THE USE OF GEOTRICHUM CANDIDUM STARTER CULTURE FOR PROTECTION OF BARLEY AND ITS INFLUENCE ON BIOTECHNOLOGICAL QUALITIES OF MALTS

Barbara Foszczyńska, Ewelina Dziuba, Regina Stempniewicz

ABSTRACT

The influence of chemical disinfection of barley grain and addition of biomass of Geotrichum candidum in the process of steeping or germinating the grain on the enzymatic and technological quality of malt was examined. Rudzik brewing barley grain disinfected using formalin or biologically protected using G. candidum strains 1, KC3 and KB5, was used as experiment material. The starter cultures were added during steeping or at the beginning of germination. It was observed that the use of formalin negatively influenced the modification of malts and resulted in, among others, lowered Kolbach index (by 4.3% avg.). Addition of starter cultures to the first steeping water was favourable for the creation of enzymatic potential of malts. As compared to the control sample, starter malts were characterised by higher activity of beta-glucanase (by 24 U·kg^-1) and alpha-amylase (by 20 U·g^-1) and by higher diastatic power (by 20 WK-u). The best enzymatic a

Key words: brewing barley, Geotrichum candidum yeasts, malting..

INTRODUCTION

In recent years, the problem of lowered, irregular technological quality and health of malts, resulting from uncontrolled development of epiphytic microflora of barley during its steeping and germinating, has been very often discussed in scientific literature. Filamentous fungi, including Fusarium, Alternaria, Aspergillus and Penicillium species, producing metabolites hazardous to human health constitute especially dangerous microflora [5, 9, 16]. Fusarium toxins: T-2, diacetoxyscirpenol (DAS) and, to a lower degree, deoxynivalenol negatively influence the process of barley germination, they inhibit the growth of rootlets, slow down protein synthesis and weaken the synthesis of alpha-amylase [15, 18, 20]. The T-2 toxin is a strong inhibitor of the growth of Saccharomyces cerevisiae yeasts, delays the initiation of fermentation and inhibits the production of ethanol.

The traditional method of protection of barley form uncontrolled growth of micro-organisms used in Polish malt-houses is disinfection of grain using such chemical compounds as formaldehyde, potassium permanganate, hydrogen peroxide or sodium hypochlorite [4, 11]. Solutions of these compounds are added during steeping stage. The use of starter cultures, slowing down the growth of undesirable micro-organisms, may be an alternative to this, not always effective, method. It is commonly used in production of cheeses, yoghurt, kefir, pickled vegetables and raw sausages [9]. The matters related to malting were the subject of numerous studies in the 90’s. Finnish scientists [12-14, 16] were examining the possibilities of the use of lactic acid bacteria for this purpose. French [3] and Polish [7, 8, 10, 17] scientists are examining the use of Geotrichum candidum yeasts. When compared with filamentous fungi, G. candidum is faster in taking up the space and using up the medium which make s them a dominant microflora of malt. In comparison with the traditional, chemical method of grain protection, it allows for obtaining malt which is characterised by recurrent and often better technological quality. For barley cultivated in the climatic and soil conditions of Poland, there is a necessity of searching for strains and methods of inoculation which are the best for malting purposes.

The aim of the study was to assess the influence of chemical disinfection of grain and the addition of Geotrichum candidum yeasts during steeping or during germination of the grain on the enzymatic and technological quality of malt.

MATERIALS AND METHODS

The following materials were used in the study:

• Rudzik brewing barley grain from the malt-house in Strzegom

• Formaldehyde – a chemical disinfecting agent

• Starter cultures of Geotrichum candidum yeasts from the Culture Collection of the Department of Biotechnology and Food Microbiology at the Agricultural University of Wrocław:

- G. candidum 1
- G. candidum KC3
- G. candidum KB5.

Rudzik barley grain was micro-malted using either chemical (formalin) or biological method of protection. For biological protection of malt, strains of G. candidum yeasts, characterised by high growth dynamics, antagonistic activity in the presence of toxin-creating fungi, low proteolytic and lipolytic activity and high cellulolytic activity were selected [21].

The following samples of malt were prepared in two repetitions:

• K – control grain without protective agent

• D – grain with an addition of disinfecting agent – formalin

• S1 – grain with an addition of Geotrichum candidum 1 starter culture

• S2 - grain with an addition of Geotrichum candidum KC3 starter culture

• S3 - grain with an addition of Geotrichum candidum KB5 starter culture.

