Palm Wine Mixed Culture Fermentation Kinetics

Transcription

1 IN: Impact Factor 1 (UJRI):.7799 ICV 1: 5.88 Palm Wne Mxed Culture Fermentaton Knetcs By Opara C.C. Ajoku G. Madumelu N.O.

2 Greener Journal of Physcal scences IN: Vol. (1), pp8-7, January 1. Research Artcle Palm Wne Mxed Culture Fermentaton Knetcs Opara C.C. Ajoku G. and Madumelu N.O. Department of Chemcal Engneerng, Unversty of Port Harcourt, Rvers tate, Ngera Emal: Tel. No: , Correspondng Author s Emal: hephzbah5@yahoo.com ABTRACT In ths work, a mathematcal model was developed descrbng the mxed culture fermentaton of palm juce based on the growth profles of the four mcroorgansms found n the juce. These mcroorgansms are Yeast, Mcrococcus, Lactc acd bactera and Leuconostoc spp. The mathematcal model was smulated usng MATLAB whch showed the relatonshp between the substrate concentraton and tme for the ndvdual mcroorgansms. Expermental data obtaned from lterature was used to obtan the growth rates and substrate saturaton coeffcent usng the Monod model. It can be seen that the cell number of Leuconostoc spp. has ncreased to whle the expermental value s at hours. The substrate concentraton at hrs was found to be.968g/1g dry matter whle ts expermental value s.8g/1g dry matter. Ths shows that there s a reasonable level of accuracy n the model developed. Keywords: Fermentaton, knetcs, mcro flora, mxed culture, yeast. INTRODUCTION Bref Hstory of Palm Juce Palm-wne s an mportant alcoholc beverage resultng from the spontaneous fermentaton of the sap of the palm, whch has been attrbuted to yeasts and bactera (Okafor, 1978). Fermented palm-juce s an exudate from palm-tree. Natural palm sap s collected from the ol palm tree_elaes guuneenses and the rapha palms, Rapha hooker and Rapha vnfera, although t could also be obtaned from other speces of palm trees that are known all over the world. Palm wne s produced when the nflorescence of the palm tree s ncsed and tapped, whch s collected n a calabash that s hung at the base of the ncson. Fresh palm-wne s sweet, clear, neutral, colorless juce contanng 1-1% sucrose, mnmal nvest sugar (less than.5%), small amounts of protens, gums and mneral. (Okafor, 1987). pontaneous fermentaton starts mmedately the sap s collected and wthn an hour or two becomes reasonably hgh n alcohol (up to %) (Bassr, 196). Organsms responsble nclude. cerevsae and schzosaccharomyces pombe, and the bactera Lactobacllus plantarum and L. mesenterods (Okafor, 1987). The equatons of reacton of fermentaton of palm-wne and oxdaton of ethanol are represented thus respectvely; C 6 H 1 O 6 yeast C H 5 OH + CO Glucose Ethanol Carbon doxde C H 5 OH + O Acetobacter CH COOH + H O Ethanol Oxygen Acetc acd water In the former fermentaton process, ethanol s produced as the sugar content dmnshes rapdly as t s acted upon by agents of fermentaton (mcroorgansms) whle n the latter, ethanol wll be on the declnng phase as t s converted to acetc acd n conons of excess oxygen. 8

