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Food Research International 40 (2007) 33–38

The effect of container type on the growth of yeastand lactic acid bacteria during production of Sethemi,

South African spontaneously fermented milk

Ameha Kebede a, B.C. Viljoen a,*, T.H. Gadaga b, J.A. Narvhus c, A. Lourens-Hattingh a

a Department of Microbial, Biochemical and Food Biotechnology, University of The Free State, P.O. Box 339, Bloemfontein, South Africab Institute of Food, Nutrition and Family Sciences, University of Zimbabwe, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe

c Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 As, Norway

Received 3 April 2006; accepted 30 July 2006

Abstract

Sethemi, a South African traditional fermented milk, was prepared in the laboratory by spontaneously fermenting raw milk in fourdifferent types of containers; namely, a clay pot, gourd, nickel jar and plastic bowl. The development of bacteria and yeasts in the dif-ferent containers was followed by taking samples at 24 h intervals for 7 days. The predominating microorganisms in all cases were theaerobic mesophilic bacteria, lactobacilli–leuconostocs (LAB on MRS) and lactococci (LAB on M17). No significant (p > 0.05) differenceswere found in the number of microorganisms between the different containers. However, there were significant (p < 0.05) differences withrespect to the types of dominant yeast species, and the diversity of yeast species. The least time to attain maximum LAB counts wasrecorded for the nickel container. In milk fermented in the clay pot and gourd the most dominant yeast species were Debaryomyces hanse-

nii and Saccharomyces cerevisiae, respectively, while Cryptococcus curvatus predominated in both the nickel and plastic containers. Thehighest yeast diversity was obtained from milk samples fermented in the clay pot represented by 15 species. Of the 23 species isolatedfrom all the containers and raw milk, only D. hansenii, Cryptococcus humicola and Kluyveromyces marxianus were isolated from bothraw and naturally fermented milks of all containers. The use of different types of containers therefore influenced the diversity of yeastsin the fermented milk and may influence the characteristics of the fermented milk product. The clay pot offered the greatest yeast diver-sity suggesting that it could be the best suited for preparing traditional fermented milk.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Fermented milk; Sethemi; Yeast; Lactic acid bacteria

1. Introduction

Fermentation is one of the oldest food processing tech-nologies in the world, which has been passed down fromgeneration to generation. According to Walshe, Grindle,Nell, and Bachmann (1991), most of the knowledge aboutfermentation has not been documented and is in danger ofbeing lost as technologies evolve and families move awayfrom traditional food preservation practices.

0963-9969/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodres.2006.07.012

* Corresponding author. Tel.: +27 51 401 2621; fax: +27 51 444 3219.E-mail address: Viljoenbc.sci@mail.uovs.ac.za (B.C. Viljoen).

Recent studies have documented the quality and diversityof African traditionally fermented foods, including naturallyfermented milks (Abdelgadir, Ahmed, & Dirar, 1998;El-Gendy, 1983; FAO, 1990; Gran, Wetlesen, Mutukumira,& Narvhus, 2002; Mutukumira, 1995; Odunfa & Oyewole,1998). Most of the milk produced in several African ruralcommunities is consumed either fresh or sour (Ganguly,Bandopadhyay, & Kumar, 1999), with only a small fractionof the milk produced entering the commercial sector(Ganguly et al., 1999; Walshe et al., 1991). For example, inSouthern and Eastern Africa, except in Kenya and Zimba-bwe, the milk that is consumed in the traditional way withinthe farming community constitutes approximately 80–90%

34 A. Kebede et al. / Food Research International 40 (2007) 33–38

of the total milk produced. It is therefore common amongsome rural African communities to convert milk into the lessperishable products such as naturally fermented milk, ghee,butter and cottage cheese (Chikoto, 1999; Kerven, 1987a,1987b; Leeuw, Bekure, & Granding, 1984; Majubwa, 1987).

