GEOTRICHUM HYDROLYTIC ACTIVITY IN MILLED MALT AND BARLEY MEDIUM

Michał Piegza, Danuta Witkowska, Regina Stempniewicz, Anita Rywińska
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

In present work we focus on biosynthesis of extracellular hydrolase by Geotrichum candidum yeasts, which can be useful in degradation of non-starch polisaccharide (cellulase, xylanase, poligalacturonase, beta-1,3-glucanase, beta-1,3(1,4)-glucanase) and also protein (protease) and acyloglicerole (lipase). As a growth medium, we use 4% milled barley or 4% milled malt suspension. We observed divergence in level of activity of hydrolase, between different strains of Geotrichum but also in used medium. The hydrolase biosynthesis by Geotrichum was much more effective in the medium containing malt than in the one with barley. This effect was not observed for lipase and protease. Strains: G.candidum PH1 and G.candidum 1 due to very high non-starch polisaccharide hydrolase activity and low protease and lipase activity could be proposed as components of starter cultures, useful in malting process.

Key words: Geotrichum candidum, cellulases, xylanases, laminarinases, lichenases, proteases, lipases.

INTRODUCTION

Geotrichum candidum yeasts have a lot of qualities, essential for a good starter culture in malting. They are distinguished by very high growth speed, good colonization of barley grain, antagonistic activity against pathogenic microflore (especially Fusarium moulds) and same known enzymatic activity [11]. The yeasts´ capability to biosynthesise extracellular hydrolase plays a very important, supporting role in the loss of grain´s cell wall structure. Responsibility for this process own enzymes of grain released during malting, could by helped by similar yeast´s enzymes [4,8,10,14,15].

Previous research in The Department of Biotechnology and Food Microbiology, with used mineral medium with hydrolase inductors, from around 50 strains of Geotrichum candidum indicate 8 strains, with the highest level of biosynthesized hydrolases. In present work selected strains we tested in capable close to natural, medium, it.m. milled malt, and milled barley water suspension. This research, presently in progress, demands further focus on enzymatic characteristic of Geotrichum and helped in compose the best inoculums for malting [2,3,11,12].

MATERIALS AND METHODS

Microorganism and culture conditions:

8 strains of Geotrichum candidum yeasts isolated from 5 sources: G.candidum 1 [OL] – collection, G.candidum KB5 [X5] and G.candidum KB6 [X6] from Brie cheese, G.candidum Sc12 [Sc12] from Camembert cheese , G.candidum MSK₃11 [3–11] and G.candidum SS32B₁ [B1] and G.candidum SS47D₂ [D2] from malt, and also G.candidum PH1 [PH] from chicken feathers was stored on YM slants at 4C.

Growth medium (4% of milled malt and barley suspension in water distillate, pH=6.0) was inoculated with arthrospore suspension of 1x10⁷ cells in 1 ml of 1% Tween 80, water soluble. The cultures were conducted in 250 ml flasks (50 ml of medium) at 28C for 7 days, at 168 rpm.

Samples for enzymatic determination were collected after 7 days, after centrifugation (10000g for 15 minutes).

Enzyme assays:

Activities of CMC-ases (carboxymethylcellulases); xylanases; poligalacturonases; beta-1.3(1,4)-glucanases; were determinated using NaCMC (1%) (Sigma), xylan from birchwood (1%) (Sigma), polygalacturonic acid (0.25%) (Sigma), laminarin (0.25%) (Sigma), lichenin (0.25%) (Sigma) respectively as a substrate in 0.05 M acetic buffer pH=4.8.

The reducing substances - products of enzymatic reaction (30min, 50⁰) were determinated colorimetrically by the Miller´s [6] method using dinitrosalicylic acid (Sigma). Activities of examinated enzymes were expressed in nKat/ml except proteases and lipases.

Activities of proteases were determined using casein (Sigma) as a substrate in pH=6.0 at 30C and expressed in micro-mol/ml/min (jP).

Activities of lipases were determined using a oil from olive (Sigma) as a substrate in pH=8.0 at 37C and expressed in micro-mol fatty acid/ml/h (jL) – The fatty acids were determined by titration 0.05 M NaOH.

