Next Article in Journal
Antibacterial and Immunostimulatory Activity of Potential Probiotic Lactic Acid Bacteria Isolated from Ethiopian Fermented Dairy Products
Next Article in Special Issue
Microbiota Succession and Chemical Composition Involved in Lactic Acid Bacteria-Fermented Pickles
Previous Article in Journal
Effect of Dioscorea Opposite Waste Supplementation on Antioxidant Capacity, Immune Response and Rumen Microbiome in Weaned Lambs
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Fermentation
Volume 9
Issue 3
10.3390/fermentation9030257
fermentation-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Biocontrol Using Pythium oligandrum during Malting of Fusarium-Contaminated Barley

by
Carlo Antonio Ng
1,
Marek Pernica
2,
Katerina Litvanova
1,
Irena Kolouchova
1 and
Tomas Branyik
2,*
1
Department of Biotechnology, University of Chemistry and Technology, Technická 5, 16628 Prague, Czech Republic
2
Research Institute of Brewing and Malting, Lípová 511/15, 12000 Prague, Czech Republic
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(3), 257; https://doi.org/10.3390/fermentation9030257
Submission received: 12 February 2023 / Revised: 24 February 2023 / Accepted: 3 March 2023 / Published: 5 March 2023
(This article belongs to the Special Issue Quality and Sensory Analysis of Fermented Products)
Versions Notes

Abstract

:
This study investigates the potential of Pythium oligandrum (strains M1 and 00X48) as a biocontrol agent in suppressing the growth of Fusarium culmorum and the production of mycotoxins during the malting of naturally contaminated barley (Hordeum vulgare). The effects of the biocontrol agent on F. culmorum-infected barley malt (BM) were evaluated through real-time PCR and its impact on mycotoxin production was determined by quantitative analysis of deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G). The effect of treatment on BM and beer quality were also determined through European Brewery Convention (EBC) standard methods. Optimal treatment with P. oligandrum strains M1 and 00X48 yielded a 59% and 48% reduction in F. culmorum contamination, by 37% and 17% lower DON, and 27% and 32% lower D3G, respectively. BM treated with both P. oligandrum strains exhibited quality enhancement; beer produced from the BM treated with P. oligandrum strain M1 resulted in no quality deterioration and with 26% and 18% less DON and D3G, respectively, transferred to the final product.

1. Introduction

Fusarium head blight (FHB) in barley is of economic concern due to crop yield losses [1] and reduced quality, leading to downgrading of grains to low-value feed use, instead of malting applications, or outright rejection for malting purposes [2]. The prevalence of FHB is important in malting and brewing with negative impacts on malt and beer quality caused by the fungal contamination, including decreased wort filterability [3] and gushing of beer [4].
Aside from quality effects, FHB is also related to food safety issues due to the production of mycotoxins in infected grains [5]. Of the mycotoxins produced by the fungi, deoxynivalenol (DON) and its modified forms, such as deoxynivalenol-3-glucoside (D3G), are the most abundant and are the greatest concern for food safety [6]. The temperature and humidity to which the grains are subjected during malting are ideal for fungal growth [7], leading to elevated mycotoxins in the final malt [8]. Variations in global temperature and humidity brought about by climate change are also expected to increase on-field Fusarium infection and to elevate mycotoxin levels in harvested cereals [9].
Barley is one of the most important cereals produced worldwide, with approximately 141 Mt produced in 2017, of which 30% was used for malting [10]. The demand for the crop is continually growing, with an estimated 54% increase in global production required to meet worldwide requirements by 2050 [11]. The supply problem is further complicated due to an increasing incidence of FHB, which causes devastating losses to malting barley. Between 1997–2014, significant economic losses resulted from FHB in barley with $18 million average annual losses in revenue reported in the United States [12]. The malting industry needs an effective biological tool to control FHB.
Due to limitations in traditional chemical and physical methods to reduce the effects of FHB in malting, biological control methods have gained interest. Chemical treatment methods are harsh treatment methods that affects malt quality and could lead to the formation of unwanted byproducts and residues, while physical treatment methods have been described to negatively affect barley germination [13]. A promising biocontrol agent against Fusarium-infection in barley malting is Pythium oligandrum, which has shown exceptional qualities against Fusarium in various crops [14,15,16] and has also shown its capability of suppressing growth of Fusarium during the malting of wheat for the brewing industry [17].
P. oligandrum is a soil-born oomycete that has exhibited antagonistic activity against a variety of pathogenic fungi. It has been observed to protect plants from fungal infections through direct and indirect mechanisms and has attracted attention as a promising biocontrol agent [13]. The microorganism has also received approval from the EU and US EPA for plant protection in agriculture applications [13].
This study explores the application of P. oligandrum during the malting of barley, the main cereal for the brewing industry. The suppression of F. culmorum growth and mycotoxin production during malting of naturally contaminated barley was determined together with the effect of the biological control treatment on malt and beer quality.

