about .18 mM of the diketone was produced within 5 h; this level remained stable over 25 h of incubation. At 100% saturation with oxygen, acetolactate synthetase and NADHoxidase activities were four and six times greater, respectively, than at 0% saturation of oxygen. Although these investigations were perfonned with citrate-fennenting lactococci, similar mechanisms may operate among leuconostocs because of the presence of NADHoxidase among leuconostocs (36). Internal Generation of Oxygen

Based on the empirical observations relating to the elevation of flavor in aerated milk cultures, Pack et al. (48) explored the effect of in situ oxygen generation on diacetyl in milk cultures of mixed strain starters containing leuconostocs. The process consisted of the following. Reconstituted NDM (10%) was autoclaved at 121'C for 12 min. After milk was cooled to room temperature (21'C), .03% of H202 was mixed into the milk, and the mixture was held at room temperature for 20 min. Catalase at .004% was then added, mixed, and allowed to react for 5 min. This treatment was experimentally found to dissipate the added H202 by

(Diacetyl Stabilization) H2 0 2 and Catalase Treatment 10% NDM Milk 12 min at 121°C

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aI. (48).

chemical analysis for the oxidant. After catalase treatment, the milk was inoculated with 1 to 2% of an active culture and held at 21·C. At various intervals, diacetyl concentrations in the culture were detennined and compared with those of an untreated control (Figure 3). In the control culture, increase in diacetyl concentration commenced after 9 h, and the maximal of 8.0 ppm was attained in 15 h, maintained for 5 h, and then rapidly declined to 1.0 ppm by 24 h. In the treated sample, diacetyl concentration rapidly increased to a maximum of 14.0 ppm in 12 h and remained constant up to 60 h (Figure 4). In another experiment, treatment of milk by H202 and catalase similarly enhanced and stabilized dicarbonyl over 5 d at 21'C and 2'C with the same mixed-strain starter. Journal of Dairy Science Vol. 77, No.9, 1994

2732

VEDAMUTHU

18+---..L---..L-----'-------'------J,

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stimulation of diacetyl synthesis by ascorbate is unknown. Further research is needed to understand the stimulatory phenomenon, especially because ascorbate has a GRAS (generally recognized as safe) status and is allowed in certain fruit juices, fruit preserves, and other beverages.

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Flavor Compounds Produced by Leuconostocs

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Figure 4. Effect of using .015% (l:.) and .03% IP) H202 (control =0) for the peroxide-catalase treatment of milk on diacetyl accumulation in milk cultures of mixed-strain starters with leuconostocs. From Pack et aI. (48).

Although diacetyl is the key flavor compound in cultured dairy products, other minor components contribute to the totality of flavor as perceived organoleptically. Of these, volatile acids, such as acetic, and other components, such as ethanol, are important in the full flavor development of cultured milks (21, 32, 61). Carbon dioxide provides the effervescence and the "lift" to cultured buttermilk as it also does in carbonated beverages (61). These compounds are by-products of carbohydrate and citrate metabolism by these heterofermentative bacteria. Keenan and Bills (32) reviewed the metabolic mechanisms by which these minor components are produced. Role of Leuconostocs In Flavor Balance

Treatment with H202-catalase to destroy undesirable flora of cheese milk in lieu of heat treatment or pasteurization is allowed by Code of Federal Regulations (7). Although such treatment of milk for cultured milks and cream is not currently permitted, this procedure could be possibly tried in the production of highly flavorful starter distillates. Effect of Ascorbic Acid

Richter et al. (54) studied the effect of added I-ascorbic acid on diacetyl production by two mixed strain starters containing Leuc. mesenteroides ssp. cremoris. Ascorbic acid was added at .1 and .25%, respectively, after heat treatment of milk in the form of 20% filter-sterilized solution. Diacetyl determinations at various intervals showed that the diketone concentrations in the ascorbic acid fortified milk cultures of both the starters were at least twice as high as in the corresponding controls from the 10th h of incubation. Lower initial pH of the samples fortified with ascorbic acid probably accounted for earlier initiation of citrate uptake. The exact mechanism of Journal of Dairy Science Vol. 77, No.9. 1994

