A Strategy to Design Efficient Fermentation

Processes for Traditional Beverages Production:

Prickly Pear Wine
lvarez, H. Jimenez-Islas, and R. Rico-Martnez
J.L. Navarrete-Bolanos, E. Fato-Aldeco, K. Gutierrez-Moreno, J.E. Botello-A

Abstract: This paper describes a methodology to establish an optimal process design for prickly pear wine production
that preserves the peculiar and unique traits of traditional products, generating at the same time, technical information for
appropriate design of both bioreactor and overall process. The strategy includes alcoholic fermentation optimization by
the mixed native culture composed by Pichia fermentans and Saccharomyces cerevisiae, followed by malolactic fermentation
optimization by Oenococcus oeni. The optimization criteria were based on multiple output functions: alcohol content,
volatile compounds profile, organic acids profile, and compound contents related to color, which were analyzed by
spectroscopychromatography methods and sensory analysis. The results showed that the mixed culture inoculated into
a bioreactor containing prickly pear juice with 20 Bx of fermentable sugars concentration, processed at a constant
temperature of 20 C for 240 h, leads to a fermented product with 9.93% (v/v) total alcohol content, and significant
abundance of volatile compounds, which provide fruity and ethereal aromatic notes, complemented by a lively but not
unpleasant acidity. This young wine was further subjected to malolactic fermentation at constant temperature (16 C)
for 192 h, decreasing malic acid, and balancing volatile compounds contents, thus resulting in a product with better
aroma and flavor perception, and a velvety feeling of long aftertaste. Repeated assays showed that the process is stable,
predictable, controllable, and reproducible. These results were used for process design and spreadsheet construction in

M: Food Microbiology
order to simulate the process, and properly select and size the equipment required for such process.

Keywords: fermentation, juice, prickly pear, wine

Introduction
Fermented foods and beverages, whether from plant or ani-
mal origin, have been produced and consumed all over the world
for a very long time span (Steinkraus 2002; Campbell-Platt 2004;
FAO 2004). Many are typical or traditional products of a specific
region where the local ecosystem allows the growth of microor-
ganisms with ability to naturally ferment local food crops and
other endemic biological resources (Blandino and others 2003;
Roy and others 2004; Achi 2005; Scott and Sullivan 2008). Of-
ten, these natural processes lead to many attractive properties in
the fermented product such as prolonged shelf-life, improved
safety, attractive flavor, nutritional enrichment, and health ben-
efits (Steinkraus 2002; Blandino and others 2003; Campbell-Platt
2004; Roy and others 2004; Achi 2005; Scott and Sullivan 2008).
These attractive traits have motivated the local producers to ac-
quire knowledge for controlling the natural fermentation process
through the experience gained by trial and error during several
generations; however, the cyclic variability on environmental con-
ditions may modify the ecosystems hindering process reproducibil-
ity, and revealing the unsuitability of natural fermentation as the
basis of any viable production process (Holzapfel 2002; Motarjemi
2002; Valyasevi and Rolle 2002; Blandino and others 2003; Achi
2005). As alternative, some producers rely on pure cultures (such
as Saccharomyces cerevisiae for wines) in order to achieve process
MS 20130212 Submitted 2/14/2013, Accepted 7/16/2013. Authors are with 2001; Holzapfel 2002; Achi 2005; Hamdi 2007; Garcia-Aguirre
Dept. de Ingeniera Qumica-Bioqumica, Instituto Tecnologico de Celaya, Av. Tec- and others 2009; Rodriguez-Lerma and others 2011; Navarrete-
nologico s/n, C.P. 38010. Celaya, Gto., Mexico. Direct inquiries to author Navarrete- Bolanos 2012). Additionally, the resulting integrated USP should
Bolanos (E-mail:luis.navarrete@itcelaya.edu.mx).
& Safety
reproducibility; however, the products obtained may not retain
the typical quality traits that are distinctive and unique in the
traditional product achieved obtained by successful natural fer-
mentations (Blandino and others 2003; Fleet 2008; Capece and
others 2011; Grieco and others 2011; Navarrete-Bolanos 2012;
Suarez-Lepe and others 2012).
Several studies have shown that inoculums selected from native
microorganisms are able to exalt the peculiarities (aroma, structure,
and color) of a wine, preserving its authenticity and uniqueness;
however, specification of the inoculums does not suffice for de-
scribing a successful process replacing the traditional preparation
of the fermentation process. The efficient fermentation process
design should also involve information regarding both upstream
processing (USP) and downstream processing (DSP) stages. The
USP is associated with factors and processes leading to and includ-
ing the fermentation, and consists of 3 main areas: (1) the starter
culture (microorganisms), (2) the culture medium, and (3) the
process conditions. The conventional DSP includes all unit pro-
cesses that follow the fermentation, which involve cell harvesting,
product purification, and finishing steps. Therefore, the starter
culture selection is only the 1st step on designing fermentation
processes, the 2nd step is the proper formulation of the culture
medium, and the 3rd step is the optimization of operation vari-
ables on bioreactors, thus defining the best processing practices
that ensure quality, authenticity, and safety on the end product
(Moo-Young and Chisti 1994; Odunfa 1998; Waites and others
also provide information for equipment selection and sizing for



