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FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Blackberry (Rubus spp.) Juice Alcoholic Fermentation: Kinetics and Aromatic
Caldern-Santoyo, M., Godina-Galindo, G., Bautista-Rosales,
Ragazzo-Snchez, J. A*.

P. U., Gutierrez-Martnez, P. and

Instituto Tecnolgico de Tepic. Laboratorio Integral de Investigacin en Alimentos. Av. Tecnolgico No.
2595 C.P. 63175. Tepic, Nayarit, MXICO, Tel. (311) 213 15 43.

Fermentations technology was used to obtain an alcoholic beverage from blackberry

juice, using different Saccharomyces cerevisiae strains. A medium alcoholic graduation
wine with good possibilities to be escalated up to industrial level was obtained, opening
an interesting option for blackberry fruit transformation. Alcoholic fermentations were
performed at 28 C, 200 rpm and non-controlled pH and Vitelevure CM 4457. Enofera
T 306, ICV K1 and Greroche Rhona L 3574 S. cerevisae strains used for different
fermentations. Kinetic and aromatic aspects of fermentation products obtained with
each strain were evaluated. Biomass quantification was determined by Abs650, substrate
consumed was reported as total sugars using Dubois method, ethanol formation was
determined by HPLC in an ion exchange column, and juice and wine aromatic
composition were determined by HS-SPME-G technique. Some kinetic parameters as
specific substrate consumed, specific product formation rate, maximum specific grow
rate, product and biomass yield were calculated. Results showed significant differences
among kinetic parameters and ethanol produced by Saccharomyces cerevisiae strains
used for the alcoholic fermentations. Similar differences were observed in aromatic
compounds produced during different fermentations.

(Keywords: Black Berry, alcoholic fermentation, Saccharomyces cerevisiae.)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Effect of Temperature, pH and Harvest Season on Polyphenols Concentrations of
Mexican Cocoa Beans Fermentation
Ruz-Garca, R., Barrios-Vias, M., Garca-Alamilla, P., Martinez-Morales, A., Urrieta-Saltijeral, J.M.*
Departamento de Ingeniera Bioqumica, Instituto Tecnolgico de Villahermosa, Carretera Vhsa-Frontera
Km 3.5 Villahermosa, Tabasco-Mxico. mailto: ;

Raw cocoa is the processed commercial form of the cocoa seed. Cocoa beans are the
principal raw material of chocolate manufacture. The major objective of fermentation
and drying is to produce beans, which will give a good chocolate flavor. in order to
obtain the optimum fermentation time in the two most common harvest season in the
region of production Tabasco, Mexico, Effects of temperature and pH on polyphenols
evolution during a traditional fermentation time (7 days) were evaluated. Freshly
harvested cocoa pods were fermented in pilot wood boxes of 100 kg for seven days with
a single turning at 24 h. Samples were taken periodically for drying treatments in an
oven at 110C, fat was eliminated from the bean by Soxhlet method and pH analysis
was determined in cotyledon, mucilage and bean. Phenol content was measured in a
spectrophotometer at 720 nm. The data were analyzed using statistical software for
analysis of variance. Temperature profiles in two different seasons of harvest show that
temperature rise slowly at first, then more rapidly, reaching 40-45C after the first 48
hours, with turning of the beans the temperature rises to 48-50C due to microbiological
and biochemical reactions inside the cotyledon beans. There was considerable variation
in temperature within the fermenting mass to the bulk of the beans. The results shown a
decrease of 80% of total phenols and 70% for tannins, with astringency diminished as a
consequence. However the results in temperature and pH showed that the time of
fermentation in the second harvest season (March-April) should be less than eight days
(6-7 days). The statistical analysis showed that tannins final content were a function of
temperature, pH and initial phenol content for this process. The present study should
allow evaluation of aromatic potential of cocoa beans in the future.

(keywords: polyphenols, cocoa beans, temperature, fermentation)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Foam Characterization of Sparkling Wines Selling in Mexico by Image Processing
Lpez-Pineda, A.,*1 Martnez-Peniche, R.,1 Castillo, E.2

Universidad Autnoma de Quertaro, Facultad de Qumica. Centro Universitario s/n Col. Cerro de las
Campanas, 76010-Quertaro, Mxico. 2 Instituto Politcnico Nacional, Centro de Investigacin en
Ciencia Aplicada y Tecnologa Avanzada, Cerro Blanco 141, Colinas del Cimatario, 76090-Quertaro,
Mxico. E-mail address:

Natural Sparkling wine is obtained through a double fermentation process of grape

must. Bubble size and its rising rate, number of trains, and collar and crown formation,
produced during pouring, determine foam quality. These parameters are commonly
evaluated by sensory analysis which could be subjective that is why image processing
as instrumental methods, has been developed. The aim of this work was analyze foam
quality of sparkling wines presents in the Mexican market by image processing and
compare these results with those obtained by sensory evaluation. Sensory evaluation
was accomplished by means of a trained-judge panel, meanwhile experimental
conditions for image processing were settled down before the analyses of the samples.
Simple correlation analysis and Principal Components Analysis (PCA) were performed
in order to find possible correlation between methods. An Italian Asti showed the best
collar and crown stability time while Champagne obtained the highest crown. Among
Mexican samples, Petillant showed good foaming characteristics, contrasting with
Chambrulet. Significant correlations between crown and collar variables measured by
image processing with the same parameters measured by sensory evaluation were
found, which doesnt occur between effervescence variables. Also, correlations between
comparable variables in the different methods were found. In the PCA samples grouped
according to their gas content, country origin or grape variety used in their fabrication.
Results show that image processing is a useful tool to measure foaming quality of
sparkling wines.

(keywords: sparkling wine, foam, image processing, sensory evaluation).


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Dynamical Characterization of Sparkling Wines Affected by their Chemical
Lpez-Pineda, A.,*1 Martnez-Peniche, R.,1 Castillo, E.2, Relkin, P.,3 Mezdour, S.3
1 Universidad Autnoma de Quertaro, Facultad de Qumica. Centro Universitario s/n Col. Cerro de las
Campanas, 76010-Quertaro, Mxico.
2 Instituto Politcnico Nacional, Centro de Investigacin en Ciencia Aplicada y Tecnologa Avanzada,
Cerro Blanco 141, Colinas del Cimatario, 76090-Quertaro, Mxico.
3 Laboratoire de Biophysique, Agro Paris Tech Massy, 1, Av. des Olympiades, 91744-Massy, France.
* Corresponding author. E-mail address:

Foam is considered one of the most important quality characteristics of sparkling wines.
Foam and collar stability are not only function of effervescence and CO2 concentration
in the liquid, but they are also dependent on physicochemical factors, such as surface
tension, and the wines chemical and colloidal composition. The composition of a
particular wine will be determined by the variety of grape, the terwar, harvest period
and winemaker among others. The aim of this study was to determine how chemical
composition affects the foaming capacity and stability of sparkling wines under
dynamical conditions. Three sparkling wines obtained in the Mexican market were used
in this study. Physicochemical characterization was achieved by OIV test methods.
Foam to be measured was generated by the gas sparging method and parameters
measured by image process techniques were: maximum height during gas injection
(HM), foam stability time (TS) and the average lifetime of a bubble at steady state (,
Bikerman coefficient). The surface tension at the air-water interface was measured by
means of an automated tensiometer. Sample with a lower alcohol content showed the
highest HM and TS. Tensiometry data exhibit that surface tension decreases faster in
the sample lower alcohol content than in other samples. Protein concentration did not
affect foam behavior.

(keywords: sparkling wine, physicochemical characterization, gas sparging method,



FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Production of Tannase by Aspergillus niger GH1 in Submerged Culture Using an
Airlift Bio-reactor
Cruz-Hernndez M.A.*, Esquivel-Contreras M.T., Ramrez-Barron S.N., Aguilar C.N.
Departamento de Investigacin en Alimentos. Facultad de Ciencias Qumicas. Universidad Autnoma de
Coahuila, Campus Saltillo, Blvd. V. Carranza e Ing. Jos Crdenas, Saltillo, Coahuila, Mxico, C.P.