Lindsay et a1. (38), in their extensive studies on flavor chemistry of butter cultures, encountered a common but serious problem with the development of "green" flavor defect. This defect is often described as "green apple" or "yogurt-like" off-flavor and is attributed to relatively high acetaldehyde concentrations. Harvey (24) reported that certain strains of lactococci produced high levels of acetaldehyde. Presence of such strains in mixed cultures results in green flavor development. Lindsay et a1. (38) examined several mixed strain butter cultures for diacetyl and acetaldehyde contents and evaluated them organoleptically. On the basis of these studies, they proposed that, for a desirable, balanced flavor in butter cultures, the ratio of diacetyl to acetaldehyde should be between 4.4: 1 to 3.2: 1. When the ratio fell below 3.2: 1, a green flavor was evident, and ratios above 5.5:1 were "harsh" and lacked balance. Essentially, then, their finding was that a small amount of the aldehyde is necessary to give a balanced flavor, but excess aldehyde leads to greenness. Generally, 1.6 to 4.0 ppm of diacetyl is needed to give a

2733

SYMPOSIUM: THE DAIRY LEUCONOSTOC

good "nut meat" flavor in cultured dairy products (50). Based on an earlier observation that indicated that leuconostocs transformed acetaldehyde produced by lactococci in mixed cultures, Lindsay et al. (38) measured the quantitative removal of added acetaldehyde by pure cultures of Leuc. mesenteroides ssp. cremoris 91404. They grew the strain in milk containing .2% sodium citrate for 18 h at 30·C. The culture was divided into two aliquots. One aliquot was adjusted to pH 4.5 with sterile phosphoric acid; no pH adjustments were made to the second aliquot (pH 6.5). Acetaldehyde was added to give 4.6 ppm in each aliquot. The cultures were incubated at 30·C for an additional 6 h. An uninoculated citrated milk control was also treated in the same way as the inoculated milk. At the end of 6 h of incubation, acetaldehyde was determined quantitatively. No reduction of aldehyde content was observed in the sterile citrated milk control. Among the inoculated aliquots, acetaldehyde in the acidified portion was reduced from 4.6 to .17 ppm; in the unacidified culture, the residual aldehyde measured .5 ppm. In another experiment, Lindsay et al. (38) showed that leuconostocs could remove acetaldehyde from milk cultures held at S·C. Further, they demonstrated that the Leuconostoc strain could reduce acetaldehyde content in a mixed lactic culture at S·c. Lindsay et al. (38) also showed that acetaldehyde stimulated the growth of leuconostocs in unacidified milk cultures (pH 6.6) in which citrate utilization does not occur and found that, compared with an unfortified control milk culture, samples that were fortified with 5.0 ppm of acetaldehyde had three times as many viable cells. The physiological basis for the observed growth stimulation was discussed. Carbohydrate metabolism by heterofermentative leuconostocs result in the formation of lactic acid, acetic acid, ethanol, and carbon dioxide. In the formation of acetic acid and ethanol, the common intermediate is acetyl phosphate. Conversion of acetyl phosphate to acetate is mediated by acetokinase, and in this step an energy-rich phosphate bond is added to ADP. The other branch of reactions involves the conversion of acetyl phosphate to acetaldehyde, which is catalyzed by acetaldehyde dehydrogenase. In this reductive reaction, the hydrogen is contributed by NADPH2.

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+

EtOH Figure 5. Tenninal reactions of carbohydrate metabolism of leuconostocs as affected by exogenous acetaldehyde: I) acetakinase (preferred). 2) acetaldehyde dehydrogenase. and 3) alcohol dehydrogenase. Reactions 2 and 3 are involved in cofactor regeneration. Exogenous acetaldehyde provides hydrogen acceptor. From Lindsay et al. (38).

