Killer yeast isolated from some foods and its biological activity

hanyehia@ksu.edu.sa Abstract Seventy eight yeasts were isolated from different foodstuffs. Out of the seventy eight isolates four yeast species namely C. parapsilosis Q3, C. solani F8, C. versatilis J3 and K. jensenii H1 were selected to study their biological activity. The four strains termed as killer yeast by observing its activity against microorganisms. Killer yeasts secrete proteinaceous killer toxins lethal effect against some of Gram positive and negative bacteria, molds and yeasts. The antagonistic effect of the four killer yeast strains on the growth of different microorganisms recorded as zone of inhibition (mm), demonstrated that C. versatilis J3 was an active stains against majority test microorganisms. Consumed sugar determined for the four strains and showed that C. versatilis J3 and C. parapsilosis Q3 reached to its maximum on the 12 hours of incubation both yeast consumed 97.5 and 95% of initial sugar (sugar utilization efficiency) while both of C. solani F8 and K. jensenii H1 sugar utilization efficiency was recorded on the 24 hours being 99.65 and 99.30%,


Introduction
The term called yeast is originated from the old Dutch word gist and the German word gischt, which refers to fermentation. There are approximately 100 genera and 800 detected species of yeasts (Kurtzman & Fell, 1998). Yeasts are distributed in different food known to contaminate and spoilage of foods and dairy products (Mushtaq et al., 2006). Yeasts are of wide distributions in the environment, and may be found as a part of the normal flora of a food product, on inadequately sanitized equipment, or as air borne contaminants. Their habitats may include not only the upper layers of the soil but also many forms of organic matter, especially of plants origin, where carbohydrates are common occurrence. Yeasts may be isolated particularly from the soil of vineyards and orchards; from the surface of grapes, apples, and most sweet fruits and from the leaves and other parts of plants (Prescott & Dunn, 1959). Mushtaq et al., (2006) have been used yeasts in the food industry principally for the production of ethanol and carbon dioxide, which are important to the brewing, wine distilling and baking industries, Yeasts are rich of proteins, lipids and vitamins (Kutty & Philip 2008). Over the years, morphological, biochemical and physiological characteristics have been used to identify yeasts (Barnett et al., 1990. This conventional methodology requires the evaluation of some 60 to 90 tests, resulting in a complex (Arias et al., 2002). From the biological activity of yeasts found a Killer yeasts which produce antimycotic compounds and form immune (Magliani et al., 1997). Yeast killer toxins are proteinaceous compounds which are active against members of the same species or closely related species, and the activities of these toxins are similar to the activities of bacteriocins in bacterial species. Some authors (Lowes et al., 2000) prefer to call yeast killer toxins mycocins and killer strains mycogenic in order to emphasize the general nature of the antagonistic interactions (Golubev, 1998). Mycocins were first found in brewing strains of Saccharomyces cerevisiae (Bevan & Makower, 1963) and since then have been shown to occur in a large number of yeast species of agronomic, environmental, industrial, and clinical interest, including Candida, Cryptococcus, Debaryomyces, Pichia, Torulopsis, and Williopsis species (Golubev, 1998;Philliskirk & Young, 1975;Young, 1987;Young & Yagiu, 1978). Certain mycotoxins have also been shown to have inhibitory effects on some pathogenic gram-positive bacteria, including Staphylococcus aureus (Izgü & Altinbay, 1997). The aim of our study is to determine the common yeast isolates in some foods and select the biologically active strain (killer strain) against some other microorganisms. Also to determine the affinity of yeast strains for glucose at different concentrations (m mole/L) and the sugar consumption for the four killer yeasts as follows (Equation 1 and 2): ( ) (2)

Samples
Samples included fresh fruits (dates, grapes, figs and strawberries), juices (carrot, orange, sugar cane and tomato), high test molasses syrup, pickles (black and green olive, carrot, lemon and cucumber), dairy products (milk, butter, ice cream, old white cheese and yoghurt), salted fish and sausage. These materials were collected from the local markets of Cairo, Egypt, and directly transferred to the laboratory for microbiological analysis.