Tannase is an enzyme that specifically breaks the galloyl ester bonds of tannins and
produces gallic acid, and therefore has been extensively used in foods, beverages,
pharmaceuticals and chemical industries. Several filamentous fungi microorganisms
have been studied for their capacity to produce high levels of tannase, namely
Aspergillus and Penicillium. Tannase production has been studied in submerged, solidstate and semisolid-state cultures; however, few contributions have been made
regarding the selection of the bioreactor. The main objective of this study was to
evaluate the kinetic production of tannase by Aspergillus niger GH1 using an airlift bioreactor. The temperature of the fermentation process was 35 C, with an initial tannic
acid concentration of 25 g/L and air flux of 0.5 L/min. Tannase activity was analysed by
HPLC and the results were then compared with those obtained by spectrophotometric
method. While in submerged culture the production of tannase is majority at an
intracellular level, in solid-state culture is at an extracellular level. Kinetic analysis were
carried out in an airlift bio-reactor for 72 h and samples were taken every 12h. After 48
h of the fermentation process a maximum value of the intracellular tannase activity was
detected (843.64 U/L), while a lower value was observed for the extracelullar tannase
activity (233.90 U/L ). The biomass production in the airlift bio-reactor was detected
at12 h (2.2 g/L) with a maximum production time at 36 h (7.2 g/L). After 36 h of the
fermentation process a significant decrease of the biomass rate was observed. The
results obtained were higher comparing with those results from submerged bioreactors
using different types of agitation. Nevertheless, the decrease in biomass production was
not observed on other bioreactors in a short time. The use of an airlift bio-reactor
represents an attractive alternative to other methods used in order to produce higher
levels of tannase. However, an optimization of the process is required.

(keywords: tannase, airlift bio-reactor, submerged fermentation)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Comparative Kinetic Study of Tannase Production by Aspergillus niger GH1
and Fusarium verticillioides.
Veana-Hernndez F.1, Aguilar C.N.2*, Cruz-Hernndez M.A.2, Rodrguez-Herrera R.2

Universidad Autnoma de San Luis Potos/ UAMZH

Departamento de Investigacin en Alimentos. Facultad de Ciencias Qumicas
Universidad Autnoma de Coahuila. Blvd. V. Carranza y Jos Crdenas. Saltillo, Coahuila. C.P.25000.

From an industrial point of view enzymes represent an important biotechnological

advance. Tannase or tannin acyl hydrolase is an enzyme widely used in several
industries. This enzyme has the ability to catalyze the hydrolysis of the gallotannins to
glucose and gallic acid, and is also used in the synthesis of trimetroprim. The present
study was conducted in order to compare two fungal sources of the enzyme, Aspergillus
niger GH1 (A. niger GH1) and Fusarium verticillioides (F. verticiloides). Tannase was
obtained by fermentation of tannic acid in submerged culture. Kinetic analysis were
made during 72 h. Crude enzymatic extracts were obtained, which were used to
determine the fungal biomass by a gravimetrical method, the substrate consumed that
was monitored spectrophotometrically, the protein content determined by Bradford
method, and the enzymatic activity that was assayed by the spectrophotometric method
of methanolic rhodanine. Mathematical models were applied, such as the Luedeking and
Piret model for substrate, Verhulst-Pearl model for biomass and Pirts model for
substrate consumption. The present results demonstrated that A. niger GH1 was a better
source of tannase than F. verticilloides, since A. niger GH1 registered an extracellular
maximal production of 172.58 U/L, with a specific activity of 4033.55 U/mg; producing
a biomass level of 6.49 g/L at 72h of fermentation time, while the substrate (acid tannic)
was degraded rapidly in the fermentative process at 24 h. An important kinetic
parameter indicated that the YP was of 1281.15 UE/gX. However F. verticiloides
registered an extracellular production of 46.89 U/L tannase activity, 1047 U/mg specific
activity and 1.33 g/L of biomass, which shows that this fungal strain also has the
capacity to produce the tannase enzyme.

(keywords: A. niger GH1, F. verticillioides, kinetic parameters)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Alcoholic Fermentation of Sotol Juice (Dasylirion spp) by Native Yeasts
Martnez-Sosa, M., De la Garza-Toledo, H.*, Aguilar-Gonzlez, C.
Departamento de Investigacin en Alimentos, Facultad de Ciencias Qumicas. Universidad Autnoma de
Coahuila. Jos Crdenas Valds s/n Col. Repblica Oriente. Saltillo, Coahuila 25280.; *

The Sotol is an alcoholic beverage obtained by the fermentation and distillation of the
cooked stalks of a plant known too as Sotol (Dasylirion spp), a Nolinaceae that grows
naturally in the Mexican deserts. The production is carried out in a traditional way and
without the knowledge of the participating microorganisms, which causes differences
on the final product. The aim of this work was to select a native yeast strain with the
adequate oenological characteristics to perform the alcoholic fermentation of Sotol, as
well as to determine the conditions to carry out the fermentation. During the first stage
of this work, alcoholic fermentations were carried out in Sotol juice using 12 native
yeasts strains and one pure strain of Saccharomyces cerevisiae used as a control. The
fermentations were carried out at 30C for 72 h; results were analyzed in base of the
CO2 production and the smell of ethanol. In order to select the conditions to perform the
alcoholic fermentation of the Sotol juice, a second set of fermentations were performed,
three temperatures (20, 25 and 30 C), three initial sugar concentrations (12, 16 and 20
Bx) and six times of incubation (12, 24, 36, 48, 60 and 72 h) were used; glucose and
fructose consumption and ethanol production were evaluated. In the first stage of the
investigation, four different strains shown the expected results, three of them were
identified as Saccharomyces cerevisiae (two natives and the control) and one as
Candida kefyr; one of the native yeasts outperforms the rest and was selected to carry
on the following fermentations. In the second set of fermentations, the major
consumption of glucose and fructose were obtained in the fermentation with 16 Bx at
30 C; the highest ethanol concentration was 2.9 %, with 16 Bx at 25 C. The selected
conditions to perform the following fermentations were: a temperature of 30 C, an
initial concentration of 16 Bx and a final time of 60 h of incubation.

(keywords: Saccharomyces cerevisiae, fermentation conditions, ethanol)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Optimization of the Engineering Parameters to Produce Zeaxanthin in a
Fluidized Bed Birreactor
Ramos-Ojeda, E. *, Escamilla-Silva, E.
Departamento de Ingeniera Qumica, Instituto Tecnolgico de Celaya , Av Tecnolgico y Av Garca
Cubas s/n Col Fovisste, Cp 38010, Celaya, Guanajuato, Mxico.

The use of bacteria in technological development has become an important economic

resource, especially for production of metabolites. The natural manufacture of
carotenoids has not been optimized to obtain high yields of production which would
minimize production costs. Particulary, in this work the procedure for zeaxanthin
production has been optimized to obtain high-level productions. The process was
carried out as an immobilized fermentation of Flavobacterium sp cells in a fluidized bed
bioreactor with recirculation. The experiment was conducted at pH 7.2, temperature 27
C with 4.5 g/l of NaCl and 3 mm diameter immobilization beads, the acide
polygalacturonate was used as material. Aeration, recirculation and pellet, with size of
2mm, 3mm and 5mm, were the analyzed variables. In the design section several nondimensional numbers were identified, particulary Reynolds, Sherwood and Schmidt
numbers. The fermentation broth behaves as a Newtonian fluid thus it facilitates the
mass transfer mass and zeaxanthin recovery process. The maximum zeaxanthin
production obtained was 1.2165 mg/ml. In difference to free cells, the immobilized cells
are reused 7 times before of suffering any damage is detectable. This project applied
techniques to obtain mathematical regression on experimental data. This process proved
to be simple with high capacity of oxygen transfer and low mechanical stress.