Acetaldehyde is further reduced to ethanol by alcohol dehydrogenase with the concomitant regeneration of NAD. Leuconostoc organisms, if possible, would preferentially shunt the terminal oxidative reactions by capturing energy via acetic acid, but, because of the need to regenerate the reduced nicotinamide cofactors for recycling, the ethanol branch is operative. If an exogenous source of acetaldehyde (hydrogen acceptor) is available, the organisms would then be able to achieve the regeneration of NAD without diverting acetyl phosphate away from the acetokinase reaction, which captures energy. The additional energy thus available could be used for cell growth (Figure 5). Keenan and Lindsay (33) found that all of the dairy Leuconostoc species were capable of acetaldehyde removal from milk cultures, but the activity was maximum with Leuc. mesenteroides ssp. cremoris. Keenan and Lindsay (30) noted that leuconostocs possess relatively high alcohol dehydrogenase activity and convert acetaldehyde to ethanol, although not quantitatively. Realizing the significance of acetaldehydescavenging abilities of leuconostocs even at S·C (38), Vedamuthu (62) suggested that these bacteria could be used to remove the aldehyde from specially fermented yogurt, which could be offered as a healthful, lowfat alternative for Journal of Dairy Science Vol. 77. No.9. 1994

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VEDAMUTHU

sour cream. Prasad and Srinivas (52) described the use of leuconostocs with conventional yogurt bacteria to modify the flavor profile of yogurt to make it acceptable to South Asian customers. Sh.lf-Ilf. Extension Using Leuconostocs

Extension of shelf-life of creamed cottage cheese prepared with a creaming mixture cultured with Leuc. mesenteroides ssp. cremoris was reported by Mather and Babel (43). They found that, compared with a control lot dressed with uncultured cream. the lot containing cultured cream dressing had a longer shelf-life because of the prevention or delay of slime formation and proteolysis by Pseudomonas tragi and Pseudomonas putre/aciens. Coliforms were also inhibited by the cultured cream dressing. Babel (2) has reviewed the reports pertaining to the inhibitory effect of Leuc. mesenteroides ssp. cremoris on psychrotrophs and foodbome pathogens. The inhibitory effect exercised by leuconostocs on psychrotrophs was mainly attributed to acetic acid. Very high concentrations of diacetyl also have a suppressive effect on Gram-negative psychrotrophic flora (31). Generation of C02 by flavor bacteria within tightly sealed cottage cheese containers (especially if citrate is added) also controls psychrotrophic growth (37). A recent study has clearly demonstrated the efficacy of C02 in controlling psychrotrophs (46). Overall, shelf-life extension of products cultured with leuconostocs is probably brought about by the combined effect of acetate, diacetyl, and C02 on aerobic, psychrotrophic flora-like pseudomonads and Alcaligenes spp. Recently, production of bacteriocins by leuconostocs active against a few Gram-positive genera has been reported (44). The role of these proteins in shelf-life extension may be minimal. Us. of L.uconostoc. In Ch.... for Inducing G.s Openn...

In certain Dutch cheese varieties, such as Edam and Gouda, small, shiny gas-induced openings, or "eyes", are desired (47). These openings are caused by occluded C02 generated within the cheese by leuconostocs included in the starters (47). Specially selected Leuconostoc strains that produce good flavor Journal of Dairy Science Vol. 77. No.9. 1994

and moderate amounts of C02 are usually included in the starters for these Dutch varieties. In Blue cheese, for proper mold veining, a good network of mechanical openings between curd cubes is necessary. This requires that the curd cubes are rigid enough to withstand the weight of the settling curd and that they do not collapse when the cheese loaf is turned several times for whey draining. The curd structure and intracurd openings are ensured by proper manufacturing procedures. To further ensure good intracurd openings, gas-producing leuconostocs are incorporated in the starters. An added benefit in using leuconostocs is in the prevention of contaminant molds from growing in the cheese. The C02 generated by flavor bacteria not only promotes intracurd openings but also helps to exclude most contaminant molds. which are sensitive to increasing C02. However, Penicillium roque/orti is relatively insensitive to prevailing levels of CO 2 within the cheese (22). In the manufacture of Danish Blue cheeses, starters of either B or BD types are used (47). Starters designated B (for betacocci) consist of lactococci and leuconostocs and BD (betacocci and diacetylactis) cultures contain leuconostocs and acid-producing and citrate-fermenting lactococci. The first reported isolation of phages for dairy leuconostocs were made from Danish Blue cheese (58). Dextran Production by L.uconostoc.