Two series of micro-malting, differing in the method of grain inoculation with starter cultures, were conducted:

- series I – starter cultures were added to the first steeping water (for 2 L of water – 10 ml of suspension containing hyphae and arthrospores at the concentration of 1.7 – 4.3x10³ cfu·ml^-1)
- series II – starter cultures were added to steeped grain at the beginning of germination (the grain was sprayed with 15 ml of suspension at the concentration of 3.2x10⁴ – 1.7x10³ cfu·ml^-1).

Formalin was added to the first steeping water at the concentration of 0.3%. A 48 hour-long water-air steeping cycle was used to reach the moisture of 44-46%. Germination of grain was conducted for 6 days at the temperature of 13-15^oC and kilning of malts at the temperature of 30-82°C.

In the malts obtained in the study, moisture, total protein and beta-glucans content and diastatic power were determined according to EBC Analytica [1] and Polish Norms [19]. The activity of beta-glucanase and alpha-amylase were determined using kits produced by Megazyme Ltd. In the worts obtained using congress method, viscosity, colour and the content of extract, nitrogen compounds and free amino nitrogen were determined. Extract yield of malts and Kolbach index were also determined.

RESULTS AND DISCUSSION

Based on the assessment of the features of malts and laboratory worts, it was observed that Rudzik barley grain, which was until recently considered a model variety of brewing barley, is characterised by high technological quality. The malts obtained from grain which was not disinfected and grain chemically and biologically protected, were characterised by standard saccharification time and high extract yield (Table 1). The malts were characterised by high protein modification (high values of Kolbach index, with the exception of the samples disinfected with formalin) and cytolytic modification (low content of beta-glucan, good friability, low viscosity of worts). It was also observed that some features of malts were influenced by the type of protective agent, method used for adding starter cultures and the conditions of malting.

The use of formalin for disinfecting grain negatively influenced the proteolysis of protein compounds which resulted in lower (as compared to the control group) content of nitrogen compounds in wort – on average by 49 mg·L^-1 – and lower Kolbach index – on average by 4.3% (Tables 1 and 2). Moreover, the use of formalin influenced cytolytic modification of malts, especially in the series I of micro-malting. The malts obtained with it were characterised by the worst friability (73 and 82%) and the highest glassiness (2.5 and 1.2%) as compared with the control sample. The malts from chemically disinfected grain in series I of malting were characterised by extract yield worse than in the control sample, higher content of beta-glucans and the wort obtained using them had higher viscosity.

The results obtained in the study allow to say that the use of formalin for disinfecting grain may, at the doses commonly used in malting (0.2 – 0.3% of water), have a negative influence on the processes of enzymatic degradation of the endosperm structure. Formalin, as a strong inhibitor, impairs the life functions of the germ during steeping and germinating which may result in disrupting the processes of synthesis and activation of enzymes responsible for the decomposition of reserve constituents of grain. The thesis is proved by the results of the assessment of amylolytic activity. The malts from grain disinfected with formalin were characterised by the lowest activity of alpha-amylase (Fig. 1) and the lowest diastatic power (Fig. 2). Lower amylolytic potential and a slightly worse cytolytic modification were probably the cause of lower extract yield obtained from these malts.

The addition of G. candidum starter cultures during steeping of grain (series I of micro-malting) had a positive influence on the enzymatic activity of malts. As compared with the control sample, the starter malts were characterised by a higher activity of beta-glucanase, by 24 U·kg^-1 on average (Fig. 3), and as a result had a lower content of beta-glucans (Table 1). Moreover, they were characterised by a higher activity of alpha-amylase, by 20 U·g^-1 on average (Fig. 1), and diastatic power higher by 20 WK-u (Fig. 2). The malt obtained from barley steeped with G. candidum 1 was characterised by the best enzymatic potential.

Haikara and Latila [13] indicated that the addition of lactic acid bacteria to the 1^st and 2^nd steeping water improved the enzymatic balance of malts. Higher activity of beta-glucanase (at 60 °C) and xylanase were observed. The malts obtained on the pilot scale with G. candidum were also characterised by an ability to a better decomposition of pentosans [3]. G. candidum yeasts show an ability to produce extracellular hydrolases induced by a proper source of carbon and energy in natural environment [22]. Cellulases, xylanases and beta-glucanases are worth special attention due to their activity in degradation of the cell wall structure. From the technological point of view, bad quality of malt resulting from a high content of grains which are low-extractive, hard and glassy may bring about such disruptions in the brewhouse as low yield of wort, difficulties in filtering the wort and beer, turbidity and lowered stability of beer [2]. This is why proper process of endosperm during malting of barley, in which the enzymes of microbiological origin are very helpful, is so important.