2. Materials and Methods

2.1. Malting Process

Malting was carried out in a micromalting device (Ravoz, Olomouc, Czech Republic) equipped with software (Proteco Ltd., Pardubice, Czech Republic) to control the malting parameters, following malting parameters described by Postulkova et al. [16]. Malting was performed in boxes containing 1 kg of barley grains (Hordeum vulgare, Lodestar variety, harvest 2020) obtained from the locality of Hrubcice, Czech Republic. The germinative capacity of used barley was 96% (EBC 2004 3.5.2), while DON and D3G contents were 1320 and 1030 µg/kg, respectively. The malting program started with a 48 h steeping step at 15 °C with alternating wet/air rest intervals at 8/12/8/12/4/4 h, followed by a 48 h germination stage with a gradual temperature decrease from 21 °C to 18 °C, ending with a 24 h kilning step starting at 45 °C for 6 h with gradual increase to a final temperature of 80 °C.

2.2. Brewing Process

Brewing trials were executed in the pilot brewery (50 L) at the University of Chemistry and Technology Prague (Prague, Czech Republic), following standard procedure from the pilot brewery with adjustments [18]. Detailed brewing conditions are summarized in Table 1. Briefly, milled grains were mashed in a single decoction process, after which the spent grains and sweet-wort were separated through a traditional lautering process. This was followed by wort boiling with the addition of hops and cooling. Fermentation was carried out with a bottom fermenting yeast, Saccharomyces pastorianus (SafLager W-34/70, Fermentis), and the beer was then left to mature.

2.3. Application of Pythium Oligandrum

Pythium oligandrum samples (strains M1 and 00X48, 1 × 106 oospores/g) were obtained from Biopreparaty Ltd. (Prague, Czech Republic). P. oligandrum suspensions were prepared by adding 0.1 to 1 g samples in 100 mL of distilled water and mixed with the barley grains (1 kg dry weight) at the following malting stages: (i) with the first steeping water, (ii) with the second steeping water and (iii) before germination. All malting experiments were performed in three biological replicates. Collected replicates were thoroughly mixed and samples for analysis were taken from the mixtures. The mixing of biological replicates is based on the industrial practice of malthouses aiming at averaging the malt properties.

2.4. Isolation and Quantification of Fungal DNA

A HighPure PCR Template Preparation Kit (Roche Applied Science, Prague, Czech Republic) was used to isolate the DNA from the samples. Isolated DNA was analyzed by real-time PCR using the LightCycler 2.0 (Roche Applied Science) to determine relative Fusarium contamination in the samples. Sample preparation, DNA isolation and real-time PCR analysis were performed according to the detailed procedure described by Ng et al. [17], with the primer sequences and Universal ProbeLibrary (UPL) probes for F. culmorum and F. graminearum as described by Postulkova et al. [16].

2.5. Mycotoxin Quantification

Deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) in the barley malt (BM) samples were determined through a Donprep immunoaffinity column (R-Biopharm AG, Darmstadt, Germany) after purification of the samples following the method described by Havelka et at. (2019) [19]. Identification and quantification of the mycotoxins were carried out by HPLC (Finnigan Surveyor) coupled to an ion trap LCQ Advantage (Thermo-Fisher, USA) with atmospheric pressure ionization, and validated according to the method described by Belakova et al. [20].

2.6. Malt and Beer Quality Parameters

Malt quality was determined based on the following technological parameters: total nitrogen of malt (EBC 2004 4.3.1), extract of malt (EBC 2004 4.5.1), diastatic power (EBC 2010 4.12), final attenuation (EBC 1999 4.11.1), free amino nitrogen (EBC 1997 4.10), soluble nitrogen (EBC 1999 4.9.3), viscosity of wort (EBC 2004 4.8), beta-glucan (EBC 2007 3.10.2) and friability (EBC 2015 4.15). Overall brewing malt quality (BMQ) of the BM was calculated based on the parameters to describe the cytolytic, proteolytic and amylolytic activities of the samples [21], 1 being the worst and 9 being the best. The following beer quality parameters were analyzed: color (EBC 2000 9.6.), original, apparent and real extract (EBC 2004 9.4), alcohol (EBC 2008 9.2.6), and apparent and real attenuation (EBC 2000 9.7) [22].

2.7. Statistical Analysis

Statistical analyses were performed with Microsoft Excel. Data were presented as means ± standard deviations. Experimental data was statistically evaluated through t-test, and all statements of significance were based on a probability of p < 0.05.

