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RESEARCH NOTES Date Syrup and Baker’s Yeast Production I. Alemzadeh* and M. Vosoughi Biochemical and Bioenvironmental Research Center, Chemical Engineering Department, Sharif University of Technology, 11365-6891 Tehran, Iran
Date syrup might be considered as one of the innovative and attractive byproducts in date processing for the habitants in west and southwest Asia. Date syrup, which is an important byproduct of dates, was obtained by water extraction and evaluated as a substrate for baker’s yeast production, in a batch laboratory-scale fermentation. Saccharomyces cerevisiae was cultivated in the date syrup (20 g/L) based medium. Batch growth was studied in shaken flasks. The diauxic growth was observed with two specific growth rates, 0.22 and 0.035 h-1, where the first relates to date sugar consumption and the second to ethanol uptake. Addition of a corn steep liquor (CSL) increased the dry cell mass, and a maximum yield was observed at 7% CSL in culture media. The effects of different carbon sources on biomass and yield indicated that date syrup could be evaluated as a favorable carbon source in baker’s yeast production. Introduction Dates are produced in abundance in west and southwest Asia. They are wasted a great deal annually. Dates contain about 60% sugar and are utilizable in many fermentation media.1,2 Date syrup is an attractive byproduct from dates. It could be replaced in different food formulations.3-6 Date syrup looks like a darker honey, presenting the same viscosity but having a very peculiar organoleptic flavor, and contains different minerals and phosphorus.5 It could be used as a substrate replacing carbon and mineral sources in single cell protein (the protein utilizable as food or feed) production. It also contains a substantial level of nutrients that are required for the growth of microorganisms.1 Baker’s yeast is an important additive among the substances, which improves bread quality. Baker’s yeast is being produced by batch, fed-batch, or continuous culture.7 Saccharomyces cerevisiae is the desired microorganism for fermentation of starch in dough, and besides giving a favorable taste, it also enriches the flour with a variety of vitamins and proteins. The main ingredient in yeast biomass production may include various carbon sources such as beet and cane molasses; because beet molasses has other major applications as alcohol production, the use of other carbohydrate sources may be considered. The aerobic batch growth of S. cerevisiae is studied by many authors.7-9 They observed a diauxic model with special sugar as the limiting carbon and energy source. The first growth phase is characterized by the assimilation of the sugar predominately by aerobic fermentation with ethanol and carbon dioxide as the major product. This is followed by a second growth phase which is characterized by respiratory growth on previously excreted ethanol. The two growth phases are separated by a diauxic lag, a period * Corresponding author. E-mail:
[email protected].
during which the organism adapts to respiratory growth. The observed specific growth rate in this phase depends on the strain and medium and other growth conditions.10 The cell cycle of S. cerevisiae was divided into an accumulation phase and a reproduction phase. Cells in the accumulation phase store carbohydrate, directly using either incoming sugar or ethanol. This phase of growth is fully or nearly respiratory, and the metabolism is primarily glycolytic, resulting in the accumulation of ethanol in the medium.9 Growth during the second exponential phase proceeds at a slow rate, on the accumulation of ethanol, and is characterized by a respiratory metabolism. The diauxic lag has been shown to be predominately a period during which the necessary enzymes for respiratory growth become rapidly depressed.10 In this study, the wasted date was turned to date syrup, and consequently, the baker’s yeast growth rate was studied in a date-based medium under the laboratory batch scale system. Also, the capability of date syrup sugar as a suitable carbon source in baker’s yeast production was investigated. Materials and Methods Microorganism: S. cerevisiae, ATCC 4126. The strain was stocked on Sabourd’s solid medium at 4 °C.11 Date Syrup Preparation. Pitted dates after stone removal were soaked in approximately 2 times their weight of water and autoclaved at 120 °C and 20 min for the extraction of nutritive ingredients, then half their weight of water was added, and the resulting solution was mixed for 1 h. The mash was centrifuged at 6000g for 20 min at 4 °C. The clear extracted juice was boiled under vacuum in a rotary vortex (Janke and Kunkel, IKA-Labor Tecknik) at 70 °C to 60° brix. Culture Preparation. The strain was grown on Sabourd’s solid medium slant at 30 °C. The liquid