The production of dextran by Leuc. mesenteroides ssp. dextranicum is well known. However, application of the glucose polymer derived from leuconostocs as a thickener or texturizer in dairy products is not wellestablished. Pucci and Kunka (53) recently described a unique dextran produced by Leuc. mesenteroides ssp. dextranicum NRRL-B18242 in a milk substrate containing sucrose. The milk culture containing dextran was dried to a powder. This powder was suitable for thickening and texturizing cultured milks, flavored milks, and fruit juices and could be used as a stabilizer in ice cream and salad dressings. The researchers also found that the dextran from this strain had antimicrobial properties. With the current interest in biopolymers, further work is needed to screen for leu-

SYMPOSIUM: THE DAIRY LEUCONOSTOC

conostocs that are hyperproducers of different types of dextrans with variable viscosities and rheological properties and to research their application in food products. Other Applications

One of the major hurdles in developing lowfat and fat-free dairy products is in obtaining sufficient flavor in the absence of full fat content. Fat has a modulating effect on flavor perception, and the intensity of flavor required in lowfat systems needs to be greater to obtain the lingering flavor sensation (64). In developing lowfat or fat-free products corresponding to sour cream, in which excessive gas production is undesirable, leuconostocs should be used in preference to citrate-fermenting lactococci. In general, for diacetyl flavor intensification, without objectionable green defect or excessive gas, leuconostocs would be the organisms of choice. Culturing cream for buttermaking is no longer widely practiced because of the greater proneness of salted, cultured butter to undergo fat oxidation and other chemical deterioration than sweet cream butter (18). Sweet cream butter is often criticized as flat and lacking in flavor. To remedy the lack of flavor, procedures have been developed to add "cultured flavor" directly to butter. In these operations, after the churning and working of butter, at the time of moisture adjustment, measured amounts of sweet cream buttermilk cultured with Leuconostoc strains is worked into the butter to adjust the moisture content to the legal limit. A suitable procedure for these operations was described by Seas et aI. (55). The authors found that butter made by their procedure had good flavor and keeping quality. Practical Considerations

In using Leuconostoc strains in pure or mixed cultures, certain practical considerations have to be taken into account to derive the maximum benefit from their usage. Trace levels of manganese are needed for proper growth and flavor production by leuconostocs. Deficiency of this mineral in the milk in certain geographical areas (Europe especially) has caused inadequate flavor development (8).

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Henning et aI. (26) found that leuconostocs are inhibited in phage-inhibitory media. When starters containing leuconostoc are propagated, media similar in composition (high phosphate content) to phage-inhibitory media should be avoided. One of the effective class of sanitizers that is used in food plants is quaternary ammonium compounds (QAC). These compounds destroy flavor bacteria (39). Although QAC have not been used in plants manufacturing cultured dairy products for some time, with the recent reports of Listeria monocytogenes in dairy plants, QAC, because of its effectiveness against this pathogen, has been reintroduced for sanitizing conveyers, crates, floors, and drains. Aerosols carried by plant air handling systems may result in distribution of sufficient concentrations of the chemicals to cause problems in cultured products manufacture. Sanitizers other than QAC, which are effective against listeria, but relatively less stable than QAC should be used in plants manufacturing and handling cultured dairy products.

