MARSCHALL RHODIA INTERNATIONAL DAIRY SCIENCE AWARD LECTURE
Adjunct Cultures: Recent Developments and Potential
Significance to the Cheese Industry1
ABSTRACT
Several previous reviews have described different
ways to enhance the flavor and texture of cheese, includ-
ing use of live cells and nonviable attenuated cells as
adjunct cultures. However, comparisons between viable
and nonviable cultures were never discussed in these
reviews. In addition, recent publications on adjunct cul-
tures have not been covered in previous reviews. This
article will survey the more recent work on adjunct
cultures—with particular attention to whether the ad-
juncts contained viable or nonviable cells—and propose
areas where additional research is needed.
(Key words: adjunct lactobacilli, attenuation, autoly-
sis, cheese ripening)
Abbreviation key: LAB = lactic acid bacteria, NSLAB
= nonstarter lactic acid bacteria.
INTRODUCTION
At the end of many cheese-making processes, a bland
rubbery mass of curd is obtained. It is during ripening
that many cheese types develop their characteristic
flavors through the gradual breakdown of carbohy-
drates, fats, and proteins. Several agents are involved
in the ripening of cheese, and the impact of the contribu-
tion of each of these agents varies according to the type
of cheese. The coagulants are principally responsible
for the first steps in protein degradation, but play a
minor role in small peptide formation (98). Indigenous
milk lipases and proteinases play a limited role during
ripening; however, plasmin hydrolyzes β-casein (48,
76). This action is more intense in highly cooked cheese
varieties (e.g., Swiss), which is probably caused by in-
creased conversion of plasminogen to plasmin under
Received July 6, 1999.
Accepted November 16, 1999.
Corresponding author: P. S. Tong. e-mail: ptong@calpoly.edu.
1
Part of this review was presented by Morsi El Soda, recipient of
the 1998 Marschall Rhodia International Dairy Science Award at the
93rd ADSA meeting, Denver, Colorado.
2000 J Dairy Sci 83:609–619
M. El Soda,† S. A. Madkor,* and P. S. Tong*
†Department of Dairy Science and Technology,
Faculty of Agriculture, Alexandria University
Alexandria, Egypt
*Dairy Products Technology Center,
California Polytechnic State University,
San Luis Obispo, California 93407
these conditions. This conversion is promoted by the
absence of the plasmin inhibitors, which are largely
removed with cheese whey or are already denatured
during cheese milk pasteurization (36, 97, 98).
It is now well established (28) that in most ripened
cheese varieties, enzymes from cheese-related microor-
ganisms and more particularly lactic acid bacteria
(LAB) are the major contributors to flavor development
through the action of their intracellular enzyme sys-
tems. This was clearly demonstrated in studies of asep-
tic cheese making where microorganism-free cheese de-
veloped little flavor (97). These organisms can originate
from the milk, or they can be intentionally or uninten-
tionally added during the cheese-making process.
MICROORGANISMS INVOLVED
IN CHEESE RIPENING
The microorganisms involved in cheese making and
cheese ripening can be divided into two major groups:
1) microorganisms that are added to the cheese milk
after being carefully selected by the starter manufac-
turer or the cheese-making company, and 2) nonstarter
lactic acid bacteria (NSLAB).
Group 1 can be further subdivided into two sub-
groups: the primary starter and the secondary starter.
The roles of these two groups have been well docu-
mented in several reviews (42, 54). The role of the pri-
mary starter culture is to ensure consistent acid devel-
opment during cheese making. This group is also in-
volved in the degradation of protein and fat during
ripening. Cultures in this group also play an important
role in the biological protection of the product (e.g.,
low pH, bacteriocin production). The secondary starter
represents cultures that are added to a limited number
of cheese varieties to provide well-defined functions,
e.g., gas production in Swiss type cheese is ensured by
Propionibacterium shermanii ssp. freudenreichii, while
Brevibacterium linens is one of the major contributors
to surface coloration in surface ripened cheese. Penicil-
lium roqueforti and Penicillium camemberti can also
be included in this group. In addition, the secondary
609
610 EL SODA ET AL.
starters, through their diverse enzymatic systems, can
be also heavily involved in the ripening process; their
contributions are indispensable for the development of
the typical flavor of many cheeses, e.g., Roquefort, Em-
mental, Camembert, and Limburger (54, 87).
Group 2 includes the nonstarter lactic bacteria. This
group has been shown to contribute to flavor develop-
ment in some varieties of cheeses and could therefore
be considered a desirable contaminant of either the
milk supply or the subsequent cheese. Lactobacillus
strains are the most common and can be found in rela-
tively high numbers; L. casei, L. paracasei, L. plan-
tarum, and L. curvatus are the predominant species.
