Beruflich Dokumente
Kultur Dokumente
TAXONOMY OF BREVIBACTERIUM LINENS ences in maximum growth temperature and salt toler-
ance (117). Another area of difference between the
Classification of the genus Brevibacterium has two proposed homology groups is that pigment
presented taxonomists with problems because of its production is light-dependent for strain ATCC 9172
close morphological similarity to other genera, such (type strain and member of DNA-DNA homology
as Arthrobacter, Caseobacter, Corynebacterium, and group 1 ) but is light-independent for other strains
Rhodococcus. The genus Brevibacterium was proposed (including strain ATCC 9175, DNA-DNA homology
initially by Breed (14), with B. linens as the type group 2 ) (94). Foissy (41), using an electrophoretic
species, and was recognized in the seventh edition of zymogram technique, examined intra- and extracellu-
Bergey’s Manual of Determinative Bacteriology (15); lar enzymatic activities in 15 strains of B. linens. On
no mention was made of coryneform morphology. the basis of the protein bands, it was possible to
However, later research showed that B. linens had divide the 15 strains into three biotypes. By use of a
coryneform morphology and showed a rod-coccus cycle similar electrophoretic zymogram technique, Sørhaug
similar to that of Arthrobacter globiformis (95, 110). (118) studied intracellular dipeptidase activities in 6
In the eighth edition of Bergey’s Manual of Deter- strains of B. linens. There were considerable differ-
minative Bacteriology (106), the genus Brevibac- ences in the number of dipeptidases observed for the
terium was listed as incertae sedis because of a num- various strains in addition to differences in substrate
ber of reports indicating its close similarity to the specificity, further indicating the heterogeneity be-
genus Arthrobacter and because the coryneform mor- tween B. linens strains. Further heterogeneity within
phology was overlooked by Breed (14); da Silva and the species was evident in another electrophoretic
Holt (23), Davis and Newton (24), and Bousfield zymogram study ( 2 9 ) of intracellular esterase activi-
( 1 0 ) even proposed that B. linens should be reclassi- ties in 18 strains of B. linens; the number of esterase
fied as Arthrobacter linens. bands varied from 2 to 6, depending on the strain. It
Later numerical taxonomic (66, 114) and is, however, necessary to mention that the expression
chemotaxonomic (74, 93, 111) studies illustrated the of these various enzymatic activities may be depen-
heterogeneity of the group and also indicated that B. dent on the growth medium and environment, and,
linens was a distinct taxon that should form the basis therefore, any attempted classification of B. linens
of a redefined genus Brevibacterium, as was sug- strains based on these reports should be treated with
gested initially by Yamada and Komagata (128) and caution.
later by Jones (65), Keddie and Cure (73), and
Sharpe et al. (117). On the basis of these and further
Characteristics of the Genus
studies, Collins et al. ( 2 1 ) redefined the genus
Brevibacterium (Breed), which forms the basis of the
Brevibacterium
genus in the first edition of Bergey’s Manual of Sys- Brevibacterium spp. exhibit a marked rod-coccus
tematic Bacteriology (67). cycle during growth on complex media; during the
At present, the genus Brevibacterium contains the exponential phase, the cells are morphologically rod-
type species, B. linens, and the species Brevibacterium shaped, but, as the cells enter the stationary phase of
iodinum, B. casei, and B. epidermidis. However, the growth, they become coccoid-shaped. Both rod and
situation is further complicated by studies of DNA- coccoid forms are Gram-positive, but some strains and
DNA homology (40), which indicate heterogeneity older colonies decolorize readily. Some important bio-
exists within the species B. linens; DNA-DNA homol- chemical and morphological properties of Brevibac-
ogy studies have shown that those strains presently terium spp. include the following: no endospore forma-
designated as B. linens constitute at least two distinct tion, nonmotile, optimum growth temperature of 20 to
species. Of the strains studied by Fiedler et al. (40), 30 or 37°C (depending on species and strain), ob-
ATCC 9172 (the type strain), ATCC 19391, ATCC ligate aerobes, slight or no acid production from glu-
9864, and strain B3 represent one homology group; cose, extracellular proteinase production, catalase-
this group represents the species B. linens because it positive, cell-wall peptidoglycan contains meso-
contains the type strain, ATCC 9172. A second homol- diaminopimelic acid (DAP) as the diamino acid, ab-
ogy group contains strains ATCC 9175, AC 251, AC sence of arabinose in the cell wall, absence of mycolic
252, AC 474, and B4. acids, and large amounts of dehydrogenated menaqui-
The presence of at least two DNA-DNA homology none (67).
groups within the species B. linens is further indi- In the first edition of Bergey’s Manual of Systematic
cated by considerable differences in the nutritional Bacteriology (67), a number of species are mentioned
requirements of different strains ( 9 4 ) and by differ- that almost certainly are not members of the genus
Brevibacterium, but for which data are insufficient to Brezina et al. ( 1 8 ) reported the partial purification
allow them to be reclassified with confidence. Such of four extracellular proteinases from B. linens. The
species are classified as incertae sedis and include pH and temperature optima of the proteinases were
Brevibacterium incertum, Brevibacterium acetylicum, 5.0 to 8.0 and 50°C, respectively. Inhibition studies
Brevibacterium oxydans, Brevibacterium halotolerans, indicated that the enzymes were serine proteinases.
Brevibacterium frigoritolerans, and Brevibacterium Juhász and Skárka ( 6 9 ) partially purified an ex-
rufescens. tracellular proteinase from a B. linens strain (iso-
lated from the cheese culture, Laktoflora 200; Laktos,
PROTEOLYTIC AND PEPTIDOLYTIC ACTIVITY Prague, Czech Republic) using a combination of
ultrafiltration and gel filtration. The pH and temper-
Extracellular, cell-wall associated, and intracellu- ature optima of the partially purified enzyme were
lar proteinases have been reported for B. linens. For 7.0 to 8.5 and 45°C, respectively. The proteinase was
convenience, the proteolytic enzymes of B. linens will completely inactivated by heat treatment at 55°C for
be discussed according to their location, as extracellu- 30 min. The molecular mass of the partially purified
lar, cell wall-associated, or intracellular. enzyme, as determined by SDS-PAGE, was 52 to 55
kDa. Inhibition studies indicated that the enzyme
Extracelluar Proteinase(s) was a serine proteinase.
