food science and food technology

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

156

FFE-01

Blackberry (Rubus spp.) Juice Alcoholic Fermentation: Kinetics and Aromatic

Aspects

Calderón-Santoyo, M., Godina-Galindo, G., Bautista-Rosales, P. U., Gutierrez-Martínez, P. and

Ragazzo-Sánchez, J. A*.

Instituto Tecnológico de Tepic. Laboratorio Integral de Investigación en Alimentos. Av. Tecnológico No.

2595 C.P. 63175. Tepic, Nayarit, MÉXICO, Tel. (311) 213 15 43.

*[email protected]

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.)


157

FFE-02

Effect of Temperature, pH and Harvest Season on Polyphenols Concentrations of

Mexican Cocoa Beans Fermentation

Ruíz-García, R., Barrios-Viñas, M., García-Alamilla, P., Martinez-Morales, A., Urrieta-Saltijeral, J.M.

*

Departamento de Ingeniería Bioquímica, Instituto Tecnológico de Villahermosa, Carretera Vhsa-Frontera

Km 3.5 Villahermosa, Tabasco-México. mailto: [email protected] ;

*mailto:[email protected]

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 110°C, 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 40-45°C after the first 48

hours, with turning of the beans the temperature rises to 48-50°C 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)


158

FFE-03

Foam Characterization of Sparkling Wines Selling in Mexico by Image Processing

López-Pineda, A.,

*1 Martínez-Peniche, R.,

1 Castillo, E.

2

1 Universidad Autónoma de Querétaro, Facultad de Química. Centro Universitario s/n Col. Cerro de las

Campanas, 76010-Querétaro, México. 2 Instituto Politécnico Nacional, Centro de Investigación en

Ciencia Aplicada y Tecnología Avanzada, Cerro Blanco 141, Colinas del Cimatario, 76090-Querétaro,

México. E-mail address: [email protected]

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 doesn‟t 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).


159

FFE-04

Dynamical Characterization of Sparkling Wines Affected by their Chemical

Composition

López-Pineda, A.,

*1 Martínez-Peniche, R.,

1 Castillo, E.

2, Relkin, P.,

3 Mezdour, S.

3

1 Universidad Autónoma de Querétaro, Facultad de Química. Centro Universitario s/n Col. Cerro de las

Campanas, 76010-Querétaro, México.

2 Instituto Politécnico Nacional, Centro de Investigación en Ciencia Aplicada y Tecnología Avanzada,

Cerro Blanco 141, Colinas del Cimatario, 76090-Querétaro, México.

3 Laboratoire de Biophysique, Agro Paris Tech Massy, 1, Av. des Olympiades, 91744-Massy, France.

* Corresponding author. E-mail address: [email protected]

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 wine‟s 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,

tensiometry).


160

FFE-05

Production of Tannase by Aspergillus niger GH1 in Submerged Culture Using an

Airlift Bio-reactor

Cruz-Hernández M.A.*, Esquivel-Contreras M.T., Ramírez-Barron S.N., Aguilar C.N.

Departamento de Investigación en Alimentos. Facultad de Ciencias Químicas. Universidad Autónoma de

Coahuila, Campus Saltillo, Blvd. V. Carranza e Ing. José Cárdenas, Saltillo, Coahuila, México, C.P.

25000

*E-mail: [email protected]

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, solid-

state 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 bio-

reactor. The temperature of the fermentation process was 35º 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)


161

FFE-06

Comparative Kinetic Study of Tannase Production by Aspergillus niger GH1

and Fusarium verticillioides.

Veana-Hernández F.

1, Aguilar C.N.

2*, Cruz-Hernández M.A.

2, Rodríguez-Herrera R.

2

1 Universidad Autónoma de San Luis Potosí/ UAMZH

2 Departamento de Investigación en Alimentos. Facultad de Ciencias Químicas

Universidad Autónoma de Coahuila. Blvd. V. Carranza y José Cárdenas. Saltillo, Coahuila. C.P.25000.