The fermentation techniques may vary from place toplace but a key element influencing the quality of the fer-mented product is the fermentation vessel. These vesselsare usually simple and are made from locally availablematerials such as woven grass, wood fiber, calabash, hol-lowed wood or animal skin bags (FAO, 1990). Calabashesmade from gourd, clay pots and wood are widely used inmost parts of Ethiopia, Kenya, Sudan and Egypt(Abdelgadir, Hamad, Møller, & Jakobsen, 2001; Bonfohet al., 2003; El-Gendy, 1983; Kimonye & Robinson, 1991;O’Mahony & Peters, 1987). In South Africa, traditionalfermented milk was historically prepared in milk-sacks, cal-abashes, clay pots, stone jars and baskets (Bohme, 1976;Fehr, 1968; Fox, 1939; Quinn, 1959). Although some ofthese may still be in use in the rural areas, many specimensare currently only available as museum collections (Bohme,1976). Amasi, one of the traditional fermented milks con-sumed by the Bantu people of Southern Africa, was tradi-tionally prepared by pouring raw milk into calabashesmade of gourd or into stone jars. According to Fox(1939), the flavor of amasi produced using the stone jarwas different from that produced using the calabash(gourd). The Sotho people also used clay pots which werebelieved to impart better flavor than calabashes (Fox,1939). Although these earlier traditional containers are cur-rently being replaced by new commercially produced andreadily available types, such as plastics and nickel jars,some gourd calabashes and clay pots are still in use inthe rural areas (Beukes, Bester, & Mostert, 2001; Coetzee,Gordeuk, Barnard, Stassen, & De Kock, 1996). The contri-bution of traditional fermentation vessels to the character-istics of indigenous fermented milks has not been fullyassessed (Narvhus & Gadaga, 2003). The current study,therefore, aimed at evaluating the influence of the type offermentation containers on the microbiological quality ofSethemi, a South African naturally fermented milk.

2. Materials and methods

2.1. Collection of milk samples

Samples of raw milk were collected from seven to eightdifferent dairy farmers (2 l from the bulk of each farmersmilk collected in sterile bottles) who supply raw milk toSethemi producers in the vicinity of Bloemfontein, SouthAfrica. All the milk farmers hand-milked a small numbersof cows (3–8) and some produced Sethemi for their ownconsumption. During each sampling trip, a total of 16 lof pooled hand-milked raw milk were purchased and trans-ported in sterile bottles to the laboratory in cooler boxeslined with ice. At the laboratory, the milk was immediatelydivided into four different types of fermentation containers,

which were then loosely covered with cloth and the milkallowed to ferment at ambient temperature.

2.2. Preparation of fermentation vessels

Four different types of containers were obtained fromthe local market. The containers used were a clay pot, agourd (made from the dried fruit wall of the plant Lagena-

ria peucantha), a nickel jar (the type that is commonly usedby the local dairy farms) and a plastic bowl. The gourd,nickel jar and the plastic bowl had been used for ferment-ing milk prior to this study. The clay pot was new and wasconditioned by fermenting three batches of raw milk in itprior to the study. With the exception of the nickel con-tainer (10 l) all containers had a volume capacity of ca. 5 l.

2.3. Fermentation

Sethemi (4 l) was prepared in each vessel by allowing themilk to ferment at ambient (25–30 �C) temperature for sixdays. Samples (20 ml) were aseptically withdrawn at 24 hintervals for chemical and microbiological analysis. Fiverepetitions of fermentation of milk in each container werecarried out.

2.4. Microbiological analysis

A portion of the fermenting milk (1 ml) was seriallydiluted using peptone water (Merck, Darmstadt, Ger-many). All microbial analyses were performed in triplicateand the results indicated as the means. Lactic acid bacteria(LAB) were enumerated by spread plating appropriatedilutions (0.1 ml) onto MRS and M17 agar (Merck) andincubating at 30 �C for 48 h. Yeasts were enumerated byspread plating appropriate dilutions on Rose BengalChloramphenicol Agar (RBCA) (Oxoid, Basingstoke,UK). The plates were incubated at 25 �C for 72 h.

Total aerobic mesophilic bacteria and coliform bacteriawere enumerated by spread plating (0.1 ml) on Plate CountAgar (PCA) (Oxoid) and Violet Red Bile Agar (VRBA)(Oxoid), respectively. The PCA plates were incubated at30 �C for 48 h, while the VRBA plates were incubated at37 �C for 24 h.

2.5. Isolation and identification of yeasts

All representative yeast colonies based on visual distinc-tion and microscopic confirmation were picked from theRBCA plates at each sampling interval from the highestdilution. These were purified by streaking at least threetimes on to Yeast Extract–Malt Extract–Glucose Agar(YM). The isolates were maintained on YM agar slantsat 4 �C before identification. All isolates were characterizedaccording to the procedure described by Van der Walt andYarrow (1984). Briefly, the yeasts were grouped accordingto production of pigments, vegetative reproduction (bud-ding, fission) and formation of pseudohyphae or true

Yeast counts

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0 1 2 3 4 5 6

Time (days)

Lo

g c

fu/m

l

Fig. 1. Changes in yeast counts during the production of Sethemi fromraw milk in a clay pot (r), gourd (n), nickel jar (m) and plastic bowl (·).Results are the means of five repetitions.

