FRUITY AROMA PRODUCTION BY Ceratocystis fimbriata IN SOLID CULTURES FROM AGRO-INDUSTRIAL WASTES

FRUITY AROMA PRODUCTION BY CERATOCYSTIS FIMBRIATA IN SOLID CULTURES FROM AGRO-INDUSTRIAL WASTES

Adriana Bramorski¹, Carlos R. Soccol^1*, Pierre Christen² and Sergio Revah³

¹Laboratório de Processos Biotecnológicos, Departamento de Engenharia Química, Universidade Federal do Paraná, Centro Politécnico, Curitiba, PR, Brasil. ²ORSTOM (Institut Français de Recherche Scientifique pour le Développment en Coopération), México, D.F., Mexico. ³Universidad Autonoma Metropolitana, Departamento IPH, Mexico, D.F., Mexico

Approved: July 23, 1998

ABSTRACT

Solid state fermentations were carried out to test the efficacy of Ceratocystis fimbriata to grow on different agro-industrial substrates and aroma production. Seven media were prepared using cassava bagasse, apple pomace, amaranth and soya bean. All the media supported fungal growth. While amaranth medium produced pineapple aroma, media containing cassava bagasse, apple pomace and soya bean produced a strong fruity aroma. The aroma production was growth dependent and the maximum aroma intensity was detected a few hours before or after the maximum respirometric activity. Sixteen compounds were separated by gas cromatography of the components present in the headspace and fifteen of them were identified as acid (1), alcohols (6), aldehyde (1), ketones (2) and esters (5).

Key words:Ceratocystis fimbriata, solid state fermentation, agro-industrial wastes, aroma

Ceratocystis

Despite of the interest in the valorization of agro-industrial wastes, not much literature is available on the production of aroma associated with the growth of molds in solid state fermentation (SSF). SSF offers several advantages on the utilization of agro-industrial substrates, such as easy handling, low cost and high productivity. In this work, we report our findings on the growth and aroma production by Ceratocystis fimbriata in solid state fermentation of four agro-industrial wastes.

Microorganism and media Ceratocystis fimbriata

Inoculum: For inoculum, a spore suspension of C. fimbriata was prepared by growing it at 30ºC for five days on PDA medium. Spores were collected with sterile water containing few droplets of Tween 80 and small glass beads to improve the harvest. Spore suspension contained 10

⁷

Substrates preparation: Four solid substrates (see Table 1) were used in various proportions, as listed in Table 2. Soya beans were previously dried and ground in order to separate the hull. Amaranth was used in popped form. All the substrates were dried, ground, sieved through a 0.8 mm screen and autoclaved at 121ºC for 15 min.

Table 1.
Composition of the substrates. Proportions are given on dry matter basis

**expressed in g/100g

Table 2.
Composition of the diferent solid media studied. Proportions are given on a dry matter base

* Salt solution as described by Christen and Raimbault (1991)

Fermentation: SSF was carried out in 250 ml Erlenmeyer flasks, covered with 6 layers of gauze, containing 15 g of dry matter. Initial water content of the media was calculated according to the maximum absorption capacity of each substrate. C. fimbriata was inoculated in all the media (pH 6.0) as described in Table 2 and incubated at 30ºC. All the experiments were done in duplicate.

Analytical procedures: Water activity (Aw) was determined using an Aqualab CX-2 apparatus (Decagon, USA). For pH measurements, samples homogeneized with deionized water and pH was measured using a potenciometric method. CO

₂et al₂^-1

The odour of the cultures was determined by sensorial evaluation using a non-trained panel consisting of six members, with no restrictions in descriptive terms.

Volatile compounds produced were characterized by injecting 2.0 ml from the headspace of the cultures to a Hewlett-Packard 5890 GC, equipped with a 5 meter Megabore HP-1 column and with a flame ionization detector. Total volatiles produced were expressed as µ mol ethanol equivalent per litre of headspace (µ mol l-1 eq.ethanol). Some compounds were also determined individually and their concentration in the headspace was expressed from standard curves as µ mol l-1.

Growth studies

As reported previously (5, 13), CO

₂Tables 34

Table 3.

Table 4.
Aroma and volatile compounds production in the headspace of cultures of C. fimbriata

In all cases, water activity was maintained at a satisfactory level for growth; in some cases the pH at the end of the fermentation was alkaline (B, C, D) which was due to the accumulation of ammonia, resulting from the hydrolysis of soya proteins (A, E, F, G), because of the liberation of organic acids. Maximum respirometric activities, represented by headspace CO

Fig. 1₂

Figure 1.
Levels of carbon dioxide in the headspace of cultures of C. fimbriata in seven solid media.