(keywords: flaviobacterium, fluidized bed reactor, hydrodynamics, immobilized cells,



FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Response of Strains of Saccharomyces cerevisiae to Conditions of Fermentative
Pez-Lerma, J.1, Rutiaga-Quiones, M. 1, Barrio-Esparducer E.2, Belloch, C.3, Querol, A.3,
Soto-Cruz, O.1*
Divisin de Estudios de Posgrado e Investigacin, Instituto Tecnolgico de Durango.
Blvd. Felipe Pescador 1830 Ote., 34080, Durango, Dgo., MXICO. *
(2) Instituto Cavanilles de Biodiversidad y Biologia Evolutiva, Universidad de Valencia. Edifcios
de Institutos, Campus de Paterna, E-46100 Burjassot, Valencia, ESPAA
(3) Instituto de Agroqumica y Tecnologa de Alimentos, Consejo Superior de investigacin
Cientfica. Edifcios de Institutos, Campus de Paterna, E-46100 Burjassot, Valencia, ESPAA

The yeasts used in the different process of fermentation are subject to different
conditions such as: changes of pH, high initial concentration of substrate, accumulation
of toxic compounds (like ethanol), and changes in the temperature along fermentative
processes. The aim of this work was to compare the 14 predominant strains of
Saccharomyces cerevisiae isolated from El Mezquital, Durango, Mxico. These strains
were compared with a reference commercial strain used in wine elaboration, which has
standardized characteristics besides a commercial patent. Yeast cells were grown in
GPY (peptone 0.5%, yeast extract 0.5%, glucose 2%). Medium was solidified by adding
1.5% agar. 48h fresh yeast cultures on GPY agar were used to inoculate 5mL of fresh
GPY and shaken overnight at 30C. The next day the cultures were adjusted to an
absorbance at 655 nm of 0.3 by dilution with fresh GPY. After shaking at 30C for
another 4 h the cells were diluted in sterile water to an A655 of 0.3 and 5 L of a 5-fold
dilution series in water were spotted onto all stress media. All plates were incubated
aerobically and checked 1 to 5 consecutive days for colony development. The stress
media included different condition such as pH from 2.8 to 3.2, temperature from 16 to
45C, ethanol concentration from 5 to 15%, and concentration of fructose and glucose
from 200 to 300 g/L. It was observed a behavior similar between the strains isolated
from the process of elaboration of mescal versus the reference strain. This result is
important because the isolated strains can be used as starter for the fermentation to
produce mescal or proved in fermentations to obtain other beverages.

(keywords: fermentation stress, Saccharomyces cerevisiae, predominant strains).


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Volatile Acidity Fraction of Cocoa Beans Roasting. Effect of Fermentation Process
Velzquez - Martnez, J.R., Robles Olvera, V, J, and Garca Alamilla, P*
*Universidad Jurez Autnoma de Tabasco (UJAT). Divisin Acadmica de Ciencias Agropecuarias
(DACA). Centro de Investigacin en Ciencias Agropecuarias (CICA). Carret. Vhsa Teapa Km. 25.
Villahermosa, Tabasco, Mxico. E- mail:
Tel: 993358-1585 fax (142 9151 y 142 91 50) ext 6604
Unidad de Investigacin y Desarrollo en Alimentos (UNIDA). Instituto Tecnolgico de Veracruz,
Veracruz, Mxico.

The process of Cocoa beans fermentation stage has a fundamental paper in the
development of chocolate flavor precursors, but is during the stage of the roasting where
these properties are developed. Nevertheless, the cocoa fermentation process varies
widely from a country to another and therefore the quality of chocolate obtained too. In
Mexico, the traditional fermentation process is carried out in a wood box; however, this
type of fermentation produces cocoa beans with undesirable volatile acidity that affects
the quality of final product. The objective of this work was to study the volatile acidity
after the process of the toasting of cacao beans being evaluated the effect that has on
this parameter the fermentation realised in a rotatory drum. Three turning intervals (9,
12, 15 revolutions manually) in a rotatory drum were compared with the traditional
fermentation process using a box wood. The fermentation process was carried out for
136, 144, 152, and 168 h samples were taken during this time and were subjected to
drying process. The samples dried were roasting to 140C during 30 minutes. The
results have shown differences between box wood and rotatory drum fermentation
process, these differences are attributed to both time and system fermentation. The
range of volatile acidity was of 0.0012 0.0062 g acetic acid / g d.m., and pH for up of
5.0 among different treatments during roasting of cocoa beans. The better intervals in
rotary system were to 15 and fermentation time of 168 h, with a pH of 5.6 and a
concentration of volatile acidity of 0.01 g / g d.m.

(keywords: volatile acidity, cocoa beans roasting, cocoa fermentation).


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Determination of Average Diffusion Coefficient During Cocoa Beans
Robles Olvera, V. J., Velzquez Martnez, J.R., and Garca-Alamilla, P.*.
*Universidad Jurez Autnoma de Tabasco (UJAT). Divisin Acadmica de Ciencias Agropecuarias
(DACA). Centro de Investigacin en Ciencias Agropecuarias (CICA). Carret. Vhsa Teapa Km. 25.
Villahermosa, Tabasco, Mxico E- mail:
Tel: 993358-1585 fax (142 9151 y 142 91 50) ext 6604
Unidad de Investigacin y Desarrollo en Alimentos (UNIDA). Instituto Tecnolgico de Veracruz,
Veracruz, Mxico.

Cocoa beans fermentation is an extremely complex process due to the diverse mass and
heat transfer mechanisms involved in its microbiological and biochemical aspects.
Nevertheless, the development of the suitable sensorial properties, fermentations main
attribute, is a mainly a function of acetic acid produced by acetic bacteria; those bacteria
dwell in mucilage and penetrates towards cotyledon by diffusion; it is necessary to
eliminate acetic acid by drying after fermentation process. In order to improve
fermentation process, the acquisition of information about the difussion processes is
fundamental. In this work, the diffusion coefficient of acetic acid was evaluated during
cacao fermentation process. For the determination of diffusion coefficient a
microanalysis of 5 internal layers of the grain from the center towards the periphery was
determined. A one-dimensional model of diffusion was used to obtain the effective
coefficient of acetic acid. The experimental results allowed quantifying the profile of
acidity from center to the periphery of the cocoa beans, being observed that two
gradients of acidity appear during the fermentation process. A gradient during the first
72 h towards the interior of the grain and later a second gradient towards the outside,
were observed. Through the kinetic of both profiles, the diffusion coefficients of acetic
acid were obtained. The values obtained for the acetic acid coefficients went 5.80x10-13
m2/s at the beginning of fermentation process and 5.35x10-14 m2/s at the end of the


cocoa beans fermentation, internal acidity profile , acetic acid, diffusion



FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Study of Combined Effect of pH and Temperature on Water-Soluble Red Pigment
Production by Penicillium purpurogenum GH2
Mndez-Zavala, A., Montas-Senz, J. C., Prez-Bermen, C., Zugasti, A., Aguilar, C. N. *
Food Research Department, Universidad Autnoma de Coahuila, Unidad Saltillo, Blvd. V. Carranza e
Ing. Jos Crdenas V. s/n. Col. Repblica, PO Box 252, ZIP 25280, Coahuila, Mxico.; *

Natural pigments are very important for many industries, and new sources of those
molecules are required. An alternative source are fungi that can produce pigments with
excellent properties. In this study it was evaluated the combined effect of pH and
temperature on water-soluble red pigment production by P. purpurogenum GH2. It was
cultured in Czapek-dox media with D-xylose (15 gL-1) as sole carbon source. An
experimental design with factorial fix was as used; three pH (5, 7 and 9) and two
temperature levels (24 and 34 C) were evaluated. The red pigment production kinetic
was monitored each 48 h and it was assayed spectrophotometrically at 500 nm, the pH
and pRedox (mV) were evaluated in a potentiometer; biomass was determined by
gravimetry of 240 h culture samples. The highest production of red pigment was
reached using a pH value of 5 and a temperature of 24C. Maximum red pigment
production was of 2.406 g/L. Biomass and red pigment production are not directly
associated. This study demonstrated that P. purpurogenum GH2 produce a pigmented
secondary metabolite of interest for food industry.

(keywords: pH, temperature, Penicillium purpurogenum, water-soluble, red pigment)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Inductive Effect Obtained from a Mixture Carbon Source in the Production of
Gibberellic Acid by Gibberella fujikuroi
Rios-Iribe, E. Y. , Escamilla-Silva E. M*.
Facultad de Qumica. Universidad Autnoma de Quertaro. Cerro de las Campanas S/N Col. Las
Campanas Apartado Postal 184 C.P. 76010. Quertaro, Qro.
Departamento de Ingeniera Qumica, Instituto Tecnolgico de Celaya
Av. Tecnolgico y Antonio Garca Cubas S/N. C.P. 38010. Celaya, Guanajuato,

The gibberellic acid (GA3) is the main secondary metabolite produced by the Gibberella
fujikuroi fungus. Actually, this plant hormone has a great importance in agriculture and
brewery industry, due to its fast and strong effects with concentrations of micrograms
on the processes of stimulation of the growth, flowering, stem elongation, and
germination of seeds, among others. Plant promoters of growth production such as the
gibberellins, specially the GA3 represent a priority to obtain better harvests in the
agricultural area and by consequence, improve the food industry. Three routes to obtain
GA3 have been reported: extraction from plants, chemical synthesis and microbial
fermentation. The last one is the most common method used to produce the GA3. The
industrial process usually used for the fermentative production of the GA3 is based on
the technique of submerged fermentation of Gibberella fujikuroi. In this investigation,
glucose-corn oil mixtures were used as carbon source on the basis of 40 grams of
carbon in a 7 L stirred tank bioreactor. A pH value of 3.5, 29 C, 600 rpm agitation and
1 vvm aeration were maintained and controlled with a biocontroller connected to the
bioreactor, throughout all culture time.. Our results shown that the mixture carbon
source affected the time and the production of the metabolite. The secondary
metabolism began around of 72 hours of culture and a production of 830 mg GA3/L
after 240 hours of fermentation was obtained when the mixture glucose-corn oil was
employed. Contrasting the 369 mg GA3/L at 240 hours of culture when only glucose
was used, with the begin of production of GA3 around 120 hours of fermentation. The
GA3 was quantified by High-Performance Liquid Chromatography (HPLC).
(keywords: Gibberellic acid, mixture carbon source, microbial fermentation).