Coliform counts and pH

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 1 2 3 4 5 6

Time (days)

Lo

g c

fu/m

l

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

Fig. 2. Changes in coliform counts (–) and pH (. . .) during thespontaneous fermentation of Sethemi from raw milk in a clay pot (r),gourd (n), nickel jar (m) and plastic bowl (·). Results are the means of fiverepetitions.

A. Kebede et al. / Food Research International 40 (2007) 33–38 35

hyphae. The conventional culture tube method was used todetermine the ability to ferment and assimilate sugars, andto assimilate nitrogen compounds. Other tests includedgrowth on vitamin free media, resistance to 0.01% and0.1% cycloheximide, urea hydrolysis, growth in 10% NaCl,growth in 50% glucose medium, growth at 37 �C and split-ting of arbutin. The yeasts were then identified by compar-ing their profiles with a computer database for yeastidentification (Barnett, Payne, & Yarrow, 1990).

Confirmation of the yeast identity was done usingsequence analysis of the D1/D2 domain using primer pairsNL-1(5 0-GCATATCAATAAGCGGAGGAAAAG) andNL-4(5 0-GGTCCGTGTTTCAAGACGG (Kurtzman &Robnett, 1998). Sequencing reactions were carried outusing the ABI PrismTM Big Dye TerminatorTM v3.1 cyclesequencing ready reaction kit. Data was collected on anABI Prism 377 DNA sequencer (Applied Biosystems,USA) and analyzed using sequencing analysis V3.3 andsequences assembled using Auto-assembler V1.4.0.

2.6. Determination of pH

The pH of raw milk, fermenting milk and the final prod-ucts was determined at 24 �C using a Cyberscan 500 pHmeter (Eutech Instruments, Singapore) fitted with an FC200 electrode (Eutech Instruments, Singapore). The pHmeter was calibrated using commercial buffers (Merck) ofpH 4 and 7.

2.7. Determination of sugars

The glucose, galactose and lactose levels in the milk dur-ing fermentation were determined using a Waters BreezeHigh Performance Liquid Chromatography (HPLC) sys-tem (Waters Corp., Milford, MA, USA), fitted with aWaters SUGARPACK1 column (Waters Corp.) and a Dif-ferential Refractive Index detector (Waters Corp.) in amodification of the method described by Bouzas, Kantt,Bodyfelt, & Torres, 1991). The mobile phase was de-ion-ised water at 84 �C at a flow rate of 0.5 ml/min. The instru-ment was externally calibrated with standard sugarsolutions (1.0%) (Sigma-Aldrich, USA) and peak areawas used to estimate the concentration.

2.8. Statistical analysis

One way ANOVA was used to evaluate differencesbetween the mean microbial counts in the different contain-ers using Analysis ToolPak (Microsoft Excel 2000) andNCSS 2000 soft ware (Hlbtze, 1998).

3. Results and discussion

3.1. Enumeration of yeasts and bacteria

The initial yeast counts in all containers wereapproximately 3.8 log cfu/ml and increased from 5.5 to

6.2 log cfu/ml during 6 days of fermentation (Fig. 1). Therewere no significant (p > 0.05) differences in the final yeastcounts between the container types. The yeast countsrecorded in this study were similar to those reported byIsono, Shingu, and Shimizu (1994) Mathara, Schillinger,Kutima, Mbugua, and Holzapfel (2004) who indicatedyeast counts ranging from 6.0 to 8.0 log cfu/ml and 4.3 to7.4 log cfu/ml in Tanzanian and Kenyan traditional fer-mented milks, respectively.