Foissy ( 4 1 ) detected extracellular proteinase ac-
The greatest number of studies have focused on the tivity in 15 strains of B. linens using an elec-
extracellular proteinase(s) of the microorganism, trophoretic zymogram technique and reported signifi-
largely because of their high activity and importance cant differences in the electrophoretic patterns
with respect to cheese ripening. In relation to the between the strains, indicating heterogeneity within
extracellular proteinase, some discrepancies exist con- the species. Frings et al. ( 4 8 ) studied the hydrolysis
cerning the number and biochemical properties of the of as1-CN and b-CN that were present in the growth
extracellular proteinase(s) of the bacterium. Ex- medium for 5 strains (ATCC 9174, DSM 20158, DSM
tracellular proteinase production was observed ini- 20425, DSM 20426, and LBT 102) of B. linens. Both
tially by Albert et al. ( 2 ) through the partial hydroly- SDS-PAGE and HPLC analysis showed qualitative
sis of protein in milk cultures. However, the first differences in the hydrolysis of the caseins between
significant study of extracellular proteinase produc- the stains. For all 5 strains, b-CN was hydrolyzed
tion by B. linens was conducted by Friedman et al. more rapidly and to a greater extent than was as1-
(47), who observed that proteinase production was CN. In a similar experiment using whey proteins in
cyclical and had 2 maxima after 2 and 8 d of growth, the growth medium, Holtz and Kunz ( 5 9 ) found that
in the absence of autolysis, possibly indicating the 4 strains of B. linens (ATCC9174, DSM20158, DSM
presence of two distinct enzymes. The proteinase 20426, and LBT 102) hydrolyzed a-LA more rapidly
produced after 2 d of growth was studied further and than did b-LG. Hayashi et al. ( 5 1 ) purified five ex-
was found to have pH and temperature optima of 7.2 tracellular proteinases from B. linens F to
and 38°C, respectively. Neither metals nor reducing homogeneity, using ammonium sulfate precipitation,
agents resulted in loss of activity. The proteinase was gel filtration, and anion-exchange chromatography.
active on a-CN and b-CN (crude preparations) with The isolated proteinases, designated A, B, C, D, and
higher activity on the former; no activity was ob- E, had molecular masses of 37, 37, 44, 127, and 325
served on BSA, g-globulins, b-LG, bovine seminal pro- kDa, respectively, as determined by gel filtration. The
teins, or a- LA. Tokita and Hosono (122) reported the enzymes were classified into two groups based on
partial purification of an extracellular proteinase their temperature optima and stability. Proteinases A
from B. linens, using ammonium sulfate precipitation and B were stable for 1 h at 35°C and had a tempera-
and gel filtration. The pH and temperature optima of ture optimum at 40°C, and proteinases C, D, and E
this partially purified proteinase were 7.0 and 25°C, were stable for 1 h at 45°C and had a temperature
respectively. The proteinase was very heat sensitive optimum at 55°C. The pH optimum for all 5 pro-
and was completely inactivated after exposure to teinases was 11.0, and proteinases A and B were
50°C for 10 min. Casein was hydrolyzed rapidly by more active at lower pH values than were proteinases
the proteinase, hemoglobin was less hydrolyzed, and C, D, and E. The 5 proteinases were stable between
ovalbumin was hydrolyzed only slightly. In agree- pH 6.0 to 11.0. Inhibition studies indicated that the
ment with the results of Friedman et al. (47), cyclical isolated enzymes were serine proteinases. Rattray et
production of the extracellular proteinase was ob- al. (100) purified an extracellular proteinase for B.
served. linens ATCC 9174 with pH and temperature optima
of 8.5 and 50°C, respectively. The molecular mass of man et al. ( 4 7 ) found that the inclusion of glucose in
the proteinase was 56 kDa by SDS-PAGE and was the growth medium resulted in no appreciable in-
126 kDa by gel filtration, indicating that the native crease in extracellular production of the proteinase.
enzyme exists as a dimer. Inhibition studies indicated Tokita and Hosono (122) observed a similar effect.
that the enzyme was a serine proteinase. The enzyme Zemanovic and Shárka (129) found that egg albumen
was activated by Mg2+ and Ca2+. The sequence of the and zein (corn gluten) in the culture medium were
first 20 N-terminal amino acids was reported. The the most effective in stimulating proteinase produc-
specificity of the proteinase on bovine as1-CN and b- tion; casein was the poorest stimulant. The effect of
CN was characterized (103, 104). The time course of temperature on proteinase production has also been
peptide formation from as1-CN indicated that His8- studied. Tokita and Hosono (122) and Zemanovic and
Gln9, Ser161-Gly162, and Gln172-Tyr173 or Phe23-Phe24 Skárka (129) observed that increasing the cultiva-
were the first, second, and third bonds cleaved, tion temperature from 25 to 30°C caused a 70%
respectively. Other cleavage sites in as1-CN included decrease in the production of extracellular proteinase
Asn19-Leu20, Phe32-Gly33, Tyr104-Lys105, Leu142- production by B. linens. However, Hayashi et al. ( 5 2 )
Ala143, Phe150-Arg151, Gln152-Phe153, Leu169-Gly170, found no significant change in proteinase production
and Thr171-Gln172. The major sites of hydrolysis of b- at elevated growth temperatures. Production of ex-
CN were Ser18-Ser19, Glu20-Glu21, Gln56-Ser57, Gln72- tracellular proteinase also appears to be influenced by
Asn73, Leu77-Thr78, Ala101-Met102, Phe119-Thr120, the pH of the growth medium prior to inoculation.
Leu139-Leu140, Ser142-Trp143, His145-Gln146, Gln167- Friedman et al. ( 4 7 ) reported that an initial pH of
Ser168, Gln175-Lys176, Tyr180-Pro181, and Phe190- 7.0 resulted in maximum proteinase production, and
Leu191. The proteinase showed broad specificity on Zemanovic and Skárka (129) reported that an initial
both as1-CN and b-CN for the amino acids present in pH of 8.0 to 8.5 was optimal; in both studies, pro-
the P1 and P′1 positions but showed a general prefer- teinase production was lowest when the initial pH of
ence for hydrophobic residues at the P2, P3, P4, P′2, the medium was 6.0.
P′3, and P′4 positions.