Media used for culturing yeasts
The following media were used throughout this work, malt extract agar (Lodder & Kreger-van-Rij, 1967), it was used or isolation and of yeasts from different foodstuffs. It has the following composition; malt extract 20, agar 20 tap water 1000 and pH was adjusted to 5.5. also the malt extract broth prepared without addition of agar. YEPD medium (Seki et al., 1985), was used for the production of high titre of killer solution. It was consisted of g/1 L: glucose (20), yeast extract (10) , peptone (20) and pH was adjusted to 4.7. Nutrient glucose agar medium (Difco Laboratories, 1988), was used for seeded bacterial strains and its composition g/1L: glucose (10), beef extract (3), peptone (5), agar (20) and pH was adjusted to 7-7.2. The medium were autoclaved at 110 or 120 °C for 20 minutes, this depends on the composition of the media and pH.

Antagonistic Effect
The Technique described by Woods & Bevan (1968) was The used throughout this investigation

Yeast yeast interaction
YEPD agar medium was used to produce the killer substances. For obtaining stable high titer of killer solution, conical flasks (250 mL in volume)containing 100 mL of YEPD broth medium was inoculated with tested yeast and incubated on an orbital shaker (150 rpm) at 30 °C for 3 days. Discs (5 mm in diameter) or sterilized filter paper were saturated with 100 µL of yeast cultures under aspect conditions. Thereafter, they were placed on the surface of 10 mL of seeded malt agar(inoculated with one mL tested yeast containing 10 3 cells, which kept for 2 hours at room temperature before incubation). The plates were incubated at 30 °C for 1-3 days and zone of inhibition was detected. Candida albicans (R12) was also used in seeded agar plates as a sensitive yeast.

Yeast fungi interaction
Malt extract agar medium was used as seeded agar plates. fungi used as seede plates (10 3 spores/mL) were Aspergillus flavus (F5), A. niger (N70) and Penicillium notatum (H3). The plates were incubated at 30 °C for 3-5 days. The yeasts, bacteria and fungi tested strains were obtained from Dep. of Microbiology, Faculty of agriculture, Ain Shams University, cairo, Egypt.

Glucose determination
Glucose was determined according to the method of Trinder (1969) using special kits. The principal of the method could be explained as follow (Equation 3) The procedure was carried out by micropiptting 10 µL of the tested sample ( or standard solution 2.0 gl -1 glucose) in a test tube (5 mL capacity), then one mL of enzymatic reagent solution (reagent 3.18.7) was added, mixed gently and incubated at 37 °C for 10 minutes. The developed color (stable for 30 minutes) was measured colurmetrically at 505 nm.

Saturation constant of glucose
Glucose is considered to be the best carbon source for propagation of yeasts. All yeasts utilize this substrate aerobically (fermented yeast). The specific growth rate of yeasts highly affected by glucose concentration. This effect is only when substrate levels become very low that the growth rate begins to be severely affected. In order to evaluate this effect, maximum specific growth rate (µmax) and saturation constant (Ks) which is inversely proportional to the affinity of yeasts for the particular substrate (glucose) were determined.
In this experiment, conical flasks (250 mL in volume) containing 100 mL fermentation broth medium. Without carbon source were autoclaved at 121 °C for 15 minutes. Different concentrations of glucose (sterilized by filtration using Millipore filter) being 0, 1, 2, 4, 8, 16, 32, 64 mmole were added. The flasks containing different concentrations of glucose were inoculated with one mL standard inoculum of tested yeasts (4 selective strains were used) and were incubated at 30 °C for 7 days using an orbital shaker (150 rpm). Yeast growth (optical density at 570 nm) was determined periodically (6-12 hours). Growth is numerically equal to the concentration of growth-limiting substrate (glucose) at half of the maximum rate (µmax/2) Hence, a plot of 1/max against 1/s. i.e. a lineweaver-Burk plot, will gave straight line with an intercept abscissa at -1/Ks. and an intercept on the ordinate 1/µmax. S = the substrate (glucose) concentration. Table 1 show the number of percentage of different yeast genera isolated from different foodstuffs. Results sin Table 1 revealed that seventy eight yeast cultures were isolated from different foodstuffs. The morphological and physiological proportion of these isolates were studied according to Lodder & Kreger-van-Rij (1967), Barnett et al. (1983) and Kreger-van-Rij (1984). All yeast isolates belonged to seven genera being Candida, Geotrichum, Hansenula, Klockera, Rhodotorula, Schizoblastosporion, and Trichosporon, represented the most dominant genera being (74.36% (58 isolates out of 78 yeast isolates). This yeasts belonged to genera represented of 74.4, 3.8, 2.6, 6.4, 8.8, 2.6 and 1.3%. Seven isolates (8.92) were found to be Rhodotorula. Other yeast genera showed the lowest percentage among the yeasts ranged from 1.28% to 6.41%. it is also interesting to notice that the yeast isolates belonging to candida were observed in all tested foodstuffs except yoghurt and milk i.e. nineteen foods out of twenty one foodstuffs. Kloeckera and Rhodotorula isolates were only recorded in five and four foodstuffs respectively. On the contrary Hansenula and Schizosaccharomyces yeasts were isolated from lemon pickles brine and sausage respectively. Active species (killer yeast) belonged to the genus of candida were C. parapsilosis Q3, C. solani F8 and C. versatilis J3, while the active specie belonged to Klocecker genus was K. jensenii H1. These four strains were used for further study throughout the following work as its activity against other microorganisms through the antagonistic effect, consumed sugar and affinity to glucose.