Pediococci and enterococci are also members of the
group but are usually present in smaller numbers (42).
Micrococci, which are not LAB, can also play a signifi-
cant role in flavor formation in certain types of
cheese (8).
OCCURRENCE OF NSLAB IN CHEESE
Typically, factory-made Cheddar cheese contains 102
to 103 NSLAB/g after pressing. They reach 107 to 108
cfu/g within about 3 mo (42). However, if high quality
raw milk is pasteurized and precautions are taken to
avoid contamination from the environment, Cheddar
cheese may remain free of detectable lactobacilli for up
to 100 d (43).
Different factors may affect the growth of NSLAB in
cheese. Lane et al. (65) demonstrated that within the
ranges normally found in Cheddar cheese the water
and salt content and the pH have little effect on the
growth and final number of NSLAB. Ripening tempera-
ture was reported to influence the rate of NSLAB devel-
opment but does not affect their final numbers in the
cheese (40). It seems that lactose, AA, and sugars from
nucleic acids are not used by NSLAB as energy sources;
however, some species can use carbohydrates associ-
ated with the milk fat globule membrane for that pur-
pose (42).
THE ROLE OF NSLAB DURING CHEESE RIPENING
In the late 1960’s, Reiter et al. (88) added groups of
NSLAB isolated from raw milk and cheese to experi-
mental cheese or cheese milk made under controlled
microbiological conditions. Although NSLAB acceler-
ated Cheddar flavor development, some NSLAB groups
developed off flavors.
More recently, Lane and Fox (64) manufactured
cheese by chemical acidification (glucono δ lactone and
lactic acid) as well as biological acidification (Lactococ-
cus lactis ssp. cremoris). A mixed culture of six lactoba-
cilli was added to biologically and chemically acidified
Journal of Dairy Science Vol. 83, No. 4, 2000
cheese milk. Cheeses without lactobacilli addition were
also made. A significant increase in proteolysis could
be measured when the lactobacilli were added to the
starter free cheese. This increase was not as obvious in
the cheese made with starter because of the lack of
available substrate for the enzymes produced by both
types of bacterial cultures. The role of lactobacilli was
also studied by Lynch et al. (73, 74) who demonstrated
that cheese with added lactobacilli developed a superior
flavor when compared to control cheese. The presence
of the lactobacilli led to increase levels of small peptides
and AA in cheese.
RATIONALE FOR USING ADJUNCT CULTURES
In recent years, procedures have been developed at
the farm and plant levels to produce milk and obtain
cheese with the lowest number of microorganisms possi-
ble. The introduction of a low-temperature pasteuriza-
tion process coupled to microfiltration led to the produc-
tion of almost sterile milk (66, 83). Although these
methods provide the consumers with dairy products
exhibiting a high degree of microbiological quality and
safety, they also lead to the disappearance of or at least
to a dramatic reduction in the number of desirable non-
starter bacteria.
For the cheese industry to offer to the consumers safe
and consistent cheeses with high organoleptic proper-
ties in a reasonable ripening time, they began to look
for new technologies such as “adjunct cultures.” Adjunct
cultures can be defined as selected strains of cheese
related microorganisms that are added to the cheese
milk to improve development of cheese sensory quality.
They were also developed to accelerate cheese ripening,
which may allow substantial cost savings to the cheese
industry. In contrast to naturally occurring NSLAB,
adjuncts are specifically selected and intentionally
added to supplement the microflora of cheese milk to
improve overall quality of finished cheese.
THE USE OF ADJUNCT CULTURES
Research in the area of adjunct cultures has followed
two main approaches: 1) nonviable (attenuated) ad-
juncts, and 2) viable (nonattenuated) adjuncts. Both
approaches appear to improve the quality of Cheddar
cheese and will be discussed in detail.
The Use of Viable Adjuncts
Viable adjunct cultures have been used in commercial
cheese making for several years. However, the need
to enhance flavor development in low-fat cheese has
spurred additional interest in their utilization. Some
 MARSCHALL RHODIA INTERNATIONAL DAIRY SCIENCE AWARD LECTURE
work was published in the 1980’s and early 1990’s on
the impact of such cultures on cheese ripening (28, 41,
67). It is only in the last 5 yr that research groups joined
efforts to select and characterize cultures. Workers in
this area have noted the importance of the activities of
adjunct cultures during ripening for the development
of the characteristic flavor of the cheese.
