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Enumeration and identification of yeasts isolated from Zimbabwean traditional fermented milk
International Dairy Journal 10 (2000) 459}466
Enumeration and identi"cation of yeasts isolated from Zimbabwean traditional fermented milk T.H. Gadaga *, A.N. Mutukumira , J.A. Narvhus Institute of Food, Nutrition and Family Sciences, University of Zimbabwe, P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe Department of Food Science, Agricultural University of Norway, P.O. Box 5036, N-1432 As s, Norway Received 28 February 2000; accepted 19 June 2000
Abstract The yeasts in 30 samples of the Zimbabwean traditional fermented milk, amasi, taken from farms, households and milk collection centres were enumerated and identi"ed. The yeast counts ranged from(2 to 8.08 log cfu g\. Yeast isolates were identi"ed using the API ID 323C test kits and the simpli"ed identi"cation method (SIM) as well as with reference to the standard taxonomic keys. From the 30 samples, a total of 20 di!erent yeast species were identi"ed. Saccharomyces (S) cerevisiae (22 isolates), Candida (C) lusitaniae (11), C. colliculosa (7) and S. dairenensis (7) were the predominant species identi"ed. Dekera (Dek.) bruxillensis, C. lipolytica and C. tropicalis were identi"ed less often. Seven of the S. cerevisiae isolates were able to assimilate DL-lactate. The strain of C. kefyr isolated could assimilate lactose and DL-lactate, but not citrate. The analysed amasi samples contained a wide variety of yeasts, but only a few species predominated and these could possibly contribute to the characteristics of the fermented milk in the 48 h fermentation. 2000 Published by Elsevier Science Ltd. Keywords: Yeasts; Fermented milk; Amasi
1. Introduction Fermented milk is an important part of the traditional diet in Zimbabwe (Mutukumira, 1995; Narvhus, "steraas, Mutukumira, & Abrahamsen, 1998). In the traditional fermentation process, raw milk is left to ferment spontaneously at ambient temperature. A mixed microbial #ora consisting of lactic acid bacteria, yeasts and moulds and enterococci plays a part in the fermentation (Mutukumira, 1995). In Zimbabwe, traditional fermented milk produced at household level is called hodzeko, amasi or mukaka wakakora (Feresu & Muzondo, 1990; Mutukumira, 1995; Gadaga, Mutukumira, Narvhus, & Feresu, 1999). This product is a result of natural fermentation of untreated raw milk in earthenware or metal pots. The fermentation is allowed to proceed for a period of up to 48 h, after which some whey is drained o! to obtain a product with a thick consistency (Mutukumira, 1995). The milk has a characteristic mild lactic acid and aromatic #avour, due
* Corresponding author. Fax: #47-64-94-37-89. E-mail address: [email protected] (T.H. Gadaga).
to the lactic acid, other organic acids, and volatile compounds produced by the mixed culture of microorganisms. The microbiological studies that have been carried out on amasi have focused on safety aspects and on the identi"cation of lactic acid bacteria (Mutukumira, 1996; Feresu & Muzondo, 1990). Lactococcus (L.) lactis subsp. lactis, Lactobacillus (Lb.) plantarum and Lb. helveticus were predominant isolates. Yeasts and moulds, as well as coliforms in amasi were also enumerated, but were not identi"ed (Mutukumira, 1995). The yeasts and moulds were considered as undesirable and a sign of poor hygiene. However, these could be an essential part of the micro#ora of amasi, hence the need to study the diversity of yeasts in the fermented milk. Apart from causing spoilage in products such as yoghurt and sour milk, yeasts are also important because they produce desirable #avours as in cheese ripening (Marshall, 1987; Fleet, 1990; Rohm, Eliskases-Lechner, & Brauer, 1992; Jakobsen & Narvhus, 1996). The low pH in the fermented milk o!ers a selective environment for yeast growth, but is unfavourable for most bacteria (Fleet, 1990; Rohm et al., 1992; Deak & Beuchat, 1996). Spoilage becomes evident when the yeast population reaches 10}10 cells g\ (Fleet, 1990).
0958-6946/00/$ - see front matter 2000 Published by Elsevier Science Ltd. PII: S 0 9 5 8 - 6 9 4 6 ( 0 0 ) 0 0 0 7 0 - 4
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Other fermented milk foods produced by traditional methods may have microorganisms other than lactic acid bacteria playing a signi"cant role in the fermentation process. There is increasing interest in the role of yeasts in dairy fermentations and especially their potential use as starter cultures (Fleet, 1990; Jakobsen & Narvhus, 1996; Loretan, Viljoen, Mostert, Vogel, & Jordaan, 1998). To establish the role of yeasts in amasi, the yeasts have to be isolated and identi"ed and then their technological properties studied. The aim of this study was to isolate, enumerate and identify the yeasts in the Zimbabwean fermented milk, amasi/hodzeko.
2. Materials and methods 2.1. Sampling Thirty samples of amasi, which had been fermented for two days (48 h), were collected from small farms, homes and milk collection centres in Zimbabwe in sterile 250 mL wide-mouthed screw-capped bottles and brought to the laboratory in a cooler box. The samples were analysed within 3 h of sampling.
2.4. Fermentation of sugars The fermentation of D-glucose, sucrose, D-galactose, lactose, maltose and ra$nose was tested, according to the description of Van der Walt and Yarrow (1984). A positive result was indicated by accumulation of gas in the Durham tubes. 2.5. Liquid assimilation of carbon compounds Distilled water (4.5 mL) in plugged 150;16 mm tubes was sterilised by autoclaving at 1213C for 15 min. An aliquot (0.5 mL) of "lter-sterilised yeast nitrogen base (Difco Laboratories, Detroit, MI, USA) containing 5% of the compound under test was aseptically added to the tubes. The tubes were inoculated by aseptically adding 0.1 mL of a visible suspension in Ringers solution (Oxoid) of an actively growing culture. The carbon compounds tested were galactose, glucose, sucrose, lactose, L-arabinose, maltose, D-mannitol, melibiose, ra$nose, soluble starch, trehalose, xylose, a-methyl-D-glucoside, cellobiose, erythritol, xylitol, citrate and DL-lactate. The tubes were inoculated as in the sugar fermentation tests. A positive reaction was detected by visual inspection for an increase in the turbidity of the solution.
2.2. Enumeration and isolation 2.6. Assimilation of nitrogen compounds The yeasts were enumerated on spread plates of yeast extract glucose chloramphenicol agar (YGCA) (IDF, 1990), after suspending the sample (10 g) in 90 mL quarter strength Ringers' solution (Oxoid, Unipath Ltd, Basingstoke, England), and making serial dilutions. The medium was sterilised by heating at 1213C for 15 min. The plates were incubated at 253C for 5 days. Colonies with distinct morphological di!erences such as colour, shape and size were picked and puri"ed by streaking at least three times on malt extract agar (MEA) (Merck, Darmstadt, Germany). The puri"ed isolates were stored on MEA slants at 43C until required for identi"cation. 2.3. Identixcation The yeasts were identi"ed based on their physiological and morphological properties as described by Deak and Beuchat (1996), Van der Walt and Yarrow (1984), and also by using the API ID32 C test strips for yeast (bioMerieux, Marcy l'Etoile, France). The tests included the fermentation of sugars, liquid assimilation of carbon compounds, liquid assimilation of nitrogen compounds, growth at 373C, growth on 50% (w/v) glucose yeast extract agar, growth in vitamin-free medium, growth in media containing 16% NaCl, resistance to 0.01% cycloheximide, and urease activity. API ID32 C strips have 30 cupules containing di!erent carbohydrates and one containing actidione (cycloheximide). The strips were used according to the manufacturer's instructions.