These various reports show that there is poor Extracellular Aminopeptidases
agreement as to the characteristics or the number of
the extracellular proteinase(s) of B. linens. Strain Purification, to homogeneity, of an extracellular
variation or autoproteolysis may provide a possible aminopeptidase from the cell-free supernatant of B.
explanation. The biochemical properties of the ex- linens ATCC 9174 was reported by Foissy (42, 43, 44,
tracellular proteinase(s) of B. linens are summarized 45); no activity of the intracellular marker enzyme,
in Table 1. glucose-6-phosphate dehydrogenase, was detected in
the cell-free supernatant, indicating that cell lysis
Induction of Extracellular had not occurred during growth and that the purified
Proteinase Production aminopeptidase was truly extracellular. The purifica-
tion protocol involved ammonium sulfate precipita-
A number of attempts have been made to stimulate tion, followed by gel filtration, reprecipitation with
the production of extracellular proteinases by B. li- ammonium sulfate, and finally, two preparative elec-
nens by modification of the growth conditions. Fried- trophoresis steps. The purified aminopeptidase had
pH and temperature optima of 9.6 and 28°C, respec- ured using casein as substrate; however, the intracel-
tively. The molecular mass of the purified extracellu- lular proteinase activity was relatively low compared
lar aminopeptidase, determined by gel filtration and with the extracellular proteinase activity. Foissy ( 4 1 )
SDS-PAGE, was 95 and 48 kDa, respectively, indicat- reported that several strains produced up to four
ing that the aminopeptidase exists as a dimer in its distinct intracellular proteinases, but some strains
native state. Activation and inhibition studies showed produced only two. Wong and Cone (127) also demon-
that Co2+ resulted in pronounced activation of the strated proteolytic activity in a crude cell-free extract
aminopeptidase. Metal-chelating agents, reducing of B. linens; enzyme activity was maximal at pH 7.9
agents, and N-bromosuccinimide inhibited the and at 45°C. The crude cell-free extract was affected
aminopeptidase. Specificity studies showed that the little by reducing agents, iodoacetic acid, N-
extracellular aminopeptidase had a strong preference ethylmaleimide, or EDTA, but activity was markedly
for leucine at the N-terminal of dipeptides with much reduced by Hg2+ and p-hydroxymercuribenzoate
less activity when phenylalanine, serine, or histidine ( PHMB) .
was at the N-terminal position. The aminopeptidase El-Soda et al. ( 3 0 ) partially purified an intracellu-
did not hydrolyze dipeptides with lysine, glycine, or lar aminopeptidase from B. linens HS using gel filtra-
proline at the N-terminus. tion. The pH and temperature optima of the partially
Hayashi and Law ( 5 3 ) reported the purification of purified intracellular aminopeptidase were 7.5 and
two extracellular aminopeptidases from B. linens F, 30°C, respectively. The enzyme was inhibited with
which were designated aminopeptidase A and B. increasing effectiveness by 1,10-phenanthroline >
Aminopeptidase A accounted for 85% of the PHMB > phenylmethylsulfonyl fluoride. The enzyme
aminopeptidase activity remaining at the end of the was active on Gly- and Ala-p-NA with greater activity
purification procedure. Both aminopeptidases had a on the former, but the enzyme did not hydrolyze Leu-,
pH optimum of 9.3 and a temperature optimum of Lys-, Pro-, or Arg-p-NA. The crude cell-free extract
40°C; aminopeptidase A showed slightly greater pH showed no dipeptidylaminopeptidase activity on Arg-
stability than did aminopeptidase B. The molecular Pro-, Gly-Pro-, or Gly-Phe-p-NA. Sørhaug (118), us-
masses of aminopeptidases A and B were estimated ing an electrophoretic zymogram technique, demon-
as 150 and 110 kDa, respectively, by gel filtration, strated intracellular dipeptidase activity in 6 strains
and as 36 and 26 kDa, respectively, by SDS-PAGE, of B. linens. A total of 18 different dipeptidase bands
indicating that the aminopeptidases exist as was observed for the 6 strains. The greatest number
tetramers in their native state. Aminopeptidases A of dipeptidases, 14, was observed for B. linens ATCC
and B were completely inhibited by EDTA and were 21330 and the least, 7, for B. linens ATCC 9172.
reactivated on incubation with Ca2+, Co2+, Mg2+, There were considerable differences in substrate
Zn2+, or Mn2+, indicating that the aminopeptidases specificity between strains. Rattray and Fox (101)
are metalloenzymes. The two aminopeptidases had purified an intracellular aminopeptidase from B. li-
similar substrate specificities with a strong prefer- nens ATCC 9174. The pH and temperature optima for
ence for Leu-p-nitroanilide ( NA) and dipeptides with this enzyme were 8.5 and 35°C, respectively. The
leucine at the N-terminus. In addition to activity on molecular mass was reported to be 59 kDa by SDS-
dipeptides, the aminopeptidases also hydrolyzed tri- PAGE and 69 kDa by gel filtration. The aminopepti-
and tetrapeptides and dipeptides with proline at the dase was strongly inhibited by PHMB, Co2+, and Zn2+
N-terminus. and hydrolyzed Ala-p-NA and Gly-p-NA but not by
Brezina et al. ( 1 8 ) reported the presence of three Val-, Phe-, Pro-, Glu-, Leu-, Lys-, Arg-, Gly-Phe-, or
extracellular aminopeptidases from a B. linens strain Ala-Pro-p-NA. The enzyme also hydrolyzed dipeptides
isolated from the cheese culture Laktoflora 200. The with an alanine residue in the N-terminal position,
partially purified aminopeptidases had pH and tem- but tripeptides were not hydrolyzed. The N-terminal
perature optima at 7.0 to 9.0 and 30°C, respectively. amino acid sequence of the first 20 amino acid
The enzymes were inhibited by EDTA, Zn2+, Cu2+, or residues of the enzyme was also reported.