The coliform counts also increased from about3.5 log cfu/ml to a maximum of 6.9 log cfu/ml after 2 days(Fig. 2). The highest coliform counts of about 6.9 log cfu/ml were recorded in the nickel jar. Similar studies by Beu-kes et al. (2001) recorded the highest coliform counts in theclay pot. The difference could suggest that the level of coli-form contamination was related to hygienic practices(Gran, Wetlesen, Mutukumira, Rukure, & Narvhus,2003). However, after 6 days, the coliform countsdecreased to about 4.7, 5.8, 5.1 and 4.6 log cfu/ml, in theclay pot, gourd, nickel jar and plastic bowl, respectively.This corresponded to a final pH range of about 4.1–4.33.Although the pH in the clay pot decreased less gradually

Galactose

0

0.5

1

1.5

2

g/L

36 A. Kebede et al. / Food Research International 40 (2007) 33–38

than the other containers, it had the lowest final coliformcount. In general, the lowest pH of Sethemi in all four con-tainers was obtained after 4 days of fermentation and themeans ranged between 4.10 ± 0.13 to 4.32 ± 0.06, whichwere higher than that reported for the Zimbabwean tradi-tional fermented milk of 3.88 ± 0.22 (Gran et al., 2003),but within the range reported for Kenyan traditional fer-mented milk (Mathara et al., 2004).

Aerobic mesophilic counts, LAB on MRS and on M17were similar, indicating that LAB were the dominatingmicroorganisms. Fig. 3 shows average values from M17agar plates. The LAB increased from about 5.2 log cfu/mlto a maximum of 9.8 log cfu/ml in the nickel jar. The claypot, gourd and plastic bowl recorded maximum LABcounts of 9, 9.2 and 9.1 log cfu/ml, respectively. Thegrowth trends in the different containers were thereforesimilar. Although the nickel jar had slightly higher finalLAB counts than the other containers, this did not trans-late into an advantage with respect to changes in pH andthe reduction in coliform counts. This is slightly differentfrom results reported by Beukes et al. (2001) where themean total aerobic mesophilic bacteria counts, LAB onMRS and LAB on M17 in the clay pot were higher thanthose in gourd. It must be pointed out, however, that theLAB counts in the clay pot and in the gourd in the currentstudy were not significantly different (p > 0.05). However, itseemed that the maximum microbial numbers attained byeach microbial group were observed one day earlier inthe nickel container than the others, suggesting that thenature of the container may influence the fermentationtime. The results also showed that at ambient temperature,a more or less similar product of Sethemi with respect tothe microbial loads can be produced with any of the fourtypes of containers.

The clay pot and the gourd had been expected to containhigher counts of the yeast and LAB considering the difficul-ties that are often encountered while cleaning these con-tainers. However, the final LAB and coliform counts inthis study seem to only reflect the growth of the microor-ganisms initially present in the raw milk. The possible roleof the microorganisms in the walls of the container needs

LAB counts

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 1 2 3 4 5 6

Time (days)

Lo

g c

fu/m

l

Fig. 3. Changes in lactic acid bacteria (LAB) counts during the produc-tion of Sethemi from raw milk in a clay pot (r), gourd (n), nickel jar (m)and plastic bowl (·). Results are the means of five repetitions.

further investigation. Habimana (2005) reported the pres-ence of yeasts, moulds and bacteria in a biofilm depositedin clay pots used for traditional fermentation.

The LAB counts reported in this study were also similarto those reported for other African fermented milkproducts (Beyene, 1994; Gran et al., 2002; Isono et al.,1994; Miyamoto, Gichuru, Akimoto, & Nakae, 1986;Mutukumira, 1995). Since the occurrence of coliformbacteria in fermented milks is often associated with poten-tial health hazards (Gran et al., 2003), the presence of highlevels of coliform bacteria in Sethemi in all containersstrongly suggests the need for adequate sanitary measures.

3.2. Changes in sugars during fermentation

In all container types, only insignificant levels of glucosewere recorded in the milk during the whole period of fer-mentation. However galactose levels increased graduallyin all container types, especially in the clay pot and plasticbowl (Fig. 4). After 5 days about 1.5 g/l galactose hadaccumulated in the clay pot, which then decreased to about1.3 g/l at day 6. The lactose concentration in the clay potdecreased from about 46 g/l to 29 g/l in 6 days, a reductionof about 36% (Fig. 4). A similar trend was observed for theother containers although the final concentration in thenickel jar was significantly lower (p < 0.05) than in allthe other containers. These trends are similar to thoseobserved during fermentation of UHT milk with yeastsand LAB (Gadaga, Mutukumira, & Narvhus, 2001), andsuggest that the microorganisms preferably utilized glucose

0 1 2 3 4 5 6

Time (days)

Lactose

0.0

10.0

20.0

30.0

40.0

50.0

0 1 2 3 4 5 6

Time (days)

g/L

Fig. 4. The changes in galactose and lactose during the production ofSethemi from raw milk in a clay pot (r), gourd (n), nickel jar (m) andplastic bowl (·). Results are the means of five repetitions.