10.1021/ie010385+ CCC: $22.00 © 2002 American Chemical Society Published on Web 01/02/2002
Ind. Eng. Chem. Res., Vol. 41, No. 1, 2002 129 Table 1. Chemical Composition of Date Syrup component
amount (g %)
component
amount (g %)
moisture ash protein total sugar reducing sugar
35.0 1.30 1.9 65.8 62.2
phosphorus calcium potassium sodium iron
0.30 0.54 0.25 0.10 0.16
culture medium contained the following for total sugar (in g/L): (NH4)2SO4, 8; NaH2PO4, 5; MgSO4, 0.25; date syrup, 20. Date syrup must be added so that the total sugar in the medium reaches 20 g/L. The medium was divided into 500 mL Erlenmeyers and sterilized at 120 °C for 20 min. Each yeast slant was mixed with 5 mL of physiological serum (9.9 g/L NaCl in distilled water prepared in test tubes and sterilized) and was transferred to culture media. So, the inoculum percent in each Erlenmeyer sample would be 5%. Fermentation was effected under shaking at 200 rpm and a temperature of 30 °C. Chemical Analysis. The sugar composition in date syrup and the culture was measured by the Nelson and Somogy method.12 Nitrogen was determined by the Kjeldahl method.13 The Kjeldahl method consisted of three basic operations: digestion for conversion of organic nitrogen compound to ammonia, steam distillation for liberation of amminia and absorption into a known volume of standard acid, and titration of the excess acid for determination of the amount of acid nutralized and the quantity of nitrogen present in the sample. The results obtained from titration help one to calculate the percentage (w/w) of nitrogen in the sample.13 The percent of nitrogen must be changed to an organic nitrogen compound by a known factor. So, the factor N × 6.25 (100/16) is utilized to convert percent nitrogen to percent organic compound or protein in this study. Ash, minerals moisture, and phosphorus were determined by the AOAC method.16 Potassium and sodium were analyzed using flame photometry (Gallenkamp flame analysis), while calcium and iron were estimated using atomic absorption spectrophotometer (Pye Unicom SP191).14-16 The dry cell weight was determined by the dry weight technique, with Millipore 0.45 µm filter paper.17 The concentration of ethanol in the culture was measured after centrifugation at 4000g for 15 min at 4 °C by a chemical method based on the oxidation of alcohol to acetic acid by acid dichromate and determination of reduced dichromate by a photometric method at 480 nm by a UV-visible spectrophotometer (Varian Techtron model 635).18 Results and Discussion Date Syrup and Growth Kinetics. The results of date syrup analysis are summarized in Table 1. The data in Table 1 present the significant quantity of fermentable sugar existing in date syrup, besides date syrup contains levels of minerals required for the growth of microorganisms. Batch growth of S. cerevisiae on a date syrup containing medium is presented in Figure 1. In Figure 1, the diauxic growth model for S. cerevisiae in a date syrup containing medium is observed. Diauxic growth kinetics on pure sugar sources such as glucose, galactose, sucrose, maltose, and ethanol by S. cerevisiae was also studied by authors.9,10 Figure 2 presents the logarithm of dry biomass production with time. The
Figure 1. Batch growth rate curve of S. cerevisiae in a date syrup medium at 30 °C and pH ) 5.4.
Figure 2. Log of dry biomass production with time. Table 2. Effect of Different CSL Percents in Culture Media on Biomass and Cell Yield CSL (%)
biomass (g/L)
yield (%)
CSL (%)
biomass (g/L)
yield (%)
0 2 4 5
3.3 4.1 5.0 5.2
16.3 19.7 24.8 26.0
6 7 8
5.3 6.6 4.9
26.1 33.1 24.7
specific growth rate is derived at the two phases:
dx µx dt µ1 ) 0.22 h-1
ln
X ) µt X0
µ2 ) 0.035 h-1
µ1 is the specific growth rate of S. cerevisiae in a date syrup containing medium, concerning date sugar uptake, and µ2 is in the case of the batch specific growth rate on ethanol consumption with time. The first phase relies largely on glycolysis for energy production. During this phase, date syrup is assimilated and ethanol is excreted. Effect of Corn Steep Liquor (CSL). CSL, the liquid waste from starch and glucose companies which are a source of nitrogen and cofactors, is a semiclear liquid with 4% solid materials and was filtered by Whatman paper no. 42 before utilization. The effect of CSL addition in a date syrup containing medium on biomass and cell yield (grams of biomass produced per grams of sugar uptake) is studied, and the results are presented in Table 2. From Table 2, it can be concluded that the addition of 7% CSL in a date syrup containing medium