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Validation of Acetobacter/Gluconobacter and Carr Media for Acetic Acid Bacteria
Enumeration During Traditional Cacao (Theobroma cacao) Fermentation
Romero-Cortes, T., Velsquez Martnez, J.R., Salgado-Cervantes, M., Ramrez-Lepe, M., GarcaAlamilla, P., Rodrguez-Jimnez, G., Robles-Olvera, V.*
Unidad de Investigacin y Desarrollo en Alimentos. Instituto Tecnolgico de Veracruz Av. Miguel Angel
de Quevedo No. 2779 Col. Formando Hogar C.P. 91897 Veracruz, Ver. Fax: (2299) 34 57 01.

The characteristic chocolate aroma is obtained after a preparation process called curing.
Cacao curing begins with cacao beans recollection. Fermentation, drying, selection and
storing are important steps in curing before roasting for chocolate production.
Fermentation is a crucial step because sensorial attributes can be strongly affected,
during this step, aroma and aroma precursors are produced. Non-fermented cacao is
bitter and astringent. Fermentation is carried out by a bacteria consortium: initial
conditions are favorable for yeast growth, when environmental conditions change as
consequence of metabolic activity to an anaerobic less acid media, lactic acid bacteria
become dominants, and in the same way, after activity of acid lactic bacteria new
environmental conditions are propitious to acetic acid bacteria growth. As consequence
acetic acid is produced. Acetic acid production is desirable for sensorial attributes, but
its excess should be eliminated during drying because high concentrations of acetis acid
mask aroma and flavor. dentified Acetic acid bacteria are: Acetobacter pasteurianus, A.
peroxydans, A. aceti, Acetobacter aceti subsp. liquefaciens and Gluconobacter oxydans
subsp. Suboxydans. In order to evaluate the evolution of these bacteria, different
specific media (acetobacter/gluconobacter or Carr media) has been used for their
enumeration. Acetobacter/gluconobacter media allow the growth of A. aceti, A.
liquefaciens, A. pasterianus, A. xylinum, Frauteria aurantia and G. oxydans, while, Carr
medium is a classic specific media for A. pasterianus. Consequently the objective of
this work was to evaluate the ability of acetobacter/gluconobacter and Carr media for
enumerate acetic acid bacteria during cacao fermentation. Cacao samples were picked
from industrial fermentation boxes, homogenized in 0.85 % NaCl, diluted if necessary,
poured in Acetobacter/Gluconobacter and Carr media and incubated at 37 C. Results
show similar counts in both media and evolution is similar to those reported in
literature. These results suggest that A. pasterianus is the dominant specie during cacao
fermentation and consequently both media can be used for acetic bacteria enumeration.
(keywords: cacao fermentation, acid cacao, acetic acid bacteria)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Effect of Saccharomyces cerevisiae Cell Wall Extract on Pathogenic Bacteria
Aguilar-Uscanga, B1*, Rodrguez, C.M1., Viveros, J.M1., Hernndez, S1., Aguilar, M.G2., Reyes-Blanco,
M1. Sols-Pacheco, J1.

Departamento de Farmacobiologa, Centro Universitario de Ciencias Exactas e Ingenieras. Universidad

de Guadalajara. Bvld. M. Garca Barragn 1458, Col. Olmpica. Guadalajara, Jalisco.
Unidad de Investigacin y Desarrollo en Alimentos, Instituto Tecnolgico de Veracruz. Miguel ngel
de Quevedo 2779, Col. Formando Hogar, Veracruz, Ver.; *

The cell wall of Saccharomyces cerevisiae is a dynamic organelle that represents 20 to

30% dry weight of the cell; it is composed of polysaccharides, 50 to 60% -glucan, 40
to 45% mannoproteins and 2 to 5 % chitin. Some studies report that the polysaccharides
of yeast cell walls mainly -glucan, have been used against various bacterial infections,
yeast, fungal, viral and parasitic diseases, besides it has been used as an ingredient for
eliminating toxins in contaminated food, the latter being a strategy to avoid product loss
with its high cost to industry. In the present study, the cell walls of three different yeasts
isolated from the tequila process (Ar5, GM and L013) were extracted. These strains
were grown in a flask containing 350 ml YPD liquid medium (250 rpm, 30C). The
cells were harvested in exponential phase and washed successively with water and TrisHCl buffer at pH 8. The cell wall was extracted by breaking the cells using a
homogenizing Mini-beadbeater, the obtained extracts were recovered by centrifugation,
washed and finally lyophilized. The study of the effect of cell wall extracts on the
growth of pathogenic bacteria was conducted using E. coli O157: H7 (10 x108 cells /
ml), suspended in a 5% saline solution, containing 100 g/mL cell wall extract. The
suspension was left to stand 5 hours at room temperature (25-27C), after which 100
L samples were taken every hour and inoculated into Petri boxes containing either
nutritive or EMB agar. The dishes were incubated at 37 C for 24 to 48 to observe
bacterial growth. Result shown that at 0, 1, 2 and 3 hours of incubation, E. coli was able
to grow, but at 4, 5 and 6 hours, bacterial growth was totally inhibited. These results
indicate that yeast cell wall extracts obtained from the tequila industry have the ability
to capture pathogenic bacteria cells, causing inhibition within 4 hours of contact.

(keywords: cell wall, yeast, polysaccharides)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Study of Volatile Compounds During Fed-Batch Fermentation
for the 100% Agave Tequila Production
Alczar-Valle, E.M.; Herrera-Lpez, J.E.; Daz-Montao, D.M.; Arellano-Plaza, M*
Centro de Investigacin y Asistencia en Tecnologa y Diseo del Estado de Jalisco A.C. Av. Normalistas
800, Colinas de la Normal, Guadalajara 44270 Jalisco,

The volatile compounds (esters and higher alcohols) are important in the aroma and
flavor characteristics of the Tequila 100% agave. Most of these compounds are
produced in the fermentation process. The aim of this work was to find the impact of
feeding agave juice on the production of volatile compounds during a fed-batch
fermentation of Tequila 100% agave. The fed rate of the diluted juice (160g fermentable
sugars L-1) was 0.099 g min-1 and the concentrate juice (370g fermentable sugars L-1)
0.088 g min-1. These juices were fortified with ammonium phosphate (0.45g L-1),
magnesium sulphate (0.08g L-1) and urea (0.09g L-1). The volatile compounds measured
in this study were acetaldehyde, ethyl acetate, methanol, 1-propanol, isobutanol, 1butanol, amyl alcohols, ethyl caprate, ethyl lactate and ethyl caproate, all of which
required in Tequila 100% agave by the official Mexican law. The fermentation was
repeated twice for the statistical validation. End point samples were analyzed using a
Hewlett Packard model HP 6890 gas chromatograph, and Head Space Hewlett Packard
model HP 7694E as faceplate. The difference of the yield coefficients of ethyl acetate
(8.7x10-5) and ethyl caproate (1.8x10-5) on carbon substrate between dilute agave juice
fed and concentrate fed fermentation were 58% and 75% respectively. Ethyl lactate and
ethyl caprate were produced only in the concentrate juice feeding. However the yield of
higher alcohols were more significant in the dilute juice fed fermentation, and the
quantities reached to 6.1x10-6 for 1-propanol, 7.3x10-5 for isobutanol, 6.3x10-5 for 1butanol and 4.3x10-4 for the amyl alcohols. The differences between dilute and
concentrated fed fermentations of these compounds were 73%, 65%, 53% and 62%
respectively. These results show that is important to know how the fermentation process
can modify the fermented must composition and change the beverages characteristics.
The analyses of these compounds are important since their concentrations are regulated
for the federal law, and they give the aroma and the flavor distinctiveness.