C 2013 Institute of Food Technologists
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doi: 10.1111/1750-3841.12237 Vol. 00, Nr. 0, 2013 r Journal of Food Science M1

Further reproduction without permission is prohibited
 Prickly pear wine production . . .
scale-up operation and, together with the DSP data, support the
process flow diagram. The main goal of this contribution is to
describe a strategy for design a technology-based production sys-
tem to replace the artisanal fermented food and beverages practice,
while retaining the unique quality features of the original tradi-
tional product. Furthermore, one should keep in mind that many
traditional fermented products are a viable alternative for dealing
with harvest surpluses, thus assisting the sustainable exploitation of
endemic natural resources (Holzapfel 2002; Blandino and others
2003; Achi 2005; Duarte and others 2010; Rodriguez-Lerma and
others 2011; Navarrete-Bolanos 2012). As an illustrative exam-
ple, let us consider the current status of prickly pear production
in Mexico. Mexico produces approximately 60% (400000 tons)
of the worlds prickly pear supply, but only 40% (160000 tons)
is commercially exploited, and the rest is lost due to the highly
perishable character of the fruits related to their nutritional value.
Prickly pears are considered one of the healthiest fruits (Piga 2004;
Salim and others 2009; Rodriguez-Lerma and others 2011). The
excess production in Mexico has prompted the development of
artisanal processes leading to unique products such as prickly pear
cheese, candies such as melcocha, and prickly pear wine (so-called
colonche). This artisanal wine is obtained by the natural fermen-
tation of red prickly pears juice (Opuntia streptacantha), and appre-
ciated for its color, flavor, and taste. However, prickly pear wine
M: Food Microbiology
is mainly consumed locally, largely due to the instability of its
production process resulting in uneven quality (Lopez-Gonzalez
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and others 1997; Rodriguez-Lerma and others 2011). Previously,
a mixed culture of Saccharomyces cerevisiae and Pichia fermentans, na-
tive microorganisms, was identified as responsible for a fermented
prickly pear wine with a rich volatile compounds mixture, re-
sulting in ethereal and fruity notes, essential on fine wine flavor
(Rodriguez-Lerma and others 2011).
Here, these efforts are brought forward one step further, by
recognizing that for some applications multistep serial process-
ing requires studying and optimizing the variables associated with
USP for alcoholic fermentation. Moreover, the malolactic fermen-
tation is also studied for increasing the overall quality of prickly
pear wine. The main idea is to enrich a framework to identify the
best processing practices to design a reliable fermentation process,
creatively engineering a wine with character and quality, by mod-
ifying fruit-derived and volatile aromas and producing additional
desirable aroma compounds as a result of the malolactic fermen-
tation. This framework may lead to the equipment design and
selection, and a process flow diagram with sufficient details for
scale-up plant operation.
Materials and Methods
Fresh material
A total of 250 kg of mature prickly pears of the Opuntia strepta- (Table 1), as described by Montgomery (2005). In all experimental
cantha variety were harvested from the La Tinaja ranch, located assays, the starter inoculum concentration (110 cells/mL) and
in the northern part of Guanajuato state, Mexico, and used as a
raw material.
Prickly pear juice
The prickly pear fruits were peeled removing their outer coat,
and the edible part was pressed and filtered to produce a juice Experimental strategy for malolactic fermentation analysis
that was free of insoluble solids, which contained approximately
130 g/L of fermentable sugars. Although, in Mexico there are perature (associated with metabolism) and V5 , agitation (associated
electromechanical equipments for peeling and pressing the prickly with mass transfer limitations on bioreactor), and 4 dependent
pear fruits, for the purposes of this research, the fruits were peeled, variables associated with the fine wine flavor and mouthfeel/body
pressed, and filtered manually.
M2 Journal of Food Science r Vol. 00, Nr. 0, 2013
Microorganisms
Saccharomyces cerevisiae and Pichia fermentans, native microorgan-
isms, previously isolated from natural fermentation of prickly pears
juice (Rodriguez-Lerma and others 2011) were used in the alco-
holic fermentation assays. Additionally, Oenococcus oeni was used
for the malolactic fermentation assays due to its acidic tolerance
and flavor profile production (Liu 2002). Oenococcus oeni is a
pure culture strain which is part of microorganisms collection of
the Bioengineering Laboratory of Celaya Inst. Technology.
Pure culture propagation and starter inoculums
Pure cultures of S. cerevisiae and P. fermentans were cultured in
agar slants (PDA, Difco, Detroit, Mich., U.S.A.) at 28 C for 48 h.
For the propagation step, biomass samples were taken from each
slant, transferred to 250 mL Erlenmeyer flasks containing 100 mL
of broth medium (PDB, Difco), and incubated at 28 C for 48
h at 120 rpm on a rotary shaker (Model 4520, Forma Scientific,
Marietta, Ohio, U.S.A.). Samples of each culture were taken and
mixed in order to obtain the starter inoculum for alcoholic fer-
mentation, which must contain 60% of S. cerevisiae cells and 40%
of P. fermentans cells according to Rodrguez-Lerma and others
(2011). In parallel, O. oeni was cultured in agar MRS (Difco) at 28
C for 48 h. Similarly, for propagation, biomass samples were taken
from the slant, transferred to 250 mL Erlenmeyer flasks containing
100 mL of broth MRS (Difco), and incubated at 28 C for 48
h at 120 rpm on a rotary shaker (Model 4520, Forma Scientific)
for culture propagation. Samples of 1106 cells/mL were taken
from this propagation step and used as pure culture for malolactic
fermentations.
Experimental design for alcoholic fermentation
optimization
An experimental strategy for maximizing both the alcohol
yield and the synthesis of volatile compounds at bioreactor level
(Autoclavable Glass Bioreactors, Applikon Dependable Instru-
ments, Schiedam, the Netherlands) was designed, taking into
consideration the following variables: V1 , sugar concentration;
V2 , potassium metabisulfite concentration; and V3 , temperature.
These variables were singled out based on their relationship with
biomass growth and metabolic activities involved in the synthesis
of products that are associated with pleasant aroma and flavor
(temperature and sugar concentration), as well as their relationship
with bacterial growth inhibition and yeast activity during the
fermentation process (potassium metabisulfite). The feasible
ranges for these variables were defined as 15 to 19 Bx for V1 , 60
to 100 mg/L for V2 , and 17 to 21 C for V3 and were based on
a preliminary screening design (not shown). A central composite
design for 3 variables with axial points ( i ) was constructed
6
the culture medium volume (2 L) were kept constant. Once the
optimum conditions for alcoholic fermentation were defined, a
batch of prickly pear wine was produced, and used as substrate
for malolactic fermentation studies.
An experimental strategy for 2 independent variables: V4 , tem-
(acidity changes, volatile compound evolution, color stability, and
Prickly pear wine production . . .

