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Alcohol Fermentation of Sweet Potato. I.

Acid
Hydrolysis and Factors Involved

A. AZHAR, Department of Biology, Georgia State University, Atlanta,


Georgia 30602, and M. K. HAMDY, Department of Food Science,
University of Georgia, Athens, Georgia 30602

Summary
Factors affecting acid hydrolysis of sweet potato powder (SPP) to fermentable sugars
were examined. These include HCI concentration, temperature, time, and levels of SPP.
Maximum reducing sugar, reported as dextrose equivalent (DE), was detected after 24 min
hydrolysis (1% SPP) in 0.034N HCI heated at 154°C. These samples also had 3.43% hy-
droxymethylfurfural (HMF) based on dry weight. A high level of HMF (9.2%) was detected
in 1% SPP heated at 154°C in O.1ON HCI for 18 min. The lowest concentration of HMF
formed (1.8%), at maximal DE of 61%, was established in samples containing 5% SPP and
heated at 154°C in 0.034N HCI for 48 min. Aqueous extracts of uncured SPP, examined by
HPLC, contained glucose, fructose and sucrose, but degraded SPP had only glucose and
fructose. Products of degraded SPP, under appropriate conditions, could be used for alcohol
fermentation.

INTRODUCTION

The use of alcohol mixed with gasoline as a motor fuel for internal
combustion engines has attracted considerable attention in recent years.
The announcement I.’ by the Brazilian government of their National Al-
cohol Program involving $400 million capital investment is the biggest
single impetus for producing alcohol by fermentation.
Due to the rapid increase in demand for grains both as food and feed,
there is an urgent need for substitution of grain starch with other sub-
strates for alcohol production as well as for various other industrial fer-
mentation~.~ This study was undertaken to examine the acid hydrolysis
of sweet potato (Zpomoea batatas), as a source of starch, to fermentable
sugars under different experimental conditions. Sweet potato is consid-
ered to be a suitable substitute for grains in this respect since it has a
higher starch yield per unit land cultivated compared to grains (corn).

MATERIALS AND METHODS


Preparation of Freeze-Dried Sweet Potato Powder (SPP)
Fresh (uncured) Georgia Jet sweet potatoes, obtained from the Hor-
ticulture Department, Tifton, Georgia, were sliced to a thickness of 0.4
Biotechnology and Bioengineering, Vol. XXIII, Pp. 879-886 (1981)
0 1981 John Wiley & Sons, Inc. CCC OOO6-3592/81/040879-08$01.OO
880 AZHAR AND HAMDY

cm and freeze-dried for 18 hr under a reduced pressure of 50-200 p.m at


a shelf temperature of 0-4°C. The moisture content of the freeze-dried
sweet potato, determined by drying samples in a vacuum oven at 80°C
to a constant eight,^ was found to be 8%. The freeze-dried sweet potato
was ground in a Wiley mill and the resulting powder was allowed to pass
through a No. 40 wire mesh screen and stored at 1-2°C until use. The
starch content of SPP is 64.4% based on dry weight.

Acid Hydrolysis
The desired concentration of SPP was prepared in hydrochloric acid
(0.034N, O.IN, and I . O N ) , sealed in glass tubes (11 mm i.d.) and heated
in an oil bath to temperatures ranging from 140 to 164°C for various time
intervals. The sealed tubes were removed from the oil bath and rapidly
cooled in an ice-water mixture for 5 min. The entire contents of each tube
was then neutralized to pH 7.0 using 0.1N sodium hydroxide, filtered
through Whatman filter paper No. 541, and then washed with 25 ml of
distilled water. The filtrate was checked for reducing sugars, type of sugar
present, and for the level of hydroxymethylfurfural.

