CyTA – Journal of Food

ISSN: 1947-6337 (Print) 1947-6345 (Online) Journal homepage: https://www.tandfonline.com/loi/tcyt20

Lactic acid from apple pomace: a laboratory

experiment for teaching valorisation of wastes

J. L. Alonso, G. Garrote, H. Domínguez, V. Santos & J. C. Parajó

To cite this article: J. L. Alonso, G. Garrote, H. Domínguez, V. Santos & J. C. Parajó (2009) Lactic
acid from apple pomace: a laboratory experiment for teaching valorisation of wastes, CyTA –
Journal of Food, 7:2, 83-88, DOI: 10.1080/11358120902906990

To link to this article: https://doi.org/10.1080/11358120902906990

Published online: 27 Oct 2010.

Submit your article to this journal

Article views: 2858

View related articles

Citing articles: 3 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tcyt20
CyTA – Journal of Food
Vol. 7, No. 2, August 2009, 83–88

ARTICLES

Lactic acid from apple pomace: a laboratory experiment for teaching valorisation of wastes
Ácido láctico a partir de pulpa de manzana: un experimento de laboratorio para la enseñanza de la
valorización de residuos
J.L. Alonso*, G. Garrote, H. Domı́nguez, V. Santos and J.C. Parajó
Department of Chemical Engineering, Faculty of Science, University of Vigo (Campus Ourense), As Lagoas, 32004 Ourense, Spain
(Received 2 July 2008; final version received 17 October 2008)

Reutilisation and valorisation of wastes and byproducts is an increasingly important topic which can be illustrated at
graduate or undergraduate level by the one-step bioconversion of apple pomace (AP) (an industrial byproduct) into
lactic acid. A simple operational procedure that can be carried out without sophisticated equipment is proposed:
starting from AP, hydrolytic enzymes and lactic acid bacteria present in yogurt, saccharification of the
polysaccharides contained in AP and their fermentation into lactic acid are simultaneously carried out. During
the process, the concentration profiles of sugars and lactic acid are measured. The experimental data provide the
information needed for discussing basic aspects such as the kinetics of lactic acid generation and the lactic acid yield.
Keywords: wastes valorisation; apple pomace; lactic acid; enzymatic hydrolysis; fermentation; simultaneous
saccharification and fermentation (SSF); chemical engineering teaching; food technology teaching

La reutilización y valorización de residuos y subproductos es un tema de creciente importancia que se puede ilustrar
a nivel de grado y postgrado mediante la bioconversión de pulpa de manzana (un subproducto industrial) en ácido
láctico en una sola etapa. Se propone un procedimiento muy simple que puede realizarse sin equipamiento
sofisticado: partiendo de pulpa de manzana (PM), enzimas hidrolı́ticas y bacterias lácticas presentes en los yogures,
se puede llevar a cabo la sacarificación de los polisacáridos de la PM y su fermentación a ácido láctico de manera
simultánea. Durante el proceso, se pueden obtener los perfiles de concentración de azúcares y ácido láctico. Los
datos experimentales proporcionan la información necesaria para discutir aspectos básicos como la cinética de
generación de ácido láctico y el rendimiento en producto.
Palabras clave: valorización de residuos; pulpa de manzana; ácido láctico; hidrólisis enzimática; fermentación;
sacarificación y fermentación simultáneas (SFS); enseñanza de la Ingenierı́a quı́mica; enseñanza de tecnologı́a de
alimentos