3. Results

3.1. Effect of Pythium Oligandrum on Fusarium Culmorum Growth and Mycotoxin Production

Real-time PCR was carried out with both F. culmorum and F. graminearum primer and probe pairs to determine which species was the dominant fungal contaminant on the grains. F. culmorum was identified as the dominant Fusarium contaminant on the barley samples, yielding exponential growth curves from the real-time PCR analysis, and the absence of a growth curve for F. graminearum. Relative levels of F. culmorum contamination were determined by real-time PCR to demonstrate the effect of P. oligandrum on inhibiting the spread of the fungi in the malting process. The addition of P. oligandrum resulted in F. culmorum growth suppression by 35–59% relative to untreated BM (Table 2). These values indicate a statistically significant reduction in fungal growth as a result of biocontrol treatment (p < 0.05). The suppression of F. culmorum growth was also observed to be the most significant when P. oligandrum was added with the second steeping water and in the germination stage (Table 2).
Together with the suppression of fungal growth during malting of barley, mycotoxin levels were also seen to drop with biocontrol treatment compared to untreated BM. Total DON levels decreased by 29% to 37% in the treated BM samples and all these decreases were statistically significant. The decrease in D3G in BM resulting from P. oligandrum treatment was 21% to 35%, being statistically significant when the biological treatment was applied to the first steeping water (Table 2).
Different concentrations of two P. oligandrum strains (M1 and 00X48) were applied in the second steeping water and similar significant reductions in F. culmorum growth were observed (Table 3). The application of strain M1 resulted in greater suppression of fungal growth than that with strain 00X48. Differences between efficiencies of the two strains were statistically significant. Reductions in fungal contamination levels with different P. oligandrum treatment concentrations were not significantly different for both the M1 and 00X48 strains (Table 3).
Total DON levels were also lower for BM samples with P. oligandrum treatment. Total DON decreased by 22% to 33% compared to untreated BM (Table 2 and Table 3). However, the decrease in DON level was statistically significant (p < 0.05) only for BM treated with 1×106 oospores M1/kg barley, while D3G reduction was only statistically significant for BM treated with 1 × 106 oospores 00X48/kg barley (Table 3).

3.2. Effect of Pythium Oligandrum on Barley Malt Quality Parameters

Aside from the decreased F. culmorum and mycotoxin concentrations, the application of the biocontrol agent resulted in improved diastatic power, apparent final attenuation and friability compared to untreated BM when added at the different malting stages (Table 4). However, the malt quality parameter differences were not significant (p > 0.05).
Different treatment concentrations of both M1 and 00X48 strains added with the second steeping water also resulted in improved diastatic power and apparent final attenuation (Table 5). The friability of BM was also greater for the treated samples, except for BM, with 1 × 106 oospores M1 per kg barley. The greatest friability was observed for the BM with 1 × 106 oospores 00X48 treatment. The beta glucan concentrations were also generally lower for the treated samples, except for BM with 1 × 106 oospores M1 per kg barley, which recorded concentrations greater than the untreated BM. Regardless of the differences, most quality parameters were not statistically different (p > 0.05).
Overall barley quality was evaluated with the system proposed by Psota and Kosar (2002) [21]. A scale from 1 to 9 was used to quantify BM quality, considering the cytolytic, proteolytic and amylolytic properties calculated from weights given to each quality parameter based on industry requirements. The overall BMQ was generally seen to improve with the addition of P. oligandrum. The best BMQ (4.1) was found for BM with both 1 × 106 oospores M1 and 00X48 per kg barley treatment compared to that of untreated BM (3.1) (Table A1 and Table A2).

3.3. Effect of Pythium Oligandrum on Beer Quality Parameters

Beer was brewed from BM samples both with and without P. oligandrum strain M1 treatment. The treated BM had a treatment dose of 1 × 106 oospores M1/ kg barley and resulted in significantly higher values (p < 0.05) in the color, apparent extract, and alcohol, while the fermentation rates and pH were statistically unchanged (Table 6).
DON and D3G transfer to the final beer samples were also measured (Table 7). The sample prepared from the P. oligandrum (M1) treated BM led to a final product with both lower DON and D3G levels by 26% and 18%, respectively, compared to the beer prepared with untreated BM. However, these differences were not statistically significant (p > 0.05).