Puchades et al. (86) compared the impact of adding
different lactobacilli such as L. casei, L. paracasei, L.
plantarum, and L. brevis during Cheddar cheese mak-
ing at a concentration of 105 cfu/ml. A desirable flavor
was obtained when homofermentative cultures were
used. The addition of L. brevis led to off flavors and gas
production. Further, Lee et al. (70) demonstrated that
although the use of some homofermentative mesophilic
lactobacilli led to more intense desirable cheese flavor,
others caused high acidity, bitterness, off flavors and
open and crumbly textures, which clearly indicates the
importance of culture selection.
Drake et al. (18) have reported on the addition of 102
or 103 cfu/ml of L. helveticus WSU 19 during Cheddar
cheese making. Cheeses containing the higher concen-
tration of adjunct obtained higher organoleptic scores
by dairy judges. Cheese with added lactobacilli exhib-
ited higher rates of proteolysis when compared to the
control cheese. In fact, a 11-kDa band could be detected
on the sodium dodecyl polyacrylamide gel electrophore-
sis gels of the cheese made with L. helveticus WSU19
after 3 and 6 mo of ripening, which was absent in the
control cheese.
The same culture (L. helveticus WSU19) was added
to 33% reduced-fat Cheddar cheese at concentrations
of 102 or 103 cfu/ml (19). The resultant cheese was sig-
nificantly less bitter and exhibited a more intense oaky/
nutty flavor. Higher levels of protein degradation were
also noticed in the L. helveticus treated cheese.
Studies involving the joint efforts of the Western Cen-
ter for Dairy Research and the Wisconsin Center for
Dairy Research compared different strains of L. casei, L.
helveticus, and Brevibacterium linens in low-fat cheese
(12). The authors demonstrated that low levels of lacto-
bacilli had a positive impact on cheese quality, while
high levels of the same culture led to flavor defects. This
work showed the importance of Br. linens to produce
methanethiol (a compound believed to be an important
contributor to the development of characteristic Ched-
dar cheese flavors). However, the role of this organism
in AA production and whether it has debitterase action
is unclear. The same group (99) also reported that a
consumer panel found that 60% reduced-fat Cheddar
cheese made with brevibacteria was preferred to cheese
made with L. casei and L. helveticus. The addition of
adjunct cultures for the enhancement of low- and re-
duced-fat cheese was also considered at the Southeast
611
Center for Dairy Research USA (20). The authors indi-
cated that the use of a combination of two adjuncts from
cultures of L. casei, L. helveticus, Lac. lactis ssp. lactis
biovar. diacetylactis and Br. linens produced best fla-
vored Cheddar cheese according to trained and con-
sumer panelists. The authors also reported that L.
helveticus alone as an adjunct significantly improved
the sensory quality of 33% reduced-fat Cheddar cheese.
An integrated culture system composed of Lac. lactis
ssp. lactis biovar. diacetylactis JVI and Lac. lactis ssp.
cremoris SKII and L. casei led to the production of a
non-bitter reduced-fat cheese with a clean acid buttery
flavor (51).
The work carried out by the previously mentioned
centers, which focused on live cells, indicated the impor-
tance of the metabolic activities of live cells during
cheese ripening especially for the production of meth-
anethiol by Br. linens. However, the role of the meta-
bolic activities of L. helveticus was not clearly explained
regarding the undesirably high concentrations of this
adjunct during ripening. The role of the intracellular
metabolic activities of the LAB, which is now well estab-
lished (28, 44, 58, 71), appears to be indispensable to
changes in cheese quality during the ripening process.
The Use of Nonviable (Attenuated) Adjuncts
This approach can be described as a means of provid-
ing packages of intracellular microbial enzymes, which
can be added to the cheese milk and subsequently re-
tained in the coagulum without interfering with the
cheese-making process. These enzymes are expected to
be well distributed and quickly released in the cheese
through the autolytic process. Because the microorgan-
isms in the adjunct cultures were originally isolated
from ripened Cheddar cheese, they are expected to con-
tribute to the formation of the typical Cheddar cheese
sensory qualities.
Early approaches to increase the number of desirable
bacteria in cheese involved increasing the amounts of
starter in the cheese milk by using a higher starter
level (92) or by adding growth stimulants (52, 53, 80).
This approach led to a cheese with lower pH, higher
moisture content, and undesirable flavors. Attempts
were therefore made to add cultures that have been
attenuated so that they could play a role during ripen-
ing without producing excess lactic acid.