(keywords: volatile compounds, fermentation, fed-batch).


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Elaboration of Kefir using Bovine Milk
Burciaga-Castillo B., Martnez Guzmn A., Muoz Ros R., Rodrguez Rosales D.J., Garca Caballero
Departamento de Ingenieras Qumica y Bioqumica, Instituto Tecnolgico de Durango, Boulevard Felipe
Pescador 1830 Ote. Colonia Nueva Vizcaya, C.P. 34080, Durango, Dgo. Mxico. Tel 01 (618) 8 29 09
19. Email:

Nowadays, elaboration and marketing of kefir in Mexico is practicality null, therefore,

its introduction to Mexican market, can be an innovation in healthy food products, with
healthy and gastronomic benefits. As a functional food, kefir has been related to health
benefits including restoration of the natural intestinal microbiota, as well as antibiotic
and antiviric properties. The aim of this work was to develop a fruit added kefir, and to
evaluate their sensorial characteristics. The experimental design was planned in two
phases. The first phase was done to evaluate the influence of dry extract, artificial
flavoring and color agents, and only the dry extract affected the product. On the second
phase the whole product was used, and the variables selected included: concentration
and type of coloring agents, in a 3x2 factorial design. Each combination was tested by
triplicate. Analysis of Kefir products included CO2, proteins, fat, ashes and humidity.
For the sensorial evaluation, a descriptive analysis was done, using a hedonic
semistructured scale, with nine points. The variables analyzed were taste, color, odor,
texture and general acceptability. Results showed a 50% carbonic gas and 0.65%
alcoholic content, showing that kefir produced is of good quality. Regarding fat (21.5
g/L) and proteins (33 g/L), their values are within official Mexican limits for fermented
dairy products. For sensorial evaluation, significant variables were olor, flavor, color,
texture and acceptability for the type of colorant used, but not for concentration used
(p<0.05). The experiment with better acceptability was pinapple-coconut flavor, with a
0.25% concentration. The development of new functional foods for the Mexican market,
such as the kefir reported here, will give additional alternatives for dairy products with
health benefits for the Mexican population.

(keywords: Kefir, fermented milk, functional foods)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Production of Saccharomyces cerevisiae Biomass on Coconut Medium in Stirred
Tank and Bubble Column Bioreactors
Hernndez-Hernndez, S.; Ricabar-Francisco, A. D.; Jimnez-Avalos, H. A. and Chaires-Martnez, L. (*)
Food Research Center. Instituto Tecnolgico Superior de Alamo Temapache (CIA-ITSAT). Km. 6.5
Carretera Tuxpan-Potrero del Llano. Xoyotitla, Alamo. Ver. CP. 92750. Tel y Fax. 017658440038.

The food yeast Saccharomyces cerevisiae was grown in batch culture on in coconut
water and on a synthetic medium containing glucose, peptone and agar, to provide
kinetic data for a feasibility study of microbial protein production in stirred tank and
bubble column bioreactors. Analyses of growth on individual carbon substrates were
made to determine sugar assimilation patterns. Yeast inoculums were prepared by
transferring dried S. cerevisiae from commercial sources to 250 mL coconut and
synthetic medium by incubation at 28o C for 24 h. Then, inoculation in 10 L stirred tank
and 2 L bubble columns bioreactors were made at 0.6 g/L using coconut and synthetic
medium separately. During fermentation, the dry biomass yield of S. cerevisiae was
determined by harvesting samples of cells into a pre-dried and pre-weighted aluminum
pan and then dried to constant weight in an oven at 95 oC. Total soluble sugars were
determined using the DNS method for monitoring substrate consumption. The values
for specific growth rate (), growth yield coefficient (Yx/s), the specific rate constants
for substrate utilization (qs), and the specific rate for cell mass formation (qx) were
calculated. Statistical analyses were performed using the SPSS statistical program
(USA) with the significance level set at P<0.05. Initial inoculums on coconut water
gave cell weight peaks higher than on synthetic medium of 3.46 and 3.86 g/L in stirred
tank and bubble column bioreactor, respectively after 24 h of fermentation. Growth on
the coconut water in bubble column produced a higher (0.09 h-1) and Yx/s (0.07 g/g)
than in stirred tank bioreactor (0.06 h-1 and 0.06 g/g). qs and qx values were 6.3 x 10-3
g/g y 5.4 x 10-3 g/g, respectively. The values of kinetic variables, notably demonstrated
that the coconut medium in bubble column provide a faster growth rate in comparison
with a synthetic medium. However, this result will be considered as preliminary
because in order to increase productivity in an industrial scale, several subjects must be
solved, as the use of supplemented coconut medium with essential nutrients, O2
concentrations, shear stress, pH regulation as reported elsewhere previously for growth
of S. cerevisiae.
(keywords: Saccharomyces, batch fermentation, stirred tank, bubble column)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Development of a Low-Alcoholic Beverage by Fermentation of Whey with
Kluyveromyces marxianus.
Escorcia Marquina A1; Dubln Garca O*
Departamento de Alimentos, Facultad de Qumica, Universidad Autnoma del Estado de Mxico. Paseo
Coln y Paseo Tollocan S/N, Toluca 50000. 1Universidad La Salle.

The whey or serum is the aqueous phase that is separated from the curd in the process of
cheese making. The cheese industry requires ten liters of milk to produce one kilogram
of cheese, thus 9 to 12 L of serum are recovered. Currently in Mexico, in most
industries, whey does not have a treatment prior to disposal, this is due to the lack of
technology and infrastructure. On the whole, the problem is similar for discharges into
the drainage system, ponds, dams and rivers, causing a serious negative environmental
impact. The purpose of this work was to develop a low-alcoholic beverage from the
fermentation of whey. The development of this drink was conducted in three stages, in
the first phase was carried to find out the optimal parameters for Kluyveromyces
marxianus growth in whey, using biochemical tests, gram stain, and morphology. At the
same time pH, acidity, Brix and speed of agitation were checked. Once confirmed the
growth of K. marxianus in whey, in the second stage, drinks were developed with 6
different flavors, added with 5 different concentrations of sweeteners. In the last stage, a
proximal analysis was carried out and a preference test was applied to determine which
combination was the most accepted by a panel of judges. Whey was a suitable substrate
for the growth of K. marxianus, reaching its stationary phase at 27.5 hrs. Moreover, the
optimal parameters for its initial growth in the whey were: acidity 0.04%, fat 0%, Brix
11, pH 6.5, lactose 4% and a stirring of 100 rpm at 30 C. It was found that pineapplecoconut was the flavor more accepted (P <0001) with a concentration of aspartameacesulfame (50:50) of 0,007%, which had 3.5 Gay-Lussac, as well: 3.35 mg/100mL
carbohydrate, 1.34 mg/100 mL protein, 0.4 mg/100 mL of fat and 0.91 mg/100mL of
mineral. Development of low-alcoholic beverages is a viable alternative to seize on the
whey derived from cheese making.

(keywords: whey, Kluyveromices marxianus, alcoholic beverage).


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Screening and Identification of Fungi Strain Lipase-Productor Isolated from
Mexican Semidesert
Y. C. Gutirrez-Alvarado, A. Ilin, J. L. Martnez-Hernndez.
Departamento de Biotecnologa. Facultad de Ciencias Qumicas. Universidad Autnoma de Coahuila.
Unidad Saltillo. Blvd. V. Carranza e Ing. Jos Crdenas V. s/n, Saltillo Coahuila CP. 25280. Mxico. Email:

Lipase (E.C. hydrolyses triglycerides to fatty acid and glycerol, and under
certain conditions, catalyses the reverse reaction forming glycerides from glycerol and
fatty acids. Some lipases are also able to catalyze both transesterification and
enantioselective hydrolysis reactions. The interest in lipase has grown over the last few
years due to their excellent catalytic properties; they have become important
biocatalysis in various industrial sectors, such as the agrochemical, pharmaceutical,
detergent and food industries. In this study lipase-producing ability of 46 strains isolated
of different sources from Mexican semidesert (soil and plants) was evaluated. To screen
strains, a mineral culture medium with sucrose as carbon source and olive oil as
inductor was used. Fermentations were carried out at 120 rpm, 30 C for 72 h. Lipase
activity was measured using para-nitrophenyl propionate (Sigma) as substrate and
reactions were followed spectrophotometrically at 410 nm. Mucor griseocyanus
H/55.1.1, strain was used as control. Three fungi strains showed higher titles with a
relative activity of 72.97, 79.72, and 98.64 % compared to the control strain.
Macroscopic methods allow the identification of two Penicillium spp and one
Aspergillus spp strains. The obtained results show that the fungi strains from Mexican
desert are the potential lipase-producers.