Table 1Central composite design for fermentation process vari- GC-MS analysis
able optimization (V1 , Bx; V2 , potassium metabisulfite concen-
tration, and V3 , temperature) seeking to maximize alcoholic
The samples were injected into a gas chromatograph coupled
yield (Y). with a mass spectrometer (GC-MS) (Clarus 500MS; Perkin-Elmer
Inc., Wellesley, Mass., U.S.A.). The separations were carried out
Assays V1 V2 V3 Y using an ATTM -WAX capillary column (30 m 0.25 mm and 0.5
1 19.0 100.0 17.0 8.54 m film thickness) (Alltech, State College, Pa., U.S.A.). Helium
2 17.0 80.0 19.0 8.78 was used as the carrier gas at a flow rate of 1.21 mL/min, oper-
3 17.0 80.0 19.0 8.72 ated at constant pressure (12 psig), and the oven temperature was
4 15.0 100.0 17.0 7.14
5 19.0 60.0 17.0 8.07 ramped from 40 to 220 C at a 7 C per min rate. The injector
6 15.0 100.0 21.0 7.45 temperature was set at 220 C in splitless mode (1.0 min), and the
7 19.0 60.0 21.0 8.12 fiber was allowed to remain in the inlet for 30 min. The MS was
8 15.0 60.0 21.0 8.58 set to electronic impact mode with ionization potential of 70 eV,
9 19.0 100.0 21.0 8.54
10 15.0 60.0 17.0 8.35
ion source temperature of 180 C, and electron multiplier voltage
11 13.6364 80.0 19.0 6.59 of 294 V, while scanning from 40 to 400 m/z with 0.1 s/scan
12 20.3636 80.0 19.0 9.47 rate. The compounds were tentatively identified by comparing
13 17.0 46.3641 19.0 7.95 their mass spectra with those in the Natl. Inst. of Standards and
14 17.0 113.636 19.0 7.28 Technologies (NIST2002) library of the MS database.
15 17.0 80.0 15.6364 7.34
16 17.0 80.0 22.3636 8.94
Sample preparation for gas chromatography (GC) analysis
Samples (20 mL) of prickly pear young wine were distilled at
alcohol content) was designed at bioreactor level, taking into con- 74 C on a Kerr-type mini-column, which simulated 5 transfer
sideration the following feasible ranges: 16 to 24 C for V4 and units, until 5 mL of distillate was obtained. Samples were prepared
0 to 50 rpm for V5 , which were defined based on preliminary by mixing equal parts of the distillate and the external standard
information for O. oeni metabolism. In all experimental assays, the for ethanol and methanol. The results were compared with the

M: Food Microbiology
starter inoculum concentration (1106 cells/mL) and the culture calibration curves for ethanol and methanol in order to obtain a
medium volume (2 L) were, once again, kept constant. quantitative concentration values.