*E-mail: [email protected]

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 Pirt‟s 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)


162

FFE-07

Alcoholic Fermentation of Sotol Juice (Dasylirion spp) by Native Yeasts

Martínez-Sosa, M., De la Garza-Toledo, H.*, Aguilar-González, C.

Departamento de Investigación en Alimentos, Facultad de Ciencias Químicas. Universidad Autónoma de

Coahuila. José Cárdenas Valdés s/n Col. República Oriente. Saltillo, Coahuila 25280.

[email protected]; * [email protected]

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 30°C 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)


163

FFE-08

Optimization of the Engineering Parameters to Produce Zeaxanthin in a

Fluidized Bed Birreactor

Ramos-Ojeda, E. *, Escamilla-Silva, E.

Departamento de Ingeniería Química, Instituto Tecnológico de Celaya , Av Tecnológico y Av García

Cubas s/n Col Fovisste, Cp 38010, Celaya, Guanajuato, México.

*[email protected]

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 non-

dimensional 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,

zeaxanthin).


164

FFE-09

Response of Strains of Saccharomyces cerevisiae to Conditions of Fermentative

Stress

Páez-Lerma, J.

1, Rutiaga-Quiñones, M.

1, Barrio-Esparducer E.

2, Belloch, C.

3, Querol, A.

3,

Soto-Cruz, O.1*

(1) División de Estudios de Posgrado e Investigación, Instituto Tecnológico de Durango.

Blvd. Felipe Pescador 1830 Ote., 34080, Durango, Dgo., MÉXICO. *soto@itdposgrado-

bioquimica.com.mx

(2) Instituto Cavanilles de Biodiversidad y Biologia Evolutiva, Universidad de Valencia. Edifícios

de Institutos, Campus de Paterna, E-46100 Burjassot, Valencia, ESPAÑA

(3) Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de investigación

Científica. Edifícios de Institutos, Campus de Paterna, E-46100 Burjassot, Valencia, ESPAÑA

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, México. 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 30ºC. The next day the cultures were adjusted to an

absorbance at 655 nm of 0.3 by dilution with fresh GPY. After shaking at 30ºC for

another 4 h the cells were diluted in sterile water to an A655 of 0.3 and 5 µL 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

45°C, 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).


165

FFE-10

Volatile Acidity Fraction of Cocoa Beans Roasting. Effect of Fermentation Process

Velázquez - Martínez, J.R., Robles Olvera, V, J, and García – Alamilla, P*

*Universidad Juárez Autónoma de Tabasco (UJAT). División Académica de Ciencias Agropecuarias

(DACA). Centro de Investigación en Ciencias Agropecuarias (CICA). Carret. Vhsa – Teapa Km. 25.

Villahermosa, Tabasco, México. E- mail: [email protected]

Tel: 993358-1585 fax (142 9151 y 142 91 50) ext 6604

Unidad de Investigación y Desarrollo en Alimentos (UNIDA). Instituto Tecnológico de Veracruz,

Veracruz, México.

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 140°C 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).


166

FFE-11

Determination of Average Diffusion Coefficient During Cocoa Beans

Fermentation.

Robles Olvera, V. J., Velázquez Martínez, J.R., and García-Alamilla, P.*.

*Universidad Juárez Autónoma de Tabasco (UJAT). División Académica de Ciencias Agropecuarias

(DACA). Centro de Investigación en Ciencias Agropecuarias (CICA). Carret. Vhsa – Teapa Km. 25.

Villahermosa, Tabasco, México E- mail: [email protected]

Tel: 993358-1585 fax (142 9151 y 142 91 50) ext 6604

Unidad de Investigación y Desarrollo en Alimentos (UNIDA). Instituto Tecnológico de Veracruz,

Veracruz, México.

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, fermentation‟s 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 m

2/s at the end of the

process.

(keywords: cocoa beans fermentation, internal acidity profile , acetic acid, diffusion

coefficient).