Five methods for cell attenuation have been described
(22, 27, 44), but most research has focused on physical
treatments: heat shocking, freeze shocking, and spray
drying. The methods described in the literature can be
summarized as follows: 1) Methods involving a physical
treatment including heat shocking, freeze shocking,
spray drying, and freeze-drying. 2) Methods involving
Journal of Dairy Science Vol. 83, No. 4, 2000
612 EL SODA ET AL.
the use of enzymes; lysozyme was the only enzyme pro-
posed for this purpose. Treatment with lysozyme does
not seem to be effective for cell attenuation because of
the difference in LAB sensitivity to lysozyme. In addi-
tion, the relatively high price of lysozyme represents
another obstacle (69). 3) Methods involving chemical
treatments (i.e., use of organic solvents). The addition
of organic solvents such as butanol (93) represents a
major drawback to the methods involving a chemical
treatment due to the concerns over residual solvent in
the cheese. 4) Methods involving genetic modifications
of the cells. In these methods, lactose negative (Lac−),
proteinase negative (Prt−), or Lac− and Prt− cells were
used (1, 37, 44, 47, 57).
We will expand on what we believe can be the most
applicable methods. The physical methods are those
most commonly used for culture cell attenuation. These
techniques also result in varying levels of cell viability,
modification of ability to produce acid, and cellular pro-
teinase and esterase activities. The autolytic properties
of the cells are also altered as a result of the physical
treatments. In previous reviews the impact of the vari-
ous physical treatments on the different intracellular
enzymatic activities were documented (27, 28, 41, 44)
and are summarized in Table 1. The focus here will
be on the impact of these treatments on the autolytic
properties of the cells. This process is of major impor-
tance during cheese ripening. Most of the bacterial en-
zymes important for cheese ripening are intracellular;
therefore, these enzymes cannot be released until autol-
ysis occurs. This release can then allow intimate contact
with desired substrates and improve flavor and texture
development during ripening (14, 15, 33, 72).
Studies on the influence of heat shocking on the rate
of cell autolysis are contradictory. Bie and Sjostrom (9)
reported an increase of autolysis when L. helveticus
cells were subjected to a heat treatment of 54, 59, or
64°C for 20 s. On the other hand, El Soda et al. (30)
showed that cells of various cheese related microorgan-
isms from the following genera Lactobacillus, Leuconos-
toc, Propionibacterium, Lactococcus, and Bifidobacter-
ium exhibited a considerable delay in cell autolysis
when subjected to a heat shocking treatment of 65°C
for 16 s. Other authors later confirmed these findings.
Thiboutot et al. (94) showed that very little autolysis
could be measured over a period of 120 h in cells of L.
casei ssp. casei if they were subjected to a heat treat-
ment of 65°C for 16 s. Most recently, El Soda et al. (32)
determined the rate of autolysis of one L. helveticus
strain and two of L. casei strains after the following
treatments: 1) heat shocking at 60, 60, and 70°C for 15
s; 2) freeze shocking at −20°C for 48 h followed by thaw-
ing at 38°C; and 3) spray drying with an outlet tempera-
ture of 82°C.
Journal of Dairy Science Vol. 83, No. 4, 2000
The results indicate that freeze-shocked cells showed
the highest rates of autolysis, followed by untreated
cells (Figure 1). The heat shock treatments significantly
affected autolysis. The rate of autolysis decreased when
the heat shock temperature was increased (Figure 2).
The decrease in autolysis after the heat shock treat-
ments may be partially attributed to a thermal denatur-
ation of the autolytic system of the cultures studied.
Spray drying using an outlet temperature of 82°C re-
sulted in autolysis rates that were lower than other
treatments. Contrary to heat shocking, the freeze
shocking process had a positive effect on autolysis. Bie
and Sjostrom (9) demonstrated a 100% increase in the
amount of DNA released from cells subjected to three
cycles of freezing at −20°C. When cells of L. acidophilus
were stored at −20°C overnight and then thawed, the
rate of autolysis was 45% higher than cells stored at
3°C for the same time period (82). In a study involving
several species of lactobacilli, El Kholy et al. (24) indi-
cated that, as a general rule, cells subjected to freezing
and thawing cycles resulted in autolysis at a faster rate
when compared to untreated cells. The differences in
the rate of autolysis between untreated cells and cells
subjected to freezing and thawing varied according to
the strain tested. Information concerning the impact of
spray drying on cell autolysis is rather limited. Johnson
and Etzel (55) and To and Etzel (95) studied the effect
of freeze drying, freezing, and spray drying on autolysis
and concluded that the slowest rate of autolysis was in
spray-dried cells.