High-performance liquid chromatography (HPLC) analysis
The main organic acids associated with wine acidity were quan-
tified via HPLC according to Frayne (1986). For this analysis, the
fermented product was filtered through Millipore membranes of
45 m in order to obtain a prickly pear young wine. Samples
consisting of 20 L of young wine were injected to the HPLC.
The equipment used for the HPLC assays was a standalone sys-
tem module that includes LC1120 advanced spindle-driven pump,
LC1205 programmable UV-vis detector, refractive index detec-
tor, and WinChrom Chromatography Management System for
computer control (GBC Scientific Equipment, Dandenong, Vic-
toria, Australia). The organic acids concentration was measured
& Safety
GC analysis
A GC system with a flame ionization detector was used to
quantify the alcohol content in the distilled samples. Samples
(1 L) were injected in split mode using a micro-syringe (Hamil-
ton, Grace Div. Discovery Science). The separations were carried
out using a capillary column ATTM -WAX (30 m 0.25 mm and
0.5 m film thickness) (Alltech), helium was used as the carrier
gas, extra-dry air was used as the flame fuel, and the oven program
temperature was ramped from 50 to 150 C at 5 C per min rate.
A comparison of the retention time to the internal standard, fol-
lowed by concentration estimation using the relative percentage
area covered in the chromatogram, was used to calculate ethanol
concentrations using the Total Chrom Workstation v. 6.3.1 soft-
R

using Prevail TM Organic Acid (Grace Div. Discovery Science, ware.

Deerfield, Ill., U.S.A.). The solvent elution was operated at 1.5
mL/min using monobasic potassium phosphate solutions with pH
of 2.5 (adjusted with phosphoric acid) using the UV-VIS detector
at 210 nm of wavelength. Retention time comparison to external
standards of malic, lactic, acetic, tartaric, and oxalic acids, fol-
lowed by concentration estimation using the relative percentage
area covered in the chromatogram, was used to calculate organic
acids concentration.
Sample preparation for gas chromatography and mass
spectrometry (GC-MS) analysis
The prickly pear young wine was distillated and treated by
solid phase micro-extraction according to Rodriguez-Lerma and
others (2011) using a polydimethylsiloxane-divinylbenzene fiber
(PDM/DVB, Supelco Bellefonte, Pa., U.S.A.). Samples (8 mL)
were collected in a vial and incubated at 28 C for 50 min. The
microfiber (PDM/DVB) was then placed inside the vial, and the
sample was incubated at 40 C for 40 min to allow for volatile
compounds adsorption. Finally, the sample was injected into the
GC-MS system.
Statistical analysis
The results from the experimental designs were analyzed using
a statistical framework based on the analysis of variance proce-
dures (Statgraphics Graphics Plus. V 5.1) followed by mathematical
model construction using linear least squares.
Results and Discussion
Variable optimization for alcoholic fermentation
Once the yeasts were propagated, these were mixed in a ratio
of 2:3 (for P. fermentans and S. cerevisiae respectively) according to
Rodriguez-Lerma and others (2011), in order to construct the
starter culture. The results, expressed as alcohol yield, are shown
in the last column of Table 1. The analysis of variance results (not
shown) revealed that for a confidence interval of 0.05, none of
the variables exhibited significant effect within their defined lim-
its, suggesting the presence of curvature and optimum proximity.
These experimental results were used to construct a 2nd-order
polynomial model using least squares (Montgomery 2005). The

Vol. 00, Nr. 0, 2013 r Journal of Food Science M3

 Prickly pear wine production . . .

