PALMYRAH PALM WINE PART 11: IMPROVEMENTS IN ALCOHOL PRODUCTION

Ahsnact: The palmyrah palm wine, a traditional mild alcoholic beverage of Northcrn Sri L.anka, is the spontallcously fcrmcnted sap of the young and mature i~~florcsccnccs of both.malc and female palmyrah (Borassus flabellifer) palms. The palnivrah totldy samplcs had a mean alcohol content of 5.8qvlv]and the efficiency. of natural fcrmcntacion process was found to hc 56%. In the present study. it was found that this cfficicncy of alcohol production can he increased by (i) the addition of inorganic salts such as NH4C1, MgS04 and KH2P04, (ii) heat sterilization of ciccalcificd palmyrah swcet toddy prior to fermentation by a preselected yeast strain ant1 (iii) the introduction of pur? yeast inoculum into collcction pots. The pcrccnragc i~lcrcasc in alcohol production over the control in each case was found to he 12%, 44% anti 2 5 30% rcspcctivcly.


Introduction
Palmyrah palm Borassus jlabcllif~~r grows naturally in the drier regions of Sri Lanka.A mild alcoholic beverage, popularly known as 'palmyrah toddy' or 'palmyrah wine' is obtained from this palm by 'tapping' the inflorescences.This palmyrah wine is 'the spontaneously fermented sap of the young and mature inflorescences of both male and female palms.

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The unfermented sap, commonly referred to as 'sweet toddy' or 'neera', contains 10 -16.5 % w/v sugar, mainly in the form of sucrose.This sugar is converted into ethyl alcohol during fermentation by wild'yeasts and bacteria usually found in 'toddy' collecting pots?o From preliminary studies it was found that the observed levels of alcohol in palmyrah 'toddy' was in the range of 5 -6 % v/v whereas the theoretical yield lies in the range of 9 -10 % v/v.I I  It appears that a considerable part of sugars in the sap is utilized by microorganisms resulting iri products other than alcohol during the early $, stages of natural f e r m e n t a t i ~n .~ The usual methods of tapping and collecting fermented coconut.toddy account for a loss of about 1 -1.5 % alcohol by injudicious handling;9 about the same percentage of alcohol can be lost 4'.
from palmyrah toddy for similar reasons.
There w i l l be a greater demand for ethanol in the near future to meet the energy crisis.Also, improvements in the efficiency of alcohol production will lead to increased production of palmyrah arrack-a product obtained by distilling palmyrah toddy.It is therefore important to formulate methods of controlled fermentation t o obtain the maximim yield of alcohol.As reported earlier, the yeasts belonging to the genus sac char om?^ces are the best fermentors isolated from naturally fermented palmyrah toddy.' Therefore Sacchurornyccs yeasts were used in these studies to improve alcohol production.

Collection of Materials and preparation of Experimental Media a). Fresh unfermented sap:
This was collected in sterile MacCartney bottles by holding thc bottle at the tip of the inflorescence for about 3 minutes.'The sample was immediately stored at 50C or was heated in a boiling water bath for 15 minutes to inactivate the microorganisms which may have contaminated thc.sap.This sample was mainly used to determine the sugar content of unfermented sap.

b) Sweet toddy:
Palmyr'h sweet toddy was collected for 14 hours in earthenware pots; the inner surface of these pots were coated with slaked lime.

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The lime used in the collection of sweet toddy was removed initially +\ .
by sedimentation and later by precipitation as calcium phosphate by addiiig *a 'i superphosphate.Precipitation was enhanced by heating to about 40 -50°C and by centrjfugation.This centrifuged, decalcified sweet toddy was a clear, colourless liquid with a pH around 6.5 -7.For experiments where sterile sap medium was required, this decalcified sweet taddy was sterilized by autoclaving at 15 Ib/in2 pressure (121%) for 15 minutes.This was obtained by collecting the palmyrah sap in earrhenware pots by adopting the traditional process of toddy collection.Usually the samples were obtained in mornings after a collection period of about 14 hours.