4. Discussion

Due to the link between FHB infection in grains and growing mycotoxin levels [23], a variety of methods have been tested to limit the spread of Fusarium and control the production of mycotoxins [6]. Chemical control agents and physical control methods have been proposed but have limited industrial applications due to the formation of unwanted byproducts and the negative affect on grain quality after treatment [13]. Because of this, biological control agents have gained increasing interest for improving the quality and safety of grains and beverages [24]. The application of biological fungicides is also in line with global food security and sustainability goals of minimizing the impact of pesticides by shifting to non-chemical alternatives [25]. Several biological agents have been proposed for malting applications to limit the spread of Fusarium and decrease mycotoxin production. Lactic acid bacteria (LAB), which are naturally present on the surface of barley, have been proposed and shown to successfully minimize the occurrence of Fusarium fungi during malting [26,27]. However, some LAB strains are undesired microbial contaminants in beer, resulting in reluctance to apply them industrially [13]. Geotrichum candidum is another microorganism used as a biocontrol agent on an industrial scale and has also shown promise in limiting Fusarium growth and in decreasing mycotoxin production in malt [28]. However, careful strain selection is required due to some strains possessing high lipase activities resulting in the formation of unwanted oxidation products [27], while some strains could form byproducts such as clavinet alkaloids, which are toxic to human health [6,29].
Pythium oligandrum is a non-pathogenic soil-born oomycete that has attracted attention as a biocontrol agent against pathogenic fungi in plants, including Fusarium species [15,30]. The biocontrol ability of P. oligandrum applied during malting is here demonstrated by decreasing the relative levels of F. culmorum on naturally contaminated barley. This is consistent with the potential of P. oligandrum to decrease artificial Fusarium infection in BM, as first described by Postulkova et al. [16]. The anti-fungal properties of P. oligandrum can be explained by its mycoparasitic properties, allowing abundant growth on Fusarium hyphae and resulting in growth inhibition of fungi [31]. Ng et al. applied P. oligandrum during the malting of wheat, resulting in greater suppression of F. culmorum contamination than observed in the case of barley [17]. This higher efficiency can be attributed to the absence of a thick husk on wheat compared to barley, which could provide shelter to fungal hyphae [32]. Fusarium spores start forming on barley husk surfaces and fungal hyphae grow over the surface of the grain and extend to the interior of the grain through crevices, which protects them against mycoparasites and allows them to thrive throughout malting [33,34].
The observed decrease in DON and D3G mycotoxin levels in P. oligandrum-treated BM were also in agreement with literature claiming a correlation between the production of mycotoxins and levels of F. culmorum contamination [35]. The decrease in F. culmorum content by P. oligandrum treatment led to decreased DON and D3G mycotoxins in the finished BM. These two Fusarium mycotoxins are the most abundant Fusarium mycotoxins and are the most frequently detected in malt and beer [8]. DON levels have also been observed to rise during the malting process, thus making them of greater concern [36].
From the BMQ data, diastatic power, apparent final attenuation and friability in P. oligandrum-treated BM samples tended to be higher than in untreated BM, although the differences were not statistically significant. Diastatic power and friability both describe the levels of amylolytic and cytolytic modification in the grains [22]. These higher values with biocontrol treatment point to increased malt modification, which can be traced to the ability of P. oligandrum to either stimulate enzymatic activity and plant growth, or/and act with its own enzymatic complement [30,37]. This is also consistent with the observed higher diastatic power obtained for the P. oligandrum-treated BM samples [38]. The enhanced proteolytic activity also resulted in the release of higher levels of free amino acids, which react with sugars, leading to the slightly darker color of the beer made from BM treated with P. oligandrum [38,39].

5. Conclusions

There is a global trend shifting away from using chemical fungicides to combat the adverse effects of Fusarium contamination on brewing barley quality. Accordingly, there is a growing interest in alternative biological control agents. A promising microorganism to control the spread of Fusarium in the malting of brewing barley is P. oligandrum, which has already received various EU and US EPA approvals for use in agriculture and human medicine. The suitably timed and dosed application of P. oligandrum during the malting of barley was able to significantly suppress the spread of Fusarium contamination and the formation of mycotoxins. Furthermore, the treatment resulted in enhanced barley malt quality and lower mycotoxin content in beer brewed from it. Although the use of biological control agents in the malting process requires the consent of the regulatory authorities, P. oligandrum is such a promising tool that major maltsters are currently seeking its registration in the Czech Republic.

Author Contributions

All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The study was supported by the Grant Agency of the Czech Republic within the project 22-13745S.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Conflicts of Interest

The authors declare that there is no conflict of interest.