The procedure was similar to the carbon compound assimilation tests except that yeast nitrogen base was replaced by yeast carbon base (Difco). The nitrogen compounds tested were nitrate, ethylamine hydrochloride, L-lysine and cadavarine. 2.7. Hydrolysis of urea This was tested using the method of Van der Walt & Yarrow (1984). Commercially produced Christensen's urea agar base (Merck) was used. The slant was inoculated from a suspension of the actively growing yeast culture using a sterile wire loop and incubated at 253C for 4 days. A positive reaction was the development of a deep pink colour in the agar. 2.8. Other tests Formation of ballistoconidia was examined on corn meal agar (Merck), by making two streaks at right angles to each other on the agar. The plate containing the corn meal agar was then inverted over another containing malt extract agar over which a sterile glass slide had been placed. The two plates were taped together along their entire circumference and incubated at 253C for up to 14 days. Ascospore formation was examined on malt extract agar (MEA) (Merck), potato dextrose agar (PDA) (Merck), and Gorodkova agar according
T.H. Gadaga et al. / International Dairy Journal 10 (2000) 459}466
to the procedure described by Van der Walt and Yarrow (1984). The plates were incubated at 253C for seven days. Formation of mycelium and pseudo-hyphae was studied on yeast morphology agar (Difco) using the Dalmau plate technique (Van der Walt & Yarrow, 1984). A three day-old liquid culture in malt extract broth was examined microscopically as a wet mount for cellular morphology.
3. Results and discussion 3.1. Enumeration of yeasts The yeasts counts ranged from less than 2} 8.08 log cfu g\ (Table 1). One sample (Number 19Amasi) had viable yeast counts of 8.08 log cfu g\. This sample could have been fermented over more than two days. The practice in that part of the country where the sample was taken is to ferment milk over more than three or four days, mixing several days' milkings and subsequent whey removal to produce a thick product, which has the consistency of cottage cheese. This product had a sharp acid taste. These viable counts, however, are comparable to those reported by Mutukumira (1995). From ten spontaneously fermented milk samples collected from Nharira milk collection centre, Lancashire, Zimbabwe, combined yeast and mould counts ranged between (3 to 5.65 log cfu g\. Loretan et al. (1998) reported yeast counts ranging between 10 and 10 cfu mL\ in ten sour milk samples collected from South African homes. The yeast and mould counts obtained from Ethiopian fermented milk were on average 6.18 log cfu mL\ for Ititu and Meomata (Fekadu, 1994) and 5.8 log cfu mL\ for fermented milk from Southern Ethiopia (Mogessie, 1990). Bankole and Okagbue (1992) also studied samples of nono, a Nigerian fermented milk, and they reported yeast counts of 1.5;10 cfu g\. These results show that yeasts constitute a signi"cant part of the micro#ora of naturally fermented milk. At such high levels, the yeast metabolism should impact on the overall quality and acceptability of these products. In our study, samples taken from the same source showed varying yeast counts. Mutukumira (1996) observed the variable quality of the naturally fermented milk, which could be indicative of an inconsistent micro#ora, and yeasts could be involved. Particularly low counts of yeasts ((2 log cfu g\) were recorded for three sources, Murehwa, Nyarungu and Nyadire. Nyarungu is a milk collection and training centre of the Zimbabwe Dairy Development Programme and the low yeast counts could possibly be explained by better hygienic practices at this centre compared to the other sources of samples. The incidence of yeasts in all the samples, however, may suggest that yeasts are a common #ora of the milking parlours, milking containers
461
Table 1 Colony counts of yeasts isolated from Zimbabwean traditional fermented milk Sample
Source
Strain ref. number
log cfu g\
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Mavambo Mavambo Mavambo Mavambo Mavambo Dr Charp Nharira Mavambo Mavambo Mavambo J. Mukurazhizha The 44 The 44 The 44 Mavambo Mavambo Tamutsa Mavambo Amasi from Bulawayo University farm University farm University farm Murehwa Nyarungu Nyarungu Murehwa Murehwa Crowhill farm Nyadire Murehwa
1, 3 2.48 4, 5, 6, 7, 8 5.32 9, 10 4.85 11, 12, 13, 14 5.20 15, 16, 17 5.71 18, 19, 20 6.41 21, 22, 23, 24, 25, 26 5.23 27, 28, 29, 30 5.83 31, 32, 33, 34, 35 5.79 36, 37, 38 5.27 39, 40, 41 6.90 42, 43, 46 4.70 * 5.30 48, 49, 52, 53 3.78 54, 55, 56 5.00 57, 58, 59 5.18 60, 61 5.60 62, 63, 64, 65 6.48 80, 81 8.08 66, 67, 68 5.40 70, 71, 72, 73 6.32 74, 75, 76, 77 5.48 78, 79 (2.0 * (2.0 * (2.0 82 5.54 83 5.00 84, 85, 86 4.48 * (2.0 * 6.18
Samples were taken from households. Milk collection centres. Farms. No isolations were carried out.
and fermentation vessels. All the samples, except one sample (No. 29), were fermented in metal pots, which could also be used for other domestic purposes. Sample No. 29, was obtained from milk fermented in a clay pot, which is the traditional fermentation vessel. 3.2. Identixcation Eighty strains of yeasts were isolated and identi"ed. Table 2 shows the yeast species identi"ed and their distribution among the samples. The identi"cation was carried out using API ID 32C kits (bio Merieux) and the SIM key (Deak & Beuchat, 1996) as well as reference to the standard taxonomic key outlined by Kutzman and Fell (1998). Twenty di!erent yeast species were identi"ed. The predominant species were S. cerevisiae (22 strains), C. lusitaniae (11), C. colliculosa (7), and S. dairenensis (7). while Dek. bruxillensis (5), C. lipolytica (4), and C. tropicalis (4) were identi"ed less often. Only one or two strains
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T.H. Gadaga et al. / International Dairy Journal 10 (2000) 459}466
Table 2 The diversity of yeasts isolated from Zimbabwean traditional fermented milks Yeast strain
Number of Number of Number of isolates samples sources
Saccharomyces cerevisiae 22 Candida lusitaniae 11 (Clavispora lusitaniae ) Candida colliculosa 7 (Torulaspora delbrueckii ) Saccharomyces dairinensis 7 Dekera bruxillensis 5 (Brettanomyces bruxillensis) Candida lipolytica 4 Candida tropicalis 4 Zygosaccharomyces spp. 2 Candida stellata 2 Candida krusei 1 Candida rugosa 1 Candida kefyr 1 (Kluyveromyces marxianus) Rhodotorula rubra 1 (mucilaginosa ) Saccharomyces pastorianus 1 Candida holmii 1 (Saccharomyces exiguus) Candida versatilis 1 Dekera anomala 1 (Brettanomyces anomalus) Cryptococcus albidus 1 Cryptococcus laurentii 1 Candida guillermondii 1 Unidenti"ed 5
11 6
6 6
5
4
6 4
2 3
3 2 2 1 1 1 1
2 2 2 1 1 1 1
1
1
1 1
1 1
1 1
1 1
1 1 1 4
1 1 1 2
Synonym. Anamorph.