The obtained results indicate an influence of G. candidum yeasts on the enzymatic activity of malts. As it was said above, Rudzik barley used in the study was of very good quality and the conditions during the whole cycle of malting brought about advanced degradation of grain biopolymers, even in control samples, i.e. in the samples which were not protected by any agent. Thus, increased enzymatic activity did not affect the technological parameters of starter malts.

The malts obtained as a result of adding G. candidum starter cultures, no matter how barley was inoculated, were characterised by parameters of protein degradation comparable to those in control malt (Table 1). The Kolbach index in control malts was high and reached 45.0% (series I of malting) and 44.4% (series II). In starter malts, it ranged within similar values of 43.7 – 46.0%. In comparison to control malts (friability 83 and 87%, glassiness 0.8 and 0.5%), starter malts were characterised by comparable or slightly worse values of friability (77-85%) and glassiness (0.6-2.0%). The values of these parameters, most similar to those in control samples, were observed in the malt obtained using G. candidum1 inoculum. Moreover, the malt contained the smallest amount of beta-glucans. It is worth noting that the content of beta-glucans in the samples of starter malts was lower than in the control sample and in the sample chemically disinfected. Numerous studi es show that starter cultures may reduce the level of beta-glucans in malt and wort [3, 13, 17]. It helps in lowering the viscosity of wort and, as a result, in improving filtration processes at breweries.

No influence of starter cultures and the method of their inoculation on the viscosity and colour of laboratory worts was observed in the present study (Table 2). It should be noted that the viscosity of all worts was very low and similar in all cases (with the exception of the sample chemically disinfected in series I of malting).

The results obtained in the study may be concluded with the observation that the use of G. candidum cultures during steeping positively influences the enzymatic apparatus of malts, which may result in better overall modification of the structure of endosperm, especially in grains of lower quality. Adding a suspension of inoculums to steeping water is a more convenient method of inoculation than spraying steeped grain, although the latter is also recommended by scientists examining the usefulness of acidifying cultures [17]. It may be supposed that the systems with prolonged period of air steeping, currently used in malt-houses, improve the colonisation of grain and enzymatic activity of starter micro-organisms. G. candidum yeasts are characterised by fast colonisation of kernels, which positively limits the development of microflora, esp. filamentous fungi. The assessment of microbiological condition of grain and malts obtained using chemical and biological protection, perform ed at the Department of Biotechnology and Food Microbiology at the Agricultural University of Wroclaw, shows that in comparison to control sample, the addition of G. candidum inoculum to steeping water resulted in reduction of Fusarium content in steeped malts higher than in case when the inoculum was added at the beginning of germination [21]. The use of cultures had protective effect comparable, and often better than the use of chemical disinfection of grain. From among the yeasts used in the study, G. candidum 1 strain was characterised by the most suitable qualities. When used during steeping of grain, it almost fully inhibited the growth of Fusarium moulds and, moreover, had a positive influence on the enzymatic activity of malts.

CONCLUSIONS

The following conclusions may be presented based on the results of the research:

1. Disinfection of barley grain using formalin may result in distortions in the modification processes during malting. The use of formalin had a negative effect on protein degradation (lowered Kolbach index), amylolytic activity of malts and, especially in series I of malting, on the parameters characterising cytolytic modification (friability, glassiness, extract yield and viscosity of wort).

2. G. candidum starter cultures added during steeping of barley grain had a positive effect on the enzymatic potential of malts. The malts obtained with the yeasts were characterised by higher glucanolytic and amylolytic activity than the control malt. The best results were obtained using G. candidum1 strain.

3. In comparison to the control malt, starter malts were characterised by similar protein and cytolytic modification. Biological protection of barley grain allows obtaining malts of technological quality comparable to that of the control malt.

4. The use of starter cultures in a form of suspension added to steeping water may, in industrial conditions, be an effective and less problematic method of inoculation than spraying the grain at the beginning of germination.

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Regina Stempniewicz
Department of Biotechnology and Food Microbiology
Agricultural University of Wrocław
ul. Norwida 25, 50-375 Wroclaw, Poland
tel. (+ 48 71) 32-05-237
fax: (+ 48 71) 32-05-273
e-mail: bfoszcz@wnoz.ar.wroc.pl

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