Appendix A. Supplemental Tables

Table
Table A1. Brewing malt quality (BMQ) parameters of barley malt (BM) and the effect of the addition of P. oligandrum (M1, 1 × 106 oospores/kg barley) oospores in different malting stages (1st STW—first steeping water, 2nd STW—second steeping water, GE—germination).
Table A1. Brewing malt quality (BMQ) parameters of barley malt (BM) and the effect of the addition of P. oligandrum (M1, 1 × 106 oospores/kg barley) oospores in different malting stages (1st STW—first steeping water, 2nd STW—second steeping water, GE—germination).
SampleBMQProteinExtractREKIDPAFAFriabilityBG
BM3.19.08.59.09.01.01.81.83.9
BM + M1 (1st STW)3.59.05.89.09.01.52.62.94.7
BM + M1 (2nd STW)4.19.08.59.09.02.83.91.72.0
BM + M1 (GE)3.59.06.99.09.01.52.12.35.4
RE—Relative extract at 45 °C, KI—Kolbach index, DP—Diastatic power, AFA—Apparent final attenuation, BG—Beta-glucans in wort.
Table
Table A2. Brewing malt quality (BMQ) parameters of barley malt (BM) and the effect of treatment with different concentrations of P. oligandrum M1 (M1) and P. oligandrum 00X48 (00X48) oospores, added in the second steeping water (number of oospores/kg barley).
Table A2. Brewing malt quality (BMQ) parameters of barley malt (BM) and the effect of treatment with different concentrations of P. oligandrum M1 (M1) and P. oligandrum 00X48 (00X48) oospores, added in the second steeping water (number of oospores/kg barley).
SampleBMQProteinExtractREKIDPAFAFriabilityBG
BM3.19.08.59.09.01.01.81.83.9
BM + 1 × 106 M1/kg 4.19.08.59.09.02.83.91.72.0
BM + 1 × 105 M1/kg3.49.05.39.09.01.22.64.07.2
BM + 1 × 106 00X48/kg4.18.94.77.65.52.14.56.49.0
BM + 1 × 105 00X48/kg3.19.06.99.09.01.01.03.24.6
RE—Relative extract at 45 °C, KI—Kolbach index, DP—Diastatic power, AFA—Apparent final attenuation, BG—Beta-glucans in wort.