Ni2+; Co2+ increased activity by 100%. Ezzat et al. ( 3 3 ) reported cell-wall–associated pro-
teinase and dipeptidase activities in B. linens CNRZ
Intracellular and Cell-Wall–Associated 944. Extraction of cell-wall–associated enzymatic ac-
Proteolytic Enzymes tivities involved washing the cell pellet initially with
a calcium-containing buffer at 4°C and subsequent
The presence of intracellular proteinase activity in washing with a calcium-free buffer at 30°C to release
B. linens has also been reported (41, 47, 48). The the enzymes. The cell wall-associated proteinase was
intracellular proteinase activity reported was meas- partially purified; preliminary characterization
demonstrated pH and temperature optima of 6.5 and range (5.5 to 8.6). Esterase 2 differed from the two
40°C, respectively. The cell-wall–associated pro- other esterases in its sensitivity to inhibitors. Ester-
teinase was inhibited by EDTA, PHMB, and ase 4b differed from esterases 2 and 4a in its sub-
phenylmethylsulfonyl fluoride. strate specificity; it hydrolyzed aliphatic and
nitrophenyl esters. The spectrum of activity of the
LIPOLYTIC AND ESTEROLYTIC ACTIVITIES other two esterases was narrower, and they hydro-
OF BREVIBACTERIUM LINENS lyzed only naphthyl esters and, in the case of esterase
2, tributyrate and ethyl butyrate. Rattray and Fox
Foissy ( 4 1 ) demonstrated extracellular esterase (102) purified and characterized an intracellular es-
activity in 14 strains of B. linens, but activity was low terase from B. linens ATCC 9174. The pH and tem-
compared with intracellular activity. However, perature optima were 7.5 and 35°C, respectively. The
Sørhaug and Ordal (119) detected no extracellular molecular mass was found to be 54 kDa by SDS-
esterase activity in 5 strains of B. linens, perhaps PAGE and 201 kDa by gel filtration. The esterase
because of low assay sensitivity. Brevibacterium li- hydrolyzed b-naphthyl esters of acetic, butyric,
nens I has been reported to have extracellular lipase caproic, caprylic, and capric acids but not lauric,
activity (108), but, again, no such activity was found myristic, palmitic, or oleic acids. The sequence of the
by Sørhaug and Ordal (119). Cell-wall–associated first 19 N-terminal amino acids of the esterase was
esterase activity in B. linens has also been reported determined.
(33, 119). Brandl and Petutschnig ( 1 3 ) found a rela-
tionship between the proteolytic and lipolytic activity PRODUCTION OF VOLATILE COMPOUNDS
for B. linens; strains with medium or strong proteo-
lytic activity also had relatively high lipolytic activity.
Sulfur-Containing Compounds
Intracellular esterase activity in 15 strains of B. li-
nens was observed by Foissy ( 4 1 ) using a-naphthyl Tokita and Hosono (120) studied the production of
acetate, b-naphthyl butyrate, and tributyrin as sub- volatile sulfur compounds by B. linens in the culture
strates. Several esterase bands were noted for all of medium. The volatile sulfur compounds were identi-
the strains when a-naphthyl acetate or b-naphthyl fied as hydrogen sulfide, mercaptans, and disulfides;
butyrate was used as substrate, but a single esterase hydrogen sulfide was at the highest concentration.
band was found when tributyrin was used (except for Methanethiol was also detected. The addition of
1 strain that had no activity on tributyrin). El-Shafei methionine to the culture medium dramatically in-
et al. ( 2 9 ) reported intracellular esterolytic activity creased the level of hydrogen sulfide produced, but
in 18 strains of B. linens. All strains tested hydro- the addition of cystine and cysteine had little effect.
lyzed o-nitrophenyl and p-nitrophenyl derivatives of Sharpe et al. (116) detected the production of
acetic and butyric acids, but the derivatives of methanethiol by 7 strains of B. linens and suggested
caprylic and palmitic acids were not hydrolyzed. It that because B. linens is a major component of smear
was also noted that the p-nitrophenyl esters of the surface-ripened cheeses, such as Limburger, Ro-
fatty acids were hydrolyzed faster than were the o- quefort, and Stilton, these methanethiol-producing
nitrophenyl derivatives. Using an electrophoretic bacteria may contribute to the aroma and flavor of
zymogram technique, it was determined that the these cheeses.
number of esterase bands varied from 2 to 6, depend- Cuer et al. ( 2 2 ) studied the production of sulfur-
ing on the strain, and most stains showed 4 active containing compounds by 8 strains of B. linens. All 8
esterases. strains produced large amounts of methanethiol,
Lambrechts et al. (82), using an electrophoretic hydrogen sulfide, dimethyldisulfide, and 2,3,4-
technique, identified 7 esterase bands in the cell-free trithiapentane. Enrichment of the culture medium
extract of Brevibacterium sp. R312. Eight esterases with methionine resulted in increased production of
were separated by anion-exchange chromatography, methanethiol, and the addition of cysteine increased
and the three principal esterases (designated as es- the production of hydrogen sulfide. Four of the strains
terase 4b, 2, and 4a) were purified to homogeneity also produced S-methylthioacetate, which is an im-
using anion-exchange chromatography, gel filtration, portant contributor to the characteristic odor of smear
and affinity chromatography. The molecular masses surface-ripened cheeses. Lamberet et al. (80, 81) exa-
of esterases 4b, 2, and 4a were 38, 45, and 56 kDa, mined in some detail the ability of B. linens to synthe-
respectively, as determined by SDS-PAGE. The three size various S-methyl thioesters by incubating resting
esterases differed in their temperature optima and cells with methanethiol in the presence and absence
thermal stability; all were active in the same pH of short-chain fatty acids; S-methyl thioacetate, S-
produced by B. linens ATCC 8377 inhibited the germi- exhibited very low deaminating activity on the other
nation of spores of Penicillium expansum NRRL 877, amino acids. The deamination of phenylalanine, tryp-
which possibly explains why there is a notable lack of tophan, and histidine could not be demonstrated.
ability of Limburger and Trappist cheeses to support Jollivet et al. ( 6 4 ) conducted a comprehensive
mold growth. Lewis ( 8 8 ) investigated the an- study on the production of volatile compounds by 4
timicrobial activity of 4 strains of B. linens against strains of B. linens. The 4 strains produced a wide
Pencillium roqueforti. Only those three strains that range of compounds belonging to different chemical
produced methanethiol were shown to be inhibitory families: fatty acids, alcohols, methyl ketones, pyra-
against the mold indicating the toxicity of zines, sulfur compounds, and cyclic compounds. Both
methanethiol toward molds. quantitative and qualitative differences existed be-
tween the compounds produced by the different
Other Volatile Compounds strains. The strains produced 2,5-dimethylpyrazine in
sufficiently high concentrations for flavor perception;
Tokita et al. (123), in a preliminary study on the
this compound is present in several cheeses, such as
production of volatile compounds by B. linens, identi-
Camembert, and gives a nutty, roasted note. The
fied the presence of volatile acids, volatile bases, and
important flavor compound, dimethyltrisulfide, was
neutral substances in the culture medium. From gas
produced by all 4 strains at concentrations higher
chromatography data, acetic, isovaleric, and caproic
than its sensory threshold; its odor is that of very ripe
acids were found to be the principal volatile com-
cheese.
