International Journal of Food Microbiology, 11 (1990) 289-304 289 Elsevier
FOOD 00340
The yeasts of cheese brines
H. Seiler and M. Busse Institute of Bacteriology, South German Dairy Research Center, Freising-Weihenstephan, F.R.G.
(Received 17 October 1989; revision received 25 May 1990; accepted 13 June 1990)
A total of 365 yeasts were isolated from the brines of soft, semihard and hard cheeses from different manufacturers. Identification was based on 131 characteristics, primarily employing a method with microtitration plates. Most brines exhibited a characteristic yeast flora. The predominant strains proved to be mainly Debaryomyces hansenii and Candida oersatilis. In a few brines Trichosporon beigelii, C. rugosa, C. intermedia, Kluyoeromyces marxianus, Saccharomyces sp. and C. tenuis/polymorpha were predominant. Also of importance were C. tropicalis, C. parapsilosis, C. zeylanoides, Issatchenkia orientalis and Geotrichum klebahnii. Not all strains could be clearly identified. Lists of characters are provided for subdividing D. hansenii and T. beigelii. The specificity of the yeast flora of brines is assumed to contribute to the sensory variety of cheeses.
Cheese is usual ly sal ted by br ining. This p rocedu re helps to remove excessive whey, s t imulates the fo rma t ion of rind, and has a regula t ive and selective effect on the microb ia l popu la t ions . The consis tency of cheeses, their r ipening, flavor, tas te and keeping qual i ty are all f avorab ly inf luenced (Kammer l ehne r , 1986; Lenoir , 1984).
Yeasts, as a pa r t of the mic rof lo ra of the cheese surface, p l ay an i m p o r t a n t role in r ipen ing of some cheese varieties. As the mos t typica l example red smear cheeses shall be ment ioned . Accord ing to Sauter (1986) , the d e v e l o p m e n t of the f inal surface f lora ent i re ly depends on the b r e a k d o w n of lact ic acid b y the yeas ts because salt to lerant co rynefo rm bacter ia , inc luding Brev ibac ter ium linens, will not grow at a p H below 5.7. Hence the v iable cell count of aerobic bac te r i a on the r ind of b r ick cheese smears does not exceed the cell count of yeas ts unt i l the 6th to 9th day.
Yeasts - owing to their s l ightly p ro teo ly t i c and l ipoly t ic ac t iv i ty - fo rm sub- s tances which s t imula te growth of the lact ic acid bac te r i a and of the ae rob ic bac te r i a on the cheese surfaces, they ferment res idual lactose, inf luence f lavor fo rma t ion by
producing volatile acids and carbonyl compounds and prevent cheese surfaces to form a ' t oad skin' (Siewert, 1986). Geotrichum candidum is said to increase evapora- tion of water by virtue of aerial mycelium formation, thus promoting the formation of rind in cheeses which contain too much whey (Lacto-Labo, 1986).
On the other hand, yeasts can also be responsible for defects in cheese. Excessive yeast growth will cause the rind to become soft and smeary, resulting in poor development of other members of the surface flora, e.g. growth of Penicillium caseicolum on cheese of the camembert type (Krapf, 1987). This condition is usually associated with an unpleasant yeasty or esterlike odor. In rare cases excessive proteolytic and lipolytic activity will cause the surface of the cheeses to run off. In hard cheese ripened in plastic bags discolorations of the rind due to yeasts have been observed.
It is an open question whether the yeast flora of brines significantly influences immature cheeses. It is usually assumed that the yeasts of the salt brine are essential for ripening. However, very little is known on these subjects. To obtain further information, as a first step, the yeast flora of cheese brines has been studied. Furthermore, this study will contribute to a reliable system for the identification of cheese yeasts. The data could form a basis for selecting the best suitable yeasts as starter cultures for the ripening of cheese.
Materials and Methods
Brine samples
The yeasts were isolated from brines of cheese varieties Limburger, Romadur, Camembert , Tilsiter and Emmentaler. The brines originated from 11 different dairies ( A - K ) in Southern Bavaria, F.R.G.
