Yeast Flora of Labaneh Produced by In-Bag Straining of Cow Milk Set Yogurt

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1 Yeast Flora of Labaneh Produced by n-bag Straining of Cow Milk Set Yogurt MOHAMMED 1. YAMAN Department of Nutrition and Food Technology Faculty of Agriculture University of Jordan Amman, Jordan MAHA M. ABUJABER Department of Food Science and Nutrition Faculty of Science and Allied Health Professions Applied Sciences University Amman, Jordan ABSTRACT The mean psychrotrophic and mesophilic yeast counts were 2.6 x lo6 and 4.4 x 106/g, respectively, in labaneh samples from 18 dairy plants directly after packaging. Labaneh was produced traditionally by straining set yogurt in cloth bags, directly after packaging. At the end of the shelf-life (14 d at 7"C), these mean counts increased to 1.1 x lo7 and 1.4 x 107/g. The percentages of samples showing yeast count >106/g were 50 for psychrotrophic and 56 for mesophilic yeasts after packaging and increased to 78 and 83 after 14 d at 7 C. Saccharomyces cerevisiae, which could be grouped in seven biovariants, was in all labaneh samples. Trichosporon brassicae, Cryptococcus cuwatus, and Kluyveromyces marxianus were in 33, 28, and 17% of labaneh samples, respectively. Trichosporon cutaneum, Debaromyces hansenii, Pichia farinosa, Geotrichum candidum, and Candida blankii were in 6% of the samples. All labaneh samples showed the characteristic signs of yeast spoilage after 14 d at 7 C. All yeast species and their biovariants were able to grow at 7'C; thus, psychrotrophic yeasts are the main cause of spoilage of traditionally produced packaged labaneh kept under refrigeration. Received February 9, 1994 Accepted June (Key words: yeast flora, labaneh spoilage, psychrotrophic yeasts, concentrated yogurt) NTRODUCTON Labaneh or labneh (from Arabic laben, milk) is the name used in Jordan and other Arab countries for the semisolid dairy product made from set yogurt by removal of part of its whey. Labaneh is widely consumed with olive oil at breakfast and supper meals or as a snack, usually a sandwich spread. The total solids and fat contents of packaged labaneh are 23 to 25% and 8 to 11%, respectively (26). Labaneh with higher total solids content (up to 40%) is also produced, especially in rural areas, where it is sold unpackaged (18). A large proportion of labaneh is still produced by the traditional method, by straining natural set yogurt in cloth bags. Because this method is labor intensive and time-consuming, and, because of growing international interest in labaneh (19, 22), new production techniques, such as the use of mechanical separators of the centrifugal type or modified nozzle separators, have been tried (27). Ultrafiltration of the milk before the production (25) and ultrafiltration of yogurt (16, 24) were also investigated. Spoilage of traditionally produced packaged labaneh kept under refrigeration may be the most important production problem. Tamime (23) studied the effect of storage on the quality of labaneh produced from hydrolyzed milk, but shelf-life was no longer than that produced by the traditional method. n an attempt to prolong the shelf-life of labaneh, Dagher and Ali (4) investigated the use of yogurt pasteurization at 60 to 80 C and the addition of.2 to 2% 1994 J Dairy Sci 77:

2 YEAST FLORA OF LABANEH 3559 hydrogen peroxide. A survey of producers in the central province of Saudi Arabia found that the major contaminants of labaneh were yeasts (20). As a product that has undergone lactic acid fermentation and is rather concentrated, labaneh is expected to be mostly suitable for the growth of fungi rather than bacteria, particularly for yeasts, when it is packaged in tight containers. This study was undertaken to determine the degree of yeast Occurrence in labaneh produced from cow milk by the traditional method, to identify the changes in yeast counts at the end of shelf-life (14 d at TC), and to study the effect of yeasts on the shelf-life of labaneh. Yeasts isolated from labaneh samples were identified. MATERALS AND METHODS Sampling and Examination Packaged labaneh samples were taken from 18 producers in Amman, Jordan. All samples were of labaneh produced from cow milk by the traditional method, as summarized in Figure 1. Each sample consisted of four sample units of four press-to-close or thermally sealed plastic containers (250 or 500 