were isolated for the other species. Although C. kefyr and C. rugosa had a low incidence in the samples, they are known to be important in dairy products (Fleet, 1990; Seiler & Busse, 1990), and their presence in the milk samples could be important. Comparing to other studies, it seems that di!erent yeast species predominate in di!erent fermented milk products. For example, S. cerevisiae together with G. geotrichum and K. marxianus were the predominant strains in makamo, a Ugandan traditional fermented milk (Sserunjogi, 1999), while other strains such as C. holmii, S. dairenensis, C. stellata and Zygosaccharomyces spp. were isolated in lesser numbers. Debaryomyces hansenii, Torulaspora delbrueckii and Klyveromyces marxianus were the predominant species found in South African traditional fermented milk (Loretan et al., 1998). Yarrowia lipolytica and Dek. anomala were found in lesser numbers. The physiological and morphological properties of some of the identi"ed yeast are shown in Table 3a and b. The 22 strains of S. cerevisiae were isolated from 11 samples and came from six di!erent sources (Table 2). S. cerevisiae was, therefore, a common species. The
strains successfully identi"ed as S. cerevisiae by both the API and SIM procedures could ferment sucrose and ra$nose in addition to fermenting glucose and galactose. They were not able to utilise lysine and cadavarine. Other reactions, which are considered diagnostic for S. cerevisiae, were lack of assimilation of ethylamine hydrochloride, D-ribose, D-mannitol and their inability to grow in vitamin-free medium (Kurtzman & Fell, 1998). Seven of the S. cerevisiae strains were able to assimilate DLlactate, but none was able to utilise lactose. Assimilation of lactate is a variable characteristic in S. cerevisiae (Kurtzman & Fell, 1998). C. lusitaniae is the anamorph (imperfect state) of Clavispora (Cl.) lusitaniae. The API identi"cation pro"le for all the strains of C. lusitaniae was recorded as very good. The 11 strains of C. lusitaniae were isolated from 6 of the samples and were not restricted to one source (Table 2). Therefore, C. lusitaniae can be said to be common in the amasi. C. lusitaniae has been mentioned as an important spoilage yeast in yoghurt (Green & Ibe, 1987; Jakobsen & Narvhus, 1996) and the species has been considered as one of the characteristic yeasts in dairies. This species has been isolated also from clinical samples but is not regarded as a human pathogen (Kutzman & Fell, 1998). This aspect, however, raises concerns about its suitability as a possible starter culture in milk fermentations. Additionally, none of the strains could utilise lactose, DL-lactate or citrate as a carbon source, which are important technological properties in milk fermentation. Torulaspora delbrueckii, the teleomorph of C. colliculosa, has been isolated frequently from dairy products (Fleet, 1990; Deak & Beuchat, 1996; Jakobsen & Narvhus, 1996; Loretan et al., 1998). The yeasts in this study were identi"ed as C. colliculosa by API ID 32C. All the strains, however, could produce ascospores (Table 3b) and it is, therefore, only appropriate to refer to the identi"ed strains with name of the holomorph, Torulaspora delbrueckii. All the strains could assimilate DL-lactate. SIM and the standard taxonomic key by Kutzman and Fell (1998) identi"ed some strains as S. dairenensis. API had classi"ed these as unacceptable S. cerevisiae because S. dairenensis is not included in the ID 32 C database. S. dairenensis is not a common isolate of dairy products, but has been isolated from silage and salads (Deak & Beuchat, 1996). The physiological and morphological characteristics listed in Table 3a seem to con"rm this identi"cation, but further study may be necessary to support this assumption. It should be noted that the API kits were developed primarily for identi"cation of clinical yeasts and the databases do not include some food borne yeasts (Heard, Fleet, Praphailog, & Addis, 1998). Four isolates were identi"ed as C. lipolytica. These were taken from three di!erent samples. C. lipolytica
Saccharomyces(S) cerevisiae
# # # # # ! ! ! # ! ! ! ! ! ! ! ! # ! nd #
Identixed as:
# # # # ! # ! ! # ! ! ! ! ! ! ! ! # ! nd !
# # # # ! # ! ! # ! ! ! ! ! ! ! ! # ! nd #
# # ! ! ! !
14
Assimilation of: Glucose Galactose Sucrose Maltose Cellobiose Trehalose Lactose Melibiose Ra$nose Erythritol MDG DL-Lactate Citrate Vitamin-free Cadavarine Lysine Cycloheximide 373C 50% glucose Ascospore Pseudohyphae
# # ! ! ! !
12
# # ! ! ! !
3, 4, 6, 7, 8, 9, 10, 11, 15, 17
Strain reference number
Fermentation of: Glucose Galactose Sucrose Maltose Lactose Ra$nose
(a) Substrate
# # # # ! # ! ! # ! ! ! ! ! ! ! ! # ! nd #
# # ! ! ! !
16
# # # # # # ! # # ! ! ! ! ! ! ! ! # ! # #
# # # ! ! !
20
# # # # ! # ! ! # ! ! # ! ! ! ! ! # ! ! !
# # # ! ! #
24
# # # ! ! # ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
# # # # ! #
29
Table 3 Identi"cation of yeasts isolated from Zimbabwean traditional fermented milk
# # # # ! # ! # # ! ! # ! ! ! ! ! # ! # !
# # # ! ! !
31, 34
# # # # ! # ! ! # ! ! # ! ! ! ! ! # ! # !
# # # ! ! !
35
# # # # ! ! ! ! # ! ! # ! ! ! ! ! # ! ! !
# # # ! ! #
71
# # # # ! ! ! ! # ! ! # ! ! ! ! ! # ! nd nd
# # # # ! #
81, 82
# ! ! ! ! ! nd !
# # ! ! ! # ! ! ! ! ! !
! # ! ! ! !
10
S. dairenensis
# # ! ! ! # ! ! ! ! ! ! ! ! ! ! ! # ! # !
# # s ! ! !
5
# # ! ! ! # ! ! ! ! ! ! ! ! ! ! ! # ! nd #
# # ! ! ! !
18, 19
# # ! ! ! # ! ! ! ! ! ! ! ! ! ! ! # ! # #
# # ! ! ! !
21
# # # ! ! # ! ! ! ! ! ! ! ! ! ! ! # ! # !
# # ! ! ! !
28
# # ! ! ! # ! ! ! ! ! # ! ! ! ! ! # ! # #
# # ! ! ! !
32
S. pastorianus
# # # # ! # ! # # # ! # ! ! ! ! ! ! ! ! #
# # ! # ! #
30
T.H. Gadaga et al. / International Dairy Journal 10 (2000) 459}466 463
C. tropicalis
Candida (C.) kefyr
Identixed as:
C. lipolytica
# # ! ! ! ! ! ! ! # ! # # # # # # ! ! ! #
! ! ! ! ! !
55, 57, 59, 67
# # # # # # ! ! ! ! # ! ! ! # # ! # # ! #
# # ! W ! !
53, 66, 68
C. lusitaniae
# # # # # # ! nd ! ! # ! ! ! # # ! # # nd #
# # ! ! ! !
63, 64
# # # # # # ! ! ! ! # ! ! ! # # ! # # nd #
# # ! ! ! !
83, 84, 85, 86
# # # # # # ! ! ! ! # ! ! ! # # ! # ! ! #
# # ! ! ! !
61, 62
# # # ! ! # ! ! ! ! ! ! ! ! ! ! ! # ! # !
# # ! ! ! !
33
C. colliculosa
# # # ! ! # ! ! ! ! ! # ! ! ! ! ! # ! # !
# # ! ! ! !
36, 37
# # # ! ! # ! ! ! ! ! # ! ! ! ! ! # ! # !
# # ! ! ! !
40
# # # ! ! # ! ! ! ! ! # ! ! ! ! ! # ! ! !
# # ! ! ! !
41
# # # ! ! # ! ! ! ! ! ! ! # ! ! ! # ! # #
# # ! ! ! !
42
# # # ! ! ! ! ! # ! ! # ! ! ! # ! # ! nd !
# # # ! ! !
70
Notes: nd"no data, s"slow, w"weak, #"positive test, !"negative test, MDG"methyl-D-glucoside.None of the isolates could assimilate inositol or nitrate. Results for soluble starch, D-xylose, L-arabinose, xylitol, D-manitol, ethylamine. HCl and growth on 16% NaCl are not included in the table.
