J. Natn. Sci. Foz~ndatzon Srz La~zlza 2000 28(1): 79-86 CHARACTERISTICS OF TWO YEAST STRAINS (CANDIDA TROPICALIS) ISOLATED FROM CARYOTA URENS (KITHUL) TODDY FOR SINGLE CELL PROTEIN PRODUCTION S. CHANDRANI WIJEYARATNE' and A. N. JAYATHILAKF: Dcpnrtmeizt of Botany, U~tzuer.szty of'Srz Jayewnrde~~epul-a, Nugegoda (Received: 03 Octobal 1999 ; accepted: 03 Marclz 2000) Abstract: Characteristics of two strains of yeast, identified as Candida tropicalis, strains NCYC 2705 and NCYC 2(i99 isolated from Caryota urens toddy were studied for their possihle utilizaticrn in single cell protein (SCP) production. Having a moisture content of 7%, ash content of 8% and crude platein content. more than 45 %, these two strains conform to oificial specification for food yeast 0fU.S. Further these two strains contain essential amino acids amounting t o 82 2 and 72 5% of ' t l~c total amino acid content. One of the saljent features ofthe amino acid cont.ents of these two strains of'C;. trol)iculis was the ligll content of cysteic acid which is noi-mally liruud in low amounts in most of the other microorganisms. Suhstantial llig-11 levels of vitamins especially thiamine and rihoflavin in these two C. tropicalis strains make them suitable candidates for SCP productic~n. Key words: Cal~didu tropicadis, Car.yota ZI.~CTZS, Single cell protein. INTRODUCTION Increasing human population and limitation In cultivable land with dwindling natural resources have made it necessary to look into alternat~ve sources for food and feed. Protein deficiency and malnutrition are consequences of limited food supplies. For centuries microorganisms have been an indirect and direct source of protein in the diet of humans. Filamentous fungi and yeast are widely used in the traditional food technologies worldwide to modify the dietary staples. Modification includes improved taste, texture and digestibility with or without any net increase in the protein content of the foodstuff. e.g. yogurt, curd, fermented fish and meat etc. Several fungi, j .e. Fusar i r~m oxysporum var. linil and Chetomium ccll~lolyticurn,~ algae i.e. Ch,lorella and Spirulina, Yeast, i.e. Candida lipotytica and Saccharomyces cerevisiaei and phototrophic bacteria such as Rhodospirilum spp..: had been explored for single cell protein (SCP) production. Nutritional quality and absence of toxicity are the primary criteria that should be satisfied before any organism can be used in SCP production. Certain yeast, bacteria and algae species have been found to meet these criteria with high protein content, high amounts of essential amino acids, vitamins and low amounts of nucleic acids. Such microorganisms also possess other characters such as high growth rate, high yield from consumed sugar and acceptable content of vitamin B-12 and carotenoids etc. to consider them for scale-up operations. 'I' Corresponding autl~or 80 S. C. WGeyaratne and A. N. Jayathilahe In this study two Candida tropicalis strains (NCYC- 2699 and NCYC-2705) isolated from fermenting phloem sap of Caryota urens E t h u U were investigated for their potential use in SCP production. These cultures were accessioned into the National Collection of Yeast Cultures (NCYC) a t Norwich, UK, as they were different from other C. tropicalis strains in the collection. As the molasses is a cheap byproduct of the sugar industry in Sri Lanka, this is a suitable substrate in SCP production. In this study, molasses was used as the carbon source for growth of yeast. METHODS AND MATERIALS Characteristics of yeast strains used: C. tropicalis, strain NCYC -2705, Cells oval to long-oval (3-5) x (4-10) p in YM broth and (2-8) x (2-9) p in YM agar, form Candida type pseudomycelium in both CMC and PDAmedia with few blastospores. C. tropicalis, strain NCYC-2699, cells oval to long oval in (4-8) x (7-10) p in YM broth and (4-8) x (5-9) p in YM agar, form well developed mycotoruloides type