Comparable studies on the impact of physical attenu-
ation of bacterial cells on cheese ripening is limited and
to some extent contradictory. Although Ezzat and El-
Shafei (35) suggested that heat shocked cells are better
candidates for the enhancement of Ras cheese flavor;
Aly (3) found no significant differences between the two
treatments on low-fat cheese quality. El-Abboudi et al.
(22) suggested that the development of typical Cheddar
cheese flavor was accelerated by addition of homoge-
nized, thermally treated cells of L. casei ssp. casei. This
treatment did not increase gross proteolysis in cheese
as measured by soluble nitrogen but accelerated the
hydrolysis of peptides, thus increasing the amount of
amino nitrogen and reducing bitterness. Johnson et al.
(56) compared the performance of freeze-shocked,
freeze-dried and spray-dried adjunct cells of L. helveti-
cus to enhance flavor and body development in reduced
fat Cheddar cheese. Cheese flavor intensity was en-
hanced in all adjunct cheeses, but cheeses made with
an adjunct spray dried at high outlet air temperature
had the lowest off-flavor intensity. Other sensory mea-
sures were not significantly different among the cheeses
in spite of differences in the cellular properties of the
 Table 1. Summary of conditions used for attenuation of dairy-related microorganisms.

Methods of physical Reduction in

Strain attenuation enzymatic activity1 Impact on cheese ripening References

MARSCHALL RHODIA INTERNATIONAL DAIRY SCIENCE AWARD LECTURE

Heat shocking
Lactobacillus helveticus 69°C/17 s 10 to 30% Increase in amount of tyrosine. Marked increase in proteolysis 84
Mixed strain lactococci 56°C/17 s 15% Increase in proteolysis, a moderate increase in flavor intensity, a less 34
bitter tase, a larger number of eyes and increase in AN level
L. helveticus 67°C/15 s AP (74%), EP (35%) Increase proteolysis. Enhance cheese flavor, free amino acids 4
L. bulgaricus 63°C/20 s N.D. Develop in flavor, high scores for body texture, proteolysis proceeded 96
rapidly
L. helveticus 64°C/18 s 10% Increase proteolysis and development of flavor of cheese 13
L. helveticus 70°C/18 s N.D. Increase is soluble N, enhance cheese flavor with decrease in bitterness 35
Increase in % of total volatile acidity
L. casei 67°C/22 s DAP (37%) Increase proteolysis, no bitter flavor 23
L. plantarum, L. casei 50°C/15 s Decrease in acidification rate, increase in AN, increase in aminopeptidase 6
activity, higher degradation of αs1-casein than of β-casein
L. helveticus, L. casei 60, 65 & 70°C/15 s 30%, 62 to 93% AP Moderate levels of proteolysis 32
0%, 40 to 64% DAP
Freeze shocking
L. helveticus −24°C/24 h Substantial increase in water soluble nitrogen. Debittering effect 7
L. helveticus, L. casei −20°C/20 h 50% reduction of Ras cheese ripening with L. helveticus 2, 3
L. helveticus −96°C/1 wk 24 to 35% EP, 13% ES 60, 61
Lac lactis subsp. lactis —20°C/20 h Mutant starter gave moderate increase in proteolysis (1–2%) 49
L. casei, Pediococcus sp. −20°C/20 h Enhanced proteolysis of Cheddar cheese. Acid flavor defect with both 29
microorganisms and calcium lactate crystals with pediococci
Pediococcus halophilus −30°C/36 h Enhancement of proteolysis and lipolysis in Ras cheese 26
L. helveticus −15°C/96 h Enhancement of proteolysis. Debittering effect 62
Leuconostoc sp. −20°C/24 h Decrease bitterness in Ras cheese 25
L. casei −24°C/24 h 75–95% 95
Journal of Dairy Science Vol. 83, No. 4, 2000

L. helveticus −20°C/2 wk AP (15%) 55

L. helveticus, −20°C/24 h High extent of enzyme release. Higher levels of water soluble nitrogen and FAA, 75, 77
L. casei enhancement in cheese flavor and prevented bitterness
Spray drying
L. helveticus 82 & 120°C AP (85%) Greater proteolysis and enhancement in cheese flavor 55, 56
1
AP = Aminopeptidase; EP = endopeptidase; N.D. = not determined; DAP = dipeptidyl aminopeptidase; ES = esterase; AN = amino nitrogen.

613
614 EL SODA ET AL.
Figure 1. Percentage of autolysis of untreated (▲), freeze-shocked
(䊊), and heat-shocked (䊉) cells of adjunct cultures of lactobacilli in
buffer system. From El Soda et al. (33).
adjunct cultures and chemical measures of cheese
ripening.