# # # # # # ! ! ! ! # ! ! # # # # # # nd #
# # # ! ! ! # ! # ! ! # ! # # # # # ! ! #
Assimilation of: Glucose Galactose Sucrose Maltose Cellobiose Trehalose Lactose Melibiose Ra$nose Erythritol MDG DL-Lactate Citrate Vitamin-free Cadavarine Lysine Cyloheximide 373C 50% Glucose Ascospores Pseudohyphae
# # # # ! !
76, 77, 78, 79
# # # ! # #
23
Strain reference number
Fermentation of: Glucose Galactose Sucrose Maltose Lactose Ra$nose
(b) Substrate
Table 3 (Continued)
464 T.H. Gadaga et al. / International Dairy Journal 10 (2000) 459}466
T.H. Gadaga et al. / International Dairy Journal 10 (2000) 459}466
(Yarrowia lipolytica) is one of the most frequently occurring species in dairy products (Fleet, 1990; Jakobsen and Narvhus, 1996). This species is capable of degrading proteins using alkaline protease and also produces lipases (Roostita & Fleet, 1996; Wyder, 1998). It has been isolated more often from lipid and protein containing substrates, such as cheese, yoghurt or salads containing meat or shrimps as well as spoiled food (Wyder, 1998). It also metabolises lactic acid. These are important technological properties in fermented milk. However, extensive proteolysis and lipolysis is undesirable in fermented milk. The role of the isolated strain of C. lipolytica in fermented milk is the subject of current investigations. Kluyveromyces marxianus var. marxianus and its anamorph, Candida kefyr, are some of the most predominant and important yeast species in milk (Fleet, 1990). In this study, only one strain (No. 23) was identi"ed as Candida kefyr. However, C. kefyr is able to assimilate lactose, and also metabolises citrate to produce ethanol, glycerol, lactic acid and propionic acid in milk (Fleet, 1990; Fleet & Main, 1987). The strain isolated in this study did not assimilate citrate. This, however, is a variable characteristic in C. kefyr (Kutzman & Fell, 1998). The strain could assimilate lactose and DL-lactate and therefore has a potential for growth in milk. Four isolates were identi"ed as C. tropicalis. These produced smooth, butyrous, cream-white colonies, which were fringed with pseudohyphae. C. tropicalis is probably the third most important yeast pathogen of humans (Ahearn, 1998). This shows that the traditionally fermented milk can be a source of pathogenic microorganisms. Hence, it emphasizes the need to develop starter cultures to produce safer fermented milk products. Feresu and Nyati (1990) and Dalu and Feresu (1996) showed that enteric pathogens such as Listeria monocytogenes and pathogenic Escherichia coli were able to survive in traditionally fermented milk, showing that current practices of naturally fermenting milk are a public health concern. Occurrence of other pathogens such as C. tropicalis may also be a cause for concern. However, C. tropicalis has also been isolated from other dairy products such as Feta cheese (Tzanetakis, Hatzikamari, & Litopoulo-Tzanetaki, 1998), and yoghurt (Rohm et al., 1992). As for the other yeast strains isolated and identi"ed in this study, C. rugosa, Rho. rubra (mucilaginosa), C. krusei (Issatchenkia orientalis), C. versatilis, C. stellata and C. holmii have been reported in yoghurt, cheese, and makamo (Suriyarachchi & Fleet, 1981; Deak & Beuchat, 1996; Jakobsen & Narvhus, 1996; Tzanetakis et al., 1998; Viljoen, 1998; Sserunjogi, 1999). C. holmii is able to grow in milk by preferentially utilising galactose. Because it has an inducible hexokinase (which phosphorylates glucose) and a constitutive galactokinase, galactose will be
465
used "rst even in the presence of glucose (Marshall, 1993). Rho. rubra is associated with products based on milk fats (Jakobsen and Narvhus, 1996). Dek. anomala was also isolated from South African sour milk, although in low frequencies (Loretan et al., 1998). Dek. bruxillensis, Cry. albidus and Cry. laurentii are lipolytic and should therefore be able to grow in milk (Viljoen, 1998). The strain of C. versatilis identi"ed in this study could assimilate lactose (results not shown) while C. stellata could assimilate citrate. Thus, although these yeast strains were isolated less frequently, they constitute the normal #ora of dairy products and should, therefore, play a role in the quality of amasi.
4. Conclusion Twenty yeast species were identi"ed in this study, but only four species were predominant. This indicates that amasi may contain a wide variety of yeasts, but possibly only a few genera are able to multiply and thereby contribute to the characteristics of the fermented milk in the 48 hours of the fermentation. The standard of hygiene in the production of the fermented milk could be a key factor in the level of contamination, although some of the yeast strains such as S. cerevisiae are known to be ubiquitous in nature. The high numbers of yeasts up to 8.08 log cfu g\ suggests that the yeasts are able to multiply in the milk and may result in spoilage or, conversely, in enhancement of the #avour of the fermented milk. Most of the identi"ed yeasts would not grow alone in milk, but are able to assimilate glucose and galactose, and some are able to assimilate DL-lactate and citrate, suggesting a possible complex community of micro#ora in the fermented milk. It is evident that strains that ferment lactose such as C. kefyr impact on the sensory pro"le of the fermented milk. However, the situation is less clear with the other strains. Investigations to ascertain their role, especially, in the production of #avours and on the growth of acid producing lactic acid bacteria, are needed.
Acknowledgements The authors thank the Norwegian Universities Committee for Research, Development and Education (NUFU, Project 26/96), through the Agricultural University of Norway and the University of Zimbabwe, for "nancial assistance. We also thank Ms C. Museza and Mr. I. Kalima of the Department of Animal Science at the University of Zimbabwe for their technical assistance and the Chief Technician in the Department of Animal Science for allowing us to use the microbiology laboratory and equipment.
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References Ahearn, D. G. (1998). Yeasts pathogenic for humans. In C. P. Kurtzman, & J. W. Fell, The yeasts: A taxonomic study (pp. 9}12). Amsterdam: Elsevier. Bankole, M. O., & Okagbue, R. N. (1992). Properties of nono, a Nigerian fermented milk food. Ecology of Food and Nutrition, 27, 145}149. Deak, T., & Beuchat, L. R. (1996). Handbook of food spoilage yeasts. Boca Raton, FL: CRC Press. Dalu, J. M., & Feresu, S. B. (1996). Survival of Listeria monocytogenes in three Zimbabwean fermented milk products. Journal of Food Protection, 59, 379}383. Fekadu, B. (1994). Present situation and future aspects of milk production, milk handling and processing of dairy products in Southern Ethiopia. Ph.D. thesis, Agricultural University of Norway, As s, Norway. Feresu, S. B., & Muzondo, M. I. (1990). Identi"cation of some lactic acid bacteria from two Zimbabwean fermented milk products. World Journal of Microbiology and Biotechnology, 6, 178}186. Feresu, S. B., & Nyati, H. (1990). Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products. Journal of Applied Bacteriology, 69, 814}821. Fleet, G. H. (1990). Yeasts in dairy products*a review. Journal of Applied Bacteriology, 68, 199}211. Fleet, G. H., & Main, M. A. (1987). The occurrence and growth of yeasts in dairy products. International Journal of Food Microbiology, 4, 145}155. Gadaga, T. H., Mutukumira, A. N., Narvhus, J. A., & Feresu, S. B. (1999). A review of traditional fermented foods and beverages of Zimbabwe. International Journal of Food Microbiology, 53, 1}11. Green, M. D., & Ibe, S. N. (1987). Yeasts as primary contaminants in yoghurts produced commercially in Lagos, Nigeria. Journal of Food Protection, 50, 193}198. Heard, G. M., Fleet, G. H., Praphailog, W., & Addis, E. (1998). Evaluation of the BIOLOG system for identi"cation of yeasts from cheese. In M. Jakobsen, J. Narvhus, & B.C. Viljoen, Yeasts in the dairy industry: Positive and negative aspects, Proceedings of the symposium organised by Group F47, 2}3 September 1996 (pp. 117}124). International Dairy Federation, Brussels, Belgium. IDF (1990). International standard 94B: Milk and milk products; Enumeration of yeasts and moulds*colony count technique at 253C. International Dairy Federation, Brussels, Belgium. Jakobsen, M., & Narvhus, J. (1996). Yeasts and their possible bene"cial and negative e!ects on the quality of dairy products. International Dairy Journal, 6, 755}768. Kurtzman, C. P. & Fell, J. W. (Eds.). (1998). The yeasts: A taxonomic study. Elsevier: Amsterdam. Loretan, T., Viljoen, B. C., Mostert, J. F., Vogel, A.-M., & Jordaan, H. F. du P. (1998). A preliminary study of the diversity and technological properties of indigenous traditional South African fermented milk (Note). In M. Jakobsen, J. Narvhus, & B. C. Viljoen, Yeasts in the dairy industry: Positive and negative aspects, Proceedings of the symposium organised by Group F47, 2}3 September 1996 (pp. 178}182). Brussels, Belgium.