Reducing Sugar Determination


The presence of reducing sugars, formed during acid hydrolysis of SPP,
was determined by their ability to reduce 3,5-dinitrosalicylic acid.’ The
data are reported as dextrose equivalent (DE) expressed in the following
equation:
reducing power of sample measured as glucose
DE = x 100
sample dry weight

Sugar Identification
High performance liquid chromatography (HPLC) was used for the
identification of sugars present before and after acid hydrolysis of SPP.
The HPLC system consisted of a Waters Associate ALC-100 equipped
with a Model 6000 A Solvent Delivery System; differential refractometer
detector R401, attentuation 4- 16; column, Waters Associates “Carbo-
hydrate,’’* 30 cm x 3.9 mm i.d. stainless steel, Waters Associate Model
U6K Universal injector. The parameters used were: eluent,
water-acetonitrile mixture [ 15 :85 (v/v)], filtered through 5 IJ.MMillipore
filter before use; flow rate, 1-2 ml/min at pressures up to 3000 psi. Sugar
standards were of high purity (Fisher Scientific Co., Pittsburgh, PA),
and were dried overnight under vacuum at 65°C. No impurities were de-

* Special purpose column of the p BONDAPAK family (monomolecular layer of orga-


nosilane permanently bonded to silica particles 10 pm in diameter).
ALCOHOL FERMENTATION OF SWEET POTATO 88 1

lo t
0 14 28 42
TIME (mid

Fig. 1. The effect of various levels of hydrochloric acid on the hydrolysis of SPP ( I % ,
(wiv); at 154"CI. The results (average of four experiments) are reported as: a ) reducing sugars
formed and measured as dextrose equivalent (DE), and b) hydroxymethylfurfural (HMF)
formed during hydrolysis.

tected during analysis. Aqueous standard sugar solutions and samples


were filtered before analysis through a 5 p M Millipore filter and then
injected (10-20 pl) with a microsyringe.

Hydroxymethylfurfural Determination
The concentration of hydroxymethylfurfural (HMF) present in each
sample was determined by absorbance measurements conducted at 285
nm6 using standard aqueous solutions of this compound (obtained from
Aldrich Chemical Company, Inc., Milwaukee, WI). The data herein is
reported as HMF/100 g dry weight of sample.

RESULTS AND DISCUSSION

Effect of Acid Concentration


Data on reducing sugar, measured as dextrose equivalent (DE), formed
during acid hydrolysis of 1% SPP at 154°C in various levels of HCI is
summarized in Figure l(a). A maximum DE value of 83% was reached
after 18 min of hydrolysis using O.1N HCI heated at 154°C. Lowering the
acid concentration from 0.10N to 0.034N had no substantial effect on the
DE values after 12 and 24 min heating at that temperature. However,
when the acid level was increased to l.ON, a rapid increase in DE value
(60%) was noted within 6 min at 154°C followed by a marked decline to
882 AZHAR AND HAMDY

DEHYDRATION

I
5-HYDROXYMETHYLFURFURAL-POLYMERS

I
L E V U L l N l C A C I D , FORMIC A C I D

Fig. 2. Starch hydrolysis in the presence of acid and the formation of different end-
products (Kerr, Ref. 8).