References

  1. Timmusk, S.; Nevo, E.; Ayele, F.; Noe, S.; Niinemets, U. Fighting Fusarium pathogens in the era of climate change: A conceptual approach. Pathogens 2020, 9, 419. [Google Scholar] [CrossRef]
  2. McKee, G.; Cowger, C.; Dill-Macky, R.; Friskop, A.; Gautam, P.; Ransom, J.; Wilson, W. Disease management and estimated effects on DON (Deoxynivalenol) contamination in Fusarium infested barley. Agriculture 2019, 9, 155. [Google Scholar] [CrossRef] [Green Version]
  3. Mastanjevic, K.; Krstanovic, V.; Mastanjevic, K.; Sarkanj, B. Malting and brewing industries encounter Fusarium spp. related problems. Fermentation 2018, 4, 3. [Google Scholar] [CrossRef] [Green Version]
  4. Shokribousjein, Z.; Deckers, S.M.; Gebrues, K.; Lorgouilloux, Y.; Baggerman, G.; Verachtert, H.; Delcour, J.A.; Etienne, P.; Rock, J.-M.; Michiels, C.; et al. Hydrophobins, beer foaming and gushing. Cerevisia 2011, 35, 85–101. [Google Scholar] [CrossRef] [Green Version]
  5. Habler, K.; Geissinger, C.; Hofer, K.; Schuler, J.; Moghari, S.; Hess, M.; Gastl, M.; Rychlik, M. Fate of Fusarium toxins during brewing. J. Agric. Food Chem. 2017, 65, 190–198. [Google Scholar] [CrossRef] [Green Version]
  6. Wolf-Hall, C.E. Mould and mycotoxin problems encountered during malting and brewing. Int. J. Food Microbiol. 2007, 119, 89–94. [Google Scholar] [CrossRef]
  7. Van Nierop, S.; Rautenbach, M.; Axcell, B.; Cantrell, I. The impact of microorganisms on barley and malt quality—A review. Am. Soc. Brew. Chem. 2006, 64, 69–78. [Google Scholar] [CrossRef]
  8. Ksieniewicz-Wozniak, E.; Bryla, M.; Waskiewicz, A.; Yoshinari, T.; Szymczyk, K. Selected trichothecenes in barley malt and beer from Poland and an assessment of dietary risks associated with their consumption. Toxins 2019, 11, 715. [Google Scholar] [CrossRef] [Green Version]
  9. Tima, H.; Bruckner, A.; Mohacsi-Farkas, C.; Kisko, G. Fusarium mycotoxins in cereals harvested from Hungarian fields. Food Addit. Contam. Part B 2016, 9, 127–131. [Google Scholar] [CrossRef]
  10. Tricase, C.; Amicarelli, V.; Lamonaca, E.; Rana, R. Economic analysis of the barley market and related uses. Grasses Food Feed. 2018, 10, 25–46. [Google Scholar] [CrossRef] [Green Version]
  11. Yawson, D.; Adu, A.; Armah, F. Impacts of climate change and mitigation policies on malt barley supplies and associated virtual water flows in the UK. Sci. Rep. 2020, 10, 376. [Google Scholar] [CrossRef] [Green Version]
  12. Wilson, W.; McKee, G.; Nganje, W.; Dahl, B.; Bangsund, D.; Economic Impact of USWBSI’s Impact on Reducing FHB. Agribusiness and Applied Economics No. 774, Sept 2017. 2017. Available online: http://ageconsearch.umn.edu/record/264672 (accessed on 21 February 2023).
  13. Ng, C.; Postulkova, M.; Matoulkova, D.; Psota, V.; Hartman, I.; Branyik, T. Methods for suppressing Fusarium infection during malting and their effect on malt quality. Czech J. Food Sci. 2021, 39, 340–359. [Google Scholar] [CrossRef]
  14. Ayed, F.; Daami-Remadi, M.; Jabnoun-Khiareddine, H.; El Mahjoub, M. In vitro and in vivo evaluation of some biofungicides for potato Fusarium wilt biocontrol. Int. J. Agric. Res. 2007, 2, 282–288. [Google Scholar] [CrossRef] [Green Version]
  15. Benhamou, N.; Belanger, R.R.; Rey, P.; Tirilly, Y. Oligandrin, the elicitin-like protein produced by Pythium oligandrum, induces systemic resistance to Fusarium crown and root rot in tomato plants. Plant Physiol. Biochem. 2001, 39, 681–698. [Google Scholar] [CrossRef]
  16. Postulkova, M.; Rezanina, J.; Fiala, J.; Ruzicka, M.C.; Dostalek, P.; Branyik, T. Suppression of fungal contamination by Pythium oligandrum during malting of barley. J. Inst. Brew. 2018, 124, 336–340. [Google Scholar] [CrossRef] [Green Version]
  17. Ng, C.; Pernica, M.; Yap, J.; Belakova, S.; Vaculova, K.; Branyik, T. Biocontrol effect of Pythium oligandrum on artificial Fusarium culmorum infection during malting of wheat. J. Cereal Sci. 2021, 100, 103258. [Google Scholar] [CrossRef]
  18. Magalhaes, P.; Dostalek, P.; Cruz, J.; Guidol, L.; Barros, A. The impact of a xanthohumol-enriched hop product on the behavior of xanthohumol and isoxanthohumol in pale and dark beers: A pilot scale approach. J. Inst. Brew. 2008, 114, 246–256. [Google Scholar] [CrossRef]
  19. Havelka, Z.; Belakova, S.; Bohata, A.; Hartman, I.; Kabelova, H.; Kriz, P.; Benesova, K.; Dienstbier, M.; Bartos, P.; Spatenka, P. The effect of low-temperature plasma discharge on mycotoxin content in barley malt. Kvas. Prumys 2019, 65, 158–165. [Google Scholar] [CrossRef]
  20. Belakova, S.; Benesova, K.; Caslavsky, J.; Svoboda, Z.; Mikulikova, R. The occurrence of the selected Fusarium mycotoxins in Czech malting barley. Food Control 2014, 37, 93–98. [Google Scholar] [CrossRef]
  21. Psota, V.; Kosar, K. Malting quality index. Kvas. Prum. 2002, 48, 142–148. [Google Scholar] [CrossRef] [Green Version]