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Table 3. Effect of Different Sugar Sources on Biomass and Cell Yield sugar source
biomass (g/L)
yield (%)
sugar source
biomass (g/L)
yield (%)
date syrup beet molasses
6.3 6.2
32.03 30.9
dextrose sucrose
4.5 3.7
22.4 18.3
Table 4. Effect of Different Total Sugar Concentrations in a Date Syrup Medium on Biomass and Yield sugar conc (g/L)
biomass (g/L)
yield (%)
sugar conc (g/L)
biomass (g/L)
yield (%)
7 12
3.87 4.88
19.04 24.19
16 20
5.68 6.34
28.27 32.62
results in optimal biomass production and cell yield in a fermentation culture. CSL addition at 8% in culture media resulted in a lower biomass concentration and a decrease in the cell yield which could be related to the inhibitory effect of chemicals existing in CSL at higher concentration. Effect of Different Carbon Sources. The effects of different pure carbon sources as dextrose and sucrose and industrial byproducts such as molasses in the same total sugar concentrations on the maximum biomass and cell yield are presented in Table 3. The concentration of total sugar in all fermentation media was 20 g/L. The data in Table 3 indicate that date syrup is the best carbon source in baker’s yeast production, and the highest cell mass and yield are observed for a date syrup based medium. Also, for a medium containing beet molasses, the biomass and cell yield are notified, but this byproduct has other applications in fermentation processes. Optimization of date syrup sugar concentration in a fermentation medium is shown in Table 4. Date syrup at a 20 g/L concentration reveals the maximum biomass yield. The best sugar concentration in a date based medium was 20 g/L; all other ingredient concentrations were the same as those in a culture medium, and 7% CSL was added. Comparing the data in Tables 3 and 4 reveals that date syrup is a suitable carbon source and the highest cell yield is observed in the case of date syrup at a sugar concentration of 20 g/L. Conclusion The batch biological process for baker’s yeast production in the culture media enriched by date syrup as the sugar source and other nutrients was investigated. The effects of different parameters as time, CSL concentra-
tion, different sugar sources, and concentrations were studied. CSL as nitrogen and cofactor sources revealed a significant effect on biomass production. Various sugar sources such as beet molasses, dextrose, and sucrose indicated lower yield compared with date syrup in the same total sugar concentration. Literature Cited (1) Kamel, B. S. Utility of Date Carbohydrate as Substrate in Microbial Fermentation. Process Biochem. 1979, June, 1. (2) Myhara, R. M.; Karkalas, J.; Taylor, M. S. The composition of maturing omani dates. Sci. Food Agric. 1999, 79, 1345. (3) Alemzadeh, I.; Vosoughi, M. Nutritive Value of date. Annual Sharif Proceedings, Tehran, Iran, 1990. (4) Alemzadeh, I.; Vosoughi, M.; Keshavarz, A. Formulation of a by-product from date. Export Development Seminar, Ahwaz, Iran, 1996. (5) Alemzadeh, I.; Vosoughi, M.; Keshavarz, A.; Maghsoudi, V. Use of Date Honey in the Formulation of Nutritious Creamy Food. Iran Agric. Res. 1997, 16, 111. (6) Sayed, M. A. Date Syrup as Sweeteners in Cake and Confectionary Products. M.Sc. Thesis, Tarbiat Modaress University, Tehran, Iran, 1985. (7) Barford, J. P. Mathematical Model for the Aerobic Growth of S. cerevisiae with a Saturated Respiratory Capacity. Biotechnol. Bioeng. 1981, 23, 1735. (8) Bijkerk, A. H. E.; Hall, R. J. A Mechanistic Model of the Aerobic Growth of S. cerevisiae. Biotechnol. Bioeng. 1997, 19, 267. (9) Hall, R. J.; Barford, J. P. Simulation the Integration of the Internal Energy Metabolism and Cell Cycle of. S. cerevisiae. Biotechnol. Bioeng. 1981, 23, 1763. (10) Pemment, N. B.; Hall, R. J.; Barford, J. P. Mathematical Modeling of Lag Phase in Microbial Growth. Biotechnol. Bioeng. 1978, 20, 34. (11) Difco Manual; Difco Laboratories: Detroit, MI, 1953. (12) Whistler, R. L.; Wolform, M. L.; BeMiller, J. N.; Shafizadeh, F. Methods in Carbohydrate Chemistry; Academic Press: New York, 1962. (13) Joslyn, M. A. Methods in Food Analysis; Academic Press: New York, 1970. (14) Yashajahu, P. E.; Clifton, E.; Meloan, C. F. Food Analysis; New York, 1994. (15) Birch, G. G. Analysis of Food Carbohydrate; Elsevier Applied Science: New York, 1985. (16) AOAC. Official Methods of Analysis, 5th ed.; Assoc. Offic. Annual Chem.: Washington, DC, 1990. (17) Moo-Young, M. Comprehensive Biotechnology; Pergamon Press: Oxford, 1985; Vol. 4. (18) Jaulmes, P.; Dieuzeide, J. C.; Hamelle, G. Sur le dosage Chimique de l’Alcohol. Trav. Soc. Pharm. Montpellier 1953, 13, 163.
Received for review April 30, 2001 Revised manuscript received October 12, 2001 Accepted October 29, 2001 IE010385+