g). The samples were purchased on the day of packaging and were brought to the laboratory within 1 h of the purchase. On arrival, two sample units were examined for psychrotrophic and mesophilic yeast counts, direct microscopic examination, ph and acidity, and organoleptic properties. The examinations were repeated on the contents of the other two sample units after 14 d at 7 C. Chemical Analysis ph and Titratable Acidity. ph was measured by immersion of the ph meter electrode (model H 8416; Hanna nstruments, Limena, taly) in labaneh. Titratable acidity, as lactic acid, was determined by titration against.1n NaOH (2). Microbiological Examination Preparation of the Subsamples. The outside of labaneh containers were wiped with a solution of 70% ethanol, and the contents of each container were thoroughly mixed using a ster- Heat milk to 85 to 95'C for 5 to 10 min Cool to 42 to 45'C noculate with 1 to 2% yogurt starter culture (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius sp. tbermophilus ncubate at 42 to 45'C until a smooth curd with desired acidity is formed Cool Add 1 to 27'0 salt and stir Fill yogurt into cloth bags (20 to 25 kg each) Stack bags on top of each other Allow whey to drain for 8 to 48 h Fill in plastic containers Figure 1. Procedure generally followed in the production of labaneh by the traditional method. ile spatula. A composite subsample was prepared by transferring from each container into a sterile beaker 50 g of labaneh, which were thoroughly mixed. Direct Microscopic Examination. Direct microscopic examination was performed from labaneh subsamples or from the first dilution (10-l) by the Gram-stained smear. Enumeration. Under aseptic conditions, 50 g of each labaneh subsample were weighed in a sterile blender jar and diluted with 450 ml of sterile peptone and water (-1%) and blended for 2 min at high speed. Suitable 10-fold dilutions were prepared. Viable counts of psychrotrophic and mesophilic yeasts were determined usinging the pour-plate technique (17). The medium used was plate count agar (Difco Laboratories, Detroit, M) to which 100 mg each of chloramphenicol and chlortetracycline-hcvl were added (14). Duplicate plates were used for every dilution. The plates were incubated at 7 C for 10 d for psychrotrophic yeast counts and at 25 C for 5 d for mesophilic yeast counts. PuriJication and Maintenance of Yeast solates. Colonies of different morphology, which revealed yeasts of different size or shape, or both, were selected from plates used in mesophilic and psychrotrophic enumerations. Pure cultures were obtained by subculturing Journal of Dairy Science Vol. 77, No. 12, 1994

3 3560 YAMAN AND ABU-JABER two to three times on malt extract agar (28). Glucose-peptone-yeast extract agar slants were used for the maintenance of yeast cultures. The cultures were stored at 5'C and subcultured at 3- to 4-mo intervals (28). dentification of the Yeast solates. dentification of yeast isolates was according to the method developed by Deak (5) and Deak and Beuchat (6). Microscopic observations of cell morphology and the production of ascospores, arthrospores, and true hyphae were used to supplement biochemical tests. A confirmation of the identification of each isolate was referred to methods of Kreger-van Rij (15). Growrh ut 5, 7, and 43'C. The ability of yeast isolates to grow at 5, 7, and 43 C was measured by inoculation on malt extract agar and incubation at 5, 7, and 43 C for 7 d (15). Organoleptic Examination Labaneh samples were examined by the investigators directly after packaging and after 14 d at 7 C for the prominent signs of yeast spoilage (yeasty flavor, gas, and surface growth of the yeasts). ph and Acidity RESULTS The ph of labaneh samples directly after packaging ranged between 3.6 and 4.0 (X = 3.7), and acidity, as lactic acid, ranged between 1.4 and = 2.16). Yeast Counts in Labaneh Directly After Packaging and After 14 d at 7'C Direct microscopic examination of labaneh directly after packaging and 14 d later showed only yogurt starter culture bacteria and yeasts, but the examination of representative colonies from the selective plates used for the counting showed only yeasts. Figure 2 shows the psychrotrophic and mesophilic yeast counts of each dairy plant directly after packaging and after 14 d at 7 C. An increase in the mesophilic yeast counts occurred in 11 labaneh samples (61%) after 14 d, although 6 samples (33%) had a decrease in the mesophilic yeast count. Psychrotrophic yeast counts increased in 11 samples (61%) after 14 d, but decreased in 7 samples (39%). The mean psychrotrophic and mesophilic yeast counts of labaneh samples directly after packaging were 2.6 x lo6 and 4.4 x 106/g, respectively. After 14 d, the mean yeast counts increased, reaching 1.1 x io7& for the psychrotrophic and 1.4 x 107/g for the mesophilic counts. Directly after packaging of labaneh psychrotrophic and mesophilic yeast counts were >ld/g in 67 and 78% of the samples, respectively, and increased to 89% for both types of yeasts after 14 d. The propor- TABLE 1. Yeast species and biovariants isolated from labaneh samples. Manufacturer Yeast species and biovariants 1 Saccharomyces cerevisiae biovar. 1, Trichosporon brassicae. and Crypfococcus curvutu~ 2 S. cerevisiae biovar. 6, and T. brassicae 3 S. cerevisiae biovar. 1 4 S. cerevisiae biovar. 1 5 S. cerevisiae biovar. 1, T. brassicae, and C. curvatus. 6 S. cerevisiae biovar. 1 and 7 7 S. cerevisiae biovar. 6, T. brassicae, and C. curvatus. 8 S. cerevisiae biovar. 1, 3, and 6 and Kuyveromyces mrxians biovar. lucfis 9 S. cerevisiae biovar. 1 and C. curvatus. 10 S. cerevisiae biovar. 1 and 6, T. brassicae, T. cutaneum, and Candida blankii 1 S. cerevisiae biovar. 1 and 6, and Debaromyces hansenii 12 S. cerevisiae biovar. 3, 6 13 S. cerevisiae biovar. 1, 4, and 5, K. murxians biovar. bulgaricus, and Pichiu furinosa var. lodderi 14 S. cerevisiae biovar S. cerevisiae biovar. 1. 2, and 6 and G. candidurn 16 S. cerevisiae biovar. 1 and 6 and C. curvatus 17 S. cerevisiae biovar. 1 and 6, T. brassicae, and K. mrxians biovar. luctis 18 S. cerevisiae biovar and 6 Journal of Dairy Science Vol. 77, No

4 YEAST FLORA OF LABANEH 3561 Pwchrotrophir. directly Pwchrotrophic, at the end 0 Mesophilic, directly after Mesophilic, at the endof the after packaging of theshelf-life packaging shelf-life O Manufacturer Figure 2. Psychrotrophic and mesophilic yeast counts of labaneh samples directly after packaging and after 14 d at 7 C. tions of the samples that had psychrotrophic and mesophilic yeast counts >106/g directly after packaging were 50 and 56%, respectively. After 14 d, these percentages increased to 78 and 83%. Yeast Species solated from Labaneh Classijication and Frequency of Yeast Species. Ninety-six yeast isolates were isolated from the labaneh samples of the 18 dauy plants. Nine yeast species were identified, and most of the samples contained more than one yeast species (Table 1). The Saccharomyces cerevisiae isolates could be grouped into seven biovariants based on differences in their fermentation of galactose and in their assimilation of maltose, raffinose, galactose, trehalose, and cellibiose (Table 2). Kluyveromyces marxianus isolates belonged to the varieties lactis and bulgaricus, and Pichia farinosa isolate belonged to the variety lodderi (15). The frequency of each yeast species isolated from labaneh is shown in Table 2. The S. cerevisiae biovariants were in all labaneh samples, but Trichosporon brassicae, Cryptococcus curvatus, and K. marxianus were in 33,28, and 17% of the samples, respectively. Trichosporon cutaneum, Debaromyces han- senii, P. farinosa, Geotnchum candidum, and Candida blankii were each in 6% of the samples. Growth of Yeasts solated from Labaneh Samples at 5, 7, and 43 C. Most of the yeast species isolated were able to grow at 5"C, but S. cerevisiae biovar. 5 and 6 were not able to grow at this temperature, and biovar. 2 and 3 showed weak growth. All nine species and their variants were able to grow at 7 C. The yeasts that were able to grow at 43 C were K. marxianus var. lactis and var. bulgaricus and S. cerevisiae biovar. 2, 5, and 6. Physical Changes in Labaneh Samples Directly after packaging, 75% of the examined samples had normal appearance and flavor; the reminder showed gassiness and yeasty flavor, the prominent characteristics of labaneh spoiled by yeasts. The mean psychrotrophic and mesophilic yeast counts in these samples were 6.8 x lo6 and 1.5 x 107/g, respectively. After 14 d at 7"C, the majority of the samples were spoiled. The mean psychrotrophic and mesophilic yeast counts in these samples were 1.3 x lo7 and 1.7 x 107/g, respectively; 80% of the samples showed gassiness and yeasty flavor, 45% had surface yeast, and 25% of sample containers were bloated. Journal of Dairy Science Vol. 77, No

