Produccin de acetato de isoamilo (aroma a pltano) por levaduras aisladas de las fermentaciones espontneas de Agave duranguensis

Hernndez Carbajal, G.R., Rutiaga Quiones, O.M., Prez Silva, A., Saucedo Castaeda, G., Soccol, C., Soto Cruz, N.O.

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Jarabes y especias saborizantes

como

Industrializacin.

1960 Identificacin de compuestos.

McCully, 2010., Schlosser., 2006

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85% se producen mtodos qumicos.

por

Quimiofobia. Altamente contaminantes

Palomares, 2000., Janssens, 1992.

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Muchos microorganismos producen aromas

Algunas levaduras producen aromas frutales Los esteres se usan en la industria de los alimentos y de los comticos

Yilmaztekin M. et al., 2008; Vadali R.V. et al., 2003.

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Acetato de isoamilo, tambin conocido como aceite de pltano. Es producto de la esterificacin del alcohol isoamilico y acetil CoA

Su uso en la industria de los alimentos es de 74,000 kg/ao.

Quilter M.G. et al., 2003; Kobayashi P. et al., 2007.

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Bajas concentraciones Problemas de recuperacin

Fermentaciones extractivas

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Justificacin

Durante los estudios en la produccin de Mezcal, se observ que haba cepas de levaduras capaces de producir aromas frutales agradables. Encontrar una cepa capaz de producir acetato de isoamilo, compuesto de inters en la industria de los alimentos. Aprovechamiento de la biodiversidad microbiana como fuentes naturales de aromas.

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Objetivo General.

Producir acetato de isoamilo por una cepa nativa seleccionada de la fermentacin espontnea de Agave duranguensis. Objetivos especficos.

Seleccionar una cepa de levadura nativa de la fermentacin espontnea de Agave duranguensis Identificar de las variables que influyen en la produccin de acetato de isoamilo. Optimizacin de las condiciones de cultivo que permitan la sobreproduccin de acetato de isoamilo.

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Materiales y mtodos

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Seleccin de cepas

Identificacin de variables

Optimizacin

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Acetato de isoamilo (g/L)

a b c g, h J G, H I, J

h, i

Levadura Figura 1. Concentracin de acetato de isoamilo producido por el sistema doble acoplado por las cepas: Candida diversa ITD00173, Hanseniaspora uvarum ITD00108,

Kluyveromyces marxianus ITD00211, Pichia fermentans ITD00165, Torulaspora

delbrueckii ITDOO233, Candida pseudointermedia ITD00101 Dekkera anomala >> 0 >> ITD00215, Saccharomyces cerevisiae ITD00015 1 >> 2 >> 3 >> 4 >>

Acetato de Isoamilo (g/L)

Recuperacin in situ

Columna empacada

Columna sin empaque

Figura 2. Comparativa entre los tres sistemas de recuperacin de acetato de isoamilo

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Figura 3. Frascos de fermentacin. Relacin altura dimetro

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12 h 120 rpm 28 C

80 mL de medio 20 mL de decano

Anlisis por cromatografa de gases

Acetato en decano

24 h 1 vvm 30 C 80 mL de medio 20 mL de soluciin

Conteo de clulas

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Resultados

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Figura 4. Concentracin de acetato de isoamilo en los frascos con estndar

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@
b

Figura 5. Produccin de acetato de isoamilo por la cepa Picchia fermentans ITD00165.

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a
a

@
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Figura 6. Picchia fermentans ITD00165.

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Conclusin

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ACTIVIDADES

ENE FEB 2010

MAR ABR

MAY JUN

JUL AGO

SEP OCT

NOV DIC

ENE 2011

Recuperacin y cuantificacin

Optimizacin

@
Artculos de investigacin. X X X X X X X

Redaccin y defensa de tesis

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Acetato de Isoamilo (g/L)

Recuperacin in situ medio melaza

Espacio de Cabeza

Yilmaztekin, 2009

Quilter 2003

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49 Strains

Doubled coupled system

Quantitative analysis

Selection

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Doubled coupled system

24 h on GPYM medium

0.2 mL from culture were spread onto the agar surface

Incubation at 30 C for 24 h

0.5 mL of decane solution were taken after 6, 12 and 24 h of incubation

Incubation at 30 C for 24 h with mild shaking

8 mL of a 1% isoamyl alcohol solution in decane was placed in agar plate

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Quantitative analysis

Analyses were carried out on HP Agilent 6890 GC-FID System Gas Chromatograph using an INNOWAX capillary column and nitrogen as carrier gas

The injection block and detector temperatures were kept constant at 200 C and 250 C, respectively. The oven temperature was programmed as follows: 80 C (6 min) to 250 C at 20 C/min

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A B C G, H J G, H I, J

H, I

Fig 2 Concentration of isoamyl acetate at hydrophobic phase in the doubled coupled system at 24 hours by the yeasts: Candida diversa ITD00173, Hanseniaspora uvarum

ITD00108, Kluyveromyces marxianus ITD00211, Pichia fermentans

Torulaspora delbrueckii ITDOO233, Candida pseudointermedia

ITD00165,
ITD00101,

Dekkera anomala ITD00215, Saccharomyces cerevisiae ITD00015

F G H

D E

E F

C G H G H I J

Fig 3. Concentration of isoamyl acetate at hydrophobic phase in the doubled coupled system at 24 hours by Kluyveromyces marxianus

A B

F G H

D E

E F

D C

G H

G H

I J

I J

Fig 4. Concentration of isoamyl acetate at hydrophobic phase in the doubled coupled system at 24 hours by Pichia fermentans

Figure 5. Concentration of isoamyl acetate at organic phase of the doubled coupled system at 0, 6, 12 and 24 hours by the strains: Pichia fermentans ITD00154, ITD00165, Kluyveromyces marxianus ITD00211 and control.

Oda (1997) reported a Pichia heedii and Pichia quercuum as high producer strains in the double coupled system. Rojas et al., (2003) reached concentrations of 2 g/L of @ isoamyl acetate. Kluyveromyces marxianus, increased concentration of isoamyl acetate in alcoholic beverages

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Conclusion

Doubled coupled system was able to identify three strains of no Saccharomyces yeast (Pichia fermentans ITD00154, ITD00165 and @Kluyveromyces marxianus ITD00211) as good candidates to produce isoamyl acetate.

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Decane (Organic phase)

Isoamyl alcohol

isoamyl acetate

Microbial film
Acetyl-CoA

Nutrients (Hydrophilic carrier)

Fig 1. Principle of the double coupling system

(Oda et al.,1996)

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R1-COOH + HOCH2-R2
cido orgnico alcohol

R1-COO-CH2-R2 + H2O
ster agua

O R1-C-O -H =
cido orgnico

@ H-O-CH2-R2
alcohol

ster

agua

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Para el cultivo de 24 horas: Medio GYMP: 40 g de glucosa, 5 g de peptona, 3 g de extracto de levadura, 3 g de extracto de malta, 1 g de sulfato de magnesio heptahidratado, 1000 mL de agua destilada, pH=6

Para cajas Petri:

Medio GYMP: 40 g de glucosa, 5 g de peptona, 3 g de extracto de levadura, 3 g de extracto de malta, 1 g de sulfato de magnesio heptahidratado, 1000 mL de agua destilada, pH=6 ms 15 g de Agar

Shinobu O. et al.,1997; Rojas V. et al., 2001.

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