Aroma production

The aroma production, as detected by olfactometry, is presented in Table 4.

The aroma production was growth dependent and the maximum aroma intensity was detected a few hours before or after the maximum respirometric activity, as reflected by CO

₂Fig. 1Table 5

Table 5. Volatile compounds identified in the headspace of 2-days cultures of Ceratocystis fimbriata

* ( none, + weak, ++ medium, +++ strong,

++++ very strong

** µ moleq.ethanol/l

Separation and identification of volatile compounds

Sixteen different compounds were detected in the gas chromatograms of the components available in the headspace from the cultures. Fifteen of these were identified by their retention time and are listed in Table 5.

The major volatile compounds found in the headspace were alcohols, esters and, in a lesser amount, ketones. The presence of the different compounds in the headspace reflected both the variations arising from each medium and the relative presence of that specific compound as determined by its vapour pressure.

The analysis on the presence of these compounds in the tested media showed that the cultures had two types of behavior: one where the esters predominated (A, B, C, D and F, representing 55 to 69% of the total volatiles) and other where the alcohols were predominant (G and E). The medium G, although predominated by the alcohols, presented production of esters too (around 10%). This was not the case in medium E, which besides showing small production of volatile compounds, produced virtually only ethanol (Fig. 2).

Figure 2.
Representation of the volatile compounds in the headspace of C. fimbriata cultures grown on seven solid media.

In all the other media, the following esters were identified through retention time comparison with a standard curve: ethyl acetate, ethyl propionate, isoamyl acetate, and ethyl butyrate. There was a clear predominance of ethyl acetate, which represented over 80% of the total esters. Among the identified alcohols, ethanol, isoamyl alcohol, 2-hexanol, 1-propanol, 2-propanol, and 1-butanol were noted. Ethanol was the leading one in the headspace of the cultures and, as a rule, was the second volatile compound most produced during the fermentations, except in medium B, due to the low concentration of cassava bagasse. Alcohols do not play a predominant role in flavours but are known to contribute to the overall flavor quality and are precursors of fruit-like flavoring esters, which are definitely present in almost all fruits (14). The presence and quantities of the different compounds in the headspace depended on their concentration in the solid medium, vapour pressure and the transfer through the cover of the flask.

C. fimbriata

RESUMO

Produção de aroma frutal por Ceratocystis fimbriata em culturas estáticas sobre resíduo sólido agro-industrial

Este estudo explorou a versatilidade de Ceratocystis fimbriata de crescer e produzir aromas naturais sobre substratos de resíduos agro-industriais. Bagaço de mandioca, bagaço de maçã, amaranto e soja em diferentes proporções compuseram os sete meios utilizados, mostrando ser substratos adequados para o crescimento e produção de aroma por este fungo em fermentação no estado sólido. Todos os meios contendo bagaço de mandioca, bagaço de maçã e soja em sua composição proporcionaram um forte aroma frutal, enquanto, o meio de amaranto produziu um agradável aroma de abacaxi. A produção de aroma foi dependente do crescimento, visto que a máxima intensidade do aroma foi detectado poucas horas antes ou depois da atividade respiratória máxima. Foram detectados dezesseis compostos pela cromatografia de gás no headspace das culturas, e quinze deles foram identificados: 1 ácido, 6 alcoois, 1 aldeído, 2 cetonas e 5 ésteres.

Palavras-chave: Ceratocystis fimbriata, fermentação no estado sólido, resíduos agro-industriais, aroma.

Indigenous fermented foods

2. Bigelis, R. Flavor metabolites and enzymes from filamentous fungi. Food Technol., 46: 151-161, 1992.

3. Christen, P.; Villegas, E.; Revah, S. growth and aroma production by Ceratocystis fimbriata in various fermentation media. Biotechnol. Lett., 16: 1183-1188, 1994.

4. Christen, P.; Raimbault, M. Optimization of culture medium for aroma production by Ceratocystis fimbriata. Biotechnol. Lett., 13: 521-526, 1991.

5. Desgranges, C.; Georges, M.; Vergoingnan, C.; Durand, A. Biomass estimation in solid state fermentation. Appl. Microbiol. Biotechnol. 35: 206-209, 1991.

6. Gervais, P. et al. Influence of water activity on aroma production by Trichoderma viride. J. Ferment. Technol., 66: 403-407, 1988.

7. Hanchmeister, K.A.; Fung, D.Y.C. Tempeh: a mold-modified indigenous fermented food made from soya beans and/or cereal grains. Crit. Rev. Microbiol., 19: 137-188, 1993.