167

FFE-12

Study of Combined Effect of pH and Temperature on Water-Soluble Red Pigment

Production by Penicillium purpurogenum GH2

Méndez-Zavala, A., Montañés-Sáenz, J. C., Pérez-Berúmen, C., Zugasti, A., Aguilar, C. N. *

Food Research Department, Universidad Autónoma de Coahuila, Unidad Saltillo, Blvd. V. Carranza e

Ing. José Cárdenas V. s/n. Col. República, PO Box 252, ZIP 25280, Coahuila, México.

[email protected]; *[email protected]

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 24°C. 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)


168

FFE-13

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 Química. Universidad Autónoma de Querétaro. Cerro de las Campanas S/N Col. Las

Campanas Apartado Postal 184 C.P. 76010. Querétaro, Qro.

E-mail: [email protected]

Departamento de Ingeniería Química, Instituto Tecnológico de Celaya

Av. Tecnológico y Antonio García Cubas S/N. C.P. 38010. Celaya, Guanajuato,

México.E.mail:*[email protected]

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 ºC, 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).


169

FFE-14

Validation of Acetobacter/Gluconobacter and Carr Media for Acetic Acid Bacteria

Enumeration During Traditional Cacao (Theobroma cacao) Fermentation

Romero-Cortes, T., Velásquez Martínez, J.R., Salgado-Cervantes, M., Ramírez-Lepe, M., García-

Alamilla, P., Rodríguez-Jiménez, G., Robles-Olvera, V.*

Unidad de Investigación y Desarrollo en Alimentos. Instituto Tecnológico de Veracruz Av. Miguel Angel

de Quevedo No. 2779 Col. Formando Hogar C.P. 91897 Veracruz, Ver. Fax: (2299) 34 57 01.

[email protected]

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)


170

FFE-15

Effect of Saccharomyces cerevisiae Cell Wall Extract on Pathogenic Bacteria

Aguilar-Uscanga, B

1*, Rodríguez, C.M

1., Viveros, J.M

1., Hernández, S

1., Aguilar, M.G

2., Reyes-Blanco,

M1. Solís-Pacheco, J

1.

1Departamento de Farmacobiología, Centro Universitario de Ciencias Exactas e Ingenierías. Universidad

de Guadalajara. Bvld. M. García Barragán 1458, Col. Olímpica. Guadalajara, Jalisco. 2 Unidad de Investigación y Desarrollo en Alimentos, Instituto Tecnológico de Veracruz. Miguel Ángel

de Quevedo 2779, Col. Formando Hogar, Veracruz, Ver.

[email protected]; *[email protected]

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% β-glucan, 40

to 45% mannoproteins and 2 to 5 % chitin. Some studies report that the polysaccharides

of yeast cell walls mainly β-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, 30°C). The

cells were harvested in exponential phase and washed successively with water and Tris-

HCl 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 (25-27°C), 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)


171

FFE-16

Study of Volatile Compounds During Fed-Batch Fermentation

for the 100% Agave Tequila Production

Alcázar-Valle, E.M.; Herrera-López, J.E.; Díaz-Montaño, D.M.; Arellano-Plaza, M*

Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. Av. Normalistas

800, Colinas de la Normal, Guadalajara 44270 Jalisco [email protected]

[email protected],

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, 1-

butanol, 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 1-

butanol 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).


172

FFE-17

Elaboration of Kefir using Bovine Milk

Burciaga-Castillo B., Martínez Guzmán A., Muñoz Ríos R., Rodríguez Rosales D.J., García Caballero

B.E.

Departamento de Ingenierías Química y Bioquímica, Instituto Tecnológico de Durango, Boulevard Felipe

Pescador 1830 Ote. Colonia Nueva Vizcaya, C.P. 34080, Durango, Dgo. México. Tel 01 (618) 8 29 09

19. Email: [email protected]

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)


173

FFE-18

Production of Saccharomyces cerevisiae Biomass on Coconut Medium in Stirred

Tank and Bubble Column Bioreactors

Hernández-Hernández, S.; Ricabar-Francisco, A. D.; Jiménez-Avalos, H. A. and Chaires-Martínez, L.