A) Routine analytical metho&:
Amount of sugar in a sample was estimated according to the Somogyi's sernimicro m e t h ~~.~' Alcohol conterit was determined using an ebulliometer.5B) Experimental procedures:

Heat sterilization of palm yrah sweet toddy and alcohol production:
Decalcified palmyrah sweet toddy medium was prepared as described in 2.l.(b); 500 ml aliquots of this m e @ m was fermented under both sterile dnd non-sterile conditions using an overnight culture of Sactharom yces cerevisiae PY 1.The inocul;urn potential was lo5 cells/rnl; the alcohol content of the experimental meda was measured after 48 hours.
-1.0 g/l.The alcohol contents of these media were determined after 48 hours of total fermentation.Sterile, decalcified palrnyrah sweet toddy was supplemented with (i) MgS04 -0.2 gll; NHdC1 -1.0 g/l and KH2P04 -1.0 g p (i) KN03 -0.5 g/l and NH4N03 -0.5 g/l.These media, supplemented with salts, were fermented using the yeast S. cerevisiae PY 1 for 48 hours and the alcohol content determined.

Effect of inoculum potential on fermentation:
500 rnl portions of the sterile, decalcified paImyrah sweet toddy were inoculated with different inoculum potentials of an overnight culture of S. cerevkiae PY 1.The initial cell density ranged from 104 to 108 cell/ ml.The sugar and alcohol contents of these experimental media were measured periodically by the routine methods.

fmprovements in'alcohol production by introducing,pure yea@ inoculum into the collection pots:
. 1 In ,this experiment pure cultures of S. cerevisiae PY 1 and Saccharomyces chevalieri PY 10 were introduced separately into clean collection pots and the2 yield of alcohol was compared with that obtairled by the usual practice.
Inoculurn for each collection pot was prepared by growing the particular yeast strain for 24 hours in 5 0 0 d of yeast exrract: peptoneglucose (2%) broth to obtain a final cell density of the order lo8 cells/ml.The yeast cells were separated by centrifugation and washed well with sterile water.This yeast residue was then transferred to clean eirtheriware pots normdy used for the collection of toddy.
The toddy samples were collected from these pots after about 15 hours and allowed to ferment for a further period of 25 hours, after which their alcohol content was determined.The results of experiment 2.1 were statistically AalYsed according to ' ~a i l e ~3 and are,presented in Table 1.
The results show that the alcohol produced from heat-sterilized sweet toddy is greater than that from nan-sterilized sweet toddy.
Although fermentation of sweet toddy is arrested during collection by the addition of calcium, there is still a large number of bacterial and yeast cells.These grow when the pH of the decalcified sweet toddy is adjusted to 6.5-7.0 due to decaIcification and compete with the inoculated yeast strains, reducing the level of alcohol production.For maxinium alcohol ,production, palmyrah sweet toddy, therefore should be sterilized prior to inoculation with the desired yeast strains.The only disadvantage in heat s t d z atibn is that the toddy may have a slightly altered, bitter flavour.This may be due to caramelisation of sugars in sweet toddy during heat sterilization.

R. Kumu th ini Chrystopher and K. Theivendirarajah
These studies reveal that the addition of NH4Cl into partly fermented palmyrah toddy significantly increased alcohol production in toddy.However, the % increase over the control in this case is approximately 12, which is much lower than that obtained for heat sterilized sweet toddy a fermented with the yeast S. cerevziiae PY 1 (44 %),without the addition of 4 'K-NH4'.This may by due to the mixed microflora present in the partially 6 fermented toddy.,Also, as reported by Nathanae19, 1 -1.5 % of the -