REFERENCES I American Public Health Association. 1992. Page 229 in Compendium of Methods for the Microbiological Examination of Foods. C. D. Vanderzant and D. F. Splittstoesser. ed. Am. Publ. Health Assoc., Washington, DC. 2 Babel, F. J. 1977. Antibiosis by lactic culture bacteria. J. Dairy Sci. 60:815. 3 Babel, F. J., and D. w. Mather, inventors. 1961. Creamed cottage cheese. Assignee, Research Corp., New York, NY. US Pat. 2,971,847. 4 Bassit, N., C. Boquieu, D. Picque, and G. Corrieu. 1993. Effect of initial oxygen concentration on diacetyl and acetoin production by Lactococcus lactis subsp. lactis biovar diacetilactis. Appl. Environ. Microbiol. 59:1893. 5 Benkerroum, N., M. Misbah, W. E. Sandine, and A. T. Elaraki. 1993. Development and use of a selective medium for isolation of Leuconostoc spp. from vegetables and dairy products. Appl. Environ. Microbiol. 59:607. 6 Brohn, 1. C., and E. B. Collins. 1970. Reduced nicotinamide adenine dinucleotide oxidase of Streptococcus diacetilactis J. Dairy Sci. 53:857. 7 Code of Federal Regulations. 1990. Title 21, Part 131. Milk and cream. Pages 152-154: 161-168. Office Fed. Reg., Nat!. Archiv. Records Admin., Washington, DC. 8 Cogan, T. M. 1976. The utilization of citrate by lactic acid bacteria in milk and cheese. Dairy Ind. Inti. 41(1): 1. 9 Cogan, T. M. 1985. The Leuconostocs: Milk Products. Bacterial Starter Cultures for Foods. S. E. Gilliland, ed. CRC Press, Inc. Boca Raton, FL. Journal of Dairy Science Vol. 77, No.9, 1994

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10 Collins, E. B. 1972. Biosynthesis of flavor compounds by microorganisms. J. Dairy Sci. 55:1022. 11 Condon, S. 1987. Responses of lactic acid bacteria to oxygen. Fed. Eur. Microbiol. Soc. Microbiol. Rev. 46: 269. 12 Cooper, R. K. and E. B. Collins. 1978. Influences of temperature on growth of Leuconostoc cremoris. 1. Dairy Sci. 61: 1085. 13 DeMan, 1. C. and J. W. Pene. 1956. Mechanism of diacety1 formation in buner and starters. 14th Int. Dairy Congr. VII 1:80. 14 EUiker, P. R. 1945. Effect of various bacteria on diacetyl content and flavor of butter. 1. Dairy Sci. 28: 93. 15 Emmons, D. B., and S. L. Tuckey. 1967. Page 77 in Conage Cheese and Other Cultured Milk Products. Pfizer Cheese Monogr. Vol. 3. Chas. Pfizer & Co, New York, NY. 16 Fabricius. N. E., and B. W. Hammer. 1937. The influence of the type of butter culture and its method of use on the flavor and keeping quality of salted butter. Pages 367 and 375 in Iowa State Coli. Agric. Exp. Stn. Res. Bull. 221, Ames. 17 Farr, S., inventor. 1969. Milk fennenting product and method of making same. Assignee, Dairy Technics Inc" Kalamazoo, MI. US Pat. 3,420,742. 18 Foster, E. M., F. E. Nelson. M. L. S~ck, R. N. Doetsch, and J. C. Olson. Jr. 1957. Pages 116 and 287 in Dairy Microbiology. Prentice-Hall Inc., Englewood Cliffs, N1. 19 Gilliland, S. E. 1972. Ravor intensification with concentrated cultures. J. Dairy Sci. 55: 1028. 20 Gilliland, S. E., E. D. Anna, and M. L. Speck. 1970. Concentrated cultures of Leuconostoc citravorum. Appl. Microbiol. 19:890. 21 Gilliland, S. E., W. Y. Cobb, M. L. Speck, and E. D. Anna. 1971. Comparison of volatile components produced by concentrated and conventional cultures of Leuconostoc citravorum. Cult. Dairy Prod. 1. 6(2):12. 22 Golding, N. W. 1937. The gas requirements of mold. I. A. preliminary repon on the gas requirements of Penicillium roqueforti (various strains of blue mold from cheese). J, Dairy Sci. 20:319. 23 Hammer, B. W., G. L. Stahly, C. H. Werkman, and M. B. Michaelian. 1935. Reduction of acetylmethyl carbinol and diacetyl to 2,3-butylene glycol by the citric acid fennenting streptococci of butter cultures. Iowa Agric. Exp. Stn. Res. Bull. 191. 24 Harvey, R. J. 1960. Production of acetone and acetaldehyde by lactic streptococci. 1. Dairy Res. 27: 41. 25 Harvey, R. 1., and E. B. Collins. 1962. Citrate transpon system of Streptococcus diaceti/actis. J. Bacteriol. 83:1005. 26 Henning, D. R., W. E. Sandine. P. R. Elliker, and H. A. Hays. 1965. Studies with a bacteriophage inhibitory medium. I. Inhibition of phage and growth of single strain lactic streptococci and leuconostoc. J. Milk Food Technol. 28:273. 27 Hoecker, W. H., and B. W. Hammer. 1941. Ravor development in salted butter by pure cultures of bacteria. Iowa Agric. Exp. Stn. Res. Bull. 290. 28 Hucker, G. J., and C. S. Pederson. 1930. Studies on the Coccaeae. XVI. The genus Leuconostoc. New York Agric. Exp. Stn. Tech. Bull. 167. Journal of Dairy Science Vol. 77, No.9, 1994