Marshall, V. M. (1987). Fermented milks and their future trends: I. Microbiological aspects. Journal of Dairy Research, 54, 559}574. Marshall, V. M. (1993). Starter cultures for milk fermentation and their characteristics. Journal of the Society of Dairy Technology, 46(2), 49}56. Mogessie, A. (1990). E!ect of curd cooking temperatures on the microbiological quality of ayib, a traditional Ethiopian cottage cheese. World Journal of Microbiology and Biotechnology, 6, 159}162. Mutukumira, A. N. (1995). Properties of amasi, a natural fermented milk produced by smallholder milk producers in Zimbabwe. Milchwissenschaft, 50, 201}205. Mutukumira, A. N. (1996). Investigation of some prospects for the development of starter cultures for industrial production of traditional fermented milk in Zimbabwe. Ph.D. thesis, Agricultural University of Norway, As s, Norway. Narvhus, J. A., "steraas, K., Mutukumira, T., & Abrahamsen, R. K. (1998). Production of fermented milk using a malty compound producing strain of Lactococcus lactis subsp lactis biovar. diacetylactis isolated from Zimbabwean naturally fermented milk. International Journal of Food Microbiology, 41, 73}80. Rohm, H., Eliskases-Lechner, F., & Brauer, M. (1992). Diversity of yeasts in selected dairy products. Journal of Applied Bacteriology, 72, 370}376. Roostita, R., & Fleet, G. H. (1996). Growth of yeasts in milk and associated changes to milk composition. International Journal of Food Microbiology, 31, 215}219. Seiler, H., & Busse, M. (1990). The yeasts of cheese brines. International Journal of Food Microbiology, 11, 289}304. Sserunjogi, M. L. (1999). Ugandan indigenous fermented dairy products, with particular focus on ghee. Ph.D. thesis, Agricultural University of Norway, As s, Norway. Suriyarachchi, V. R., & Fleet, G. H. (1981). Occurrence and growth of yeasts in yogurts. Applied and Environmental Microbiology, 42, 574}579. Tzanetakis, N., Hatzikamari, M., & Litopoulo-Tzanetaki, E. (1998). Yeast of the surface micro#ora of Feta Cheese. In M. Jakobsen, J. Narvhus, & B. C. Viljoen, Yeasts in the dairy industry: Positive and negative aspects, Proceedings of the symposium organised by Group F47, 2}3 September 1996 (pp. 34}43). IDF, Brussels, Belgium. Van der Walt, J. P., & Yarrow, D. (1984). Methods for the isolation, maintenance, classi"cation and identi"cation of yeasts. In N. J. W. Kreger-van Rij, The yeasts: A taxonomic study (pp. 45}105). Amsterdam: Elsevier Science. Viljoen, B. C. (1998). The ecological diversity of yeasts in dairy products. In M. Jakobsen, J. Narvhus, & B. C. Viljoen, Yeasts in the dairy industry: Positive and negative aspects, Proceedings of the symposium organised by Group F47, 2}3 September 1996 (pp. 70}77). IDF, Brussels, Belgium. Wyder, M. T. (1998). Identixcation and characterisation of the yeast yora in kefyr and smear ripened cheese: Contribution of selected yeasts to cheese ripening. Ph.D. dissertation, ETH, Zurich, Switzerland.
Enumeration and identi"cation of yeasts isolated from Zimbabwean traditional fermented milk T.H. Gadaga *, A.N. Mutukumira , J.A. Narvhus Institute of Food, Nutrition and Family Sciences, University of Zimbabwe, P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe Department of Food Science, Agricultural University of Norway, P.O. Box 5036, N-1432 As s, Norway Received 28 February 2000; accepted 19 June 2000
Abstract The yeasts in 30 samples of the Zimbabwean traditional fermented milk, amasi, taken from farms, households and milk collection centres were enumerated and identi"ed. The yeast counts ranged from(2 to 8.08 log cfu g\. Yeast isolates were identi"ed using the API ID 323C test kits and the simpli"ed identi"cation method (SIM) as well as with reference to the standard taxonomic keys. From the 30 samples, a total of 20 di!erent yeast species were identi"ed. Saccharomyces (S) cerevisiae (22 isolates), Candida (C) lusitaniae (11), C. colliculosa (7) and S. dairenensis (7) were the predominant species identi"ed. Dekera (Dek.) bruxillensis, C. lipolytica and C. tropicalis were identi"ed less often. Seven of the S. cerevisiae isolates were able to assimilate DL-lactate. The strain of C. kefyr isolated could assimilate lactose and DL-lactate, but not citrate. The analysed amasi samples contained a wide variety of yeasts, but only a few species predominated and these could possibly contribute to the characteristics of the fermented milk in the 48 h fermentation. 2000 Published by Elsevier Science Ltd. Keywords: Yeasts; Fermented milk; Amasi
1. Introduction Fermented milk is an important part of the traditional diet in Zimbabwe (Mutukumira, 1995; Narvhus, "steraas, Mutukumira, & Abrahamsen, 1998). In the traditional fermentation process, raw milk is left to ferment spontaneously at ambient temperature. A mixed microbial #ora consisting of lactic acid bacteria, yeasts and moulds and enterococci plays a part in the fermentation (Mutukumira, 1995). In Zimbabwe, traditional fermented milk produced at household level is called hodzeko, amasi or mukaka wakakora (Feresu & Muzondo, 1990; Mutukumira, 1995; Gadaga, Mutukumira, Narvhus, & Feresu, 1999). This product is a result of natural fermentation of untreated raw milk in earthenware or metal pots. The fermentation is allowed to proceed for a period of up to 48 h, after which some whey is drained o! to obtain a product with a thick consistency (Mutukumira, 1995). The milk has a characteristic mild lactic acid and aromatic #avour, due
* Corresponding author. Fax: #47-64-94-37-89. E-mail address: [email protected] (T.H. Gadaga).
to the lactic acid, other organic acids, and volatile compounds produced by the mixed culture of microorganisms. The microbiological studies that have been carried out on amasi have focused on safety aspects and on the identi"cation of lactic acid bacteria (Mutukumira, 1996; Feresu & Muzondo, 1990). Lactococcus (L.) lactis subsp. lactis, Lactobacillus (Lb.) plantarum and Lb. helveticus were predominant isolates. Yeasts and moulds, as well as coliforms in amasi were also enumerated, but were not identi"ed (Mutukumira, 1995). The yeasts and moulds were considered as undesirable and a sign of poor hygiene. However, these could be an essential part of the micro#ora of amasi, hence the need to study the diversity of yeasts in the fermented milk. Apart from causing spoilage in products such as yoghurt and sour milk, yeasts are also important because they produce desirable #avours as in cheese ripening (Marshall, 1987; Fleet, 1990; Rohm, Eliskases-Lechner, & Brauer, 1992; Jakobsen & Narvhus, 1996). The low pH in the fermented milk o!ers a selective environment for yeast growth, but is unfavourable for most bacteria (Fleet, 1990; Rohm et al., 1992; Deak & Beuchat, 1996). Spoilage becomes evident when the yeast population reaches 10}10 cells g\ (Fleet, 1990).
0958-6946/00/$ - see front matter 2000 Published by Elsevier Science Ltd. PII: S 0 9 5 8 - 6 9 4 6 ( 0 0 ) 0 0 0 7 0 - 4
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Other fermented milk foods produced by traditional methods may have microorganisms other than lactic acid bacteria playing a signi"cant role in the fermentation process. There is increasing interest in the role of yeasts in dairy fermentations and especially their potential use as starter cultures (Fleet, 1990; Jakobsen & Narvhus, 1996; Loretan, Viljoen, Mostert, Vogel, & Jordaan, 1998). To establish the role of yeasts in amasi, the yeasts have to be isolated and identi"ed and then their technological properties studied. The aim of this study was to isolate, enumerate and identify the yeasts in the Zimbabwean fermented milk, amasi/hodzeko.