A. Kebede et al. / Food Research International 40 (2007) 33–38 37

as an energy source than galactose, and hence the latteraccumulated in the milk. Since the trends in the containerswere the same, it can be deduced that the growth character-istics of the microorganisms in the different containers weresimilar. This is also in agreement with the trends shown inthe growth of yeasts and lactic acid bacteria (Figs. 1 and 2).

3.3. Yeast diversity

A total of 112 yeast strains were isolated from raw milkand Sethemi produced in the laboratory using the four dif-ferent containers. Of these, 29, 35, 14 and 16 isolates wereobtained from milk fermented in the clay pot, gourd, nickeland plastic bowl, respectively, whereas the remaining 18isolates were obtained from raw milk. The isolatesbelonged to 12 genera and 23 different species (Table 1).Eight different yeast species were isolated and identifiedfrom raw milk, namely; Candida albicans, C. ernobii,C. parapsilosis, Clavispora lusitaniae, Cryptococcus albidus,Cr. curvatus, D. hansenii and K. marxianus (Table 1). How-ever, in the clay pot, 15 different species were identified sug-gesting that some of the strains in the final productoriginated from the container. Of the original strains inthe raw milk, only two (C. ernobii and Cr. albidus) couldnot be isolated from the clay pot. The biofilm reportedby Habimana (2005) showed the presence of such yeastsin the clay pot. The gourd, nickel jar and plastic bowlhad nine, four, and six different species, respectively. Thenickel jar therefore had the least diversity of yeasts. How-ever, the wider yeast diversity did not necessarily translate

Table 1The diversity of yeasts isolated and identified from raw milk and Sethemi pro

Yeast species Raw milk Fermented m

Clay pot

Candida albicans + +Candida ernobi + �Candida parapsilosis + +Candida rugosa � +Candida sake � +Candida tenuis � �Candida tropicalis � �Clavispora lusitaniae + +Cryptococcus albidus + �Cryptococcus curvatus + +Cryptococcus laurentii � �Debaryomyces anomala � �Debaryomyces hansenii + +Kluyveromyces marxianus + +Pichia anomala � +Pichia pini � +Pichia scolyti � +Rhodotorula glutinis � +Rhodotorula mucilaginosa � +Saccharomyces cerevisiae � �Sporobolomyces roseus � +Torulaspora delbrueckii � �Yarrowia lipolytica � +

into higher final yeast counts in the clay pot, suggestingthat whatever yeasts were present, adapted to the condi-tions in the fermentation vessel and multiplied to the levelsobserved. The final numbers may also be dependent on theavailability of nutrients and interaction with other groupsof microorganisms such as LAB (Gadaga et al., 2001).

D. hansenii and K. marxianus are frequently isolatedfrom milk and milk products and have the ability to assim-ilate lactose and lactic acid (Fleet & Mian, 1987; Viljoen &Greyling, 1995). These two species were isolated from rawmilk as well as all the containers showing their adaptationto the milk environment. Pichia anomala, P. pini, P. scolyti,Rodotorula glutinis, R. mucilaginosa, C. rugosa and C. sake

were only isolated from the clay pot, while S. cerevisiae andTorulospra delbrueckii were only isolated from the gourd.Although Cr. laurentii was found in the milk fermentedin the plastic bowl, it was not present in the raw milk.

4. Conclusion

The use of different containers, namely clay pot, gourd(calabash), nickel jar and plastic bowl, for the productionof Sethemi had no significant effect on the final microbialcounts in the product. However, Sethemi prepared in theclay pot had a wide diversity of yeasts, including speciesthat are known to utilize milk constituents such as lactoseand lactic acid. While it seemed possible to produce Set-hemi with similar characteristics from all the containersused, the clay pot provides prospects for a product withdiverse flavour characteristic because of the many different

duced in a clay pot, nickel jar, calabash gourd and plastic bowl

ilk

Gourd Nickel jar Plastic bowl

+ � +� � �+ � �� � �� � �� � ++ � �+ � �� � �+ + +� � ++ � �+ + ++ + +� � �� � �� � �� � �� � �+ � �� � �+ � �� + �

38 A. Kebede et al. / Food Research International 40 (2007) 33–38

microorganisms isolated. The nickel jar and the plasticbowl, on the other hand, provide opportunities for produc-ing a safer fermented milk product than the gourd or claypot.

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