(keywords: Lipase, fermentation, fungi, relative activity).


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Effect of Feeding Non-Sterilized Agave Juice in a Continuous Tequila
Hernndez-Corts G., Crdova-Lpez J., Herrera-Lpez E.J., Daz-Montao D.M.*
CIATEJ: Centro de Investigacin y Asistencia en Tecnologa y Diseo del Estado de Jalisco. Av.
Normalistas 800, Col. Colinas de la Normal
Guadalajara, 44270 Jal.; *

In recent years, the shortage of Agave tequilana Weber and the increase of tequilas
international demand, have led tequila producers to search for new alternatives that
could increase their process efficiency and productivity. Traditional tequila
fermentations are carried out using batch cultures, which present low productivities.
Continuous and fed-batch cultures have been used on alcoholic fermentation processes
due to their higher productivities; nevertheless, in the tequila industry these approaches
have not been attempted. Continuous cultures have been used at CIATEJ to ferment
agave juice, showing a significant increase on the productivity and the conversion yield
of sugar to ethanol. These fermentations have been performed using sterilized agave
juice at laboratory scale; however, in a large scale, sterilizing the medium could
seriously increase process costs. The main purpose of this work was to evaluate the
effect of the pH (controlled at 4 or not controlled, pH = 2.8), the aeration (0, 0.01 and
0.02 vvm) and the agave juice fed (sterilized or non-sterilized) on the fermentative
capacity of two strains of Saccharomyces cerevisiae (GU4 and AR5) recently isolated
by our research group, cultured in continuous. In fermentations fed with sterilized or
non-sterilized agave juice and performed at both pH conditions and with no aeration, the
sugar to ethanol yields and productivity parameters were not significantly different and
were approximately 0.46 gg-1 and 0.56 gl-1h-1, respectively. Nevertheless, when aeration
was supplied to those fermentations (0.01 and 0.02 vvm), substrate to product yields
and productivity parameters were significantly augmented when sterilized medium was
fed using both strains. These results indicated that the control of pH and the sterilization
of agave juice are not significant factors influencing the continuous anaerobiosis
fermentation. On the other hand, aeration showed to be an important parameter,
improving the fermentation efficiency on pure cultures. These results are giving key
information for the implementation of large scale continuous fermentations for the
production of tequila and work is going on to carry out the scale up.

(keywords: Tequila, continuous culture, Saccharomyces cerevisiae, Agave tequilana



FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

A Multiplex PCR Technique for Simultaneous Detection of Different Annexed
Sequences and Transgenic Events in Commercial Foods
Hernndez-Orona, V. L.1, Rodrguez Herrera, R1*. , Aguilar Gonzlez, CN1., and Reyes-Valds M.

Departamento de Investigacin en Alimentos, Facultad de Ciencias Qumicas, Universidad Autnoma de

Coahuila. Blvd. V. Carranza e Ing. Jos Crdenas V. s/n. Col. Repblica Ote. C.P. 25280. Saltillo,
Coahuila. *
Departamento de Fitomejoramiento, Universidad Autonoma Agraria Antonio Narro, Buenavista Saltillo
25000 Coahuila

In Mexico, at the present time, there are a high percentage of commercial foods with
transgenic residues but these foods do not have any label about these residues. The
objective of this work was to perform the simultaneously detection of different
transgenic residues from DNA of commercial and home-made foods using multiplex
polymerase chain reaction (PCR). First, DNA from maize, soybean and cotton plants
was extracted using the CTAB (Cetil Trimethyl Ammonium Bromide) technique. After
that transgenic sequences from each vegetal sample were individually amplified by
PCR. Specific primer pairs for the epsps, cry 1Ab/1As, and als genes and for the
annexed sequences nos, luc and ntp II were used for PCR amplification. The soybean
and maize samples identified as transgenic were used for processing of six home-made
foods (tortillas, snacks and tamales based on maize and milk, sausage and tofu based on
soybean). In addition, 25 foods with at least 1 soybean or corn ingredient and
commercialized in a Saltillo Coahuila market were analyzed too. DNA was isolated
from each food and detection of transgenic residues was performed by multiplex PCR.
The same specific primer pairs described above were used in this step. Using multiplex
PCR, it was possible to detect simultaneously three transgenic events (epsps, als and cry
1 A/b) and the annexed sequences (luc, ntp II and nos) using DNA from plants, homemade and commercial foods.

(Keywords: ntpII, luc, nos, epsps; cry 1Ab/1As, als)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Identification of Yeast in the Natural Fermentation of Mezcal Duranguense and its
Relations to Chemical Composition.
Martell-Nevrez M.A.*, Sols-Soto A., Soto-Cruz N.O., Rodrguez-Herrera R., Rutiaga-Quiones O.M.
Departamento de Investigacin y Posgrado en Ingeniera Bioqumica. Instituto Tecnolgico de Durango.
Blvd. Felipe Pescador 1803 ote. Durango, Dgo., Mxico. 34080. Departamento de Investigacin y
Posgrado en Alimentos. Facultad de Ciencias Qumicas. Universidad Autnoma de Coahuila. Blvd. V.
Carranza s/n Col. Repblica, ote. Saltillo, Coahuila.*

The mezcal is a traditional Mexican alcoholic beverage, the production process involves
4 stages: cooking, milling, fermentation and distillation, in most cases the development
of this drink, takes place on an artisanal way, without knowledge or control of
microorganisms present during the process. Currently in Mexico, the State of Durango
has the Protected Designation of origin for the production of mezcal, it is important to
generate knowledge that will help to improve production, and therefore quality.
Isolation and identification of microbial flora is one of the most important steps for this
improvement, because microorganisms are largely responsible for the chemical
composition of mezcal. In general, the identification of microorganisms and specifically
yeast can be made through its morphological characteristics, physiological, and through
the use of molecular techniques such as RFLP (Restriction Fragment Length
Polymorphism), as well as through the sequence of bases of a particular fragment of
DNA. The objective of this study was to isolate and identify microorganisms prevalent
in the natural fermentation of mezcal and their relation to the chemical composition of
the product of 2 major producers of the State of Durango, Mezquital and Nombre de
Dios. The microorganisms isolated in the two predominantly vinatas analyzed were 5
different strains, one of them, Klyuveromices marxianus was found in both
fermentations. The chemical composition of mezcales was very similar, we found these
major components such as ethanol, methanol, acetic acid, Isoamilic alcohol. The vinata
that presented the highest number of strains, showed the highest concentration of
Isoamilic alcohol. The vinata of Mezquital only showed two different strains including
identifying the gender Saccharomyces cerevisiae, had a higher concentration of ethanol
in the final product. The microbial diversity is a determining factor in the chemical
composition of mezcal.

(Keywords: mezcal, fermentation, yeast, PCR.)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Effect of the Dilution Rate and the Aeration Conditions on the Fermentative and
Aromatic Capacities of S. cerevisiae GU4 in Continuous Culture
Morn-Marroqun, G. A., Crdova-Lopez, J., Estarrn-Espinoza, M., Daz-Montao, D. M*.
Centro de Investigacin y Asistencia en Tecnologa y Diseo del Estado de Jalisco, Av. Normalistas 800,
Col. Colinas de la Normal.Guadalajara 44270 Jal. ;*

Tequila is the distilled beverage elaborated from the fermented juice of cooked Agave
tequilana Weber (blue variety). In the tequila industry, strains of S. cerevisiae
(indigenous or commercial) are cultured in batch fermentations, due to their high
fermentative efficiency, ethanol tolerance and osmotolerance. Nevertheless, from these
fermentations, beverages with low aromatic qualities are generally obtained. The aim of
this work was to study the effect of the aeration and the dilution rate (D) on the volatile
compounds and ethanol production, the substrate consumption, and yields of ethanol
and biomass in continuous cultures. The experiments were performed using a recently
isolated yeast strain (S. cerevisiae GU4) and agave juice (containing 100 gL-1 of
reducing sugars), supplemented with ammonium sulphate and ammonium phosphate;
this media was feed at dilution rates (0.04, 0.08, 0.12, 0.16 and 0.2h -1). When nonaerated cultures were used, the highest productions of ethanol (40.1 gL-1) and biomass
(5.7 gL-1), consumption of substrate (91.6 gL-1) and yields of ethanol (0.44) and
biomass (0.06) were reached at 0.08h-1 Nevertheless, when the continuous cultures (at D
= 0.08h-1) were aerated at 0.01 VVM, the productions of ethanol (44.0 gL-1) and
biomass (7.2 gL-1), consumption of substrate (98.6 gL-1) and yields of ethanol (0.45)
and biomass (0.07) were higher. Similar results were obtained when the culture was
aerated at 0.02 VVM. Furthermore, significant differences in the concentrations of
volatile compounds were observed at different aeration conditions.