model describes the relationship that exists among the variables. Furthermore, the GC-MS analysis shows the presence of 12
In this case, the regression model obtained is the following: volatile compounds, 10 of which (Ethyl acetate; 2-methyl-1-
propanol; 1-Butanol 3-methyl acetate; Hexanoic acid, ethyl es-
YOH = 23.0966 + 1.4525 V1 0.0428 V2 + 1.9344 V3
ter; 3-Methyl-1-butanol; Octanoic acid, ethyl ester; Decanoic
0.0508 V1 + 0.0101 V1 V2 0.0153 V1 V3
2
acid, ethyl ester; Ethyl 9-decenoate; Acetic acid, 2-phenylethyl
0.00097 V2 + 0.00009 V2 V3 0.4109 V3
2 2
ester; Phenylethyl alcohol) seemingly can be considered as the
Here, the variables are specified in their real units. In the equa- major volatile compounds because they represent the 98.25% of
tion, it is implicitly implied that response variable dependence on all volatile compounds. Of these, the esters (ethyl acetate; Hex-
the factors is nonlinear and that the effects of these factors are anoic acid, ethyl ester; Octanoic acid, ethyl ester; Decanoic acid,
additive. The statistical analysis of the model indicates that these ethyl ester; Ethyl 9-decenoate; and Acetic acid, 2-phenylethyl es-
assumptions are adequate. The location of the optimum is a simple ter), produced by the yeast during alcoholic fermentation, are
exercise for extreme locating on a multidimensional system, and it largely responsible for the pleasant aromatic notes (fruity, herbal,
is achieved via the derivation of the 2nd-order model and solving and ethereal) that are considered to be essential for fine wine, and
the resulting set of linear equations: together with the synthesized alcohols make a pleasant mixture of
aromas, flavor, and taste. The remaining volatile compounds ((S)-
[YOH ] 3,4-Dimethylpentanol; Acetic acid, Lauric acid, ethyl ester) that
= 1.4525 0.1016V1 + 0.0101V2
V1 were detected comprise only 1.75% of the total, and are consid-
ered secondary to the beverage characteristics (Table 2). However,
0.0153V3 = 0
the aroma, flavor, and taste profile is the result not only of the
[YOH ] presence but also of the volatile compounds abundance. A spe-
= 0.0428 + 0.0101V1 0.001946V2
V2 cific compound should be present beyond its detection threshold
+ 0.00009V3 = 0 level, also known as odor activity value in order to contribute
to the wine aroma, flavor, and taste (Grosch 2001). For exam-
[YOH ] ple, isopentanol is present in an amount 12 times greater than the
= 1.9344 0.0153V1 + 0.00009V2
V3
M: Food Microbiology

ethyl 9-decenoate, but it also has a 20000 higher threshold level

0.8218V3 = 0 (22 ppm compared with 1.1E-4 ppm), therefore, the contribution
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of ethyl 9-decenoate to the aroma, flavor, and taste is more signif-

icant than that of isopentanol (Etievant 1991; Marsili and others
The system solution is the following: V1 = 20.36, V2 = 94.07, 2007). From the profile of the GM-MS analysis, it appears that a
and V3 = 19.85, which are the conditions that should allow for fruity and sweet wine can be considered the predominant traits in
the production of a fermented product containing 9.17% (v/v) the fermented product.
alcohol (analytical solution). This procedure indicates that the
optimum conditions for alcohol yield maximization are: V1 = Sensory analysis
20.36 Bx, V2 = 94.07 mg/L of potassium metabisulfite, V3 = The prickly pear wine was evaluated by local sommeliers based
19.85 C. Using these experimental conditions, confirmation as- on the 4 recognized stages of wine tasting: the appearance, the in
says were performed and the results yielded 9.93% (v/v average) glass aroma, the in mouth sensations, and the finish (after-
alcohol, which compares favorably against the predicted model taste). The results qualified the fermented product as a young red
value (9.17% v/v). Moreover, the yield value achieved shows a wine with a deep purple-red garnet color, unusual and attractive,
95.9 of efficiency according to mass balance. with floral aroma, fruity notes and a lively acidity, but with gentle
The fermented product was analyzed by GC and GC-MS, as character.
well as sensorial analysis, in order to assess its quality. The GC Based on the above, the young wine was analyzed by HPLC in
analysis showed the following profile: 96.28% ethanol, 0.14% order to quantify organic acids content and associated them with
methanol, 0.12% 1-propanol, 0.7% 2-methyl-1-propanol, 2.5% wine acidity. The HPLC revealed the presence of the following
3-methyl-1-butanol, and 0.26% Phenyl ethyl alcohol. Methanol is organic acids: oxalic, tartaric, malic, lactic, acetic, and 2 more
undesirable because it causes blindness or even death when at toxic unidentified acids (Figure 1). The slightly elevated wine acidity,
levels. The safety limit, or maximum permitted concentration of and in particular the presence of malic acid, leads us to propose
methanol, in wines is regulated to avoid these risks to consumers a 2nd fermentative stage for the prickly pear wine production, in
health. While currently a norm for prickly pear wine does not an attempt to decrease malic acid content without significantly
exist, International norms can be used as reference. These regula- altering the flavor, aroma, and mouthfeel/body.
tions set as maximum limits of 15 mg/100 mL for white and 30
mg/100 mL for rose and red (Wang and others 2004; Paine and Variable optimization for malolactic fermentation
Davan 2001). The prickly pear wine obtained has 10.84 mg/100 The 1st fermented product (wine young) was used as substrate
mL methanol, which compares favorably against these norms. The to develop experimental assays in order to optimize variables in a
other alcohols: 1-propanol, 2-methyl-1-propanol, and 3-methyl- 2nd, malolactic, fermentation (Table 3). The objective is the con-
1-butanol, so-called higher alcohols also are in low concentrations version of L(-) malic acid to monocarboxilic (L-) or D(-) lactic acid
well below toxicity levels, thus contributing to aroma, flavor, and and carbon dioxide to reduce the titratable acidity and increase pH
taste, and together with the phenyl ethyl alcohol give pleasant traits values, but without losing the presence of other desirable com-
to the wine (Etievant 1991; Rapp 1998; Lambrechts and Pretorius pounds, seeking a rounder, softer, mellower mouthfeel wine. In
2000). As preliminary conclusion from the GC analysis, the alco- general, the goal is to preserve color and alcohol content, and en-
holic fermented product has balanced alcohols content, without rich volatile compounds profile, thus accentuating flavor, aroma,
placing any risk to consumers health, while possessing pleasant and mouthfeel/body, and increasing wine quality perception. Ac-
notes associated with its aroma, flavor, and taste. cordingly, the optimization criteria are as follows: higher values for