3 Greener Journal of Physcal scences IN: Vol. (1), pp8-7, January 1. Mxed Culture Fermentaton Mxed culture fermentaton s a bochemcal process nvolvng two or more mcroorgansms n a nutrent substance n specally controlled conons for scentfc, medcal or commercal purposes (Mcrosoft Encarta, ). The nteractons of mxed culture systems are dffcult to nterpret because there s no good reference pont when so many conons and concentratons may be changng at once. Research Objectve The purposes of ths research are: - To obtan from lterature the mcroorgansms that prolferates durng the mxed-culture fermentaton of palm wne. - To determne the process conons for the fermentaton. - To determne the yeld coeffcents of the respectve mcro-organsms n the fermentaton process wth respect to tme. - To estmate knetc parameters of the mcroorgansms present based on relable expermental data from lterature. - To determne the substrate utlzaton profle from the start of fermentaton (th hour) to the end of fermentaton (7 nd hour) at ntervals of two hours. - To wrte a mathematcal model that descrbes the process, ths wll be an ad to Bochemcal and Chemcal Engneers n desgnng a large-scale fermenter of the process. - To wrte a computer program that wll solve the mathematcal model and smulate the process. Mcroorgansms Responsble for Fermentaton tudes on the mcrobology of palm wne have shown that palm wne contan both yeast and bactera (Okafor, 1978). Yeast Okafor (1978) solated and dentfed yeasts from palm wne from varous parts of Ngera and concluded that the dstrbuton of yeast n palm wne s not dctated by the type of palm or the localty n whch t grows. In hs study, all the samples were domnated by saccharomyces and candda rrespectve of the type of palm or the area n whch t s grown. Bactera Bactera encountered n palm wne nclude Lactobacllus, Leuconostoc, Bacllus, erratc, treptococcus, Zymomonas mobls, Mcrococcus, Brevabacterum, Pedacoccus, Corynebacterum, Klebsella (Bassr, 196; Faparus, 1966). UCCEION of MICRO FLORA DURING FERMENTATION In ths regard, Bassr and Faparus (1971) reported the presence of Lactobacllus and Leuconostoc n the early stages of fermentaton. Okafor (1975) found that whle lactc acd bactera as a group were mportant, there were no consstent patterns of the dstrbuton of the varous lactc. Accordng to hm, streptococcus was found throughout the 7days perod he carred out hs work whereas lactobacllus remaned only durng the frst days of fermentaton. Okafor (1975) reported the presence of enterobacteracae such as serrata and klebsella early n fermentaton. Accordng to hm, yeasts and mcrococcus seem to occur consstently n many samples of palm wne. Faparus and Bassr (1971) also reported the same n ther nvestgatons. Authortes who have studed the successon of mcro flora n palm wne consstently reported the emergence of Acetobacter after about to days of fermentaton, at whch tme; alcohol was present n reasonable quanttes (Faparus and Bassr, 1971; Okafor, 1975). 9

4 Greener Journal of Physcal scences IN: Vol. (1), pp8-7, January 1. Factors Affectng Rate of Fermentaton Despte ts complexty, the rate of fermentaton s largely dependent upon certan parameters namely yeast, wort composton (.e. Fermentable fltrates normally nutrents for yeast) and the processng conon such as tme, temperature, volume, vessel shape and sze, ph, acd, water actvty, carbon sources are also complementary varables. These conons are n general appled to all classes of fermentaton, be t batch process or the contnuous process. Fermentaton Process In fermentaton, an accurate mathematcal model s a prerequste for the control, optmzaton and the smulaton of a process. The knetcs for growth s generally modeled as frst order. A batch fermentaton process such as that for palm wne has a growth rate equaton modeled as = (1.1) Where = concentraton (cell number) of the th organsm = specfc growth rate of the th organsm t = tme Where d Y = (1.) = ubstrate concentraton Y = yeld coeffcent for ndvdual organsm The assumpton made n generatng ths model s smply that the organsms compete for the common substrate wthout preyng on one another. Ths can be used as an analogy for the mxed culture fermentaton of palm juce as there are no serous prey-predator relatonshps among the organsm. The growth rate, takes the form max = (1.) km + The equaton above s known as the Monod model Where max = maxmum reacton rate Km = aturaton constant of the mcro-organsm To solve ths set of dfferental equatons, t s mportant to note that the values of km and max must be determned for ndvdual mcro-organsm. A soluton of ths wll provde an approxmate model for ths process. METHODOLOGY Mathematcal Model Development In developng ths model, several assumptons were made so as to smplfy ths complex process. One of the major assumptons s that the specfc growth rate of the cells s exponental. Ths assumpton leads to takng the Monod Model as an approxmate model for the growth rate.,max = (1.) km +