42.5% after 12 min. When the aforementioned acid hydrolyzed and heated
SPP samples were examined for 5-hydroxymethylfurfural (HMF) content,
the data [Fig. l(b)] revealed that this compound was formed rapidly within
minutes and its level was related to both the acid concentration and time
of exposure. For example, the HMF formed in 1.ON HCI increased rapidly
from 1.4 to 7.5% within only 3 min, followed by a sharp decline upon
further heating. However, when hydrolysis was carried out at a lower
acid level (O.O34N), only a slow and gradual increase in HMF content
was observed. Hydrolysis performed at 0.10N HCI for 18 min resulted
in maximum HMF content of 9.2%.
In addition to acid hydrolysis of starch yielding D-glucose units, other
reactions (inter- and intramolecular elimination of water between the hem-
iacetal and the primary hydroxyl group) have been noted at the same time
leading to the formation of a variety of end-products.' Kerr' summarized
the various reactions observed during the hydrolysis of starch (Fig. 2).
Miller and Cantor' reported that D-glucose formed during starch hydrol-
ysis was dehydrated, in the presence of acid, to 5-hydroxymethylfurfural
as a main end-product and lesser amounts of 2-hydroxymethylfuran. Peat
et al. l o quantitatively analyzed the reversion products formed (maltose,
isomaltose, etc.) following acid treatment of glucose. The maximum DE
value reached [Fig. l(a)], at each acid level, was followed by a decline
upon further heating. Such a decline could be attributed to the faster
conversion of glucose to reversion products or dehydration of HMF as
compared to the formation of this sugar from starch. Decrease in HMF
content at 0.10N and 1 .ON HCl, after a maximum has been reached [Fig.
l(b)], could also be attributed to the rapid decomposition of HMF to
levulinic acid and formic acid' than to the formation of HMF from glucose.
The starch hydrolysis is affected by both the kind and concentration
of acid used. A marked difference between the hydrolysis rate with hy-
drochloric acid and with sulfuric acid was noted by Hollo and Szejtli,"
when both acids were used in equivalent amounts. The rate constant was
larger for hydrochloric acid than for sulfuric acid and depended on both
ALCOHOL FERMENTATION OF SWEET POTATO 883

the acid concentration and temperature. Investigating the cause of the


different rates of hydrolysis, Litvak" stated that sulfuric acid formed salts
with the nonstarchy substances which could inhibit the rate of hydrolysis;
chloride salts had no inhibitory effect and hydrochloric acid in excess
was necessary to guarantee a normal hydrolysis rate and complete hy-
drolysis.

Effect of Ternperuture
The effect of acid on hydrolysis of SPP, at various reaction tempera-
tures as a function of time, is summarized in Figure 3(a). A maximum D E
value of 83% was reached upon heating (at 154°C) the slurry of SPP (1%)
for 24 min in 0.034N HCI. The increase in temperature resulted in a
marked effect on the hydrolysis of sweet potato as evidenced by the re-
ducing sugar content of samples heated for varying times. Increasing the
temperature from 140°C to 154°C increased the DE values. However,
when the temperature was further increased to 164"C, a considerable
decline in DE values was observed. This is in agreement with the con-
clusions reported by Kerr,x who stated that the loss of D-glucose by
recombination and destruction increases more rapidly with rising tem-
perature than its appearance by hydrolysis. Further heating after a max-
imum DE value had been reached, at each reaction temperature, resulted
in a decline in reducing sugar content which was indicative of faster
glucose decomposition through recombination and dehydration than its
formation as the result of acid catalysis.

j
' A
140 O C
154 'C
o 164 O C

0 20 40 60
TME (mid
Fig. 3. The effect of heating temperature on the acid hydrolysis of SPP [1% (wiv); in
0.034N HCI]. The results (average of four experiments) are reported as: a) reducing sugars
formed and measured as dextrose equivalent (DE), and b) hydroxymethyfurfural (HMF)
formed during hydrolysis.
884 AZHAR A N D HAMDY

6t b
4
8
LL
f 2

0
0 10 2 0 30 40 50 60 70
TIME (mid

Fig. 4. The effect of various levels of SPP (in 0.034N HCI, at 154°C) on a) reducing
sugars formed and measured as dextrose equivalent (DE) and b) hydroxymethylfurfural
(HMF) formed during hydrolysis. Data represent an average of three experiments.

The effect of acid (0.034N) at various temperatures on the HMF content


of samples was also determined and the results are shown in Figure 3(b).
A gradual but steady increase in total HMF content of samples was ob-
served at both 140 and 154"C, with the highest level (4.6%) after 36 min
heating at the latter temperature. Hydrolysis at higher temperatures
(164"C), however, showed detrimental effects on HMF content which
might be due to the degradation of HMF to levulinic acid, formic acid,
and possibly, formation of colored compounds.