Microbial counts
The brine or the cheese mass scraped off from the rind was transferred to a sterile bottle and diluted about ten times in sterile tap water, treated with an Ultra Turrax to give an even suspension, and tenfold dilutions were prepared. The yeast counts were recorded on yeast extract glucose chloramphenicol bromphenol blue agar (YGCB agar: 0.5 g yeast extract, 2 g glucose, 0.01 g chloramphenicol, 2 g NaC1, 0.001 g bromphenol blue, 1.5 g agar, 100 ml demineralized water, p H 6.0-6.4) at 27°C. For maintenance and storage of pure yeast cultures, yeast extract malt extract agar (YM agar: 0.3 g yeast extract, 0.3 g malt extract, 0.5 g casein peptone, 1 g glucose, 1.5 g agar, 100 ml demineralized water, pH 6.0-6.4) was used with incubation at 27 ° C and storage at refrigeration.
Tests carried out Microtitration plates were used for examination of utilization of carbon sources,
utilization of nitrogen sources, cycloheximide resistance, growth without vitamins, growth with NaC1 and urease activity (Seiler and Busse, 1988). For the remaining
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physiological tests standard methods for yeast identification were used (Kreger-van Rij, 1984).
All isolates were examined for their production of a killer factor, as described by Philliskirk and Young (1975). The sensitive test strain, Saceharomyces cerevisiae 381, was kindly provided by Dr. P. Pfeiffer, University of Mainz.
Ascospore formation was examined on McClary's acetate agar, potato glucose agar, Gorodkowa agar, corn meal agar, malt extract agar and 2% glucose-yeast autolysate agar (Kreger-van Rij, 1984). The plates were incubated at 18-22°C for 4 weeks.
Colony morphology was examined on Wort agar (Merck), pH 5.5. The plates were incubated at 27°C for 2 weeks.
For investigation of cell morphology potato dextrose agar (Merck) was used. The Dalmau plate technique (Kreger-van Rij, 1984) was applied with incubation at 27°C for 2 weeks.
Numerical grouping The methods used were described in a previous paper (Seiler, 1983). As reference
material for the identifications we used the data provided by Barnett et al. (1983).
Results and Discussion
The yeast counts of the brines investigated varied considerably. In most cases the concentrations per ml were between 1 x 10 4 and 1 x l0 s. The lowest value was approximately 5 x 10 2 and the highest about 1 x 10 6. The reasons for this variation were differences in the age and condition of the salt brines. Some brines looked quite clear, while others were turbid, or even with a thick layer of a fat-casein mixture floating on the surface. In one dairy (plant A) experiencing problems with Mucor spp., the brine was heated up to 85-90°C for 45 s once a week, using slaked lime after cooling to precipitate the protein. Only the clear supernatant was reused. With the exception of brine from one dairy (plant G), none of the 11 dairies had inoculated either the brine or the cheese with yeast cultures. The salt brine from plant G contained yeast cultures selected for their distinctive flavor. The salt concentration in the various plant brines was 19-20% (w/w), the temperatures varied between 13 and 16°C, and pH ranged from 4-6.
A total of 365 yeast strains were isolated from the brines, with care being taken to include representatives of all colony types. The quantitative share of all colony types was also recorded. The strains were tested for 131 characters. All strains were positive for the characters growth without inositol, p-aminobenzoic acid and riboflavin and growth at 25 and 30°C. The strains were all negative for the characters growth at 42°C, utilization of glucuronate, methanol, ethanol, tartaric acid, adipic acid, D-fUcose and glycogen, and for the formation of starch, acetic acid and ascospores. With the exception of glucose, the fermentation characters tested were not included in the evaluation (i.e. gas from galactose, maltose, methyl-D-glu- coside, sucrose, trehalose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin
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and starch). It was found that, if a strain was principally able to ferment, the results obtained in the fermentation tests were the same as those obtained in the corre- sponding assimilation tests. The diazonium blue B reaction (Barnett et al., 1983) was not determined because it is positively correlated with the decomposition of urea. This resulted in 103 characters to be used for the taxonomic grouping.