Bartels et al. (7) found that Gouda cheese made with
freeze-shocked adjunct cultures of L. helveticus had bet-
ter flavor quality than cheese with untreated cultures.
Our work (77) on the impact of attenuated adjunct cul-
tures on cheese quality indicated that the attenuated
adjunct cultures with enhanced autolytic properties
provide a more controlled and consistent ripening re-
sulting in flavor and texture improvement. The stimu-
lating effect of attenuation, in particular, freeze shock-
ing on release of enzymes from lactobacilli can be of a
Figure 2. Effect of heat shocking temperature (60, 65, or 70°C for
15 s) on the rate of autolysis of adjunct cultures of Lactobacillus
helveticus strains I (*), M (䊐), U (䊏), and L (◆) and Lactobacillus
casei strains A (▲) and T (䊉) after incubation for 48 h at 30°C. From
El Soda et al. (30).
Journal of Dairy Science Vol. 83, No. 4, 2000
great importance in assuring the desired aminopeptido-
lysis early during cheese maturation (7, 24, 77).
SELECTION CRITERIA FOR ADJUNCT CULTURES
The selection of appropriate adjuncts is becoming
widely used for successful cheese production. However,
if they are not making a contribution through peptidase
and esterase activities, some adjunct cultures may con-
tribute little to cheese flavor development. In fact, in a
study in which several commercial adjuncts were com-
pared, El Soda et al. (31) demonstrated that some of the
adjuncts exhibited little peptidase and esterase activity
and autolyzed at a very slow rate. We believe that in
addition to production of specific flavor compounds, the
selection of adjuncts should be based on enzyme profiles
(enzyme level and enzyme specificity) and autolytic
properties.
Selection Based on Enzyme Profile
The literature indicates that there are wide differ-
ences in the concentrations of proteinases, peptidases,
and esterases among genus, species, and strains of LAB
(85). It was shown (21) that X-prolyldipeptidyl pepti-
dase may not play a decisive role during Cheddar cheese
ripening, while the debitterase role of pep N is now well
established in LAB.
Amino acid catabolism during ripening is thought to
play an important role in flavor development. Gao et
al. (45) reported that dead unlysed cells of Lac. lactis
51 were able to degrade aromatic AA, and they sug-
gested that cell lysis may not be important for AA catab-
olism by some lactic culture strains. However, Yvon et
al. (100) reported that little alteration of the cyto-
plasmic membrane is needed to increase penetration of
AA in the cells. These authors also suggested that after
cell rupture and the progressive release of the intracel-
lular contents of the cell in the cheese matrix, the ami-
notransferases from Lac. lactis could still be active since
the co-factors appear to be tightly bound to the enzyme.
The enzyme was still active at the pH and salt concen-
tration of cheese. Further work is however needed for
a better understanding of the positive and negative
impact of AA catabolism on cheese flavor and to deter-
mine the role of autolysis during this event.
Laan et al. (63) studied the effect of pH and NaCl
and CaCl2 concentration on the aminopeptidase activity
of lactococci and lactobacilli to assess the potential role
of these enzymes on proteolysis during cheese ripening.
The authors concluded that the aminopeptidase activity
of the various cultures was partially inhibited at pH
5.2. The inhibition was restored by the addition of NaCl
or CaCl2 at concentrations found in the moisture phase
 MARSCHALL RHODIA INTERNATIONAL DAIRY SCIENCE AWARD LECTURE
of Cheddar cheese. Nonstarter lactic acid bacteria seem,
therefore, to play a significant role during the ripening
process, and their presence is essential for the develop-
ment of a full-flavored cheese. Several reviews (27, 28,
44, 67) reported methods to improve the enzyme poten-
tial of LAB using genetic approaches. Recently Arnau
et al. (5) expressed bovine plasmin in Lac. lactis, and
the plasmin produced in Lac. lactis was biologically
active. The authors did not apply the genetically modi-
fied culture in a cheese system.
Several intracellular enzymes from nonstarter ad-
junct cultures have been investigated and demon-
strated the powerful ability to enhance cheese flavor
development and quality due to their specific aminopep-
tidolytic action on casein during cheese maturation (22,
50). Adjunct culture of lactobacilli were reported to have
5 to 100 times higher intracellular enzyme activity than
lactococci with high potential to degrade hydrophobic
AA and reducing bitter off-flavor (50, 63). Recently, our
group has investigated (77) proteolysis and flavor devel-
opment in Cheddar cheese as function of addition of
some strains of lactobacilli. The study revealed that
cheese made with adjunct strains selected for high pep-
tidolytic potential especially those with low acidifica-
tion ability (33) exhibited the highest levels of proteoly-
sis and flavor appreciation without detrimental effect
on cheese-making procedure or cheese quality. This in-
dicated the important role of high intracellular enzyme
activities of lactobacilli, in particular those appropri-
ately selected for their high aminopeptidase specificity.