Introduction material for lactic acid (LA) manufacture and (owing

Even though undergraduate science curricula generally
focus upon discipline-oriented courses that emphasise
quantitative rigor and mastery of scientific principles,
the instructors usually bring in modern applications
and examples to motivate students, allowing them to
learn how the science they are studying relates to the
world we live in (Mathews, 2007).
The need for innovation of educational tools and
strategies is particularly evident in the scientific and
technological fields, where classic teaching methods
have very often proved inadequate (Santucci, Mini,
Ferro, Martelli, & Trabalzini, 2004).
Apple pomace (AP), the solid byproduct from
apple pressing in cider and juice making industries, is
produced in large amounts. AP is used as a feed
component (a low added-value application) and in
pectin manufacture. Owing to its polysaccharide
content, it can be considered as a potential raw
to its renewable character) provides an opportunity to
introduce some basic principles of sustainable devel-
opment to the students.
LA is a chemical commodity with applications in
food technology, pharmaceuticals and chemicals
(Gullón, Garrote, Alonso, & Parajó, 2007). The world
market for LA is estimated at 54,500–59,000 tons per
year, but the demand is expected to increase due to the
growing production of polylactic acid, a biodegradable
polymer which will replace a part of conventional
plastics (Neureiter et al., 2004).
LA is mainly produced by fermentation of sugar-
containing media obtained by enzymatic hydrolysis
of starch-containing substrates. However, due to
economical reasons, waste products from agriculture
and forestry have been proposed as alternative raw
materials for LA production (Hofvendahl & Hahn-
Hägerdal, 2000). Owing to the amount and nature of

*Corresponding author. Email: xluis@uvigo.es

ISSN 1947-6337 print/ISSN 1947-6345 online

Ó 2009 Taylor & Francis
DOI: 10.1080/11358120902906990
http://www.informaworld.com
84 J.L. Alonso et al.
the polysaccharides present in AP (which are highly
susceptible to enzymatic hydrolysis), LA can be
produced by enzymatic hydrolysis and fermentation
(which can be carried out consecutively or simulta-
neously). The general principle of both types of
processes is shown in Figure 1: polysaccharides (for
instance, glucose polymers, including cellulose and
starch) are first converted into monosaccharides
(glucose) by the action of enzymes (cellulases and
amylases), and the glucose obtained in the reaction is
employed as a carbon source by LA bacteria under
anaerobic conditions, generating LA.
Simultaneous saccharification and LA fermenta-
tion (SSF) proceeds in a single vessel, avoiding
handling and reducing the reaction time respect to
sequential hydrolysis and fermentation, when the
operational conditions (pH, temperature) are suitable
for assuring both the activity of hydrolytic enzymes
and the metabolism of LA bacteria. As the increase in
LA concentration would result in pH drop, calcium
carbonate can be added to the media for
neutralisation.
These general ideas highlight the basic concepts
involved in the laboratory experiment described in
this work. Since a sound assessment of aspects such as
Figure 1. General principle of lactic acid production from cellulose and/or starch.
Figura 1. Principio general de la producción de ácido láctico a partir de celulosa y/o almidón.
substrate composition, hydrolysis of the various
polysaccharides to sugars, sterile inoculation, opera-
tion and sampling, consumption kinetics of the
various carbon sources and kinetics of LA generation
would be too complicated for being carried out by
students, we describe a simplified operational method
involving limited analytical duties and requiring
limited skills. Enzymes are obtained from commercial
firms, yogurt samples are used as inocula of LA
bacteria, and special working conditions (including
total anaerobiosis and sterility) are not major
requirements, owing to the experimental conditions
employed. This laboratory experiment is proposed for
Chemical Engineering, Chemistry, Food Technology,
Biotechnology, Bioengineering or Industrial Micro-
biology undergraduate students, who can learn valu-
able techniques, acquire practical skills and practice
process calculations. As in other laboratory experi-
ments dealing with fermentation (Vullo & Wachsman,
2005) the experimental data can be used for high-
lighting basic aspects of the whole conversion pro-
cess, including the kinetics of product generation and
the product yield. In this context, the experiment
described here provides to the students the oppor-
tunity of handling, observing and checking major
 CyTA – Journal of Food 85

scientific concepts, whereas the instructor or teacher

may contribute to a dynamic learning process also by
posing questions, promoting discussions, proposing
experimental and modelling tasks, drawing conclu-
sions and making real-world connections. In this way,
a critical thinking from the evaluation of the results
achieved in the experimental work can be better
achieved.

Materials and methods

Raw material
Apples were cut in pieces and processed in a home
centrifugal juice extractor to obtain juice and AP
separately. In order to avoid degradation, AP was
partially dried at 60 8C for 24 h. Aliquots from this
dried material were employed in SSF experiments.

Chemical analysis of the raw material

An aliquot of the AP was used for determining its
chemical composition. This step is omitted for simpli-
city, but detailed information on the analytical
methods employed can be found in an article by
Gullón et al. (2007).