Antagonistic effect of isolates
It has been known for many years that antagonisms can exist between microorganisms growing in a common environment. Some organisms may produce metabolic products or specific toxic substrate which inhibit or kill other microorganisms. This phenomenon is widely studied in Table: Inhibition of microbial growth by yeast strains. is widely studied in bacteria and fungi. Certain yeast strains termed killer yeasts produce an extracellular toxin which is lethal to another yeasts (sensitive yeasts). This killer interaction is restricted between strains of species within one genus but reactions and different genera have been reported (Bevan & Makower, 1963). In this work, the interaction between seventy eight yeast isolated from some foodstuffs were studied. The antagonistic effect of these yeasts against certain bacteria and fungi was elucidated. Results in Table 2 showed that (3.5%) C. solani F8, C. parapsilosi Q3 snd K. jensenii H1 out of 78 yeast isolates showed inhibitory effect against Schizoblastosporon strakeii P2, Sch. starkeii P3 and C. parapsilosis A3, respectively. The area of inhibition 6, 3, and 1 mm respectively. Regarding to the interaction between yaeast isolates and other microorganisms, the results showed that four yeast strains had a deleterious effect on some microorganisms associated with foods. Additionally, the inhibitory effect was highly varied from one yeast to another. Besides, C. versatilis J3 was capable to retard the growth of all tested microorganisms except Aspergillus flavus (F5) and penicillium notatum (H3). Moreover, the highest inhibition zone was recorded in the case of Staphylococcus aureus (A3) being 8 mm whereas the the lowest value was shown in the case of E. coli (1 mm, however, K. jensenii H1 did not inhibit the growth of C. albicans (R12) and Penicillium notatum (H3), while highly inhibited Proteus vulgaris (H9), (8 mm inhibition   On the other hand, all tested yeasts had antagonistic effect against Staph. aureus (A3), while A. flavus (F5) was inhibited by only one yeast strain (K. jensenii). It could be concluded that, the occurrence of these yeasts in foods may play a role in retardation of some undesirable microorganisms associated with food. On the other hand, the the inhibitory effect of these yeasts may be due to some metabolic products which was excreted from the cells to the media. These results are in line with those observed by Yokomori et al. (1988) and Palpacelli et al., 1991. Killer activity was recorded in some yeast strains such as Hansenula saturn (Bussey & Sherman, 1973), Candida galabrata (Bussey & Skipper, 1975), Saccharomyces cerevisiae (Palfree and bussey, 1979), Klyveromyces lactis (Sugisaki et al., 1984), H. markeii (Ashida et al., 1983), Candida sp. (Yokomori et al., 1988) and Metscenikowia pulcherima (Farris et al., 1991). On the other hand, Wilson & Chalutz (1989) explained the role of antagonistic yeast to biocontrol of Penicillium rots of citrus. McLaughlin et al. (1990) studied the effect of inoculum concentration and salt solutions on the biological control of postharvest disease of apple with Candida sp. and Debaromyces hansenii (ascosporogenous yeast) was also used to biocontrol of green and blue mold and sour rot of citrus by . Izgü & Altinbay (1997), mentioned that certain mycocins have been shown to have inhibitory effects on some pathogenic gram-positive bacteria, including Staphylococcus aureus and these results were agreed with us. There is evidence that interactions between mycogenic yeasts and sensitive yeasts are widespread in natural habitats and are probably ecologically significant (Stumm et al., 1977). The researches of mycogenic yeasts based on the activity assays in vitro focused on the molecular aspects of production, properties of the mycocins, and the mechanisms of action. Little attention has received about the role of killer yeasts in ecological community structure and it is assumed that these organisms have an advantage over sensitive competitors. When mycogenic yeasts are present in natural communities, a single killer strain usually predominates (Starmer et al., 1987). The action between killer yeast and sensitive yeast in mixed culture lead to predominant the killer and can be detrermined through kinetic studies (Petering et al., 1991;Ramon-Portugal et al., 1998). It is essential to develop an understanding of the interactions of killer strains in communities if mycocins are to be used as biological control agents. Starmer et al., (1987) found that mycogenic yeasts are widespread in natural populations of fruit and decaying vegetable matter and concluded that these organisms have an important effect on the development and composition of the yeast flora. Generally it could be concluded that our yeast strain termed killer strains and may delay some pathogenic bacteria,, yaest and fungi and these all enhancement it role in food preservation from contaminant microorganisms Table 3 declared the amount of consumed sugar by the tested yeasts in a 100 mL medium containing 2.0% glucose. During the first ,12 hours of incubation, consumed glucose by C. versatilis J3 and C. parapsilosis Q3 was increased rapidly reaching to 19.5 and 19.0 g/L on the 12 th hours respectively, which means that both yeast consumed 97.5 and 95% of initial sugar (sugar utilization efficiency) respectively, during this period (12 hours). Besides, the corresponding figures of specific sugar consumption rate were 0.248 and 0.447 h -1 . In contrast, C. solani F8 and K. jensenii H1 did not exhibit the same trend where the highest sugar utilization efficiency (SUE) was recorded on the 24 hours being 99.65 and 99.30%, respectively. These yeasts also showed the lowest specific sugar consumption rate (0.0484 and 0.114 h -1 ) as compared with other two strains. There are direct proportional between the consumed sugar and the percentage of fermentation. This could be due to the utilization of sugar for the formation of other products and depends on the strain efficiency in utilized sugar and other factor intrinsic and extrinsic factor as the type of sugar and its concentration, pH, temperature, nitrogen source agitation rate.