Selection Based on the Rate of Cell Autolysis
The work of Kawabata et al. (59) and Lepeuple et al.
(71) confirmed that early lysis of Lactococcus cultures
allowed the release of intracellular peptidases, which
are then active in cheese. These publications also dem-
onstrated that the extent of starter lysis could be re-
lated to the level of proteolysis (59). Furthermore, it
was shown that starter strain lysis resulted in higher
free AA levels and a decrease in bitterness (74).
Studies on the autolytic properties of both starter
and nonstarter LAB have shown that the autolytic prop-
erties of a cell are strain dependent (Figure 3) (14, 15,
30, 58, 72, 89).
The environmental conditions and the physiological
conditions affected the rate of autolysis. Sodium chlo-
ride at a concentration of 0.2 M was reported to enhance
the autolysis of lactococci (58). El Soda et al. (30) re-
ported that concentration of 0.5 M and up to 1 M of
NaCl was required to obtain higher rates of autolysis
from Lactobacillus cultures. Several studies indicated
that cells harvested during log phase autolyzed faster
when compared to late stationary phase cells (10, 11,
615
Figure 3. Rate of autolysis of different strains of Lactobacillus
helveticus (H, K, J, U, I, L, M, G) and Lactobacillus casei (A, B, C,
D, E, R, T). ◆: A, H; 䊏: B, K; ▲: C, J; 䉭: D, U; 䊐: E, I; 䊉: R, L; 䊊:
T, M; *: G. From El Soda et al. (29).
14, 58, 72). The previously described studies were car-
ried out in a buffer system, which may not reflect what
happens in a cheese system. More recently, Boutrou et
al. (15) followed the autolysis and proteolytic activities
of lactococci in a pseudo-curd. The authors classified
their cultures into three groups: low, moderate, and
high lytic capacity. Boutrou et al. (10) also demon-
strated that cell lysis positively influenced the ripening
of cheese. Proteolysis was more advanced in cheese
made with the highly autolytic cultures.
El Soda et al. (31) compared the autolytic properties,
peptidase and esterase activities of commercially avail-
able adjunct cultures. The cultures were classified into
four groups according to their levels of autolysis and
aminopeptidase and esterase activities. Some cultures
exhibited high rates of autolysis combined with high
levels of aminopeptidase and esterase activities,
whereas others possessed high autolytic activity with
relatively low enzymatic activity. The third group was
low in autolysis and high in enzymatic activity. The
fourth group was composed of cultures low in autolysis
and enzyme activity. The corresponding cheese slurry
experiments carried out by the same group (75) con-
firmed the data obtained by Boutrou et al. (10, 11) for
the lactococci. The slurries with added lactobacilli ex-
hibited considerably higher aminopeptidase and ester-
ase activity when compared to the control. The slurries
made with cultures selected for their high enzymatic
potentials and high autolytic properties exhibited the
highest concentrations of released peptidases and ester-
ases. On the other hand, slurries to which the poorly
autolytic strain was added displayed very little enzyme
Journal of Dairy Science Vol. 83, No. 4, 2000
616 EL SODA ET AL.
activity. Higher concentrations of water soluble nitro-
gen and free fatty acids could also be measured in the
slurries made with the cultures showing high levels of
enzymatic activities combined with high autolysis (75).
The autolysis of starter and adjunct during the ripen-
ing process is assumed to result in the release of intra-
cellular enzymes into the curd matrix leading to en-
hancement of flavor development. A selection could
therefore be made based on the autolytic properties of
the cells to ensure a sufficient release of intracellular
enzymes.
Methods to Enhance Cell Autolysis
Among the methods used for cell attenuation, freeze
shocking seems to be the most promising for high autol-
ysis and high enzyme activity (24, 30, 35). Approaches
have also been suggested to enhance and control lysis
in live cells. Freitag and McKay (38, 39) isolated a Lac-
tococcus strain harboring a thermoinducible prophage,
which exhibited normal growth at 30 to 32°C and lysed
when the cells were shifted to 38 to 40°C. Although this
approach seems attractive because of the possibility of
releasing the intracellular content of the cells at a given
time during the cheese-making process neither cheese-
making trials nor further work with the cultures was re-
ported.