Effect of Sweet Potato Concentration


Figure 4(a) illustrates the acid hydrolysis (0.034N HCl, at 154°C) of
various levels of SPP as a function of heating time. Maximum reducing
sugar (DE = 83%) formed was evidenced after 24 and 36 min in both
samples containing 1 and 2% SPP, respectively. Further heating resulted
in a decrease in DE values. It is possible that such a decrease could be
due to decomposition of glucose formed during starch hydrolysis. This
decomposition could be brought about by recombination type reaction.
As can be seen in Fig. 2, the recombination of D-glucose is an equilibrium
reaction and, therefore, is dependent on the concentration of D-glucose.
Litvak and Rudenko-Grit~yuk'~ studied the kinetics of glucose conversion
in dilute acid and reported that the duration of heating required to attain
an equilibrium in the reversion reaction depended on the concentrations
of D-glucose and the acid as well as the temperature. Thus the rate of acid
ALCOHOL FERMENTATION OF SWEET POTATO 885

hydrolysis of SPP was affected by both SPP concentration and heating


time. For example, lowering SPP concentration from 5 to 2 to 1% resulted
in an increase in the corresponding DE values up to 24 min. These results
are similar to those reported by Litvak and R~denko-Gritsyuk,'~ who
showed that the loss of glucose during the hydrolysis of starch, brought
about by the reversion and decomposition of glucose, could be decreased
by lowering the concentration of starch.
Data obtained for HMF formed during this experiment [Fig. 4(b)] re-
vealed that both SPP concentration and heating time affected the level
of HMF present. A rapid increase in HMF content in samples containing
1% SPP resulted in a maximal value of 4.6% after 36 min. However, when
the SPP level was raised to 5%, only a very slow increase of HMF was
detected reaching a level of 1.9% after 48 min. The lowest HMF formed
(0.41%) was observed after 12 min heating in 0.034N HCI at 154°C. As
HMF concentrations are expressed in terms of gram HMFI100 g dry
weight of sample:
4.6- -
- concentration of HMF --CH
100 concentration of dry sample C,
4.6/100 = CHI1 .o .'. CH 0.05%
1.9/100 = C ~ l 5 . 0... CH 0.10%
Therefore, in spite of the relative reduction of HMF, we could have in-
hibition problems with wort prepared at 5% SPP but not with wort pre-
pared with 1% SPP. Hydroxymethylfurfural has been found to inhibit
microbial fermentation. Wilson and BrownI4 observed that the growth of
Saccharomyces ellipsoides was retarded in sucrose solutions which had
been heated, and that this inhibition increased with the degree of heating.
These authors suggested that the inhibition might be caused by furfural
or hydroxymethylfurfural. Ingram et al.I5 reported that the addition of
0.1% HMF to a medium containing fructose as sole carbon source inhib-
ited growth and fermentation by Saccharomyces cerevisiae.

Sugar Identfication
High performance liquid chromatographic analysis of SPP revealed the
presence of glucose, fructose, and sucrose as the only mono- and dis-
accharides in sweet potato tubers. Data obtained in this investigation con-
firmed the results reported by LambouI6 and McDonald and Newson."
Miyake" excluded the presence of pentose, galactose, manose, and mal-
tose in the sweet potato. Quantitative analysis of sugars could not be
accurately accomplished due to the tailing of the peaks.
When 1% SPP was heated at 154°C in 0.034N HCI for 6 min and then
examined for sugars present, only glucose and fructose were detected
indicating that sucrose was completely hydrolyzed to its components,
glucose and fructose.
886 AZHAR AND HAMDY

Presented at the 39th Annual meeting of the Institute of Food Technologists, June 10-13,
1979. The authors thank Dr. S. J. Harmon, Horticulture Department, University of Georgia
at Tifton, Georgia for supplying us with sweet potatoes.

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Accepted for Publication July 28, 1980