Simultaneous saccharification and fermentation (SSF)

and analysis
SSF experiments were carried out in 250-mL Erlen-
meyer flasks containing 100 mL of media, which were
made of AP, water, yeast extract, enzymes, yogurt and
calcium carbonate. Experiments were performed with
3.59 g of wet AP (3.33 g dry AP), 78.91 g water and
2 g of calcium carbonate (for LA neutralisation).
Simultaneously, 10 mL of solution of 133 g yeast
extract/L was prepared for nutrient supplementation.
Then both preparations were autoclaved separately
(121 8C, 20 min), mixed and cooled. Figure 2 sum-
marises the experimental procedure. After sterilisation,
the commercial cellulases (from Trichoderma reesei)
and b-glucosidase (from Aspergillus niger) were added,
with a cellulase to dry AP ratio of 25 FPU g71
(corresponding to 0.44 mL of ‘‘Celluclast 1.5L’’ per
experiment) and a b-glucosidase to cellulase ratio of 5
IU/FPU (corresponding to 0.65 mL of ‘‘Novozym
188’’ per experiment). In experiments, the media were
inoculated with 10 mL of yogurt and incubated in a
shaker at 37 8C at 50 rpm. At given fermentation
times, samples from the media (0.5 mL) were with-
drawn and centrifuged (5500 rpm, 3250 g, 5 min).
Aliquots of the supernatants were assayed for glucose,
fructose, xylose, mannose and galactose, disaccharides
(lactose and saccharose) and LA by HPLC using a
1100 series Hewlett Packard chromatograph fitted
with a refractive index detector (temperature, 50 8C).
Other analysis conditions were as follows: column,
ION-300 (Transgenomic, USA); mobile phase,
Figure 2. Experimental procedure followed in this work.
Figura 2. Procedimiento experimental seguido en este
trabajo.
0.003 M H2SO4; flow, 0.6 mL/min. In case the HPLC
analysis cannot be carried out, the main goals of the
experiment can be achieved just by spectrophotometric
measurements: determinations of saccharose, glucose
and fructose can be achieved, for example, using the
Boheringer-Mannheim enzymatic kit reference number
10716260035, lactose (contained in the yogurt) using the
Boheringer-Mannheim enzymatic kit reference number
10176303035 and L(þ)-LA using the Boheringer-
Mannheim enzymatic kit reference number
11112821035. If more simplicity is needed, the total
sugar concentration can be estimated spectrophotome-
trically by the Somogyi-Nelson method (Somogyi, 1952)
or DNS method, which would introduce only minor
errors derived from the presence of non-reducing sugars
(such as saccharose) in the SSF media.
Safety considerations
Students must be informed about the rules of
behaviour and safety to be adopted in the laboratory,
as well as about the specific risks derived from the
utilisation of the reagents and equipment employed in
this experiment.
86 J.L. Alonso et al.
Results and discussion
AP components
In order to establish a basis for quantitative calcula-
tions, an aliquot of the partially dried AP (see ‘‘raw
material’’ section) was oven-dried at 105 8C until
constant weight. The observed dry content was
0.929 g oven-dried material/g wet AP (moisture ¼
0.071 g water/g wet AP).
As the composition of AP is difficult to measure
and involves a variety of different laboratory techni-
ques, this point is not considered within the experi-
mental work to be carried out by students. However,
the instructor can explain that about 40% of the dry
AP corresponds to ethanol-extractable material, which
is mainly made up of mono- and disaccharides. The
remaining 60% corresponds to joint contribution of
lignin (made up of phenolic units), pectins (made up of
uronic acid moieties), polysaccharides (cellulose, made
up of b-glucose units, hemicelluloses, made up of
xylose, mannose and galactose, and starch made up of
a-glucose units). Figure 3 shows the approximate
composition of AP that can be shown and commented
to students. Fructose and glucose were the most
important free sugars (contained in extractives frac-
tion), and only 22% of the alcohol insoluble fraction is
made up of non-sugar compounds.
SSF production of LA
Under the action of enzymes, AP polysaccharides can
be converted into monosaccharides, which are suitable
carbon sources for LA fermentation. Hexoses
(C6H12O6, the most abundant sugars from AP
Figure 3. Composition of apple pomace.
Figura 3. Composición de la pulpa de manzana.
hydrolysis) are converted into LA (C3H6O3) by lactic
bacteria according the equation:
C6 H12 O6 ! 2 C3 H6 O3
The total amount of monosaccharides that could be
obtained upon total hydrolysis (called ‘‘potential
sugars’’) can be measured experimentally by total
hydrolysis followed by monosaccharide quantification
using spectrophotometric methods (as reducing sugars)
or HPLC. Details of this methodology can be found in
the article by Gullón et al. (2007).
Using the simple experimental set-up described
in the methods section, the concentration profiles
shown in Figure 4 were obtained using HPLC analysis.
In case the utilisation of HPLC is not desired, the
whole process can be followed by spectrophotometric
methods (see the Materials and Methods section).
The concentrations of monosaccharides first in-
creased to reach a maximum value, and then decreased.
The increasing part of the curve corresponds to
operational conditions in which the sugar generation
by saccharification was predominant over the sugar-
consuming, fermentative step. Once the sugar con-
centration reached its maximum value, the curve
decreased due to both the lower availability of
unconverted polysaccharides (which result in decreased
hydrolysis rate) and the faster fermentation kinetics
derived from the increased concentration of the carbon
source and microbial biomass. Xylose was not
metabolised by bacteria, remaining unconverted in
the medium at the end of the process. This sugar is
included in the (Fructose þ others) series of data.
The concentration data shown in Figure 4 can be
easily converted into results referred to the initial
amount of potential sugars (when known) or referred
to the initial amount of oven-dry AP, enabling the
Figure 4. Time course of glucose, fructose and other sugars,
disaccharides and lactic acid concentrations determined in
the SSF assay.
Figura 4. Curvas de evolución en el tiempo de la
concentración de glucosa, fructosa y otros azúcares,
disacáridos y ácido láctico obtenidas en el experimento de
SFS.
 CyTA – Journal of Food 87