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6 YEAST FLORA OF LABANEH 3563 DSCUSSON The traditional method of labaneh production (Figure 1) is still used on a large-scale in Jordan and other Middle Eastern countries. When this method is used, especially when general good manufacturing practices are not thoroughly followed, contamination of labaneh by yeasts and their subsequent growth cannot be prevented. Yeast Occurrence in Labaneh Yeasts apparently are indigenous to labaneh, and labaneh and its microbial flora form an interesting microbial ecosystem. The high acid content of labaneh, which is enough to inhibit bacterial growth, and the limited access of air to the labaneh in the containers during refrigerated storage make it a special microbial habitat that is most suitable for the growth of the yeasts. The high yeast counts in abaneh produced by the traditional method directly after packaging suggest a high degree of contamination and mishandling of the product. This problem could be attributed to many factors related to the processing of labaneh, such as the use of yogurt starter cultures, yogurt, or both that were contaminated by yeasts or to the poor hygienic conditions prevailing during straining of the whey, especially cleanliness of the bags and apparatus used. Lack of refrigeration during straining, which could be extended up to 48 h, and poor hygienic practices during packaging may also contribute to the contamination by yeasts. The presence of yeasts in dairy products is not uncommon. Yeasts were reported in many products, such as in yogurt (21), cottage cheese (13), cream (12), and kefir (10). n quarg, a dairy product similar to labaneh in composition, yeasts seem to play a major role as spoilage microorganisms (1 1); yeasty flavor developed when counts reached lo5 yeast cells/g of quarg. Engel (9) studied the growth of yeasts in quarg kept under refrigeration (6 C) and found that yeast spoilage of quarg would sometimes occur during storage at 6 C within the given shelf-life (18 d). To avoid spoilage, quag should be packed yeast-free, and the designated shelf-life should be reduced. Yeast Species solated from Labaneh All yeast species isolated from labaneh samples could be considered to be psychrotrophic because of their ability to grow at 7 C. The predominance of S. cerevisiae in labaneh (Table 2) may be explained by its ability to ferment glucose and galactose and to assimilate glucose, galactose, and lactic acid, which occur in appreciable amounts in yogurt whey (29). solation of S. cerevisiae from labaneh is inconsistent with its isolation from other related dairy products, yogurt starter culture (l), and yogurt (21). The isolation of the lactose-fermenting yeast, K. marxianus, is in agreement with previous studies on yeasts in yogurt starter culture (l), yogurt (12, 13), kefir grains (lo), and quarg (7). The remaining yeast species encountered in this study were previously isolated from different dairy products: G. candidum and T. curaneum were isolated from raw milk (3, 8) and quarg (7); C. blankii was isolated from pasteurized milk (13); and D. hansenii was isolated from yogurt (21). The presence of these yeast species at the different stages of yogurt production and their predominance in labaneh suggest that milk, yogurt starter culture, and yogurt could play a role as a source of contamination of labaneh by yeasts. CONCLUSONS Psychrotrophic yeasts are the main cause of spoilage of traditionally produced packaged labaneh. Use of alternative methods of production, instead of the traditional in-bag straining, may help avoid the adverse effects of yeast growth. The use of yeast-free yogurt starter cultures, application of strict hygienic measures during processing and packaging, and maintenance of an effective cold chain from yogurt production until labaneh consumption could help control labaneh yeasts. A shortening of the anticipated shelf-life to 7 to 10 d may also be useful. REFERENCES 1 Baroudi, A.A.G., and E. B. Collins Microorganism and characteristics of laban. J. Dairy Sci. 59: Case, R. A., R. L. Bradley, and R. R. Williams Chemical and physical methods. Page 327 in Standard Methods for the Examination of Dairy Products. G. H. Journal of Dairy Science Vol. 77. No. 12, 1994

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