5 Greener Journal of Physcal scences IN: Vol. (1), pp8-7, January 1. From the expermental data by Brauman et al. (), the followng were the cell number and the substrate concentraton of the four major mcro-organsms common n the mxed culture fermentaton of palm juce. These four mcro-organsms are Yeast, Mcrococcus, Lactc Acd bactera (LAB) and Leuconostoc spp, (LEU). The result s gven below. Table.1: Change n Cell number and ubstrate Concentrate of the Indvdual Mcro-organsm wth Tme Tme (hr) Yeast Mcrococcus Lactc Acd Leuconostoc spp ubstrate Concentrate Bactera (LAB) (LEU) (g/1g of dry matter) E+9 1E+7 1E E+1 1. E E E+1.9 E+9.51 E E+1 5 E+9 E ource: Brauman et al. () A plot of substrate concentraton aganst tme was made n order to have an approxmate value for the substrate concentraton from to 7 hours at hours nterval usng the equaton below. y = 8.85e -.56x ubstrate Concentraton(g/1 g) Plot of ubstrate Concentraton aganst tme 1 5 y = 8.85e -.56x 6 8 Tme(hr) ubstrate Concentraton(g/1g) Plot of ubstrate Concentraton aganst tme Tme(hr) Fg.1: Plot of ubstrate Concentraton aganst tme at hrs nterval The cell numbers of the mcroorgansms s gotten from the followng equatons Yeast: y = 65e.67x Mcrococcus: y = 58e.17x Lactc Acd Bactera, LAB: y = 186e.161x Leuconostoc spp, LEU: y = 78.88e.16x The cell growth rate of the ndvdual mcroorgansms can be estmated usng Where ln( N f ) ln( N) = (.1) t t f N f = cell number of an organsm at tme, t f N = cell number of an organsm at tme, t = pecfc growth rate The calculaton s done for each of the mcro-organsms and shown n the table below. 1

6 Greener Journal of Physcal scences IN: Vol. (1), pp8-7, January 1. Table.: Cell Growth rate of Mcroorgansm wth Tme Tme (hrs) yeast mcrococcus LAB LEU Knetc Constant Estmaton It s mportant to determne the knetc constants for the ndvdual mcro-organsm n order to get an approprate functonal expresson form for the Monod Models. The technques used n achevng ths nclude 1 Lneweaver-Bulk Plot Eade Hoftee Plot Hanes Plot The choce of choosng whch technque to use s dependent on the nature of the process. The am s to obtan a lnear form of the Monod model and an estmate of the knetc constant. The knetc parameters of Yeast, Leuconostoc spp., and Lactc acd bactera was determned usng Lneweaver-Burk plot from a plot of 1/ aganst 1/. Mcrococcus was determned usng Hanes plot from a plot of / aganst. The plots are shown below. 6 Plot of 1/ aganst 1/ for Yeast Plot of / aganst for Mcrococcus 1/ / / / Plot of 1/ aganst 1/ for LAB / 1/ Plot of 1/ aganst 1/ for LEU / Fg : Plot of the Knetc parameter for the four () mcroorgansms

7 Greener Journal of Physcal scences IN: Vol. (1), pp8-7, January 1. Table.: Knetc Parameter for Indvdual Mcro-organsms wth Tme Km (g/1g) m (hr -1 ) Yeast Mcrococcus Lactc Acd bactera, LAB Leuconostoc spp, LEU Yeld Coeffcent The yeld coeffcent of the four mcroorgansms s calculated from equaton.1 usng the formula below. Yeld coeffcent = Where = f f (.) f = the cell number of a partcular organsm at tme, t f = the cell number of a partcular organsm at tme, t = the substrate concentraton at tme, t f = the subtrate concentraton at tme t f Table.: Yeld Coeffcent of the Indvdual Mcro-organsm wth Tme Tme ubstrate Yeast Mcrococcus LAB LEU (hrs) E+9 5.E+7 5.E E E+7 1.E E+9.1E E+1 6.E+9 7.9E We plot the respectve yeld coeffcents of the mcroorgansms wth respect to tme usng Mcrosoft Excel. The plots are shown below. Plot of Yeld coeffcent of Yeast aganst tme Plot of Yeld coeffcent of Mcrococcus aganst tme Change n yeld coeffcent of Yeast E+1 E+1 1E+1-1E Tme(hr) Change n Yeld coeffcent of Mcrococcus Tme(hr)