2. Materials and methods 2.1. Sampling Thirty samples of amasi, which had been fermented for two days (48 h), were collected from small farms, homes and milk collection centres in Zimbabwe in sterile 250 mL wide-mouthed screw-capped bottles and brought to the laboratory in a cooler box. The samples were analysed within 3 h of sampling.
2.4. Fermentation of sugars The fermentation of D-glucose, sucrose, D-galactose, lactose, maltose and ra$nose was tested, according to the description of Van der Walt and Yarrow (1984). A positive result was indicated by accumulation of gas in the Durham tubes. 2.5. Liquid assimilation of carbon compounds Distilled water (4.5 mL) in plugged 150;16 mm tubes was sterilised by autoclaving at 1213C for 15 min. An aliquot (0.5 mL) of "lter-sterilised yeast nitrogen base (Difco Laboratories, Detroit, MI, USA) containing 5% of the compound under test was aseptically added to the tubes. The tubes were inoculated by aseptically adding 0.1 mL of a visible suspension in Ringers solution (Oxoid) of an actively growing culture. The carbon compounds tested were galactose, glucose, sucrose, lactose, L-arabinose, maltose, D-mannitol, melibiose, ra$nose, soluble starch, trehalose, xylose, a-methyl-D-glucoside, cellobiose, erythritol, xylitol, citrate and DL-lactate. The tubes were inoculated as in the sugar fermentation tests. A positive reaction was detected by visual inspection for an increase in the turbidity of the solution.
2.2. Enumeration and isolation 2.6. Assimilation of nitrogen compounds The yeasts were enumerated on spread plates of yeast extract glucose chloramphenicol agar (YGCA) (IDF, 1990), after suspending the sample (10 g) in 90 mL quarter strength Ringers' solution (Oxoid, Unipath Ltd, Basingstoke, England), and making serial dilutions. The medium was sterilised by heating at 1213C for 15 min. The plates were incubated at 253C for 5 days. Colonies with distinct morphological di!erences such as colour, shape and size were picked and puri"ed by streaking at least three times on malt extract agar (MEA) (Merck, Darmstadt, Germany). The puri"ed isolates were stored on MEA slants at 43C until required for identi"cation. 2.3. Identixcation The yeasts were identi"ed based on their physiological and morphological properties as described by Deak and Beuchat (1996), Van der Walt and Yarrow (1984), and also by using the API ID32 C test strips for yeast (bioMerieux, Marcy l'Etoile, France). The tests included the fermentation of sugars, liquid assimilation of carbon compounds, liquid assimilation of nitrogen compounds, growth at 373C, growth on 50% (w/v) glucose yeast extract agar, growth in vitamin-free medium, growth in media containing 16% NaCl, resistance to 0.01% cycloheximide, and urease activity. API ID32 C strips have 30 cupules containing di!erent carbohydrates and one containing actidione (cycloheximide). The strips were used according to the manufacturer's instructions.
The procedure was similar to the carbon compound assimilation tests except that yeast nitrogen base was replaced by yeast carbon base (Difco). The nitrogen compounds tested were nitrate, ethylamine hydrochloride, L-lysine and cadavarine. 2.7. Hydrolysis of urea This was tested using the method of Van der Walt & Yarrow (1984). Commercially produced Christensen's urea agar base (Merck) was used. The slant was inoculated from a suspension of the actively growing yeast culture using a sterile wire loop and incubated at 253C for 4 days. A positive reaction was the development of a deep pink colour in the agar. 2.8. Other tests Formation of ballistoconidia was examined on corn meal agar (Merck), by making two streaks at right angles to each other on the agar. The plate containing the corn meal agar was then inverted over another containing malt extract agar over which a sterile glass slide had been placed. The two plates were taped together along their entire circumference and incubated at 253C for up to 14 days. Ascospore formation was examined on malt extract agar (MEA) (Merck), potato dextrose agar (PDA) (Merck), and Gorodkova agar according
T.H. Gadaga et al. / International Dairy Journal 10 (2000) 459}466
to the procedure described by Van der Walt and Yarrow (1984). The plates were incubated at 253C for seven days. Formation of mycelium and pseudo-hyphae was studied on yeast morphology agar (Difco) using the Dalmau plate technique (Van der Walt & Yarrow, 1984). A three day-old liquid culture in malt extract broth was examined microscopically as a wet mount for cellular morphology.
3. Results and discussion 3.1. Enumeration of yeasts The yeasts counts ranged from less than 2} 8.08 log cfu g\ (Table 1). One sample (Number 19Amasi) had viable yeast counts of 8.08 log cfu g\. This sample could have been fermented over more than two days. The practice in that part of the country where the sample was taken is to ferment milk over more than three or four days, mixing several days' milkings and subsequent whey removal to produce a thick product, which has the consistency of cottage cheese. This product had a sharp acid taste. These viable counts, however, are comparable to those reported by Mutukumira (1995). From ten spontaneously fermented milk samples collected from Nharira milk collection centre, Lancashire, Zimbabwe, combined yeast and mould counts ranged between (3 to 5.65 log cfu g\. Loretan et al. (1998) reported yeast counts ranging between 10 and 10 cfu mL\ in ten sour milk samples collected from South African homes. The yeast and mould counts obtained from Ethiopian fermented milk were on average 6.18 log cfu mL\ for Ititu and Meomata (Fekadu, 1994) and 5.8 log cfu mL\ for fermented milk from Southern Ethiopia (Mogessie, 1990). Bankole and Okagbue (1992) also studied samples of nono, a Nigerian fermented milk, and they reported yeast counts of 1.5;10 cfu g\. These results show that yeasts constitute a signi"cant part of the micro#ora of naturally fermented milk. At such high levels, the yeast metabolism should impact on the overall quality and acceptability of these products. In our study, samples taken from the same source showed varying yeast counts. Mutukumira (1996) observed the variable quality of the naturally fermented milk, which could be indicative of an inconsistent micro#ora, and yeasts could be involved. Particularly low counts of yeasts ((2 log cfu g\) were recorded for three sources, Murehwa, Nyarungu and Nyadire. Nyarungu is a milk collection and training centre of the Zimbabwe Dairy Development Programme and the low yeast counts could possibly be explained by better hygienic practices at this centre compared to the other sources of samples. The incidence of yeasts in all the samples, however, may suggest that yeasts are a common #ora of the milking parlours, milking containers
461
Table 1 Colony counts of yeasts isolated from Zimbabwean traditional fermented milk Sample
Source
Strain ref. number
log cfu g\
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Mavambo Mavambo Mavambo Mavambo Mavambo Dr Charp Nharira Mavambo Mavambo Mavambo J. Mukurazhizha The 44 The 44 The 44 Mavambo Mavambo Tamutsa Mavambo Amasi from Bulawayo University farm University farm University farm Murehwa Nyarungu Nyarungu Murehwa Murehwa Crowhill farm Nyadire Murehwa
1, 3 2.48 4, 5, 6, 7, 8 5.32 9, 10 4.85 11, 12, 13, 14 5.20 15, 16, 17 5.71 18, 19, 20 6.41 21, 22, 23, 24, 25, 26 5.23 27, 28, 29, 30 5.83 31, 32, 33, 34, 35 5.79 36, 37, 38 5.27 39, 40, 41 6.90 42, 43, 46 4.70 * 5.30 48, 49, 52, 53 3.78 54, 55, 56 5.00 57, 58, 59 5.18 60, 61 5.60 62, 63, 64, 65 6.48 80, 81 8.08 66, 67, 68 5.40 70, 71, 72, 73 6.32 74, 75, 76, 77 5.48 78, 79 (2.0 * (2.0 * (2.0 82 5.54 83 5.00 84, 85, 86 4.48 * (2.0 * 6.18
Samples were taken from households. Milk collection centres. Farms. No isolations were carried out.