pounds. Volatile amines were also identified, includ-
ing histamine, tyramine, dibutylamine, monoethyla-
mine, monomethylamine, diethylamine, and cada- CATABOLISM OF AROMATIC AMINO ACIDS
verine. The neutral volatile substances included for-
Brevibacterium linens may participate in the for-
maldehyde, acetaldehyde (acetoaldehyde), acetone
mation of flavor compounds and their precursors
(aceton), ethanol, isopropanol, n-propanol, and
through the catabolism of aromatic amino acids. Such
isobutanol.
compounds include phenol and indole, both of which
Hosono and Tokita (61, 63) studied the production
are found at high concentrations in Limburger cheese
of volatile flavor compounds by B. linens, Candida
(96).
mycoderma, and Debaryomyces kloekeri in broth
The transport of radioactively labeled phenylala-
medium. When grown in milk broth, B. linens
produced higher levels of hydrogen sulfide, volatile nine, tyrosine, and tryptophan by B. linens has been
fatty acids (particularly n-butyric acid), and volatile studied (11). Based on the differential behavior of
carbonyl compounds (formaldehyde, acetaldehyde, the transport in terms of concentration-dependent ki-
acetone, pentanone-2, and heptanone-2) than did C. netics, pH and temperature optima, structural and
mycoderma or D. kloeckeri. It was also shown that B. stereospecificity, and responses to metabolic inhibi-
linens produces acetone, acetaldehyde, and acetic acid tors and sulfydryl reagents, it was concluded that the
from citric acid. Hosono and Tokita ( 6 2 ) reported transport of aromatic amino acids by B. linens is
that larger amounts and a greater number of volatile determined by three high affinity permeases. The
carbonyl compounds were produced by B. linens from transport of phenylalanine was optimal at pH 7.5 and
casein than from milk fat or glucose, indicating that 25°C and that of tyrosine and tryptophan at pH 8.0
casein is an important source for the production of and 35°C. Tryptophan noncompetitively inhibited the
volatile carbonyl compounds. transport of tyrosine; similarly, phenylalanine and
In another study, Hosono and Tokita ( 6 0 ) studied tyrosine noncompetitively inhibited the transport of
the decarboxylation of 13 amino acids and subsequent tryptophan. All combinations of the aromatic amino
amine production by a crude cell extract from B. acids resulted in noncompetitive inhibition of the
linens. The amines produced from lysine, alanine, transport mechanism. Transport was almost totally
leucine, glutamate, and tyrosine were cadaverine, inhibited by carbonyl cyanide-m-chloro-
monomethyl amine, isoamyl amine, g-amino butyric phenylhydrazone and 2,4-dinitrophenol, indicating
acid, and tyramine, respectively; lysine was decarbox- that the prime energy source for transport was proton
ylated at the highest rate. Hemme et al. ( 5 6 ) studied conduction.
the deamination of amino acids by 23 strains of B. The transport of phenyalanine by B. linens was
linens isolated from the smear of Comté and Beaufort studied further by Boyaval et al. ( 1 2 ) using high
cheeses. Serine, glutamine, and asparagine were ac- resolution autoradiography. It was shown that la-
tively deaminated by certain strains. Most strains beled phenylalanine rapidly penetrates the cells and
is localized in the cytoplasm; the labeled phenylala- ATCC 9175) to inhibit the germination of spores of
nine did not remain bound to the cell wall or mem- Clostridium botulinum type A. The inhibitory agent
brane. It was not possible to identify the precise present in the culture fluid was not identified, but the
localization of the transported phenylalanine in the maximum inhibitor level was produced after 7 d of
cytoplasm because of the small size of the cells. It was growth. The unidentified inhibitory substance was
also noted that chromatography of bacterial extracts active after heat treatment at 121°C for 15 min; it
gave two radioactive spots, one that was due to was soluble in 70% ethanol and was always as-
phenylalanine and the other due to a small peptide. sociated with a yellow-red color. Culture superna-
Lee and Desmazeaud ( 8 4 ) reported that transami- tants from strain ATCC 9175 were more active than
nation was the first step in the utilization of aromatic were those from strain ATCC 9174. The production of
amino acids as sole nitrogen sources by B. linens. The this inhibitory substance may explain why surface-
deaminated metabolites of the amino acids were iden- ripened cheeses are free of C. botulinum.
tified in culture supernatants, and the enzyme ac- Bacteriocin production by B. linens was demon-
tivity was identified in cell-free extracts. They also strated by Kato et al. (72). The bacteriocin, desig-
reported that the cells contained increased aromatic nated as Linecin, was produced by B. linens ATCC
amino acid aminotransferase activity when aromatic 9175 and another B. linens strain (unspecified) and
amino acids were used as sole nitrogen sources. In was found to be inhibitory to B. linens ATCC 8377
addition, two aromatic amino acid aminotransferases and B. linens ATCC 9172. Linecin was not inhibitory
from a cell-free extract were resolved by ion exchange to other species within the genus Brevibacterium,
chromatography. One of these aromatic amino acid Corynebacterium, or Micrococcus. In a later investiga-
aminotransferases was purified and had pH and tem- tion, Kato et al. ( 7 0 ) reported the purification and
perature optima of 8.5 to 9.0 and 37 to 40°C, respec- characterization of Linecin A from the culture super-
tively (85). The molecular mass of the enzyme was natant of B. linens ATCC 9175. Linecin A consisted
estimated by gel filtration to be 126 kDa. The cofactor mostly of protein with an estimated molecular mass
pyridoxal 5-phosphate was tightly bound to the en- of 95 kDa (as determined by gel filtration). Linecin A
zyme. was also sensitive to proteolytic attack, thermolabile,
Lee et al. ( 8 7 ) investigated the enzymatic mechan- and totally inactivated by heating at 45°C for 60 min.
isms involved in the catabolism of phenylalanine and Linecin A inhibited B. linens strains ATCC 8377,
tyrosine by some coryneform bacteria isolated from ATCC 9172, and ATCC 9174.
cheese; the two key steps studied were deamination Valdés-Stauber and Scherer (124) isolated and
and benzene ring cleavage. It was found that orange characterized a bacteriocin, designated Linocin M18,
coryneforms catabolized phenylalanine and tyrosine from the culture supernatant of B. linens M18 (iso-
by transamination, and the benzene ring was cleaved lated from the surface of red smear cheese). The
by 3,4-dihydroxyphenylacetate-2,3-dioxygenase; both bacteriocin consisted of a single protein subunit with
enzymes appeared to be inducible. It was also noted a molecular mass of 31 kDa by SDS-PAGE, but in its
that yellow and white coryneforms (in contrast to
native state formed aggregates of extremely high
orange-pigmented coryneforms) possessed uninduci-
molecular masses (>2000 kDa). Linocin M18 was
ble low aminotransferase activity and lacked enzymes
heat labile (totally inactivated after 5 min at 80°C )
for benzene ring cleavage. In a further study (86),
and was sensitive to a wide range of proteinases. The
the quantitative importance of the 3,4-
N-terminal amino acid sequence was determined.