pseudomycelium with many blastospores in both CMA and PDA media. Production of Yeast cells for analyses: Yeast cells were grown in flasks containing yeast extract, peptone, dextrose (YPD) medium. The cultures were aerated using a shaker a t a speed of 200 rpm for three days. The cell mass was harvested by centrifugation and freeze dried after washing with water to remove the contaminated medium. In all the following determination procedures, the final result obtained'was the mean val.ue of three replicates. Determination of moisture corlter~t of cells: One gram of freeze dried sample was weighed into a crucible and placed in an oven a t 105" C for 4 to 5 h. After drying, the samples were cooled in a desiccater and weighed. Drying and cooling was done several times, until a constant weight was obtained. Determir~ation of ash conlent: To determine the ash content 2.00 g of sample was weighed into a porcelain crucible with a lid and ignited in a muffle furnace a t 550°C untjl gray ash was obtained. The crucible with ash was left to cool in a desiccator and weighed. The sample was transferred back into the furnace and left for further 112 an hour. Then cooled and weighed again. This procedure was repeated until a constant weight was obtained. Determination of total nitrogen. and protein content: The freeze- dried cells were used to determine the total nitrogen content using micro-Kjeldahal, method as described in AOAC4. Crude protein content of the cells was determined by multiplying the total, nitrogen by the factor 6.25. Cajzdida. tropica.1i.s fb7- single cell protein p~ .~dz~c t i on . 81 Growth at difire7i.t temperatl~res: The two selected strains were tested for their ability to grow a t different temperatures 30", 35'and 40" C using the synthetic ~nedirnn.~ Altnown amowit of cells (1x10 %ells/ml) were inoculated and allowed to grow statically at different. temperatures as above. The growth of these cultures was estimated by measuring the optical density at 660 nm at different intervals of time. G r o ~ v t h i n m.olasses med i z~n l : The molasses was diluted three-fold to give approxjmately 20% fermentable sugars and added 0.5% magnesium sul.phate and 0.1 '% urea. The pH was adjusted to 4.8 before autoclaving. Flasks containing 100ml of'the above rnedjum were inoculated with a lonp-full of yeast cells and incubated a t room temperature for three days on a shaker a t 200 rpm. After three -days, the medium was filtered and d r y mass of cells was determined. Dctermin.u.tio71. oj' antir~o acid conter1.t: Amino acid content was determined using Pheiiylthiocarbamvl (PTC) derivative of samples by HPLC as described by Bidljngmeyer et al." Dctcrm.il~.ntioii. of ' Thtaruii~e ai1.d RibofZauir~ conten.ts: Thiamj.ne and riboflavin corlt,ent,s were determinecl by the HPLC method developed a t the Laboratory of Fovd Chemistrg: Agrjcultural Research Center of Finland by Hagg.' RESULTS Moisture content: Both strains of C. tropicalis contained about 6-774 moisture (Table 1). Ash content: Both strains of C. tropicalis had about 7% ash. (Table 1). Total nitrogen and protein contents: Both strains of C. tropicalis contained 9%) total nitrogen and more than 50'5, crude protein on dry weight basis. St,rain NYCY 2699 had a higher crude protein content of 57.7 <% on dry weight basis. (Table 1). Table 1: Moisture, Ash, Total nitrogen and Crude Portein contents of two C. tropicalis strains NCYC 2705 and NCYC 2699. St,l.~i.n No Moisture corlt.ent:'' Ash c o n t , e n t : V o t , a l nitrogen" Prot,ein content? 'Xt dry wt. '5) dl:y wt.. % dry ~ t , . '%! dry wt.. NCYC 2705 fj.58 7.12 9.10 56.9 NCYC 2699 7.33 6.92 9.23 57.7 Mean value ol't111.ee replicates Growth a t different temperatures: Figs. l a and I b show the growtl~ curves of' two strains under this study at different temperatures. Both cultures showed good g ~ o w t l ~ at 35' C. At 40" C both cultures showed a higher growth rate initially but t11is dec.r.easetl 1at.e~. OD 660 nm 1 . 