and fermentation vessels. All the samples, except one sample (No. 29), were fermented in metal pots, which could also be used for other domestic purposes. Sample No. 29, was obtained from milk fermented in a clay pot, which is the traditional fermentation vessel. 3.2. Identixcation Eighty strains of yeasts were isolated and identi"ed. Table 2 shows the yeast species identi"ed and their distribution among the samples. The identi"cation was carried out using API ID 32C kits (bio Merieux) and the SIM key (Deak & Beuchat, 1996) as well as reference to the standard taxonomic key outlined by Kutzman and Fell (1998). Twenty di!erent yeast species were identi"ed. The predominant species were S. cerevisiae (22 strains), C. lusitaniae (11), C. colliculosa (7), and S. dairenensis (7). while Dek. bruxillensis (5), C. lipolytica (4), and C. tropicalis (4) were identi"ed less often. Only one or two strains
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Table 2 The diversity of yeasts isolated from Zimbabwean traditional fermented milks Yeast strain
Number of Number of Number of isolates samples sources
Saccharomyces cerevisiae 22 Candida lusitaniae 11 (Clavispora lusitaniae ) Candida colliculosa 7 (Torulaspora delbrueckii ) Saccharomyces dairinensis 7 Dekera bruxillensis 5 (Brettanomyces bruxillensis) Candida lipolytica 4 Candida tropicalis 4 Zygosaccharomyces spp. 2 Candida stellata 2 Candida krusei 1 Candida rugosa 1 Candida kefyr 1 (Kluyveromyces marxianus) Rhodotorula rubra 1 (mucilaginosa ) Saccharomyces pastorianus 1 Candida holmii 1 (Saccharomyces exiguus) Candida versatilis 1 Dekera anomala 1 (Brettanomyces anomalus) Cryptococcus albidus 1 Cryptococcus laurentii 1 Candida guillermondii 1 Unidenti"ed 5
11 6
6 6
5
4
6 4
2 3
3 2 2 1 1 1 1
2 2 2 1 1 1 1
1
1
1 1
1 1
1 1
1 1
1 1 1 4
1 1 1 2
Synonym. Anamorph.
were isolated for the other species. Although C. kefyr and C. rugosa had a low incidence in the samples, they are known to be important in dairy products (Fleet, 1990; Seiler & Busse, 1990), and their presence in the milk samples could be important. Comparing to other studies, it seems that di!erent yeast species predominate in di!erent fermented milk products. For example, S. cerevisiae together with G. geotrichum and K. marxianus were the predominant strains in makamo, a Ugandan traditional fermented milk (Sserunjogi, 1999), while other strains such as C. holmii, S. dairenensis, C. stellata and Zygosaccharomyces spp. were isolated in lesser numbers. Debaryomyces hansenii, Torulaspora delbrueckii and Klyveromyces marxianus were the predominant species found in South African traditional fermented milk (Loretan et al., 1998). Yarrowia lipolytica and Dek. anomala were found in lesser numbers. The physiological and morphological properties of some of the identi"ed yeast are shown in Table 3a and b. The 22 strains of S. cerevisiae were isolated from 11 samples and came from six di!erent sources (Table 2). S. cerevisiae was, therefore, a common species. The
strains successfully identi"ed as S. cerevisiae by both the API and SIM procedures could ferment sucrose and ra$nose in addition to fermenting glucose and galactose. They were not able to utilise lysine and cadavarine. Other reactions, which are considered diagnostic for S. cerevisiae, were lack of assimilation of ethylamine hydrochloride, D-ribose, D-mannitol and their inability to grow in vitamin-free medium (Kurtzman & Fell, 1998). Seven of the S. cerevisiae strains were able to assimilate DLlactate, but none was able to utilise lactose. Assimilation of lactate is a variable characteristic in S. cerevisiae (Kurtzman & Fell, 1998). C. lusitaniae is the anamorph (imperfect state) of Clavispora (Cl.) lusitaniae. The API identi"cation pro"le for all the strains of C. lusitaniae was recorded as very good. The 11 strains of C. lusitaniae were isolated from 6 of the samples and were not restricted to one source (Table 2). Therefore, C. lusitaniae can be said to be common in the amasi. C. lusitaniae has been mentioned as an important spoilage yeast in yoghurt (Green & Ibe, 1987; Jakobsen & Narvhus, 1996) and the species has been considered as one of the characteristic yeasts in dairies. This species has been isolated also from clinical samples but is not regarded as a human pathogen (Kutzman & Fell, 1998). This aspect, however, raises concerns about its suitability as a possible starter culture in milk fermentations. Additionally, none of the strains could utilise lactose, DL-lactate or citrate as a carbon source, which are important technological properties in milk fermentation. Torulaspora delbrueckii, the teleomorph of C. colliculosa, has been isolated frequently from dairy products (Fleet, 1990; Deak & Beuchat, 1996; Jakobsen & Narvhus, 1996; Loretan et al., 1998). The yeasts in this study were identi"ed as C. colliculosa by API ID 32C. All the strains, however, could produce ascospores (Table 3b) and it is, therefore, only appropriate to refer to the identi"ed strains with name of the holomorph, Torulaspora delbrueckii. All the strains could assimilate DL-lactate. SIM and the standard taxonomic key by Kutzman and Fell (1998) identi"ed some strains as S. dairenensis. API had classi"ed these as unacceptable S. cerevisiae because S. dairenensis is not included in the ID 32 C database. S. dairenensis is not a common isolate of dairy products, but has been isolated from silage and salads (Deak & Beuchat, 1996). The physiological and morphological characteristics listed in Table 3a seem to con"rm this identi"cation, but further study may be necessary to support this assumption. It should be noted that the API kits were developed primarily for identi"cation of clinical yeasts and the databases do not include some food borne yeasts (Heard, Fleet, Praphailog, & Addis, 1998). Four isolates were identi"ed as C. lipolytica. These were taken from three di!erent samples. C. lipolytica
Saccharomyces(S) cerevisiae
# # # # # ! ! ! # ! ! ! ! ! ! ! ! # ! nd #
Identixed as:
# # # # ! # ! ! # ! ! ! ! ! ! ! ! # ! nd !
# # # # ! # ! ! # ! ! ! ! ! ! ! ! # ! nd #
# # ! ! ! !
14
Assimilation of: Glucose Galactose Sucrose Maltose Cellobiose Trehalose Lactose Melibiose Ra$nose Erythritol MDG DL-Lactate Citrate Vitamin-free Cadavarine Lysine Cycloheximide 373C 50% glucose Ascospore Pseudohyphae
# # ! ! ! !
12
# # ! ! ! !
3, 4, 6, 7, 8, 9, 10, 11, 15, 17
Strain reference number
Fermentation of: Glucose Galactose Sucrose Maltose Lactose Ra$nose
(a) Substrate
# # # # ! # ! ! # ! ! ! ! ! ! ! ! # ! nd #
# # ! ! ! !
16
# # # # # # ! # # ! ! ! ! ! ! ! ! # ! # #
# # # ! ! !
20
# # # # ! # ! ! # ! ! # ! ! ! ! ! # ! ! !
# # # ! ! #
24
# # # ! ! # ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
# # # # ! #
29
Table 3 Identi"cation of yeasts isolated from Zimbabwean traditional fermented milk
# # # # ! # ! # # ! ! # ! ! ! ! ! # ! # !
# # # ! ! !
31, 34
# # # # ! # ! ! # ! ! # ! ! ! ! ! # ! # !
# # # ! ! !
35
# # # # ! ! ! ! # ! ! # ! ! ! ! ! # ! ! !
# # # ! ! #
71
# # # # ! ! ! ! # ! ! # ! ! ! ! ! # ! nd nd
# # # # ! #
81, 82
# ! ! ! ! ! nd !
# # ! ! ! # ! ! ! ! ! !
! # ! ! ! !