(*)

Food Research Center. Instituto Tecnológico Superior de Alamo Temapache (CIA-ITSAT). Km. 6.5

Carretera Tuxpan-Potrero del Llano. Xoyotitla, Alamo. Ver. CP. 92750. Tel y Fax. 017658440038.

Email: [email protected]

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)


174

FFE-19

Development of a Low-Alcoholic Beverage by Fermentation of Whey with

Kluyveromyces marxianus.

Escorcia Marquina A

1; Dublán García O*

Departamento de Alimentos, Facultad de Química, Universidad Autónoma del Estado de México. Paseo

Colón y Paseo Tollocan S/N, Toluca 50000. 1Universidad La Salle.

*[email protected]; [email protected]

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 pineapple-

coconut was the flavor more accepted (P <0001) with a concentration of aspartame-

acesulfame (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).


175

FFE-20

Screening and Identification of Fungi Strain Lipase-Productor Isolated from

Mexican Semidesert

Y. C. Gutiérrez-Alvarado, A. Iliná, J. L. Martínez-Hernández.

Departamento de Biotecnología. Facultad de Ciencias Químicas. Universidad Autónoma de Coahuila.

Unidad Saltillo. Blvd. V. Carranza e Ing. José Cárdenas V. s/n, Saltillo Coahuila CP. 25280. México. E-

mail: [email protected]

Lipase (E.C. 3.1.1.3) 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, 30º 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).


176

FFE-21

Effect of Feeding Non-Sterilized Agave Juice in a Continuous Tequila

Fermentation

Hernández-Cortés G., Córdova-López J., Herrera-López E.J., Díaz-Montaño D.M.

*

CIATEJ: Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco. Av.

Normalistas 800, Col. Colinas de la Normal

Guadalajara, 44270 Jal. [email protected]; *[email protected]

In recent years, the shortage of Agave tequilana Weber and the increase of tequila‟s

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-1

h-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

juice)


177

FFE-22

A Multiplex PCR Technique for Simultaneous Detection of Different Annexed

Sequences and Transgenic Events in Commercial Foods

Hernández-Orona, V. L.

1, Rodríguez – Herrera,

R

1*. , Aguilar – González, CN

1., and Reyes-Valdés M.

2,

1Departamento de Investigación en Alimentos, Facultad de Ciencias Químicas, Universidad Autónoma de

Coahuila. Blvd. V. Carranza e Ing. José Cárdenas V. s/n. Col. República Ote. C.P. 25280. Saltillo,

Coahuila. * [email protected] 2Departamento 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, home-

made and commercial foods.

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


178

FFE-23

Identification of Yeast in the Natural Fermentation of Mezcal Duranguense and its

Relations to Chemical Composition.

Martell-Nevárez M.A.*, Solís-Soto A., Soto-Cruz N.O., Rodríguez-Herrera R., Rutiaga-Quiñones O.M.

Departamento de Investigación y Posgrado en Ingeniería Bioquímica. Instituto Tecnológico de Durango.

Blvd. Felipe Pescador 1803 ote. Durango, Dgo., México. 34080. Departamento de Investigación y

Posgrado en Alimentos. Facultad de Ciencias Químicas. Universidad Autónoma de Coahuila. Blvd. V.

Carranza s/n Col. República, ote. Saltillo, Coahuila.*[email protected]

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.)


179

FFE-24

Effect of the Dilution Rate and the Aeration Conditions on the Fermentative and

Aromatic Capacities of S. cerevisiae GU4 in Continuous Culture

Morán-Marroquín, G. A., Córdova-Lopez, J., Estarrón-Espinoza, M., Díaz-Montaño, D. M*.

Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, Av. Normalistas 800,

Col. Colinas de la Normal.Guadalajara 44270 Jal. [email protected] ;* [email protected]

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 non-

aerated 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).


180

FFE-25

Alcoholic Fermentation of Agave duranguensis. Study of the Population Dynamics

of Native Yeasts Using RFLP´s

Páez-Lerma, J.

1, Diaz Campillo M.

1, Rutiaga-Quiñones, M.