The phenogram for the 365 isolates and 46 reference strains from international yeast collections is shown in Fig. 1. The 365 strains isolated are clustered in 23 groups of varying size. Debaryomyces hansenii formed the largest group with 83 strains (groups 1 and 7), followed by Trichosporon beigelii (44 isolates), Candida rugosa (25 isolates), C. oersatilis (24 isolates), C. polymorpha (20 isolates), C. parapsilosis (17 isolates), Issatchenkia orientalis (15 isolates), C. tropicalis (13 isolates), C. tenuis (12 isolates), C. intermedia (12 isolates) and Saccharomyces sp. (11 isolates). All other species were represented by less than 10 isolates. These included yeast genera that are typical representatives of the yeast flora of milk and milk products, such as Geotrichum, Kluyueromyces or Torulaspora.
Identification was carried out by comparing the test results with the table of Barnett et al. (1983) in 70 characters. Although morphological data were established for all strains, they only served as identification criteria in cases where several species exhibited the same degree of similarity based on numerical comparison.
For all characteristics used Table I gives a detailed picture of the frequencies of positive reactions for the groups shown in Fig. 1. The quality of the identifications varied considerably. Group 1 was clearly identified as D. hansenii, although only one strain showed no deviation from Barnett's data (1983). The majority of strains exhibited 1 to 4 differences. The species next in order of similarity were C. tenuis, C. sake, C. intermedia, and Metschnikowia bicuspidata. As D. hansenii appears to be a highly heterogeneous yeast species (Haridi, 1987), some characters which might be valuable for internal subgrouping are listed in Table II. As can be seen, these are primarily colony morphology and utilization of erythritol, amygdalin, D-gluco- samine or D-ribose.
Identification of group 2 presented considerable difficulties. In group 2a C. polymorpha was cited as the species with the greatest similarity, followed by C. tenuis, C. atrnospherica, D. hansenii and M. bicuspidata. The strains of group 2b exhibited the highest physiological similarities to C. tenuis. The species with similar characteristics were C. atmospherica, D. hansenii, C. fennica, M. bicuspidata and Pichia nakazawae.
Group 3 was identified as (7,. intermedia. The differences as compared to D. hansenii were minor.
Identification proved to be difficult in group 4, too. The species C. parapsilosis and C. haemulonii are possible candidates with roughly the same level of similarity.
Fig. 1. Condensed phenogram based on simple matching coefficient and average linkage sorting 365 isolates from cheese brines and 46 reference strains tested for 103 characters. Figures in parentheses indicate the culture collection number of a reference strain clustered together with isolates. DSM = Deutsche Sammlung von Mikroorganismen, Braunschweig, F.R.G.; CBS = Centraalbureau voor Schim-
melcultures, Baarn, The Netherlands.
% Distance
,,~6.37.~8.~4.2° ~ 6 ~2. ~ , ~ , Phe- No. of , i nmn strains
46 ) 17
I I
10 5
20 12
i 12 17
i i
13 6 3 4 i
i i i 4 I I i I I 1 I i i I
31 6 5 i i 1 i
10 10 1
I la 4 11b 10
I 12 5
1 ]
1 25
5 I0
3 15
i 8 I I I i 9
15 I i 9 I i
~ | 2 2 6 i ]
1 I
24 1 I 1
[ , ~ 2 3
t ' • , , , . . . . . . . . . ' . . . . . . .
36 32 28 24 20 16 12 8 4
Presumptive i d e n t i f i c a t i o n Debaryomgces hansen i i (DSM 3428) Debaryomgces hansenii
Debaryomyces hansenii DSM 7 0 2 3 8
Debargomyces hansenii CBS 1 1 7 Debaryomyces hansenii
Trichosporon beigelii CBS 2466 Lipomyces tetrasporus CRS 5910 Klugveromgces l a c t i s (CBS 2360; DSM 70807) Klugveromyces lactds x. ma. CBS 712; DSM 70292, 70343, 70804 K. mar×ianus (CBS 397; DSM 70344) un ident i f ied 11ansenula jadinii Dekkera intermedia CDS 4914 un ident i f ied Candida kefyr DI Candida rugosa
70471, 70509, 70514, 7047~, 70547, s. cez. 70449, 70470, 70511; P. 381, 967) s . sp. Saccharomyce: spp, Saccharomgces sp. un ident i f ied Yarrowia lipolgtica
Yarrowia lipolytica Yarrowia lipolytica CDS 6124-i u n i d e n t i f i e d u n i d e n t i f i e d Caf~dJda versatJlis
un ident i f ied un ident i f ied un ident i f ied
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TABLE I
Frequency of positive characters, expressed as percentage positive in the phenons 1 through 7, 9 through 15, and 17 through 23 as described in Fig. 1
Less likely are the species which come next in order of similarity, C diddensiae, D. hansenii, C. sake and M. bicuspidata.