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Prickly pear wine production . . .

Table 2Relative volatile compounds abundance found on the fermented products obtained after alcoholic (A.F), and malolactic
fermentation (M.F).

Compounds: IUPAC
Retention name (CAS Aromatic
Nr. time(min) Registry Nr) note A.F M.F Ref.
1 3.63 Ethyl acetate (141-78-6) Etherial, fruity, sweet, 5.98 2.69 Mosciano 1994
grape, and rum-like
2 11.07 2-methylpropan-1-ol Wine, solvent, bitter 3.16 3.69 Ferreira and others 2001
(/78-83-1)
3 11.53 3-methylbutyl acetate Fruity, particularly banana 11.19 0 Ruth 1986; Mosciano
(29732-50-1) 1995; Mosciano 1998
4 14.63 Ethyl hexanoate Fruity, strawberry, anise, 4.21 0 Kiyoshi and others 1988
(8068-81-3) Wine gum
5 14.66 Isopentanol Fusel, alcoholic, pungent, 49.97 42.82 Mosciano 1993
3-methylbutan-1-ol etherial, cognac, fruity,
(6423-06-9) banana and molasse
6 19.59 Ethyl octanoate Fruity, floral, green leafy, 7.58 19.18 Mosciano 1997
(106-32-1) menthol, anise
7 20.32 3,4-dimethylpentan-1-ol Not determined 1.27 1.39
(6570-87-2)
8 23.68 Ethyl decanoate Grape, fruity, particularly 10.21 12.23 Mosciano 1990
(110-38-3) apple
9 24.68 Ethyl dec-9-enoate Wine, fruity, sweet 1.09 3.51 Atienza and others 1998
(67233-91-4)
1 27.21 2-phenylethyl acetate Sweet, honey, floral rosy, 2.13 1.18 Mosciano 2001
(103-45-7) cocoa, and balsamic
nuance
11 27.40 Ethyl dodecanoate Fatty and fruity 0.48 1.43 Nykanen 1986

M: Food Microbiology
(106-33-2)
12 28.86 2-phenylethanol Sweet, honey-like, 2.73 10.28 Mosciano 1993
(60-12-8) yeast-like, floral, spicy

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13 30.82 Octanoic acid unpleasant rancid sour 0 1.17 Mosciano 1998
(8006-77-7) fatty
14 33.95 Decanoic acid Citrus, fat, nasty, sour 0 0.43 Christensen and
(52627-73-3) Reineccius 2006

malolactic acid (YM.A ) and volatile compounds (YV.C ), as well as
lower values for color compounds (YC.C ), ethanol content (YOH ).
Statistical analysis for the main variables and their interactions was
performed (not shown), revealing that low temperatures and in-
tense agitation decrease malic acid content, minimum variations
on color and alcohol content can be obtained at low temperatures
independently of agitation intensity, and volatile compounds con-
centrations increase when the process is performed at low temper-
atures again independently of agitation intensity. Additional assays
based on these observations were performed at low temperature
with and without agitation, revealing that the fermented prod-
uct obtained without agitation exhibits better balance between
flavor (reduced acidity, broad organic acids profile, and alcohol
content), aroma (broad aromatic compounds profile and increased
content), and appearance (no significant change in appearance as-
sociated with the color). Therefore, considering the malolactic fer-
mentation a process beyond young wine deacidification, the best
conditions for its development are low temperatures and without
agitation. With this perspective, further fermentation assays at low
temperatures on static systems (without agitation) were performed
(Table 4). Once again, the results are expressed as output functions
that are differences between initial and end values, and reveal that
16 C is the best temperature for malolactic fermentation because
wine acidity is decreased (59.25% average) without significantly

Figure 1HPLC chromatogram showing organic

acid profile found for prickly pear young wine.

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 Prickly pear wine production . . .