  22. EBC Analytica. Available online: https://brewup.eu/ebc-analytica (accessed on 23 February 2023).
  23. Spanic, V.; Zdunic, Z.; Drezner, G.; Sarkanj, B. The pressure of Fusarium disease and its relation with mycotoxins in the wheat grain and malt. Toxins 2019, 11, 198. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Oliveira, P.M.; Brosnan, B.; Furey, A.; Coffey, A.; Zannini, E. Lactic acid bacteria bioprotection applied to the malting process. Part I: Strain characterization and identification of antifungal compounds. Food Control 2015, 51, 433–443. [Google Scholar] [CrossRef]
  25. European Commission. Report from the Commission to the European Parliament and the Council. Available online: https://ec.europa.eu/food/system/files/2020-05/pesticides_sud_report-act_2020_en.pdf (accessed on 23 February 2023).
  26. Lowe, D.P.; Arendt, E.K. The use and effects of lactic acid bacteria in malting and brewing with their relationship to antifungal activity, mycotoxins and gushing: A review. J. Inst. Brew. 2004, 110, 163–180. [Google Scholar] [CrossRef]
  27. Mauch, A.; Dal Bello, F.; Coffey, Z.; Arendt, E. The use of Lactobacillus brevis PS1 to in vitro inhibit the outgrowth of Fusarium culmorum and other common Fusarium species found on barley. Int. J. Food Microbiol. 2010, 141, 116–121. [Google Scholar] [CrossRef]
  28. Boivin, P.; Malanda, M. Inoculation by Geotrichum candidum during Malting of Cereals or Other Plants. U.S. Patent 5,955,070, 21 September 1999. [Google Scholar]
  29. Anderson, H.E.; Santos, I.C.; Hildenbrand, Z.L.; Schug, K.A. A review of the analytical methods used for beer ingredient and finished product analysis and quality control. Anal. Chim. Acta 2019, 1085, 1–20. [Google Scholar] [CrossRef]
  30. Takenaka, S. Studies on biological control mechanisms of Pythium oligandrum. J. Gen. Plant Pathol. 2015, 81, 466–469. [Google Scholar] [CrossRef]
  31. Benhamou, N.; Rey, P.; Picard, K.; Tirilly, Y. Ultrastructure and cytochemical aspects of the interaction between the mycoparasite Pythium oligandrum and soilborne plant pathogens. Phytopathology 1999, 89, 506–517. [Google Scholar] [CrossRef] [Green Version]
  32. Krstanovic, V.; Habschied, K.; Mastanjevic, K. Research of malting procedures for winter hard wheat varieties-part I. Foods 2021, 10, 52. [Google Scholar] [CrossRef] [PubMed]
  33. Jin, Z.; Solanki, S.; Ameen, G.; Gross, T.; Poudel, R.; Borowicz, P.; Brueggeman, R.; Schwarz, P. Expansion of internal hyphal growth in Fusarium head blight-infected grains contributes to the elevated mycotoxin production during the malting process. Mol. Plant-Microbe Interact. 2021, 34, 793–802. [Google Scholar] [CrossRef] [PubMed]
  34. Oliveira, P.; Mauch, A.; Jacob, F.; Arendt, E.K. Impact of Fusarium culmorum-infected barley malt grains on brewing abd beer quality. J. Am. Soc. Brew. Chem. 2012, 70, 186–194. [Google Scholar]
  35. Krstanovic, V.; Mastanjevic, K.; Velic, N.; Pleadin, J.; Persi, N.; Spanic, V. The influence of Fusarium culmorum contamination level on deoxynivalenol content in wheat, malt and beer. Rom. Biotechnol. Lett. 2015, 20, 10901–10910. [Google Scholar]
  36. Janssen, E.; Liu, C.; van der Fels-Klerx, H. Fusarium infection and trichothecenes in barley and its composition with wheat. World Mycotoxin J. 2018, 11, 33–46. [Google Scholar] [CrossRef]
  37. Gibson, T.; Solah, V.; Holmes, M.; Taylor, H. Diastatic power in malted barley: Contributions of malt parameters to its development and the potential of barley grain beta-amylase to predict malt diastatic power. J. Inst. Brew. 1995, 101, 277–280. [Google Scholar] [CrossRef]
  38. Yousif, A.; Evans, D. Changes in malt quality during production in two commercial malt houses. J. Inst. Brew. 2020, 126, 233–252. [Google Scholar] [CrossRef]
  39. Krstanovic, V.; Mastanjevic, K.; Nedovic, V.; Mastanevic, K. The influence of wheat malt quality on final attenuation limit of wort. Fermentation 2019, 5, 89. [Google Scholar] [CrossRef] [Green Version]
Table
Table 1. Summary of the brewing process.
Table 1. Summary of the brewing process.
Brewing StageExperimental Conditions
MashingMalt grist (5.3 kg) was mixed with 25 L water and a single decoction process was carried out. First rest of the mash was at 52 °C and the second rest was at 73 °C
LauteringTraditional lautering was carried out to separate the spent grains and wort. A total of 35 L sweet-wort was collected including one sparging with hot water (70 °C)
Wort boilingSaaz hop pellets (70 g, T90, Bohemia Hop, Czech Republic) were added to the boiling wort. A total of 60 min boiling was performed and approximately 30 L were obtained after the process
Wort coolingTrub was removed and the wort was cooled to between 14–15 °C prior to yeast pitching