1, Barrio-Esparducer, E.

2, Querol, A

3,

Soto-Cruz, O.1*

(1) División de Estudios de Posgrado e Investigación, Instituto Tecnológico de Durango. Blvd.

Felipe Pescador 1830 Ote., 34080, Durango, Dgo., MÉXICO.

*[email protected]

(2) Instituto Cavanilles de Biodiversidad y Biologia Evolutiva, Universidad de Valencia. Polígono

La Coma s/n 46980 Paterna – València, ESPAÑA

(3) Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de investigación

Científica. Polígono La Coma s/n 46980 Paterna – València, ESPAÑA

Mescal is a traditional spirit beverage produced in some regions of México. 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 (www.yeast–id.com). 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).


181

FFE-26

Effect of the Amino-acids Supplementation on the Agave tequilana Juice

Fermentation by Kloeckera africana in Batch and Continuous Cultures

Valle-Rodríguez, J.O., Córdova López, J.A., Hernández-Cortés, G., Díaz-Montaño, D.M.*

CIATEJ Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, Av.

Normalistas 800. Guadalajara 44270 Jal. [email protected]; *[email protected]

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

acids)


182

FFE-27

Biogas Production from Agro-Industrial By-products

Melgoza-Palma. N., Munguía-Pérez, R*., Álvarez-Rodríguez, S., Zenteno-Méndez, J., Hernández-

Arroyo, M., Gonzáles-Salome, F., Santacruz-Vázquez, C.

Benemérita Universidad Autónoma de Puebla. Edificio 76 Complejo de Ciencias. C.U. C.P. 72570

*[email protected]

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 by-

products 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 by-

products is not a profitable manner of producing combustible gas.

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


183

FFE-28

Optimization of hydrolyser process using “B” molasses by enzymatic method

López-Zamora L.*Cerecero-Enríquez R., Reyes-Grajales L.M.

Departamento de Posgrado e Investigación. Instituto Tecnológico de Orizaba

Oriente 9 No. 852. Col. Emiliano Zapata, Orizaba, Veracruz., 94320. [email protected]

“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 25ºBrix). 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)


184

FFE-29

Physicochemical and Sensory Evaluation of Seven Red Wines Obtained from

Three Varieties Established in Dolores, Guanajuato, Mexico

Silva-Juárez, A. L., Canónico-Franco, M.*, Martìnez-Peniche, R.

Departamento de Investigación y Posgrado en Alimentos, Facultad de Química. Universidad Autónoma

de Querétaro, CU, Cerro de las Campanas s/n Col. las Campanas, Querétaro, 76010 Qro. México

[email protected] ; [email protected];* [email protected]

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

Ćabernet Sauvignon´ (CS), ´Merlot´ (M) and ´Syrah´ (S), and their mixes: Ćabernet

Sauvignon-Syrah´ (CS-S), Ćabernet Sauvignon-Merlot´ (CS-M), ´Merlot-Syrah´ (M-S),

and Ćabernet 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).


185

FFE-30

Effect of Fructans of Agave duranguensis on the Growth of Probiotic Bacteria

Orozco-Cortés, A. D., Rutiaga-Quiñones, O. M., López-Miranda J., Soto-Cruz O.*

División de Estudios de Posgrado e Investigación, Instituto Tecnológico de Durango. Blvd. Felipe

Pescador 1830 Ote., 34080, Durango, Dgo., MÉXICO. *[email protected]

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 80º 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)


186

FFE-31

Determination of the Phenotype Killer in Native Yeasts Isolated from the Alcoholic

Fermentation of Agave duranguensis

Nuñez-Guerrero, M. E., De los Rios-Deras, G., Rutiaga-Quiñónez O. M., Ochoa-Martínez A., Soto-Cruz,

O.*

División de Estudios de Posgrado e Investigación, Instituto Tecnológico de Durango. Blvd. Felipe

Pescador 1830 Ote., 34080, Durango, Dgo., MÉXICO. [email protected]; soto@itdposgrado-

bioquimica.com.mx

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 28°C 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)

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