Affinity of yeast strains for glucose
Glucose is considered to be the best carbon source for propagation of yeasts. All yeasts utilize this substrate aerobically (fermented yeast). The specific growth rate of yeasts highly affected by glucose concentration. In this study C. versatilis J3, C. parapsilosis Q3, C. solani F8 and K. jensenii H1 were grown at different glucose concentration ( 1,2,4,8,16 and 64 mmol/L) to determine saturation constant (Ks) for each strain. Tables 4, 5, 6, 7 showed gradual increase of yeast of yeast growth, during the exponential growth pahse (24 hors) where the growth increased from 0.43 to 3.8 O.D for C. solani F8, from 1.0 to 3.6 O.D for C. parapsilosis Q3 and from 0.47 to 2.8 O.D for C. versatilis J3 and from 1.0 to 3.3 O.D for K. jensenii H1 where Table 3. Consumed sugar, sugar utilization efficiency and specific consumption rate of sugar during propagation of yeast strains in medium containing 2% glucose at 30 C using shake flasks as a batch culture.      Original Article utilized the lowest amount of sugar to produce a unit of growth.
On the contrary C. parapsilosis Q3 showed the higest value of Ks being 3.3 * 10 -4 mole, i.e. it utilize higest amount of sugar per a unit of sugar per unit of growth. Generally, these yeasts had different saturation constant, i.e. their effecieny to utilze glucose varied from one strain to another, where the highest efficiency was recorded in the case of C. solani F8. Higgins et al., (1985) reported that Ks values for carbon energy substrat are usually 10 -5 mole. Rose & Harrison (1970) also reported thaty the µmax and Ks for some yeasts were found to be 0.37 ± 0.03 h -1 and 3.6 ± 0.5 * 10 -4 mole at 30 °C and pH value 4.0.