calculation of product yields. In this case, a product
yield of 0.693g LA/g of dry AP was obtained after 30 h
of fermentation time.
Additionally, empirical modeling of LA produc-
tion can be easily performed: as it can be seen in
Figure 4, the LA concentration profile shows a fairly
sigmoidal shape, suggesting that the product con-
centration, P(t) can be assessed by the following
equation:
0
A  Pm  eP0 t
PðtÞ ¼ 0 ð1Þ
Pm  A þ A  eP0 t
where Pm is the maximum LA concentration, A is a
regression parameter with units of concentration and
P00 is defined as the ratio between the initial volumetric
rate of product formation and A. This equation is
formally equivalent to the one proposed by Morain
and Rogovin, which was used to correlate data from
LA fermentation in batch mode (Gullón, Alonso, &
Parajó, 2008; Téllez-Luis, Moldes, Vázquez, & Alonso,
2003). The values of these parameters can be obtained
by non-linear fitting of experimental data. In this
case, values of 1.3 g/L, 23.6 g/L and 0.163 h71 were
calculated for A, Pm and P00 , respectively, and a
R2 ¼ 0.993 was obtained, thereby demonstrating the
suitability of the model for this kind of processes.
Additionally, this equation can be directly employed to
calculate the mean volumetric productivity at a given
fermentation time. This parameter can be calculated
using following the Equation (2)
PðtÞ  P0
Q0p ðtÞ ¼ ð2Þ
t
where Qp0 (t) is the mean volumetric productivity at
time t, P(t) is the product concentration achieved at the
considered time, and P0 is the initial product concen-
tration. In this experiment (see data in Figure 4), a
Qp0 ¼ 0.769 g/(L h) was observed after 30 h of
fermentation.
Other parameters such as the maximum volumetric
productivity and the reaction time at which it was
achieved can also be determined. From the Equation
(1), the following expression for the volumetric
productivity, Qp(t), can be deduced
 2  0 0
dPðtÞ APm  A2 Pm P0 eP0 t
Qp ðtÞ ¼ ¼ h i2 ð3Þ
dt 0
Pm  A þ AeP0 t
The maximum volumetric productivity can also be
calculated from Equation (3) by solving the equation
dQp ðtÞ d2 PðtÞ
¼ ¼0 ð4Þ
dt dt2
As an example, a maximum volumetric productiv-
ity of 1.286 g/(L h) (achieved at 12.93 h) was
mathematically obtained from Equation (3) using the
data shown in Figure 4.
Conclusions
This simple laboratory experiment, for which no
special bioconversion equipment or microorganisms
are necessary, provides a suitable framework for
assessing multiple major topics, including
. Sustainable development, based on the conver-
sion of byproducts and wastes into high value-
added products,
. Polysaccharides and natural product
composition,
. Enzyme-based reactions (saccharification),
. LA fermentation,
. Consecutive reactions (saccharification/fermen-
tation) and limiting factors,
. Material balances and process calculations
. Mathematical modelling (multiple regression,
least square method)