RESULTS AND DISCUSSION

The use of two different media, with milled malt (M) and milled barley grain (B), in Geotrichum culture resulted in different levels of the biosynthesis of hydrolytic enzymes. Generally, medium M in all researched strains was more favorable for the biosynthesis of the enzymes of polysaccharides than medium B. It is understandable because of higher amount of hydrolyzed polysaccharides in malt than in barley. The polysaccharides are not only a source of carbon for the growth of yeasts, but also inductors necessary for biosynthesis of specific enzymes. We observed that all tested strains were capable of diverse activity of enzymes.

G.candidum PH1 was most capable of cellulase biosynthesis and also had the largest diversity of the activities in both tested media. The activity in medium M was 12 times higher than in medium B. The remaining Geotrichum strains had cellulase activity within 0.5-3.3 nKat/ml in medium M, which was 1-6 times higher than in medium B.

In xylanase biosynthesis in medium M, the strains G.candidum MSK₃11 (4.9 nKat/ml) and G.candidum KB5 (4nKat/ml) were especially productive and their activity was 10 and 4 times higher than the activity observed in grain medium. In the other strains this difference was less significant in both tested media.

Polygalacturonase was also intensively biosynthesized by Geotrichum strains. Activity in medium M for most of tested strains exceeded 5 nKat/ml (max to 9 nKat/ml), and was exceptional for G.candidum Sc12 (5.9 nKat/ml) and G.candidum KB6 (8.8 nKat/ml).

Figure 1. Activity of extracellular hydrolase of G.candidum strains in submerged cultures in milled malt and barley: {A}–cellulases, {B}–Xylanases, {C}–Poligalacturonases, {D}–Laminarinases, {E}–Licheninases, {F}–Proteases, {G}–Lipases

Enzymes which hydrolyze beta-1.3 bonds (laminarine) and beta-1.3; beta-1.4 (lichenin) play a very important role in the direct hydrolysis of beta-glucan. The tested strains were characterized by capability to biosynthesize those enzymes on different levels. Only 4 out of 8 strains presented laminarinase activity within 3.8-8.5 nKat/ml, which was from 2 to 9 times higher than in the other strains.

Less diversity was observed in case of licheninase in medium M, where most of strains had the activity of ca. 3.5 nKat/ml. In medium B, the biosynthesis was within ca. 0.1-0.6 nKat/ml.

In our cultures of the selected Geotrichum strains we also examined the protease and lipase activities. We did not observe the same regularities as in case of other enzymes. From among 8 strains, 6 biosynthesized protease much more effectively in medium B than in medium M, but the level of this activity was not high, which is a very important quality of strains suggested as starter cultures. Only G.candidum SS32B₁ showed high activity in barley medium, and G.candidum KB6 in malt medium.

We showed that in all tested enzymes, lipase activity was least variable, comparing both types of growth media. With the exception of G.candidum KB6, the activity of the strains was very low and did not exceed 20 jL. This is also a perfect quality for a starter culture strain.

The use of Geotrichum candidum strains in malting as starter cultures is rather a new problem. There is a few information about growth of Geotrichum on barley or malt and it´s influence on malt quality, but it´s also hard to find any information about enzymatic characteristic of those yeasts important for their activity in malting. Information about capability of those yeasts to biosynthesis lipases or proteases is easily available in world´s literature, especially in cheese industry [4] and also xylanases [13]. There is also a lot of information about poligalacturonase and cellulase characterized especially in terms of the citrus waste storage and investigation at their biochemical properties [7, 8].

In the literature there can be found evidence for the very differentiation in hydrolase´s expression by strains extracts from different sources. The tested strains were able to biosynthesize all investigated extracellular enzymes, including amylase, which activity we don´t find in culture medium. Amoa and et.a. [1] showed strain capability to synthesize beta-glicosydase and cellulase but not pectinase, Falih [6] found G.candidum strain with very low amylolytic activity.

CONCLUSIONS

1. Diversity in the level of activity of the tested enzymes, both between Geotrichum strains, and between the growth media was observed.

2. Geotrichum candidum strains biosynthesized hydrolases of non-starch polysaccharides much more effectively in medium with malt than with barley. This effect was not observed in lipase and protease.