4 1 Time (hours ) I (a) Growth curves of strain NCYC 2 7 0 5 Time ( hours ) 1 (b l Growth curves of strain NCYC 2699 Figure 1: Growth curves of two yeast strailis at dilferent temperatures. Calzdida tropicalrs fb7- si~zgle cell proteilz prodztctio?s 83 Amino acid content: Percentage composition of amino acid of two strains of' C. tropicalis under this study, calculated from chromatographs are given in Table 2. Both these strains contained appreciable amounts of cysteic acid , 8.9% in strain NCYC 2705 and 10.2% in strain NCYC 2699. Glutamic acid, theronine and i soleucine were found in higher amounts in strain NCYC 2699 than i-n strain NCYC 2705, while serine, histidine, alanine, valine and cystine were found in higher amounts in strain NCYC 2705 than in strain NCYC 2699. Strain NCYC 2705 had aspartic, tyrosine, methionine and lysine in amounts less than (I($,) while strain NCYC 2699 had lesser amounts of serine, p r o b e and leucine. Table 2: Percentage Composition of Amino Acid of two C. tropicalis strains NCYC 2705 aild NCYC 2699 Amino Acid % Composition of Amino Acid NCYC 2705 NCYC 2699 Cysteic acld 8.9 10.2 Aspartic acid 0.27 2.2 Glutam~c acid 4.7 8.7 Serlne 4.5 0.92 Glycme 1 . G 1.9 H~stidine 4.7 1.1 Arginine Threonin Alanine Proljne 'Pyrosine Valine Methionine Cystei~le Iso-leucine Leucine Pherlylalallirle Lys~ne Vitamin B content: Both strains contained 0.1 to 0.2 mg of riboflavin per 1.0 g of cells on dry weight basis. The thiamine and riboflavin conte1lt.s of the two strains are giver1 in the Table 3. Table 3: Riboflavin and Tlliamine contents of C. tropicalis NCYC 2705 and NCYC 2699 Strain No. Riboflavin mg/g Thiamine mg/g NCYC 2705 0.213 0.178 NCYC 2699 0.175 0.10 DISCUSSION Use of microbes in the production of protein gives many advantages over the conve~~tional methods due to following reasons. Microbes have shorter generation t>irne, allow easy genetjc transformation, utilize maIly substrates, have no requjrernerlt for arable land or any particular season to grow, and have the possibility of contilluoes production anywhere in the world. The cell yield (g dry we~ght cells/g) varies according to the substrate and. the yeast species employed. The theoretical yield to be expected fro111 one gram of glucose is approxin~ately 0.45 - 0.5 g of' cells, i.e. 45 - 50% of substrate is converted to cells under aerobic rnetabolisnl. Both strains of' C. tropicalis used in this study yielded 0.2 g /g of ferirlentable sugars in rxlolasses medium. This is a moderate amount which could be improved by mechanical aeration and supplementing the medium with vitamins etc. Accordjrig to official specification for food yeast in US, a yeast species to be ~ l sed in SCP production should have the following requirements: maximum mloisture content of 7%, a maximum ash content of Sf%;, and a niinimum crude protein content of' 45'Xj." Single cell proteins produced from these strains of C. t7.opica~dis conform, very well to the above-mentioned standard.^. Protein co~ltent is an important criterion that is used to determine the suitnbilit,y of microorganisms for SCP productiou. Fungi when grown a t higher growth rate normal.1~ have higher protein contents as well as increased levels of nucleic acids. As far as potential use of SCP in human feeding is concerned, the nucleic acid is undesjrable because their metabolism leads to unacceptably high level R of' uric acid .!' Two Car~dida strains studied had high protein .content (5591 on dry weight basis) and this may include some non-protein nitrogen also. However, i t is possible to limit the amount of nucleic acid formed (especially RNA) by controll~ing the growth rate of the culture. Caizdida tl-opicalis for siizgle cell protein production 85 The nutritional value of a protein is dependent on its amino acid pattern and is judged to