(keywords: Alcoholic fermentation; continuous cultures; volatile compounds; S.

cerevisiae; tequila).


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Alcoholic Fermentation of Agave duranguensis. Study of the Population Dynamics
of Native Yeasts Using RFLPs
Pez-Lerma, J.1, Diaz Campillo M.1, Rutiaga-Quiones, M. 1, Barrio-Esparducer, E.2, Querol, A3,
Soto-Cruz, O.1*
(1) Divisin de Estudios de Posgrado e Investigacin, Instituto Tecnolgico de Durango. Blvd.
Felipe Pescador 1830 Ote., 34080, Durango, Dgo., MXICO.
(2) Instituto Cavanilles de Biodiversidad y Biologia Evolutiva, Universidad de Valencia. Polgono
La Coma s/n 46980 Paterna Valncia, ESPAA
(3) Instituto de Agroqumica y Tecnologa de Alimentos, Consejo Superior de investigacin
Cientfica. Polgono La Coma s/n 46980 Paterna Valncia, ESPAA

Mescal is a traditional spirit beverage produced in some regions of Mxico. It is

produced by traditional methods in the state of Durango using Agave duranguensis. The
aim of this study was to describe the population dynamics through isolation and
identification of native yeast present along the alcoholic fermentation of Agave
duranguensis. To analyze the strains present in this process the 5.8S-ITS genomic
region was used, as it is conserved at species level and highly variable among genus.
After amplification tree restriction enzymes (CFO, HaeIII and HinfI) were used, in
order to obtain specific digestion profiles for each strain. The obtained digestion
patterns were identified using the YEAST ID database ( It was
observed that at the beginning of the fermentation yeast population was formed by
Candida, Saccharomyces, Kluyveromyces, Torulaspora, Naumovia, and Pichia genus.
Saccharomyces cerevisiae, Torulaspora delbruekii and Kluyveromyces marxianus
increased their population afterwards, while the population of Candida, Naumovia,
Pichia genus were diminished. At the end of the fermentation, Saccharomyces
cerevisiae was the only yeast specie present in the culture, being the predominant strain
during alcoholic fermentation of Agave duranguensis. Saccharomyces cerevisiae is the
native predominant strain, because it supports the stress conditions at the end of the
fermentation, particularly the high concentration of ethanol.

(keywords: mescal, population dynamics, Saccharomyces cerevisiae).


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Effect of the Amino-acids Supplementation on the Agave tequilana Juice
Fermentation by Kloeckera africana in Batch and Continuous Cultures
Valle-Rodrguez, J.O., Crdova Lpez, J.A., Hernndez-Corts, G., Daz-Montao, D.M.*
CIATEJ Centro de Investigacin y Asistencia en Tecnologa y Diseo del Estado de Jalisco, Av.
Normalistas 800. Guadalajara 44270 Jal.; *

Tequila is an alcoholic beverage elaborated from juice extracted extracted from Agave
tequilana (Weber). Kloeckera yeasts are very important in tequila production because
they synthesize a great variety of volatile compounds, contributing decisively to the
beverage bouquet. These yeasts proliferate in the first fermentation stage but their
population is dramatically reduced due to a supposed low ethanol tolerance and/or a
nutritional limitation; however, studies exploring this phenomenon are scarce. For this
research, a Kloeckera africana strain (TE4) isolated from spontaneous tequila
fermentations was selected and its nutritional requirements were studied when it was
cultured in agave juice. Firstly, the effect of the addition of organic or inorganic
nitrogen sources to the agave juice, on the growth and fermentative capability of K.
africana was studied. In continuous cultures (dilution rate of 0.04 h-1), the agave juice
(containing 100 gL-1 of reducing sugars) was supplemented either with ammonium
sulfate or with 20 amino-acids (added at the same N concentration of 220 mgL-1),
obtaining respectively: 0.6 and 2.5 gL-1 of biomass concentration, 13.3 and 32.6 gL-1 of
ethanol production and 66 and 34 gL-1 of residual sugars. Then, an unifactorial
experiment was performed to determine the importance of each aminoacid for the
growth and fermentative capability of K. africana. It was found that the amino-acids
that showed the most significant effect were (in descending order): asparagine,
glutamine, glutamic acid, aspartic acid and arginine. Finally, a kinetic of growth and
ethanol production in a batch bioreactor was performed using agave juice supplemented
with asparagine. For this culture, final concentrations of biomass and ethanol of 2.5 and
53.1 gL-1, respectively; and an efficiency in ethanol production of 95% were found. The
results showed that asparagine is the main limiting nutrient that must be added to the
agave juice to perform an efficient fermentation by culturing K. africana. Remarkably,
concentrations and yields of ethanol obtained in this study were higher than those
reported in the scientific literature and in the tequila industry, which suggest that the
low growth observed for Kloeckera in previous reports for the last stage of fermentation
is due to a nutritional limitation, instead of a low ethanol tolerance.

(keywords: tequila, Kloeckera africana, ethanol tolerance, nutritional limitation, amino



FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Biogas Production from Agro-Industrial By-products
Melgoza-Palma. N., Mungua-Prez, R*., lvarez-Rodrguez, S., Zenteno-Mndez, J., HernndezArroyo, M., Gonzles-Salome, F., Santacruz-Vzquez, C.
Benemrita Universidad Autnoma de Puebla. Edificio 76 Complejo de Ciencias. C.U. C.P. 72570

Biogas is known as the mixture of different kind of gases that are gotten from the
fermentation of the organic residues in an anaerobic environment. Starting material like
animal dung and vegetable by-products are commonly used in this process. Bacteria
from different species are involved in the production of biogas and they release a gas
mixture that is composed by methane (the main component of biogas), carbon dioxide,
hydrogen, nitrogen and sulfhidric acid. Methane can be produced from a wide variety of
biodegradable organic materials (biomasses) such as agricultural harvest residues
(stems, leaves, husk), gramineous, vegetables and fruits that have a high energy
potential. The objective of this work is to produce biogas from agro-industrial byproducts from fruits like apple, prickly pear, melon, banana, papaya, mango and orange,
mixed with an inoculum of animal dung. Gas chromatography was used to identify each
component in the biogas and its percentage composition. When we compared the
average composition of biogas and the gas produced in this project it is possible to see
following results: the biogas generated from papaya has the following composition: CH4
3.06% mol, CO2 0.5594% mol, N2 0.4402% mol; that from mango: CH4 0.0011% mol,
CO2 0.4043% mol, N2 0.5945% mol; that from orange: CH4 0.0024% mol, CO2
0.4526% mol, N2 0.5449% mol; that from melon: CH4 0.0% mol, CO2 0.4673% mol,
N2 0.5326% mol; and, that from mango-banana: CH4 0.0% mol, CO2 0.4762% mol, N2
0.5237% mol, while apple used during the fermentation only manages to produce a
minimum amount of methane and other gases. The results demonstrate that the gas
contains nitrogen and carbon dioxide in an important proportion; methane was found in
a smaller proportion. Because of this low yield, biogas produced by such fruit byproducts is not a profitable manner of producing combustible gas.

(keywords: Biogas, Agro-industrial by-products, methane)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Optimization of hydrolyser process using B molasses by enzymatic method
Lpez-Zamora L.*Cerecero-Enrquez R., Reyes-Grajales L.M.
Departamento de Posgrado e Investigacin. Instituto Tecnolgico de Orizaba
Oriente 9 No. 852. Col. Emiliano Zapata, Orizaba, Veracruz., 94320.