Table 3Physical effectors (V4 , temperature; V5 , agitation) on cess (V4 = 16 C), a suitable pilot fermenter was designed. The
malolactic fermentation on prickly pears wine using O. oeni. stainless steel vessel was proposed with a height to diameter 3:1
The results show the percentage changes between the start and
the end of the process. ratio, 2 inlet water ports (hot and cold) for temperature control
with sensors coupled to an automatic control system, one inlet
Variables Output functions steam port (for sterilization purposes), one feeding port, drain at
Assay V4 V5 YM.A YC.C YE.C YV.C the bottom, and a CO2 removal port (Figure 2). The fermenter
design was used as the basis for production simulation in order
1 16 0 36.39 54.78 15.83 18.63
2 16 50 74.58 54.18 13.13 25.82 to built the process flowsheet database based on the mass balances
3 20 25 32.02 54.67 21.32 35.97 (Table 5), required for equipment dimensioning for upstream and
4 24 0 16.24 63.64 31.14 18.58 downstream stages (Figure 2).
5 20 25 12.81 55.62 23.08 4.83
6 24 50 16.46 47.68 32.55 2.57
Global process assays at optimal conditions
The process begins with the harvest of ripe prickly pears, fol-
Table 4Temperature effect on malolactic fermentation on
prickly pears wine using O. oeni. The results show the percentage lowed by peeling, pressing and filtering to obtain the prickly pear
changes between the start and the end of the process. juice. The juice is adjusted to a concentration of 20.2 (1) g/L fer-
mentable sugars and is mixed with 90.4 mg/L sodium metabisul-
Variable Output functions fite. The resulting solution is allowed to stand for 24 h and is then
Assay V4 YM.A YC.C YE.C YV.C inoculated with mixed cultures comprised of 4105 cells/mL of
1 14 36.02 62.93 17.95 13.90 P. fermentans and 6105 cells/mL of S. cerevisiae at 10% inoculum
2 15 27.41 66.06 10 6.23 solution to substrate solution ratio (v / v). The inoculated juice
3 16 59.67 54.81 15.74 4.98 is kept at 20 () C for 7 d to allow the alcoholic fermentation
to proceed. Once this 1st fermentation process is finished, the
fermented product is filtered, and the liquid fraction, which is
altering the color and alcohol content, and increasing the content the young prickly pear wine, collected. Fermented product can
M: Food Microbiology

of volatile compounds. be packaged in bottles or again fermented using 1106 cells/mL

The fermented product was, once again, analyzed by GC, GC- of O. oeni and kept at 16 (1) C for 8 d to allow malolactic
& Safety

MS and sensory analysis. The GC analysis showed the follow-
ing profile: 96.27% ethanol, 0.00% methanol, 0.09% 1-propanol,
0.74% 2-methyl-1-propanol, 2.67% 3-methyl-1-butanol, and 0.23
Phenyl-ethyl alcohols. Seemingly the main alcohols profile is
conserved, but note the positive methanol depletion. On the
other hand, GC-MS analysis shows the presence of 12 volatile
compounds, 7 of which (Ethyl acetate; 2-methyl-1-propanol; 3-
Methyl-1-butanol; Octanoic acid, ethyl ester; Decanoic acid, ethyl
ester; Ethyl 9-decenoate; Phenylethyl alcohol) seemingly can be
considered as major volatile compounds because they represent
94.4% of all volatile compounds that have pleasant aromatic notes.
The remaining 5 volatile compounds ((S)-3,4-Dimethylpentanol;
Acetic acid, 2-phenylethyl ester; Lauric acid, ethyl ester; Caprylic
acid; Capric acid) comprise only 5.6% of the total. They are of
secondary importance to beverage characteristics (Table 2). Again,
esters ethyl acetate; Octanoic acid, ethyl ester; Decanoic acid, ethyl
ester and ethyl 9-decenoate, are largely responsible for pleasant aro-
matic notes (fruity, herbal, and ethereal) related to fine wine, and
together with the synthesized alcohols make a pleasant complex
mixture of aroma, flavor, and taste.
Sensory analysis
The product from the malolactic fermentation was, once again,
evaluated by local sommeliers based on appearance, in glass
aroma, in mouth sensations, and finish (aftertaste). This sen- fects the aroma and adds complexity to the flavor. As a result,
sory analysis indicated that the prickly pear wine retains its unusual
and attractive purple-red garnet color although not as deep, but
exhibits increased fruity and floral aromatic notes, emphasizing
its gentle character. Based on their traits, the sommeliers rec-
ommended it to be consumed at a temperature between 10 and
15 C.
Fermenter design
Based on the optimum conditions for alcoholic fermenta-
tion process (V1 = 20.24 Bx, V2 = 90.43 mg/L of potassium
metabisulfite, V3 = 19.96 C) and malolactic fermentation pro-
fermentation to proceed. Once this 2nd fermentation process is
finished, the fermented product is filtered, and the liquid fraction,
which is the prickly pear wine, collected and packaged in bottles.
Several repetitions of the process, based on the protocol described
above, showed that the process is stable, predictable, controllable
and reproducible, for the 2 types of prickly pear wine.
Conclusions
A procedure to optimize the physical effectors associated with
sequential alcoholic and malolactic fermentations at bioreactor
level has been described. The 2-tier design intends to enrich a
methodological platform to improve traditional fermented bever-
ages production. First, an optimized alcoholic fermentation pro-
cess, using a mixture of selected native microorganisms, was de-
veloped to obtain a fermented product with a deep purple-red
garnet color, floral and fruity aromatic notes, slight acidity, and
gentle character. Described by local sommeliers as unusual and
attractive, with a unique and distinctive style that is the result
of native microorganisms metabolisms. The 2nd (malolactic) fer-
mentation performed at the end of alcoholic fermentation, went
beyond a deacidification process, emphasizing the wine gentle
character by modifying volatile compounds profile and decreas-
ing alcohol content, without significantly altering its color. Thus,
the secondary malolactic fermentation using O. oeni positively af-
2 prickly pear wine styles can be obtained, each one with their
relative merits or attributes. It is difficult to assess which one is
the best, because aroma, flavor, and taste perception in humans are
strongly dependent on a persons preferences and cultural back-
ground. Wine appreciation is entirely subjective; as a result there
are several styles and wine cultivars on the market trying to satisfy
all tastes.
Several assays of the 2 sequential fermentations performed in a
bioreactor at optimal conditions demonstrated that the process is
stable, predictable, controllable, and reproducible. The proposed
protocol serves as a platform for the design of reliable processes,