be better the more closely i t resembles the amino acid content of whole egg. There are nine essential amino acids, valine, methionine, phenylalanine, iso-leucine, leucine, histidine, threonin, tryptophan and lysine ." The strain NCYC 2699 contained 41% essential amino acids while NCYC 2705 contained 42%) essential amino acids from the total amino acid content of each of the strains. In SCP sources sulphur-containing amino acids are usually limiting.ll However in the two strains studied cysteine content was fairly high when com- pared with other published results, although methionine content was low. In addition to valuable proteins, these two yeast strains contained substantial levels of vitamins, especially those of the B group. Thiamine and riboflavin are water soluble vitamins which are needed for carbohydrate metabolism and protein metabolism respectively. The two strains of yeast contained about 200 pg of riboflavin /g and 100 pg of thiamine / g of dried cells which is adequate to fulfill the daily requirement. The values are substantially higher than those reported for C utilis grown on sulphur waste liquor, viz 45 pg and 50 pg /g dry weight of cells. l' These results reveal that the two strains of C. tropicalis used in this study, if grown more efficiently using molasses as the substrate, are promising yeast strains for the production of single cell protein. As such these strains could be recommended for the production of animal feeds using cheaper substrate such as molasses. Acknowledgement This work was supported by the University of Sri Jayewardenepura. The authors are grateful to Dr S. S. E. Ranawana of the Veterinary Research Institute, Gannoruwa for providing the HPLC facilities for amino acid and vitamin analyses and the National Collection ofyeast Cultures, Norwich , UK, for identifying yeast cultures. 1. Silva M. E. S. T. & Nicoli J. R. (1985). Production of nutritive value of single cell protein from Fusariunr oxysporurn var. 1in.i grown in vinasse. Journal of'Fermentatiolz Teciznology 63(1): 91-94. 2. Moo-Young M., Chahal D. S. & Vlach D. (1978). Single cell protein from various chemically pretreatzed wood substrate usi~lg Chetomium cellulolyticum. Biotechn,ology and Bioelzgineering xx, 107-118 3. Sasikala CH. & Ramana CH. V. (1995). Biotechnological potential. of anoxgenic phototrophic bacteria. Advances of Applied Microbiology 4: 173 -235, S. C. Wjeyaratne and A. N. Jayathilake Offcial Methods of Analysis, Association of Analytical Chemist, 4t" edition, (1984). Published by Association of Official Analytical Chemists, Inc. Virginia, U. S. A. Lodder J. (1984). "The Yeast" A taxonomic study. 3'" Edition pp 1385, North Holland Publishing Company, Amsterdam. Bidlingmeyer Brian A., Cohen Steven A. & Tarvin Thomas L. (1984). Rapid analysis of amino acids using pre-column derivative. J o u r n a l o f Chronzatogrczphy 336: 93-104. Hagg Margareta & Kumpulainen Jorma (1994). Thiamine and riboflavin contents of Finnish breads and their corresponding flours. Journal ofFood Conzposition and Analysis 7: 94-101, National Formulary (NF XII), American Pharmaceutical Association, Washington D. C. Edozien J. C., Udo U. V., Young V. R., & Scrimshaw N. S. (1970). Effects of high levels of yeast fceding on uric acid metabolism of young men. Nature, London, 228: 180. Coricise Encyclopedia of Biochemistry (German- Brockhaus ABC Biochemje) Translated and revised by Thomas Scott & Mary Brewer, Published by Walter de Gruyter, Berlin. Mateles Richard I. (1979). The physiology of single cell protein (SCP) production. In: Microbial Tech.nology : Crcrrent State, Futu.re Prospects. (Eds. A.T.Bu.11, D.C.Ellwood and C. Ratledge) Twenty-ninth syinposium of the Society for General Microbiology. JNSF 28_1_79.pdf JNSF 28_1_79 (2).pdf JNSF 28_1_79 (3).pdf JNSF 28_1_79 (4).pdf JNSF 28_1_79 (5).pdf JNSF 28_1_79 (6).pdf JNSF 28_1_79 (7).pdf JNSF 28_1_79 (8).pdf