The strains of group 5 were classified as C. tropicalis, although they might also be C. haemulonii or C. maltosa. Species M. reukauffii, C. oleophila and D. hansenii were named on the next highest level of similarity.
The strains of group 6 (Clavispora lusitaniae) were physiologically very similar to D. hansenii, M. reukauffii, M. bicuspidata and M. lunata.
Groups 7 ( D. hansenii), 8 ( Rhodotorula glutinis or R. mucilaginosa), 9 (Tri- chosporon beigeilii), 10 and 11 (Kluyverornyces marxianus), 12 ( P. jadinii), 16 (G. capitatum), 18 (C. zeylanoides), 19 (Torulaspora delbrueckii), 20 (S. cerevisiae), 22 (Yarrowia lipolytica) and 23 (Candida versatilis) could all be clearly identified.
The species most similar to group 13 was C. rugosa. However, always reveafing 4 to 6 differences between the characteristic data determined by us and those of Barnett et al. (1983), it is not surprising that in the comparison by computer several other species were indicated for the next similarity level (Zygosaccharomyes rouxii, P. rnembranaefaciens, Z. bailii, Z. bisporus, C. apis, C. magnoliae, M. bicuspidata, T. delbrueckii, S. cerevisiae, C apiculata and C. silvae ).
Group 14 had an almost equal degree of physiological similarity to the species P. dispora, C silvae, C. vini and S. cerevisiae. The reference strain P. norvegensis CBS 6639 was included at 14% aberration. However, in the computer-aided identification this species name appeared only on the second highest similarity level. The species names P. kluyveri and P. membranaefaciens were listed before it.
Group 15 was identified as G. klebahnii, G. penicillaturn or G. armillariae, although strain G. candidum DSM 1240 had established itself there. The reason for this identification was a negative test result that we rated as deviation as against a
a Frequency of positive strains expressed as a percentage.
The species Issatchenkia orientalis and Pichia membranaefaciens indica ted on the highest similarity level in group 17 cannot be dis t inguished on the basis of
physiological data. All efforts to locate ascospores were in vain. The ident i ty of this
group must therefore remain open. Species with very similar physiology are I.
occidentalis and L scutulata. Strains of group 21 must be assigned to the species S. cereuisiae, S. dairensis or a
Saccharornyces species not yet described.
Aside f rom D. hansenii, only T. beigelii was divided into several subclusters.
Characters suitable for d i f ferent ia t ion are shown in Table III.
In another numerica l analysis our data were compared with a shor tened vers ion
of Barnet t ' s table (1983). The table included only species which, according to data
by Deak and Beuchat (1987), are typical of milk and milk products . Our analysis
yielded essentially the same species. Except ions were M. bicuspidata (cluster 2a) and
C. zeylanoides (group 15). For ty -one characters that were largely either posi t ive or negat ive in the individual
groups were selected and again compared with Barnet t ' s data (1983) . As it tu rned
out, the qual i ty of ident i f icat ion was not substant ial ly lower. An ident i f ica t ion key
T A B L E IV
Yeast species isolated f rom soft cheese brines and an exper imenta l br ine
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Yeast Dai ry
A Ba a Bb C D E1 b E 2 E 3 E 4 F (G) ':
C a n d i d a i n t e r m e d i a + d _ _ _ + + + . . . . .
C a n d i d a p a r a p s i l o s i s - + + . . . . . . .
C a n d i d a t e n u i s /
p o l y m o r p h a - + + + + + . . . . . . .
C a n d i d a t r o p i c a l i s . . . . . . + . . . .