8. Janssens, L. et al. Production of flavours by microorganisms. Proc. Biochem., 27: 195-215, 1992.

9. Hang, Y.D. Improvement of the nutritional value of apple pomace by fermentation. Nutrit. Rep. Intern., 38: 207-210, 1988.

10. Hang, Y.D.; Woodmans, R.H. Apple pomace: A potencial substrate for citric acid procuction by Aspergillus niger. Biotechnol. Lett. 6 : 763-764, 1984.

11. Hesseltine, C.W. The future of fermented foods. Nutr. Rev., 41: 293-301, 1983.

12. Lanza, E.; Palmer, J.K. Aroma production of cultures of Ceratocystis moniliformis. J. Agric. Food Chem., 24, 1247-49, 1976.

13. Mitchell, D.A. Biomass determination in solid-state cultivation. In Solid state cultivation. H.W. Doelle; D.A. Mitchell & C.E. Rolz, Elsevier Applied Science: London, U.K., 1992, pp. 53-63.

14. Senemaud, C. Les champignons filamenteux producteurs d’aromes fruités. Estudes de faisabilité sur substrats agro-industriels. Ph.D. Thesis, Université de Bourgogne, France, 1988, p. 170.

15. Welsh, F.W.; Murray, W.D.; Willians, R.E. Microbiological and enzymatic production of flavor and fragance chemicals. Crit. Rev. Biotechnol., 9: 105-169, 1989.

16. Soccol, C.R.; Krieger, N. Brazilian Experiments for the valorization of agro-industrial residues by solid state fermentation. In: Advances in Biotecnology. A. Pandcy, Educational Publishers & Distributors, New Delhi, India, 1998, pp. 25-40.

• ²
  Bigelis, R. Flavor metabolites and enzymes from filamentous fungi. Food Technol., 46: 151-161, 1992.
• ³
  Christen, P.; Villegas, E.; Revah, S. growth and aroma production by Ceratocystis fimbriata in various fermentation media. Biotechnol. Lett., 16: 1183-1188, 1994.
• ⁴
  Christen, P.; Raimbault, M. Optimization of culture medium for aroma production by Ceratocystis fimbriata Biotechnol. Lett., 13: 521-526, 1991.
• ⁵
  Desgranges, C.; Georges, M.; Vergoingnan, C.; Durand, A. Biomass estimation in solid state fermentation. Appl. Microbiol. Biotechnol. 35: 206-209, 1991.
• ⁶
  Gervais, P. et al Influence of water activity on aroma production by Trichoderma viride. J. Ferment. Technol., 66: 403-407, 1988.
• ⁷
  Hanchmeister, K.A.; Fung, D.Y.C. Tempeh: a mold-modified indigenous fermented food made from soya beans and/or cereal grains. Crit. Rev. Microbiol., 19: 137-188, 1993.
• ⁸
  Janssens, L. et al. Production of flavours by microorganisms. Proc. Biochem., 27: 195-215, 1992.
• ⁹
  Hang, Y.D. Improvement of the nutritional value of apple pomace by fermentation. Nutrit. Rep. Intern., 38: 207-210, 1988.
• ¹⁰
  Hang, Y.D.; Woodmans, R.H. Apple pomace: A potencial substrate for citric acid procuction by Aspergillus niger. Biotechnol. Lett. 6 : 763-764, 1984.
• ¹¹
  Hesseltine, C.W. The future of fermented foods. Nutr. Rev., 41: 293-301, 1983.
• ¹²
  Lanza, E.; Palmer, J.K. Aroma production of cultures of Ceratocystis moniliformis. J. Agric. Food Chem., 24, 1247-49, 1976.
• ¹³
  Mitchell, D.A. Biomass determination in solid-state cultivation. In Solid state cultivation. H.W. Doelle; D.A. Mitchell & C.E. Rolz, Elsevier Applied Science: London, U.K., 1992, pp. 53-63.
• ¹⁴
  Senemaud, C. Les champignons filamenteux producteurs d’aromes fruités. Estudes de faisabilité sur substrats agro-industriels. Ph.D. Thesis, Université de Bourgogne, France, 1988, p. 170.
• ¹⁵
  Welsh, F.W.; Murray, W.D.; Willians, R.E. Microbiological and enzymatic production of flavor and fragance chemicals. Crit. Rev. Biotechnol., 9: 105-169, 1989.

Publication Dates

• Publication in this collection
  26 Feb 1999
• Date of issue
  Sept 1998

History

• Accepted
  23 July 1998
• Reviewed
  04 June 1998
• Received
  01 Jan 1998