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alcohol would have been lost during the collection of the partially fermented toddy.
The of M ++, NH: and Po4---increases the yield of alcohol significantly over k e control.In the case of natural toddy, ad&tion of these salts has led to an 8.26 % increase in alcohol production over the control whereas in the case of sweet toddy fermentation, this increase was approximately 12 %.However, this 12 % increase over the control can be achieved by the additiop of NH4Cl alone into the fermenting medium.
Jansz6 reports that the addition of NH4c1 at a prefermentation stage improves the flavour of toddy, but it does not affect si@icantly the yield of alcohol.JSalyananda7 states that the effect of NH4Cl on fe&ention depends on when it is added.It has been reported that the NH4Cl supplies the yeasts with' an easily digestible source of nitrogen, resulting in increased sugar utilization and higher yields of alcdhol?
From the results presented in Table 2, it is obvious that the addition of nitrates suppresses the production of alcohol.It may be that palmyrah sap has sufficient levels of nitrates and any addition would lead to inhibitory levels of nitrate.It may be also due to the fact the S. cerevisiae strains cannot utilize nitrate since they lack the ability t o reduce it to NH4+ ions These studies reveal that the rate of fermentation can be significantly increased by increasing the inoculum potential.It was found that an inoculum potential of l o 7 cells/ml used up all the sugar within 48 hours of fermentation thus suggesting that the maximum alcohol can be obtained within 48 hours with a l o 7 cellslml inoculum potential.
An initial cell density of lo7 cells/ml is easier to handle than 108 cells/ml, whereas better results can be obtained quicker by using an inoculum potential of 108 cells/ml.The present studies also suggest that fermentation proceeds rapidly when the same quality of medium 5s used for both the preparation of the starter culture of yeast and for the subsequent fermehtation.This may be explained by the shorter lag period.
When a lesser number of cells are inoculated into the fermenting medium, they will utilize 1 -2 % of the sugar for their biomass production and increase in number, after which alcohol production starts.This will reduce the efficiency of fermentation.But when a higher density of yeast is inoculated not much sugar is needed for the growth of yeasts and therefore the percentage conversion of sugar to alcohol will be more, thus resulting in higher yields of alcohol.

Improvements in Alcohol Production by Introducing Pure Yeast
Inoculum into the collection Pots: Statistically analysed results of the investigation described in 2.4 are presented in Table 3. used for two weeks with an appreciable increase in the yield of alcohol over the control.Kalyananda' suggests thqt instead of introducing yeast inoculum into conection pots, the addition af NH4+ into the collection * ' pots would significantly increase the alcohol content of coconut toddy.
Though these experiments indicate that the production of alcohol in toddy can be increased by (i) the fermentation of heat-sterilized sweet toddy with pre-selected, efficient yeast strains in the presence of NH4+ salts and (ii) the addition of pure yeast inoculum into the collection pots, there are Limitations in applying these two methods.The Limitations in the fermentation of heat-sterilized sweet toddy are (i) the risk of altered flavour and aroma from the caramelization of sugars in sweet toddy (ii) the need to have the sweet toddy samples collected and to have the lime present in those samples removed by the addition lof superphosphate and (iii) the difficulties arising from large quantities of palrnyrah sweet toddy having to be sterilized without delay.
Introduction of pure yeast inoculum into collection pots also involves the preparation of a high density of yeast inoculum in a convenient form so that it can be easily handled by the tapper.
c) Partly fermented toddy: Control samples were obtained by collecting the toddy from the usual pots having a sediment of wild yeasts and bacteria, including the test strains S. cerevziiae PY 1 and S. chevalien' PY 10.Toddy samples were collected from the same pots and .analysedagain on the 7th and 15th day after the introduction of pure yeast inocula This experiment was camed out from Ma& 1983 to June 1983 with samples of toddy from two male palmyrah palms, and the procedure was .repeated 5 times.

Figure 1 : 1 RFigure 2 :
Figure 1: Effect of inoculum potential on total sugar content of palmyrah sweet toddy medium fermented by the yeast Saccharomy ces cerevisiae PY 1

Effect of Inorganic Salts on Alcohol Production: Results of the Experiment 2.2 were statistically analysed according to Bailey3 and are presented in Table 2.Table 2 .Effect of inorganic salts on alcohol production
'Control,:.iSxp&,,jen&:.