3. G.candidum 1 and G.candidum PH1 strains may be proposed as components of starter cultures useful in malting because of high activity of enzymes hydrolyzing non-starch polysaccharides and very low activity of protease and lipase.

ACKNOWLEDGEMENT

This research was supported by the Agriculture University of Wrocław within the project no. GW/509/2003.

REFERENCES

1. Amoa-Awna W.K., Sefa-Dedeh, Jakobsen M., 1997. The contribution of moulds and yeasts to the fermentation of agbelina cassava dough, Journal of Applied Microbiology, 83, 288–296.

2. Boivin P., Malanda M., 1997. Improvement of malt quality and safety by adding starter culture during the malting process, Technical Quarterty, vol 34, no 2, 96–101.

3. Boivin P., Malanda M., 1996. Microflora management during malting, Lectures Conference, 97-108.

4. Chrzanowska J., Stempniewicz R., Wojtatowicz M., 2001. Ocena aktywności proteolitycznej wybranych szczepów Geotrichum candidum. [Evaluation of proteolytic activity of selected Geotrichum candidum strains]. Praca zbiorowa pod red. T.Habera, H.Porzucek. ISBN83-915774-0-6.Warszawa, (płyta CD) [in Polish].

5. Dziuba E., Foszczyńska B., 2001. Biological protection of barley grain and its influence on selected features of malt, Polish Journal of Food and Nutrition Sciences, vol 10, no 4, 49–53.

6. Falih A.M.: 1998. Effect of heavy-metals on amylolytic activity of the soil yeast Geotrichum capitatum and Geotrichum candidum, Bioresource Technology, 66, 213–217.

7. Guessous Z., LebbarS., Ouhssine M., Mokhatari A., Yachioui MEl., 2001. Endo and exopolygalacturonase from Geotrichum candidum: partial purification and characterization, Science Letters, vol 3, no 1, 1–5.

8. Hang Y.D., Woodams E.E., 1992. Production and characterization of polygalacturonase from Geotrichum candidum. World Journal of Microbiology., 8, 480–482.

9. Miller G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem., 31, 426–428.

10. Mo K., Hayashida S., 1988. Production and characteristics of two types of endocellulase from Geotrichum candidum. Agric.Biol.Chem., 52(7), 1675–1682.

11. Olesieniewicz A., Grajek W., 2003. Enzymy w browarnictwie [Enzymes in brewing]. VIII Szkoła Technologii Fermentacji [in Polish].

12. Piegza M., Stempniewicz R., 2002. Ocena antagonizmu drożdży Geotrichum candidum w stosunku do toksynotwórczych grzybów rodzaju Fusarium, [Evaluation of the Geotrichum candidum antagonim towards toxigenic fungi of Fusarium sp.]. Żywność, Nauka, Technologia Jakość – suplement, nr3 (32), 136–148 [in Polish].

13. Radionowa N.A., Dubavaya N.V., Eneiskaja E.V., Martinovich L.I., Gracheva I.M., Bezborodov A.M., 2000. Purification and characterization of endo (1–4)–b-xylanase from Geotrichum candidum 3C, Appl. Biochemistry and Microbiology, vol 36, no 5, 406

14. Tahoun M. K., Mostafa E., Mashaly R., Abou–Donia S., 1982. Lipase induction in Geotrichum candidum. Milchwissenschaft.,37(2), 86-88.

15. Witkowska D., Żarowska B., Stempniewicz R., Rodziewicz A., 2001. Aktywność enzymów hydrolitycznych Geotrichum candidum [Hydrolytic activity of Geotrichum candidum]. Praca zbiorowa pod red. T.Habera, H. Porzucek. ISBN83-915774-0-6.Warszawa, (płyta CD) [in Polish].

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Michał Piegza
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
phone +48 71 320 7737
fax +48 71 320 7794
51-630 Wrocław
Poland
email: michal.piegza@upwr.edu.pl

Danuta Witkowska
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
Poland
email: wit@ozi.ar.wroc.pl

Regina Stempniewicz
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Norwida 25, 50-373 Wrocław, Poland
Fax. 4871- 3284124
Phone: 48-71-3205116

Anita Rywińska
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
Poland
email: anita.rywinska@wnoz.up.wroc.pl

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