29 Hugenholtz, J. 1993. Citrate metabolism in lactic acid bacteria. Fed. Eur. Microbiol. Soc. Microbiol. Rev. 12:165. 30 Hugenholtz, 1., and MJ.C. Starrenburg. 1992. Diacetyl production by different strains of Lactococcus lactis ssp. /actis var. diacetilacris and Leuconostoc spp. Appl. Microbiol. Biotechnol. 38:17. 31 Jay, J. M. 1982. Antimicrobial propenies of diacetyl. Appl. Environ. Microbiol. 44:525. 32 Keenan, T. W., and D. D. Bills. 1968. Metabolism of volatile compounds by lactic starter culture microorganisms. A review. J. Dairy Sci. 51:1561. 33 Keenan, T. W., and R. C. Lindsay. 1966. Removal of green flavor from ripened buner cultures. 1. Dairy Sci. 49:1563. 34 Kempler, G. M., and L. L. McKay. 1981. Biochemistry and genetics of citrate utilization in Streptococcus lactis ssp. diacetylactis. J. Dairy Sci. 64: 1527. 35 King, N. 1948. Modifications of the Voges-Proskauer test for rapid colorimetric determination of acetylmethyl carbinol plus diacetyl in butter cultures. Dairy Ind. 13:800. 36 Koike, K., T. Kobayashi, S. Ito, and M. Saitoh. 1985. Purification and characterization of NADH oxidase from a strain of Leuconostoc mesenteroides. 1. Biochern. 97:1297. 37 Kosikowski, F. V.. and D. P. Brown. 1973. Influence of carbon dioxide and nitrogen on microbial populations and shelf-life of cottage cheese and sour cream. J. Dairy Sci. 56: 12. 38 Lindsay, R. c., E. A. Day, and W. E. Sandine. 1965. Green flavor defect in lactic starter cultures. 1. Dairy Sci. 46:863. 39 Lundstedt, E. 1950. Don't let quats ruin that flavor. Food Ind. 22:2056. 40 Lundstedt, E. 1962. Citrated whey starters. I. Growth patterns of starters and their aroma bacteria when cultivated in rennet whey or conage cheese whey, citrated with the addition of five percent trisodium citrate, pentahydrate. J. Dairy Sci. 45:1320. 41 Lundstedt, E.• and E. A. Corbin. 1983. Controlled fennentation of buttermilk. Cult. Dairy Prod. J. 18:6. 42 Lundstedt. E., and W. B. Fogg. 1962. Citrated whey starters. II. Gradual formation of flavor and aroma in creamed conage cheese after the addition of small quantities of citrated cottage cheese whey cultures of Streptococcus diaceti/actis. J. Dairy Sci. 45:1327. 43 Mather. D. W., and F. 1. Babel. 1959. A method for standardizing the biacetyl content of creamed cottage cheese. J. Dairy Sci. 42:1045. 44 Mathieu, F., I. S. Suwandhi, N. Rekhif, J. B. Milliere, and G. Lefebvre. 1993. Mesenterocin 52. a bacteriocin produced by Leuconostoc mesenteroides ssp. mesenteroides FR 52. 1. Appl. Bacteriol. 74:372. 45 Michealian, M. B., R. S. Farmer, and B. W. Hammer. 1933. The relationship of acetylmethyl carbinol and diacetyl to butter cultures. Iowa Agric. Exp. Stn. Res. Bull. 155. 46 Moir. C. J., M. J. Eyles. and 1. A. Davey. 1993. Inhibition of pseudomonads in cottage cheese by packaging in atmospheres containing carbon dioxide. Food Microbial. 10:345. 47 Nath, K. R. 1993. Cheese. Dairy Science and Technology Handbook. Vol. 2. Product Manufacturing. Y. H. Hui. ed. VCH Pub\., Inc .• New York, NY. 48 Pack, M. Y., E. R. Vedamuthu, W. E. Sandine and P.