C a n d i d a v e r s a t i l i s - - - + + - + + + + + + + + + + - -
C l a v i s p o r a l u s i t a n i a e . . . . . . . + - - -
D e b a r y o m y c e s h a n s e n i i - + + + + + + + . . . . . + + + -
G e o t r i c h u m k l e b a h n i i . . . . . . . + + - -
K l u y v e r o m y c e s m a r x i a n u s + + + . . . . . . . . . .
S a c c h a r o m y c e s sp. - . . . . . . . . . + + +
T r i c h o s p o r o n b e t g e l i i + - + - - + + + + + + - -
a Ba = first sample; B b = second sample af ter 10 months .
b E 1 _ E 4 = four different br ines in dairy E.
c (G) = an exper imenta l brine.
d + + + = 50-100%; + + = 30-49%; + = 1 0 - 2 9 % ; -- = 0 -9% of the total yeast flora.
based on this set of characters was suggested previously (Seiler and Busse, 1988). The set of characters corresponding to Barnett's data (1983) was expanded to include the characters assimilation of D-lyxose, D-turanose, gentibiose, N-acetyl-t)- glucosamine and D-arabitol. For differentiating more clearly between physiologi- cally very similar species it might also prove useful to test for the characters growth at 37 and 42°C.
The quantitative data as to the size of the clusters for the various species in Fig. 1 do not reflect the actual conditions in the brines. These are shown in Tables IV and V. As can be seen in Table IV, D. hansenii was by no means always predominant on soft cheese. In the case of dairy B, C. tenuis/polymorpha and C. parapsilosis were found in addition to D. hansenii; the second analysis after 10 months showed only minor differences compared to the first one. In dairy C we isolated C. versatilis, a species also predominant in all brines of dairy E. The brines of dairies A, D, F and G contained only one type of yeast each (K. marxianus, C. intermedia, D. hansenii, Saccharomyces sp.). Significant quantities of T. beigelii were present only in the brines 1 and 2 of dairy E.
Species D. hansenii and C. versatilis were predominant also in the brines of semihard and hard cheese (Table V). In dairy I only C. rugosa was found, while dairy L contained a large number of different yeast species in roughly equal quantities. The yeast floras of cheese rinds I and J did not correspond to the respective brines. Interestingly, these cheeses did not meet production standards. Further detailed studies would be required to determine the degree of correspon- dence between the yeasts in the brines and the yeast flora on the rinds of cheese,
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TABLE V
Yeast species isolated from semi-hard and hard cheese brines and cheese rinds
" s = semi-hard cheese brine. b h = hard cheese brine. c + + + = 50-100%; + + = 30-49%; + =10-29%; - = 0-9% of the total yeast flora.
and to clarify whether there are any t ime-changes in the yeast popula t ions occurr ing
on the rinds.
C o n c l u s i o n s
Yeast species found in br ines are more or less the same reported for the yeast flora of cheese r ind (Siewert, 1986). However, there is one exception. In br ines C.
versatilis dominates quite often. This apparent ly does not apply to the cheese rind. Other yeasts typical for br ines are D. hansenii, C. rugosa, T. beigelii and C. polymorpha . T h e yeast flora in br ines of different dairies seems to be quite specific. In two cases brines of a given dairy were analysed repeatedly. In these cases the
composi t ion of the yeast flora varied only slightly. As to the quest ion whether br ines are an impor tan t source for the yeast flora of
cheese, very little can be said at the moment . Compara t ive studies on cheese and br ine are necessary. Our material included only two comparisons with faulty hard cheeses r ipened in plastic bags. In these cases the yeast flora of the cheeses differed significantly from the flora of the brines. Evidently, yeast con tamina t ion of br ines are derived from immature cheese and dairy equipment . Since, however, immature cheese, r ipening cheese and brines differ in their ecology, the typical yeast flora of
those three biotops most p robab ly may have specific traits. Some manufac turers market starter cultures conta in ing K. marx ianus or D.
hanseni i , either to be sprayed on the cheese or added to the solut ions used as inoculum. It remains to be clarified whether this might promote un i formi ty in the cheese aroma. It seems quite conceivable that it is precisely the great differences i n
303
the yeast flora observed between the brines of different cheese dairies that represent an essential part of the sensory variety of cheeses.
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