The experiment can be run at different levels,
depending on the instructor’s choice (determination or
not of compositional data) and on the analytical
equipment available (HPLC and/or spectrophotometry).
The results can be modeled by equations proposed
in literature, and quantitative conclusions (product
yield, LA productivity, etc.) can be easily calculated
based on material balances.
Additional information: equipment needed
Granny Smith apples
Yogurt
Home centrifugal juice extractor
Centrifuge and test tubes
Orbital shaker
Autoclave (o thermostatic bath to keep temperature at
100 8C + 3 8C)
Commercial enzymes (Celluclast 1.5 L and Novozym
188)
Erlenmeyer flasks (250 mL)
Pasteur pipettes
Distilled water
Yeast extract
Flasks
Cotton
Membranes (0.20 mm) and membrane filter devices
Calcium Carbonate
Spectrophotometer or HPLC fitted with a refractive
index detector
Acknowledgements
The authors are grateful to the Ministry of Education and
Science of Spain (Project ref: CTM2005-04604 partially
financed by the FEDER funds of the European Union) for
the financial support of this work.
88 J.L. Alonso et al.
References
Gullón, B., Garrote, G., Alonso, J.L., & Parajó, J.C. (2007).
Production of L-lactic acid and oligomeric compounds
from apple pomace by simultaneous saccharification and
fermentation: a response surface methodology assess-
ment. Journal of Agricultural and Food Chemistry, 55,
5580–5587.
Gullón, B., Alonso, J.L., & Parajó, J.C. (2008). Experimental
evaluation of alternative fermentation media for L-lactic
acid production from apple pomace. Journal Chemical
Technology and Biotechnology, 83, 609–617.
Hofvendahl, K., & Hahn-Hägerdal, B. (2000). Factors
affecting the fermentative lactic acid production from
renewable resources. Enzyme and Microbial Technology,
26, 87–107.
Mathews, C.K. (2007). The news of science, a Colloquium-
style course designed to promote lifelong scientific
awareness. Biochemistry and Molecular Biology Educa-
tion, 35, 101–104.
Neureiter, M., Danner, H., Madzingaidzo, L., Miyafuji, H.,
Thomasser, C., Bvochora, J., Bamusi, S., & Braun, R.
(2004). Lignocellulose feedstocks for the production of
lactic acid. Chemical and Biochemical Engineering Quar-
terly, 18, 55–63.
Santucci, A., Mini, R., Ferro, E., Martelli, P., & Trabalzini,
L. (2004). Innovative tools for scientific and technological
education in Italian secondary schools. Biochemistry and
Molecular Biology Education, 32, 78–83.
Somogyi, M. (1952). Notes on sugar determination. Journal
of Biological Chemistry, 195, 19–23.
Téllez-Luis, S.J., Moldes, A.B., Vázquez, M., & Alonso, J.L.
(2003). Alternative media for lactic acid production by
Lactobacillus delbrueckii NRRL-B445. Food and Biopro-
ducts Processing, 81C, 250–256.
Vullo, D.L., & Wachsman, M.B. (2005). A simple laboratory
exercise for ethanol production by inmobilized bakery
yeast (Saccharomyces cerevisiae). Journal of Food Science
and Education, 4, 53–55.