Addition of bacteriocin-producing cultures was
shown to promote autolysis in lactococci. Lactococcus
lactis ssp. lactis DPC 3286, which produces bacteriocins
A, B, and M, was shown to exhibit a lytic effect on other
lactococcal strains. Cheese made with the bacteriocin-
producing culture showed higher levels of AA and
higher sensory evaluation scores than the control. The
mechanism of cell lysis is, however, not understood and
it is thought that a combination of the three bacteriocins
may be necessary to initiate the lysis process (79). De
Ruyter et al. (17) used the lytic genes of bacteriophage
δ US3 (Lyt A and Lyt H) to accomplish controlled lysis
of lactococcal cells. This expression system has the ad-
vantage of allowing the gene of interest to be present
and silent in the host until the system is triggered by
sublethal amounts of nisin. The authors suggest the
use of an adjunct culture containing Lyt A and Lyt H
genes under control of the nisin-incisive promoter.
Other L. lactis present in the cheese will lyse after
induction with nisin. The presence of nisin, which is
essential to trigger the lytic system, can be obtained
through the addition of a nisin producing culture.
Meijer et al. (78) have reported the production of a
nisin-immune transconjugant producing nisin, which
could serve that purpose.
Bacteriophage genes were also cloned and expressed
in lactococcal strains by Shearman et al. (90, 91), which
Journal of Dairy Science Vol. 83, No. 4, 2000
led to the normal growth of the cells through exponen-
tial phase then lysed prematurely in the stationary
phase. Studies were also carried out to construct sys-
tems in which change in temperature could trigger gene
expression. Naut et al. (81), using comparative molecu-
lar modeling, isolated a derivative that became totally
inactive at 42°C. Such a system can be useful in cheese
making, where temperature shift constitutes an initial
step in the process.
The bacteriophage-based methods may have poten-
tial for cheese-ripening enhancements but may be unac-
ceptable to cheese manufacturers, and more particu-
larly when the actual phage is needed to assist the lytic
process (15, 59, 68). While the methods described above
have improved our understanding of autolysis, their
practical application has not been demonstrated. Thus,
it appears that more traditional physical approaches
to enhancing autolysis will be more widely used.
In addition to their role in cheese flavor enhance-
ment, adjunct cultures were also used as probiotics.
Gardiner et al. (46) manufactured Cheddar cheese with
L. salivarius or L. paracasei, which were isolated from
the human small intestine and have been characterized
for their probiotic potential; L. paracasei was found
to grow and sustain high viability in cheese during
ripening, but the L. salivaricus cultures declined over
the ripening period. The authors suggest that Cheddar
cheese can be an effective vehicle for delivery of some
probiotics organisms to the consumer.
CONCLUSION
The literature survey on the use of adjunct cultures
for cheese quality enhancement clearly indicates the
need for more research on the difference between live
and attenuated (nonviable) adjuncts for the improve-
ment of the sensory quality of cheese. Although live cells
favor the production of desirable metabolic products of
adjuncts, the production of free fatty acids and AA will
be delayed because these activities can only occur after
cell autolysis. Live cells can also hardly be involved in
the debittering process known to be important in cheese
(22, 50). However, the productions of compounds like
methanethiol are unlikely to be obtained if 100% atten-
uated (nonviable) cells are used as adjuncts.
A significant role of viable cells in improving the fla-
vor and texture of mild Cheddar beyond methanethiol
production is unlikely. Viable cells may have a larger
impact on full-flavor Cheddar if the combined effects of
their metabolic activity and associated enzyme activity
from subsequent cell lysis are important. In young, mild
cheeses, Cheddar flavor is less complex. Here, early
high enzymatic activity may be desirable. Therefore
nonviable, attenuated adjunct cells could rapidly re-
 MARSCHALL RHODIA INTERNATIONAL DAIRY SCIENCE AWARD LECTURE
lease intracellular enzymes to accelerate flavor and tex-
ture development. However, there is little information
to support or refute such an approach.
A study of the same adjuncts added in viable and
nonviable attenuated forms could help to address these
questions. Studies that clarify the role of lysed cells
versus metabolically active cells and the impact of both
types of adjuncts on the rate of flavor and texture
changes are needed.
ACKNOWLEDGMENTS
The authors appreciate the financial support pro-
vided by Rhodia, Inc., Dairy Management, Inc., and
California Dairy Research Foundation.
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Journal of Dairy Science Vol. 83, No. 4, 2000