SYMPOSIUM: THE DAIRY LEUCONOSTOC R. Elliker. 1968. Hydrogen peroxide-catalase milk treatment for enhancement and stabilization of diacetyl in lactic starters. 1. Dairy Sci. 51:511. 49 Pack, M. Y., E. R. Vedamuthu, W. E. Sandine, P. R. Elliker, and H. Leesment. 1968. The effect of temperature on growth and diacetyl production by aroma bacteria in single- and mixed-strain lactic culture. 1. Dairy Sci. 51:339. 50 Parker, R. B., and P. R. Elliker. 1953. Effect of spoilage bacteria on biacetyl content and flavor of cottage cheese. 1. Dairy Sci. 36:843. 51 Pette, J. W. 1949. Some aspects of butter aroma problem. Proc. 12th. Int. Dairy Congr., Stockholm 2: 572. 52 Prasad, D. N., and K. Srinivas. 1987. Perfonnance of Leuconostoc species in the manufacture of yoghurt. Cult. Dairy Prod. J. 22(3):10. 53 Pucci, M. J. and B. S. Kunka, inventors. 1990. Novel dextran produced by Leuconostoc dextranicum. Assignee, Microlife Technics, Sarasota, FL. US Pat. 4,933,191. 54 Richter, R. L., W. S. Brank, C. W. Dill and C. A. Watts. 1979. Ascorbic acid stimulation of diacetyl production in mixed-strain lactic acid cultures. J. Food Prot. 42:294. 55 Seas, S. W., W. F. Stoll, D. F. Breazeale, and R. J. Baker. 1960. Manufacture and sale of cultured butter.

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South Dakota State Call. Agric. Exp. Stn. Bull. 492. 56 Seitz, E. W., W. E. Sandine, P. R. Elliker, and E. A. Day. 1963. Distribution of diacetyl reductase among bacteria. 1. Dairy Sci. 46: 186. 57 Seitz. E. W., W. E. Sandine, P. R. Elliker, and E. A. Day. 1963. Studies on diacetyl biosynthesis by Streptococcus diautilactis. Can. J. Microbial. 9:431. 58 Shin, C., and Y. Sato. 1979. Isolation of Leuconostoc bacteriophages from dairy products. Jpn. 1. Zootechnol. Sci. 50(6):419. 59 Speckman, R. A., and E. B. Collins. 1968. Diacetyl biosynthesis in Streptococcus diacetylactis and Leuconostoc citravorum. J. Bacteriol. 95: 174. 60 Vedamuthu, E. R. 1985. What is wrong with cultured buttermilk today? Dairy Food Sanit. 5(1):8. 61 Vedamuthu, E. R. 1988. Engineering flavor into fermented foods. Page 664 in Handbook of Anaerobic Fermentations. L. E. Erickson and D.Y.C. Fung, ed. Marcel Dekker Inc., New York, NY. 62 Vedamuthu, E. R. 1992. The yogurt story-past, present and future. Part X. Dairy Food Sanit. 12:351. 63 Walker, D. K., and S. E. Gilliland. 1987. Buttermilk manufacture using a combination of direct acidification and citrate fermentation by Leuconostoc cremoris. J. Dairy Sci. 70:2055. 64 Yackel, W. C., and C. Cox. 1992. Application of starch-based fat replacers. Food Technol. 46(6):146.

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