C molasses are frequently used in the fermentation industry because of their low
price. However, reduction of total sugars in this by-product, due to improvements in
process crystallization within sugar mills, have dramatically affected several industries
using this material, such as those producing amino acids, alcohol, yeast, citric acid, etc.
Therefore, pre-treated B molasses can be an interesting alternative for the
optimization of fermentation processes. The purpose of this investigation was to design
a process to hydrolyze the sugar content in B molasses in a short time. The B
molasses composition was determined to know the initial invert sugar content, and the
variation in different samples. Tests were conducted at laboratory and pilot scale level
using stainless steel equipment and agitation system. A 20 g/l yeast invertase suspension
(Nutriase 208WB strain) was prepared by dissolving in water at 40 C. B molasses were
then diluted using water from 85 Brix to two concentrations (45 and 25Brix). The
inversion process was carried out in a reactor mixing the yeast invertase with diluted
molasses under agitation and aeration systems. The control temperature was 60 C, the
inversion percentage was estimated each 15 min. The initial invert sugar content was
around 30-35%, and after 90 min total inversion time it increased to 94.3%. The main
parameters involved in inversion time reduction were temperature and agitation. In the
fermentation industries where continuous processes are used, short pretreatment times
for raw materials are very important to obtain material available for the following steps.
B molasses usage is a good option due to its relatively high sugar content and purity
compared with C molasses. On the other hand, its price is low compared to other
carbon sources, such as dextrose or raw sugar. For example, at industrial level, L-Lysine
production cost with C molasses and raw sugar using the acid inversion method is US
$ 11,311 /ton, and if produced from B molasses and raw sugar using the enzymatic
inversion method the cost will be US $ 10,238/ton, which represents a reduction of
approximately 10%.

(keywords: molasses, sugar inversion, yeast invertase)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Physicochemical and Sensory Evaluation of Seven Red Wines Obtained from
Three Varieties Established in Dolores, Guanajuato, Mexico
Silva-Jurez, A. L., Cannico-Franco, M.*, Martnez-Peniche, R.
Departamento de Investigacin y Posgrado en Alimentos, Facultad de Qumica. Universidad Autnoma
de Quertaro, CU, Cerro de las Campanas s/n Col. las Campanas, Quertaro, 76010 Qro. Mxico ;;*

The wine industry in Mexico is growing, and high quality wines, produced from
selected varieties, compete with imported wines and are also exported. Mexico counts
on ecological conditions to produce acceptable table wines but few efforts to introduce
noble grape varieties and to evaluate the quality of their wines have been realized. The
aim of this study was to evaluate the sensory and physicochemical quality of wines
elaborated from fine varieties produced in Central Mexico. Seven treatments consisting
in monovarietal and mixed wines were generated with three base wines elaborated from
Cabernet Sauvignon (CS), Merlot (M) and Syrah (S), and their mixes: Cabernet
Sauvignon-Syrah (CS-S), Cabernet Sauvignon-Merlot (CS-M), Merlot-Syrah (M-S),
and Cabernet Sauvignon-Merlot-Syrah (CS-M-S), extracted from grapes harvested in
Dolores Hidalgo, Guanajuato, Mexico in 2006. Malolactic fermentation (MLF) was
performed with Leuconostoc oenos 3-X bacteria. Main physicochemical analyses
performed were alcohol, total residual sugars (RTS), total titratable acidity (TTA),
volatile acidity (VA) and SO2. Wines were sensory evaluated by a hedonic scale and a
Kramer test. All wines obtained a relatively low alcohol degree (from 9.03 to 10.03%
Vol.) due to a fairly low concentration of sugar in the grapes. Monovarietal M wine and
M-CS-S obtained the highest alcohol values (10.04% and 9.8% respectively) contrasting
with CS (8.15 %) and CS-S (9.04 %). Significant difference in TTA were also found and
M wine registered the lowest TTA (6.79 g/L of tartaric acid), in contrast with CS-S
(7.63 g/L of tartaric acid). This variation is probably due to the fact that bacterial
development during MLF was not similar for the different wines. VA was high in S and
CS-S (more than 0.8 g/L), but did not surpass the legal limit, neither did RTS surpass
the limit for dry wines. The highest concentration of free SO2 was obtained by S (34.5
mg/L) and its combinations CS-S, M-S (33.2 mg/L both). Finally, the hedonic tests
showed that CS-S was the best accepted wine while S obtained the lowest value and, a
high correlation (R = 0.83) between taste and general acceptance was also revealed. In
the Kramer test panelists also considered CS-S superior than the other wines (P 0.05),
while S was classified below the average. These results suggest that mixed wines have
better quality and a better general acceptance than monovarietal red wines.

(keywords: Red wine, noble varieties, mixed wine, sensory evaluation).


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Effect of Fructans of Agave duranguensis on the Growth of Probiotic Bacteria
Orozco-Corts, A. D., Rutiaga-Quiones, O. M., Lpez-Miranda J., Soto-Cruz O.*
Divisin de Estudios de Posgrado e Investigacin, Instituto Tecnolgico de Durango. Blvd. Felipe
Pescador 1830 Ote., 34080, Durango, Dgo., MXICO. *

Probiotics are live microorganisms that are added as a supplement in the diet, while;
prebiotic is a non-digestible sugar, inert for the human, which improves the growth of
probiotic bacteria in the gut. Fructans, main reserve carbohydrates of agave plants, are
good candidates to be used as prebiotic. The purpose of this study was to determine the
effect of fructans extracted from Agave duranguensis on the growth of the probiotic
bacteria Lactobacillus reuteri and Bifidobacterium longum. Fructans were extracted
from agave by 3 h maceration in 80 C water. Liquid MRS medium containing 20 g/l of
inulin or glucose as sole carbon source were used as positive and negative control
respectively. Additional media were prepared replacing 1/3, 1/2 and 2/3 of glucose in
MRS medium by agave fructans. Biomass growth in each media was determined by dry
weight. L. reuteri show a better growth with glucose as sole carbon source, while
growth was slower and scarce in the presence of fructans and inulin. B. longum grew
better on fructans rather on inulin, because reached a growth similar to that obtained
when grown on glucose, but in a longer time. Fructans of A duranguensis can be
consider a carbon source similar than inulin for probiotic bacteria.

(keywords: probiotics, prebiotics, agave, lactic fermentation)


FSFB, 3rd International Congress, 14-17 October, 2008, Quertaro

Determination of the Phenotype Killer in Native Yeasts Isolated from the Alcoholic
Fermentation of Agave duranguensis
Nuez-Guerrero, M. E., De los Rios-Deras, G., Rutiaga-Quinez O. M., Ochoa-Martnez A., Soto-Cruz,
Divisin de Estudios de Posgrado e Investigacin, Instituto Tecnolgico de Durango. Blvd. Felipe
Pescador 1830 Ote., 34080, Durango, Dgo., MXICO.;

Mescal is a mexican traditiona beverage, which is produced by a spontaneous

fermentation with the participation of native yeast. An antagonistic interaction between
yeasts is the so-called killer phenotype, which involves the secretion of a toxic protein
of low molecular weight. The toxin is lethal to sensitive strains, while the producing
yeast is immune to its own toxins. The objective of this study was to determine the
killer activity of native yeasts isolated from the alcoholic fermentation of Agave
duranguensis, which is performed to produce mescal in Durango, Mexico. The native
strain were previously identified as Saccharomyces cerevisiae, Torulaspora delbrueckii,
Kluyveromyces marxianus, Candida culliculosa y Candida kefyr. Plates of YEPD-MB
(0.5% yeast extract, 1% peptone, 2% glucose, 2% agar, 0.003% methylene blue, 0.1 M
sodium citrate, at pH of 4.2) were seeded with the killer-sensitive strain Saccharomyces
cerevisiae CECT 1890 (pre-grown for 48 h on YEPD-agar slants). Strains to be tested
for killer activity were loaded onto the seeded agar. Colonies exhibiting clear halos on
the sensitive lawns after 3-7 days of incubation at 28C were considered to be killer
positive. To test killer-sensitive traits, YEPD-MB plates were seeded with the strains to
be tested and overlaid with the killer strain Saccharomyces cerevisiae CECT 1893.
Strains exhibiting clear halos on the plates were considered to be killer sensitive
towards the respective reference killer strains. Finally, it was determined the possible
cross inhibition between native strains, using the same methodology. The native strains
were not inhibited by the killer strain, nor were able to inhibit the growth of the
sensitive strain. Moreover, no cross inhibition was observed. Then, the native strains are
considered neutral with respect to the phenotype killer.

(keywords: killer toxin, mescal, native yeasts)