8 Greener Journal of Physcal scences IN: Vol. (1), pp8-7, January 1. Plot of yeld coeffcent of LAB aganst tme Plot of yeld coeffcent of LEU aganst tme Change n Yeld coeffcent of LAB Tme(hr) Change n Yeld coeffcent of LEU Tme(hr) The respectve equaton from the graphs n gven below. Ths equaton s used to generate the yeld coeffcent of the ndvdual mcroorgansms at hours nterval. For yeast Y 1 = 5E+8x -E+1x +7E+11 E+11 For Mcrococcus Y = 1756x +56x -E+7x +8E+7 For Lactc Acd Bactera, LAB Y = 187x 1E + 7x + E + 8x 6E + 7 For Leuconostoc spp Y = -.16 x + 5.8x 1.x MODEL The approprate model for the process s as follows: 1 1. = 11 = (.) 1.55 = =.17 + (.).588 = = (.5).19 = = (.6) d 1 = Y Y Y Y (.7) Where 1,,, and are the ntal cell numbers of the ndvdual mcroorgansms Numercal oluton to the Model F (, 1 = = F 1 ) (.8) F (, = = F ) (.9)

9 Greener Journal of Physcal scences IN: Vol. (1), pp8-7, January 1. F (, = = F ) (.1) F (, = = F ) (.11) d F 5 = = F( 1,,,, Y1, Y, Y, Y, ) (.1) The dfferental equatons can be solved by MATLAB whch provdes functons called solvers that mplement Runge Kutta methods. The solver ode 5 uses a combnaton of fourth and ffth order Runge kutta. Therefore the solver ode 5 s used to solve the dfferental equatons from -7 hours. DICUION OF REULT AND CONCLUION REULT AND DICUION The results from the mathematcal models are approxmate values based on the assumpton that the relatonshp between the ncreases n the cell number of a mcro-organsm s exponental wth respect to the equvalent usage of substrate. The mathematcal model s useful, n that t shows consderable relaton of the cell growth trend n the fermentaton process. The ntal substrate concentraton of the medum s 6.5g/1g dry matter and the correspondng values of 1,, and for the respectve mcro-organsm n successon that they occur are 11.6 cells,. cells, 1 cells, cells respectvely. An exponental yeld coeffcent for each of the mcro-organsm was estmated as Y1 = 5E+8 -E+1 +7E+11-E+11 cells/g substrate Y = E+7+8E+7 cells/g substrate Y = 187-1E+7 +E+8-6E+7 cells/g substrate Y = cells/g substrate From ths nputs, the result on the MATLAB plots at hours of the fermentaton process, the cell number of Leuconostoc has ncreased to whle the expermental value at ths tme s , the equvalent substrate concentraton at ths pont s.968g/1g dry matter whle ts expermental value s.8g/1g dry matter. Ths shows that there s reasonable level of accuracy n the model developed. The result also suggests that towards the end of the fermentaton process, the cell number of the ndvdual mcro-organsm remans constant whle the substrate concentraton contnued depletng. 5

10 Greener Journal of Physcal scences IN: Vol. (1), pp8-7, January 1. Fg : MATLAB Plots for substrate and the four mcroorgansms aganst tme CONCLUION Four mcro-organsms were found to be frequently present durng the mxed culture fermentaton of palm-juce, these mcro-organsms n the order of successon are; Yeasts, Mcrococcus, Lactc Acd bactera and Leuconostoc spp; t has also been shown that the fermentaton process s acdc as t progresses and there s prolferaton of mcroorgansms dependng on what the conon of the medum s. Knetc parameters for the varous organsms were estmated based on expermental data from lterature. The yeld coeffcent of the ndvdual mcroorgansm was obtaned. The yeld coeffcent was found to be exponental from to 7 hrs. Ths s because the yeld coeffcent goes through a lag phase before t ncreases. A mathematcal model has also been developed whch represents the relatonshp of the mcrobal cell number and the substrate concentraton wth tme. A computer program was also developed whch solves and helps n smulatng the model from to 7 hours. REFERENCE Anon O (199). Applcaton of Botechnology to Traonal Fermented Foods, Report of an Ad Hoc Panel of the Board on cence and Technology for Internatonal Development,, Natonal Academy Press, Washngton D.C., UA. P 15-. Bassr O (196). Observaton of the Fermentaton of Palm-wne, West Afrcan Journal of Bologcal Chemstry.6:5-1. Brauman A, Keleke, Malonga MO, Mavoungou, Ampe F and Mab E (). Cassava Lactc Fermentaton n Central Afrcan; Mcrobologcal and Bochemcal Aspect, n Cassava Flour and tarch Progress n Research and Development, Centro Internatonal de Agrcultural Tropcal, P Chnnarasa E (1968). The Preservaton and Bottlng of Palm-wne, Federal Insttute of Industral Research Oshod, Research Report No. 8. Eseche HA (1979). Fermentaton tudes on Ngera Palm-wne Ng. Agrc. Journal. 16: Faparus I and Bassr O (1971). Mcroflora of Fermentng Palm-wne ; J. Fd. c. Technology. 8: 6. Fellows P (1997). Traon Food, Intermedate Technology Publcaton, UK. P 68. Ihekoronye A and Ngoddy P (1985). Integrated Food cence and Technology for the Tropcs, Macmllan Educaton Ltd, London, P 1-7. Krk-Othmer (198). Encyclopeda of Chemcal Technology, 5 th Ed, Vol. 1, John Wley and ons Inc., P Ngeran Insttute of Ol Palm Research (NIFOR) (1981). Informaton Bulletn No. Octave L (197). Chemcal Reacton Engneerng, nd Ed., John Wley and ons Inc., pp