M6 Journal of Food Science r Vol. 00, Nr. 0, 2013

 Prickly pear wine production . . .

Figure 2Process flow diagram (PFD) for prickly pear wine production.

Vol. 00, Nr. 0, 2013 r Journal of Food Science M7

M: Food Microbiology
& Safety
 & Safety
M: Food Microbiology

Table 5Spreadsheet showing the mass balances for prickly pears wine production.
Stream A B C D E F G H I J K L M N O P Q R S T U
Whole Prickly Pulp Juice with Juice Inoculums fermen- First Young Second Inoculums Second Refined
Prickly pear wine production . . .

Name prickly pears and residual Residual free Effluent (mixed tation fermented Residual wine fermentation (pure fermentation Residual wine
stream pears Skin shelled seeds pulp pulp pulp Sucrose Metabisulfite feed culture) feed product biomass (packing) feed culture) Sucrose product biomass (packing)
1 Mass flow A1 0.37 A1 0.63 C1 0.155 C1 84.5 E1 0.042 E1 0.958 (G1(G3- G1 0.09 G1+H1 G1 8.7E-3 J1+K1 L1-M12 M5+M6 O2 0.6 S13
(K/batch) I3))/ I3-1000

2 Volumetric flow G1/G7 H1/H7 I1/I7 G2+H2 K1/K7 J2+K2 L2- M2 0.05 M2-N2- O2 R1/R7 (O2-Q1) S2 0.05 R2

(L/batch) (M12/1.977) M12/1.977) 0.95

M8 Journal of Food Science r Vol. 00, Nr. 0, 2013

3 Sugar content 125 1587 202.4 J3 0 1587 0
(g/L)
4 Total sugar (kg) (G2 G3) (H2 H3) (J2 J3)/1000 J4 0 (R2 R3)/1000 0
/1000 /1000
5 Pichia fermentans K1 0.18 K5 K5 1.17
(k)g/batch
6 Saccharomyces K1 0.82 K6 K6 2.25
cerevisiae
(kg/batch)
7 Density 1.05 1.587 1.12 3.43 1.587
8 Alcohol content 2.547 M8 M8 2.27
(g/L)
9 Alcohol content 9.93 M9 M9 9.78
(%V/V)

10 Fermentation (M8/2.654) 100 (S2 14/O2

efficiency O14) 100

11 Unprocessed L4-(L4 0.959)
sugar into
alcohol
(kg/batch)
12 Carbon dioxide ((L4-M11)/180.16)

content 2 44
(kg/batch)
13 Oenococcus oeni O2 0.001 Q13 0.7
(kg/batch)
14 Malic acid 1.85 O14 1.16
content (g/L)
Prickly pear wine production . . .
illustrated by producing a fermented product with a unique aroma,
flavor and taste profiles, representative of prickly pear wine.
Based on the results presented above, a detailed process flowsheet
has been prepared including the flow diagram (Figure 2) and the
mass balance (Table 5), which are the basis for equipment sizing
and selection. This information is also used to define the plant
layout and instrumentation required for process control in order
to obtain wine with uniform quality. At laboratory level, overall
process efficiency was of 46.5 for young wine and 44.2% for
refined wine, based on the whole fruit. That is, 2.2 kg (average)
of fruit are required to obtain 1 L of wine. For a fruit that has
little market value, the results obtained (efficiency and quality)
constitute a viable alternative for sustainable harvesting of prickly
pears.
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