dihydroxyphenylacetate meta-cleavage pathway (the
Usually, bacteriocins inhibit only closely related bac-
principal pathway of tyrosine catabolism) in the
teria, but Linocin M18 exhibited an extraordinarily
catabolism of phenylalanine in B. linens 47 was
broad activity spectrum with activity against species
evaluated using tyrosine-negative mutants. Less than
of the genera Bacillus, Arthrobacter, Corynebac-
5% of phenylalanine was catabolized through the
tyrosine pathway, indicating that in B. linens the two terium, Micrococcus, and Listeria. Of particular in-
structurally analogous amino acids are catabolized terest is the inhibition of Listeria spp., which may be
principally by different pathways. important in the biological control of pathogenic
Listeria spp. in smear surface-ripened cheeses.
Oligonucleotide probes based on the N-terminal
PRODUCTION OF BACTERIOCINS
amino acid sequence have been used to locate the
AND ANTIMICROBIAL SUBSTANCES
gene coding for Linocin M18 (125). A single copy of
Grecz et al. ( 5 0 ) demonstrated the ability of cul- the gene, lin, was located on chromosomal DNA. The
ture supernatants from B. linens (ATCC 9174 and amino acid composition, N-terminal sequence, and
molecular mass derived from the nucleotide sequence ent from Linencin A and Linocin M18 and is not a
of an open reading frame of 798 nucleotides coding for bacteriocin in the strict sense. The antibacterial sub-
266 amino acids found on a 3-kb BamHI restriction stance was inhibitory toward foodborne pathogens,
fragment correspond closely to those obtained from including Staphylococcus aureus and L.
the purified bacteriocin. The taxonomic distribution of monocytogenes, but not against Gram-negative bac-
lin was also studied; 52 isolates of different species of teria. Hemolytic activity of the substance on sheep
the genera Brevibacterium, Arthrobacter, and Coryne- erythrocytes was also demonstrated by Boucabeille et
bacterium were probed for lin by polymerizing chain al. ( 9 ) . The molecular mass of the native Linenscin
reaction. The gene could be amplified in a surpris- OC2 was estimated with gel filtration to be 285 kDa;
ingly wide distribution of bacteria; it was amplified in however, SDS-PAGE and mass spectrometry indi-
12 of 26 B. linens strains isolated from different cated molecular masses of 1196.7 Da and 2412 Da,
cheeses, in 1 strain of Brevibacterium flavum, 1 strain
respectively, indicating that the native Linenscin
of Brevibacterium lyticum, 5 of 6 Arthrobacter spp.,
OC2 exists as large aggregates (90). About 50% of
and 5 of 9 Corynebacterium spp. In a model cheese
the activity remained after heating for 10 min at
ripening system, the antimicrobial activity of B. li-
nens M18 toward Listeria strains has been demon- 100°C. The antibacterial activity of crude Linenscin
strated with reduction of Listeria ivanovii and OC2 was reduced but not eliminated by incubation
Listeria monocytogenes counts by 1 to 2 log units with various proteolytic enzymes, a-amylase, and li-
(31). pase (90). The biochemical mode of action of Linen-
In another study, 3 isolates of B. linens (isolated scin OC2 is believed to be similar to that of bacterio-
from the brine used to salt red smear cheese) have cins such as nisin, which in addition to their
been shown to produce an antimicrobial agent active cytoplasmic membrance-disruptive action induce au-
against Listeria spp. (92). The antimicrobial agent tolysis ( 9 ) . The biochemical properties of the various
was dialyzable through a membrane with a 1-kDa bacteriocins and antibacterial agents produced by B.
cutoff, was stable at pH 4.0 to 9.0, and was stable to linens are summarized in Table 2.
heat treatment for 30 min at 80°C at acid pH. The One aspect of particular importance with respect to
agent also remained active after treatment with pro- the production of these various bacteriocins and an-
teinase, catalase, or lipase. The antimicrobial agent tibacterial substances by B. linens is the extrapola-
was bacteriostatic or bacteriocidal, depending on the tion of the inhibitory effects from a model buffer
strain of L. monocytogenes tested. In a similar study, system to that of a smear surface-ripened cheese. The
Ryser et al. (107) isolated and identified from cheese degree to which a bacteriocin produced at the surface
an orange coryneform resembling B. linens that of the cheese by B. linens can diffuse toward the
produced a bacteriocin-like agent that was inhibitory center obviously influences any inhibitory effect on
for Listeria spp. including L. monocytogenes. The microorganisms in the interior of the cheese. The
bacteriocin-like agent was resistant to 5 different pro- molecular masses of the bacteriocins reported from B.
teolytic enzymes. linens are probably too large to result in significant
Maisnier-Patin and Richard ( 9 0 ) purified to diffusion from the surface to the interior, and their
homogeneity an antibacterial substance, designated effects are, therefore, likely to be confined to the
Linenscin OC2, from the cell-free supernatant of B.
surface only.
linens OC2. This antibacterial substance was differ-
structure between microorganisms can be used for High oxygenation rates, cultivation temperature, and
taxonomic discrimination. However, the analysis of medium ingredients are significant factors affecting
peptidoglycan is expensive and laborious. the final cost of these products to the industrial
producer.
Insecticide Degradation Famelart et al. ( 3 4 ) studied the effect of tempera-
ture, pH, and dissolved oxygen on the growth of B.