11 Greener Journal of Physcal scences IN: Vol. (1), pp8-7, January 1. Odunfa A (1985). Afrcan Fermented Foods n Mcrobology of Fermented Foods Elsever Appled cence Publshers UK, P 5-7. Okafor N (1975). Prelmnary Mcrobologcal tudes on the Preservaton of Palm wne. Journal Applcaton Act 8, P 81. Okafor N (1987). Industral Mcrobology, Unversty of Ile Ife Press. P Okafor N (199). Traonal Alcoholc Beverages of Tropcal Afrca: trateges for cale-up, Process Bochemstry Internatonal journal UA, P Opara CC (). Bochemcal and Mcrobologcal Engneerng, Chjoke Consultant publcaton. Ikenegbu, Owerr. P Mountney GI and Gould WA (1988) Practcal Food Mcrobology and Technology AVI Books, Van Nostrand Renhold Compand, New York, UA. P 51. tenkraus KH (1996). Handbook of Indgenous Fermented Foods, Marcel Decker Inc., New York. P 6. Walker PMB (1988). Chambers cence and Technology Dctonary, Chambers Cambrdge Unversty Press, UK. Vol 5. NOTE: The followng authors were cted n the content but not lsted out as references Okafor, 1978; Mcrosoft Encarta, ; Faparus, 1966; APPENDI A Functon fle functon dx = smulatonfeoma (t, x) % for Yeast Y1= (5*(1^8)*(t^)) - (*(1^1)*(t^)) + (7*(1^11)*t) - (*(1^11)); % for Mcrococcus Y= (1756*(t^)) + (56*(t^)) - (*(1^7)*t) + (8*(1^7)); % for Lactc Acd Bactera (LAB) Y= (187*(t^)) - (1*(1^7)*(t^)) + (*(1^8)*t) - (6*(1^7)); % for Leuconostoc spp Y= (-.16*(t^)) + (5.8*(t^)) - (1.*t) + 6.6; dx1= ((1.*x(5))/(6.668+x(5)))*x(1); dx= ((.55*x(5))/(.17+x(5)))*x(); dx= ((.588*x(5))/(9.88+x(5)))*x(); dx= (((-.19*x(5))/(-1.78+x(5))))*x(); dx5= -(((1.*x(5))/(6.668+x(5))*x(1))/Y1)-(((.55*x(5))/(.17+x(5)*x())/Y))-... (((.588*x(5))/(9.88+x(5))*x())/Y)-(((-.19*x(5))/(-1.78+x(5))*x())/Y); dx=[dx1;dx;dx;dx;dx5] crpt Fle [t,x]= ode5('smulatonfeoma',[::7],[ ]) % cell number of Yeast 1=x (:,1) % cell number of Mcrococus =x (:, ) % cell number of Lactc Acd Bactera (LAB) =x (:, ) % cell number of Leuconostoc spp =x (:, ) % substrate ubstrate=x (:, 5) Plot (t, ), xlabel ('tme(hours)'), ylabel ('change n cell number'),... ttle ('plot of change n Leuconostoc spp aganst tme') 7