10
S. dairenensis
# # ! ! ! # ! ! ! ! ! ! ! ! ! ! ! # ! # !
# # s ! ! !
5
# # ! ! ! # ! ! ! ! ! ! ! ! ! ! ! # ! nd #
# # ! ! ! !
18, 19
# # ! ! ! # ! ! ! ! ! ! ! ! ! ! ! # ! # #
# # ! ! ! !
21
# # # ! ! # ! ! ! ! ! ! ! ! ! ! ! # ! # !
# # ! ! ! !
28
# # ! ! ! # ! ! ! ! ! # ! ! ! ! ! # ! # #
# # ! ! ! !
32
S. pastorianus
# # # # ! # ! # # # ! # ! ! ! ! ! ! ! ! #
# # ! # ! #
30
T.H. Gadaga et al. / International Dairy Journal 10 (2000) 459}466 463
C. tropicalis
Candida (C.) kefyr
Identixed as:
C. lipolytica
# # ! ! ! ! ! ! ! # ! # # # # # # ! ! ! #
! ! ! ! ! !
55, 57, 59, 67
# # # # # # ! ! ! ! # ! ! ! # # ! # # ! #
# # ! W ! !
53, 66, 68
C. lusitaniae
# # # # # # ! nd ! ! # ! ! ! # # ! # # nd #
# # ! ! ! !
63, 64
# # # # # # ! ! ! ! # ! ! ! # # ! # # nd #
# # ! ! ! !
83, 84, 85, 86
# # # # # # ! ! ! ! # ! ! ! # # ! # ! ! #
# # ! ! ! !
61, 62
# # # ! ! # ! ! ! ! ! ! ! ! ! ! ! # ! # !
# # ! ! ! !
33
C. colliculosa
# # # ! ! # ! ! ! ! ! # ! ! ! ! ! # ! # !
# # ! ! ! !
36, 37
# # # ! ! # ! ! ! ! ! # ! ! ! ! ! # ! # !
# # ! ! ! !
40
# # # ! ! # ! ! ! ! ! # ! ! ! ! ! # ! ! !
# # ! ! ! !
41
# # # ! ! # ! ! ! ! ! ! ! # ! ! ! # ! # #
# # ! ! ! !
42
# # # ! ! ! ! ! # ! ! # ! ! ! # ! # ! nd !
# # # ! ! !
70
Notes: nd"no data, s"slow, w"weak, #"positive test, !"negative test, MDG"methyl-D-glucoside.None of the isolates could assimilate inositol or nitrate. Results for soluble starch, D-xylose, L-arabinose, xylitol, D-manitol, ethylamine. HCl and growth on 16% NaCl are not included in the table.
# # # # # # ! ! ! ! # ! ! # # # # # # nd #
# # # ! ! ! # ! # ! ! # ! # # # # # ! ! #
Assimilation of: Glucose Galactose Sucrose Maltose Cellobiose Trehalose Lactose Melibiose Ra$nose Erythritol MDG DL-Lactate Citrate Vitamin-free Cadavarine Lysine Cyloheximide 373C 50% Glucose Ascospores Pseudohyphae
# # # # ! !
76, 77, 78, 79
# # # ! # #
23
Strain reference number
Fermentation of: Glucose Galactose Sucrose Maltose Lactose Ra$nose
(b) Substrate
Table 3 (Continued)
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(Yarrowia lipolytica) is one of the most frequently occurring species in dairy products (Fleet, 1990; Jakobsen and Narvhus, 1996). This species is capable of degrading proteins using alkaline protease and also produces lipases (Roostita & Fleet, 1996; Wyder, 1998). It has been isolated more often from lipid and protein containing substrates, such as cheese, yoghurt or salads containing meat or shrimps as well as spoiled food (Wyder, 1998). It also metabolises lactic acid. These are important technological properties in fermented milk. However, extensive proteolysis and lipolysis is undesirable in fermented milk. The role of the isolated strain of C. lipolytica in fermented milk is the subject of current investigations. Kluyveromyces marxianus var. marxianus and its anamorph, Candida kefyr, are some of the most predominant and important yeast species in milk (Fleet, 1990). In this study, only one strain (No. 23) was identi"ed as Candida kefyr. However, C. kefyr is able to assimilate lactose, and also metabolises citrate to produce ethanol, glycerol, lactic acid and propionic acid in milk (Fleet, 1990; Fleet & Main, 1987). The strain isolated in this study did not assimilate citrate. This, however, is a variable characteristic in C. kefyr (Kutzman & Fell, 1998). The strain could assimilate lactose and DL-lactate and therefore has a potential for growth in milk. Four isolates were identi"ed as C. tropicalis. These produced smooth, butyrous, cream-white colonies, which were fringed with pseudohyphae. C. tropicalis is probably the third most important yeast pathogen of humans (Ahearn, 1998). This shows that the traditionally fermented milk can be a source of pathogenic microorganisms. Hence, it emphasizes the need to develop starter cultures to produce safer fermented milk products. Feresu and Nyati (1990) and Dalu and Feresu (1996) showed that enteric pathogens such as Listeria monocytogenes and pathogenic Escherichia coli were able to survive in traditionally fermented milk, showing that current practices of naturally fermenting milk are a public health concern. Occurrence of other pathogens such as C. tropicalis may also be a cause for concern. However, C. tropicalis has also been isolated from other dairy products such as Feta cheese (Tzanetakis, Hatzikamari, & Litopoulo-Tzanetaki, 1998), and yoghurt (Rohm et al., 1992). As for the other yeast strains isolated and identi"ed in this study, C. rugosa, Rho. rubra (mucilaginosa), C. krusei (Issatchenkia orientalis), C. versatilis, C. stellata and C. holmii have been reported in yoghurt, cheese, and makamo (Suriyarachchi & Fleet, 1981; Deak & Beuchat, 1996; Jakobsen & Narvhus, 1996; Tzanetakis et al., 1998; Viljoen, 1998; Sserunjogi, 1999). C. holmii is able to grow in milk by preferentially utilising galactose. Because it has an inducible hexokinase (which phosphorylates glucose) and a constitutive galactokinase, galactose will be
465
used "rst even in the presence of glucose (Marshall, 1993). Rho. rubra is associated with products based on milk fats (Jakobsen and Narvhus, 1996). Dek. anomala was also isolated from South African sour milk, although in low frequencies (Loretan et al., 1998). Dek. bruxillensis, Cry. albidus and Cry. laurentii are lipolytic and should therefore be able to grow in milk (Viljoen, 1998). The strain of C. versatilis identi"ed in this study could assimilate lactose (results not shown) while C. stellata could assimilate citrate. Thus, although these yeast strains were isolated less frequently, they constitute the normal #ora of dairy products and should, therefore, play a role in the quality of amasi.
4. Conclusion Twenty yeast species were identi"ed in this study, but only four species were predominant. This indicates that amasi may contain a wide variety of yeasts, but possibly only a few genera are able to multiply and thereby contribute to the characteristics of the fermented milk in the 48 hours of the fermentation. The standard of hygiene in the production of the fermented milk could be a key factor in the level of contamination, although some of the yeast strains such as S. cerevisiae are known to be ubiquitous in nature. The high numbers of yeasts up to 8.08 log cfu g\ suggests that the yeasts are able to multiply in the milk and may result in spoilage or, conversely, in enhancement of the #avour of the fermented milk. Most of the identi"ed yeasts would not grow alone in milk, but are able to assimilate glucose and galactose, and some are able to assimilate DL-lactate and citrate, suggesting a possible complex community of micro#ora in the fermented milk. It is evident that strains that ferment lactose such as C. kefyr impact on the sensory pro"le of the fermented milk. However, the situation is less clear with the other strains. Investigations to ascertain their role, especially, in the production of #avours and on the growth of acid producing lactic acid bacteria, are needed.
Acknowledgements The authors thank the Norwegian Universities Committee for Research, Development and Education (NUFU, Project 26/96), through the Agricultural University of Norway and the University of Zimbabwe, for "nancial assistance. We also thank Ms C. Museza and Mr. I. Kalima of the Department of Animal Science at the University of Zimbabwe for their technical assistance and the Chief Technician in the Department of Animal Science for allowing us to use the microbiology laboratory and equipment.
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