The degradation of 1,1,1,-trichloro-2,2-bis(p- linens ATCC 9175 in a fermentor. It was found that
chlorophenyl)ethane (DDT) and 1,1-dichloro-2,2- maximum growth occurred when the dissolved oxygen
bis(p-chlorophenyl)ethylene (DDE) by two strains of was maintained at 50% saturation; growth was in-
B. linens (ATCC 9172 and ATCC 9175) has been hibited when the pO2 was at 40 or 60%. Famelart et
demonstrated in media augmented with these insecti- al. ( 3 5 ) studied the metabolism of B. linens in a
cides (83). Strain ATCC 9175 degraded the insecti- fermentor on a basal medium supplemented with
cides to a greater extent than did strain ATCC 9172; amino acids, sodium L-lactate, NaCl, and vitamins.
both strains degraded 1,1,1,-trichloro-2,2-bis(p- Biomass yields indicated that amino acids are the
chlorophenyl)ethane more extensively than 1,1- limiting factor for B. linens. The most rapidly con-
dichloro-2,2-bis(p-chlorophenyl)ethylene. Geotrichum sumed amino acids were tyrosine, phenylalanine, ar-
candidum, which is present on smear surface-ripened ginine, proline, glutamate, and histidine and ap-
cheeses, also has the ability to degrade these insecti- peared to be the limiting substrates. The degradation
cides. of arginine in ripening cheeses was reported (112) to
occur via the production of citrulline, ornithine, and
Antibiotic Resistance putrescine. Tokita and Hosono (121) noted the
production of large quantities of ornithine by B. linens
In a study on the resistance of various dairy when oxygen concentration was low. Ammonia was
microorganisms to a range of 30 different antibiotics, also produced, but large quantities were produced
B. linens was found to be reasonably resistant (105). only when the medium contained amino acids alone
The various dairy microorganisms included in this or in a large proportion compared with the carbon
study could be listed in order of decreasing resistance source.
to the antibiotics tested: Leuconostoc dextranicum, B.
linens, Streptococcus faecalis, Streptococcus durans,
Potential Biotechnological Applications
Lactobacillus bulgaricus (now Lactobacillus bulgari-
cus ssp. delbrueckii) , Streptococcus lactis (now Lac- In addition to the commercial production of B. li-
tococcus lactis ssp. lactis) , Streptococcus diacetylactis nens for use as a cheese-ripening agent by the previ-
(now citrate-utilizing Lactococcus lactis ssp. lactis) , ously mentioned culture companies, a number of pa-
Streptococcus thermophilus, Streptococcus cremoris tents have been published involving the use of B.
(now Lactococcus lactis ssp. cremoris) , Staph. aureus, linens in biotechnological applications. They are
Staph. epidermidis, and Micrococus varians. The an- mostly Japanese patents, which describe the use of B.
tibiotics to which B. linens was found to be resistant linens in waste water treatment, vitamin production,
were methicillin, nafcillin, cloxacillin, oxacillin, and pharmaceutical processes. It is not known
furadantin, and nalidixic acid. whether any of these patents are used commercially.
These biotechnological applications are summarized
Fermentation Studies in Table 3.
manufacture from raw milk, 3 ) manufacture with ate the ripening of Ras cheese (32). During ripening,
genetically modified organisms, 4 ) addition of adjunct the cheeses treated with either Neutrase alone or
cultures, 5 ) addition of exogenous enzymes, and 6 ) with the cell-free extract had a significantly more
addition of flavor precursors. The adoption of any of intense Ras cheese flavor but were more bitter at the
these techniques requires that the ripening time be end of ripening (as judged by a trained taste panel).
reduced without leading to any negative flavor or Hayashi et al. ( 5 4 ) used the partially purified
textural characteristics. extracellular proteinases produced by B. linens to
The use of B. linens as a cheese-ripening accelerant accelerate the ripening of Cheddar cheese. Cheddar
can be either direct or indirect. The direct use of the cheese was manufactured in the conventional way
microorganisms involves the addition of viable cells or with the addition of the proteinase to the milled curd.
cell extracts to the curd or cheese milk. Indirect After ripening for 2 mo at 12°C, the cheeses were
methods involve the addition of the extracellular pro- assessed for flavor, bitterness, and proteolysis. The
teinase or extracellular aminopeptidase to the cheese flavor scores (judged by a trained taste panel of 17
milk; some success has been reported with both persons) for the treated cheeses were increased sig-
methods; however, some caution is necessary in the nificantly without significant amounts of bitterness.
interpretation of these results due to the difficulty The TCA-soluble nitrogen increased in the treated
associated with the accuracy and reliability of the cheeses, and b-CN was degraded extensively. In a
various taste panels employed in these studies. further study by Hayashi et al. (55), a partially
Chen et al. ( 1 9 ) used lyophilized extracts from purified extracellular aminopeptidase from B. linens,
several cheese-related microorganisms, including B. combined with a commercial neutral proteinase, was
linens, to accelerate the ripening of low fat Cheddar. used to accelerate Cheddar cheese ripening. After
The lyophilized extracts, prepared by grinding the ripening for 2 and 3 mo at 12°C, the flavor intensity
cells, were added to the low fat Cheddar curd after (judged by a trained taste panel of 24 persons) of the
milling. The cheeses were then assayed monthly for enzyme-treated cheese had increased significantly
flavor (no details of taste panel) and for proteolysis compared with the untreated control; no significant
up to 6 mo. At the end of the ripening period, the bitterness was detected. Protein hydrolysis to pep-
treated cheese showed significantly higher phos- tides and amino acids, as measured by TCA-soluble
photungstic acid-soluble nitrogen and TCA-soluble and sulfosalicylic acid-soluble nitrogen, was signifi-
nitrogen than did the control. Bitterness and other cantly increased compared with the untreated control.
off-flavors were not detected, but the treated cheese Significant differences in casein degradation, as
did not exhibit pronounced Cheddar cheese flavor. An measured by urea-PAGE, between the control and
intracellular cell-free extract from B. linens has been treated cheese were also observed. Hayashi et al.
used individually and in combination with Neutrase ( 5 5 ) suggested that the ripening period for Cheddar
( a commercial neutral proteinase, Novo Enzyme could be reduced from 4-6 mo to 2 mo using this
Products Ltd., Windsor, United Kingdom), to acceler- system.
Brezina et al. ( 1 7 ) accelerated the ripening of lated with B. linens or encapsulated separately with
Dutch-type cheese (variety not specified) by the B. linens and mixed together. The production of
separate and combined addition of extracellular pro- hydrogen sulfide from cysteine in microcapsules was
teinase and extracellular aminopeptidase from B. li- slightly reduced by the methanethiol produced from
nens. There was a very good correlation between the methionine. However, the presence of hydrogen sul-
amount of proteinase added and water-soluble nitro- fide stabilized the methanethiol produced from
gen; unfortunately, there was also a good correlation methionine in milkfat-coated microcapsules and, con-
between the amount of proteinase added and bitter- sequently, increased the level of methanethiol. It was
ness (judged by a trained taste panel of 15 persons). concluded that this microencapsulation system is
However, bitterness was successfully reduced in capable of producing adequate amounts of
cheeses manufactured using a combination of the ex- methanethiol for the development of Cheddar cheese
tracellular proteinase and the extracellular flavor, but no cheese trials were performed to support
aminopeptidase, and the ripening process was ac- this claim.
celerated. Recently, B. linens cultures have been used as
The proteinase of B. linens has also been used to adjunct cultures to accelerate the flavor development
accelerate flavor development in Ras cheese slurries of Cheddar cheese (B. Weimer, 1997, personal com-
(89). Addition of the proteinase to the slurry in- munication). The addition of B. linens to the cheese
creased the ratio of soluble nitrogen to total nitrogen, had a very positive effect with a significant reduction
the ratio of nonprotein nitrogen to total nitrogen, in the ripening time required to produce a mature
soluble tyrosine, and tryptophan as incubation flavor in Cheddar cheese. The cultures were also ef-
progressed. Pronounced flavor development (no de- fective at increasing the flavor intensity of low fat
tails of taste panel) was noticed in the proteinase- Cheddar cheese.
treated slurry. In a study involving the use of B.
linens as a debittering agent, Brezina et al. ( 1 7 )
CONCLUSIONS
found that it was possible to eliminate the bitterness
of peptides almost completely by using an intracellu- A large number of questions remain to be answered
lar aminopeptidase preparation from a strain of B. in many areas of the physiology, metabolism, genet-
linens. A process for the prevention of bitterness in ics, and taxonomy of B. linens. Although the commer-
Camembert cheese using a mutant of B. linens was cial importance of B. linens is small compared with
also developed. that of the lactic acid bacteria, it is nevertheless an
One of the obvious limitations of employing the important dairy microorganism. The precise role of B.
extracellular proteinase or aminopeptidases from B. linens in relation to smear surface-ripened cheese will
linens is that a large amount of purified or partially remain difficult to define until more knowledge is
purified enzyme is required for the treatment of small available about its metabolite production and en-
quantities of curd. However, ample scope remains for zymatic activities.
increasing the proteolytic capacity of B. linens by Most research to date on B. linens has demon-
variation in medium composition, different cultiva- strated that the physiological and metabolic activities
tion conditions, and the use of genetically modified of the bacterium are significantly strain dependent.
organisms. The reported differences in the biochemical properties
Kim and Olson (76, 78) evaluated the production and the number of its extracellular proteinases is a
of methanethiol and hydrogen sulfide in milk fat- clear example; differences are also reported for its
coated microcapsules containing B. linens and methio- aminopeptidase, esterase, and lipase activities. Fur-
nine or cysteine for possible flavor development of ther strain variation is evident by differences between
Cheddar cheese. Production of methanethiol from the biochemical properties and target microorganisms
methionine occurred aerobically and anaerobically, of the bacteriocins produced by B. linens. The heter-
but the production was three- to fourfold greater un- ogeneity among different stains of B. linens has also
der aerobic conditions; optimum pH and temperature been confirmed by studies of DNA-DNA homology
for methanethiol production was 8.0 and 26°C, respec- (40), which indicate those strains at present desig-
tively. Production of hydrogen disulfide from cysteine nated as B. linens constitute at least two distinct
required anaerobic conditions (77). The interactive groups. This degree of strain variation within the
effect of methanethiol and hydrogen sulfide produc- species B. linens must be addressed by taxonomists to
tion was also examined (75), in which the two sub- facilitate further research of strains currently classi-
strates, cysteine and methionine, were coencapsu- fied as B. linens.
In the area of enzymology, several areas of future 9 Boucabeille, C., D. Mengin-Lecreulx, G. Henckes, J. M. Simo-
net, and J. van Heijenoort. 1997. Antibacterial and hemolytic
research of B. linens are of particular interest. The activities of linenscin OC2, a hydrophobic substance produced
purification and characterization of further pepti- by Brevibacterium linens OC2. FEMS Microbiol. Lett. 153:
dases and esterases would improve the understanding 295–301.
10 Bousfield, I. J. 1972. A taxonomic study of some coryneform
of the role of B. linens in the ripening of smear bacteria. J. Gen. Microbiol.71:441–455.
surface cheeses. Furthermore, the purification of the 11 Boyaval, P., E. Moreira, and M. J. Desmazeaud. 1983. Trans-
various amino acid converting enzymes would be of port of aromatic amino acids by Brevibacterium linens. J.
Bacteriol. 155:1123–1129.
benefit. In the area of carbohydrate metabolism, the 12 Boyaval, P., M. Rousseau, and M. J. Desmazeaud. 1986.
metabolic pathways and enzymes involved have not Phenylalanine transport by Brevibacterium linens studied
yet been established in B. linens. There is a paucity of with high resolution autoradiography. Milchwissenschaft 41:
219–221.
information on most aspects of the genetics of B. 13 Brandl, E., and K. Petutschnig. 1972. Orientierende unter-
linens. It would be of interest to identify and charac- suchungen über die proteolytische und lipolytische aktivitaẗ
terize the genes that code for the extracellular pro- einiger Brevibacterium linens-stämme verschiedener herkunft.
Die Osterreichische Milchwirtschaft, Wissenschaftliche
teinase and aminopeptidase, bacteriocins, L- Beilage 3, 27:17–25.
methionine-g-demethiolase, carotenoid production, 14 Breed, R. S. 1953. The families developed from Bacteriaceae
and antibiotic resistance. Cohn with a description of the family Brevibacteriaceae Breed
1953. Proc. 6th Int. Congr. Microbiol., Rome, Italy 1:10–15.
15 Breed, R. S. 1957. Family IX. Brevibacteriaceae Breed, 1953.
ACKNOWLEDGMENTS Pages 490–503 in Bergey’s Manual of Determinative Bacteriol-
ogy. 7th ed. R. S. Breed, E.G.D. Murray, and N. R. Smith, ed.
This research has been part-funded by grant aid William and Wilkins, Baltimore, MD.
16 Brezina, P., D. Cikánek, S. Pavlı́ková, J. Zemanovic, and
under the Food Sub-Programme of the Operational O. Valentová. 1990. Acceleration of ripening process in cheese
Programme for Industrial Development, which is ad- by proteolytic enzymes of Brevibacterium linens. 23rd Int.
ministered by the Department of Agriculture, Food Dairy Congr., Montreal, Canada. Int. Dairy Fed., Brussels,
and Forestry (Dublin, Ireland) and is supported by Belgium 2:331.(Abstr.)
17 Brezina, P., D. Cikánek, M. Plocková, I. Schovánková, and
national and European Union funds. J. Kopecny. 1989. Properties and degradation of bitter peptides
in cheese. Prumysl Potravin 39:303–304. (Cited from Dairy Sci
Abstr. 51:151.).
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