FermentationBottom fermentation was carried out at 12 °C for 5 days
MaturationMaturation at 1 °C for 21 days
Table
Table 2. Relative F. culmorum contamination, deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) content in barley malt (BM) samples treated with P. oligandrum strain M1 (M1) oospores added at different malting stages (1st STW—first steeping water, 2nd STW—second steeping water, GE—germination). Each addition contained 1 × 106 oospores/kg barley.
Table 2. Relative F. culmorum contamination, deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) content in barley malt (BM) samples treated with P. oligandrum strain M1 (M1) oospores added at different malting stages (1st STW—first steeping water, 2nd STW—second steeping water, GE—germination). Each addition contained 1 × 106 oospores/kg barley.
SampleRelative F. culmorum ContaminationDON (μg/kg)D3G (μg/kg)Decrease in Total DON Concentration
BM100% a701 ± 131 a2260 ± 445 a
BM + M1 (1st STW)65% ± 6% b501 ± 94 b1470 ± 290 b33%
BM + M1 (2nd STW)41% ± 1% c444 ± 83 b1660 ± 327 ab29%
BM + M1 (GE)45% ± 2% d467 ± 87 b1780 ± 351 ab24%
a–d values: Values with different letters in columns are significantly different (p < 0.05).
Table
Table 3. Relative F. culmorum contamination, deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) in barley malt (BM) samples treated with different concentrations of P. oligandrum M1 (M1) and P. oligandrum 00X48 (00X48) oospores added in the second steeping water (number of oospores/kg barley).
Table 3. Relative F. culmorum contamination, deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) in barley malt (BM) samples treated with different concentrations of P. oligandrum M1 (M1) and P. oligandrum 00X48 (00X48) oospores added in the second steeping water (number of oospores/kg barley).
SampleRelative F. culmorum ContaminationDON (μg/kg)D3G (μg/kg)Decrease in Total DON Concentration
BM100% a701 ± 131 a2260 ± 445 a
BM + 1 × 106 M1/kg41% ± 1% b444 ± 83 b1660 ± 327 ab29%
BM + 5 × 105 M1/kg39% ± 3% b603 ± 113 ab1730 ± 341 ab22%
BM + 1 × 105 M1/kg41% ± 6% b629 ± 118 ab1660 ± 327 ab23%
BM + 1 × 106 00X48/kg52% ± 1% c587 ± 110 ab1540 ± 303 b28%
BM + 5 × 105 00X48/kg52% ± 1% c624 ± 117 ab1680 ± 331 ab22%
BM + 1 × 105 00X48/kg52% ± 1% c670 ± 125 ab1610 ± 317 ab23%
a–c values: Values with different letters in columns are significantly different (p < 0.05).
Table
Table 4. Malt quality parameters of barley malt (BM) and the effect of the treatment with P. oligandrum M1 (M1) oospores added at different malting stages (1st STW—first steeping water, 2nd STW—second steeping water, GE—germination). Each addition contained 1 × 106 oospores/kg barley.
Table 4. Malt quality parameters of barley malt (BM) and the effect of the treatment with P. oligandrum M1 (M1) oospores added at different malting stages (1st STW—first steeping water, 2nd STW—second steeping water, GE—germination). Each addition contained 1 × 106 oospores/kg barley.
SampleViscosity (mPa∙s)AFA (%)DP (u∙WK)FAN (mg/L)BG (mg/L)Extract (%)RE (%)KI (%)Friability (%)
BM1.442 ± 0.01079.3 ± 1.0220 ± 10183 ± 20195 ± 4982.9 ± 0.340.5 ± 1.345.4 ± 6.479.7 ± 2.4
BM + M1 (1st STW)1.445 ± 0.01079.6 ± 1.0225 ± 10189 ± 21180 ± 4582.4 ± 0.341.0 ± 1.445.5 ± 6.480.7 ± 2.4
BM + M1 (2nd STW)1.442 ± 0.01080.0 ± 1.0234 ± 10191 ± 21216 ± 5482.6 ± 0.341.9 ± 1.446.5 ± 6.580.7 ± 2.4
BM + M1 (GE)1.440 ± 0.01079.4 ± 1.0225 ± 10180 ± 20168 ± 4282.6 ± 0.340.3 ± 1.343.2 ± 6.080.1 ± 2.4
AFA—Apparent final attenuation, DP—Diastatic power, FAN—Free amino nitrogen, BG—Beta-glucans in wort, RE—Relative extract at 45°C, KI—Kolbach index. Note: Values in columns are not statistically different (p > 0.05).
Table
Table 5. Malt quality parameters of barley malt (BM) and the effect of the treatment with different concentrations of P. oligandrum M1 (M1) and P. oligandrum 00X48 (00X48) oospores added in the second steeping water (number of oospores/kg barley).
Table 5. Malt quality parameters of barley malt (BM) and the effect of the treatment with different concentrations of P. oligandrum M1 (M1) and P. oligandrum 00X48 (00X48) oospores added in the second steeping water (number of oospores/kg barley).
Sample Viscosity (mPa∙s)AFA (%)DP (u∙WK)FAN (mg/L)BG (mg/L)Extract (%)RE (%)KI (%)Friability (%)
BM1.442 ± 0.010 a79.3 ± 1.0 a220 ± 10 a183 ± 20 a195 ± 49 a82.9 ± 0.3 a40.5 ± 1.3 a45.4 ± 6.4 a79.7 ± 2.4 a
BM + 1 × 106 M1/kg1.450 ± 0.010 a80.1 ± 1.0 a238 ± 10 a187 ± 21 a231 ± 58 a82.9 ± 0.3 a41.7 ± 1.4 a46.6 ± 6.5 a79.6 ± 2.4 a
BM + 1 × 105 M1/kg1.446 ± 0.010 a79.6 ± 1.0 a222 ± 10 a199 ± 22 a133 ± 33 ab82.3 ± 0.3 b45.0 ± 1.5 b47.8 ± 6.7 a81.6 ± 2.4 a
BM + 1 × 106 00X48/kg1.431 ± 0.010 a80.3 ± 1.0 a231 ± 10 a204 ± 22 a78 ± 20 b82.2 ± 0.3 b48.9 ± 1.6 c50.2 ± 7.0 a83.7 ± 2.5 a
BM + 1 × 10500X48/kg1.444 ± 0.010 a78.5 ± 1.0 a220 ± 10 a186 ± 20 a183 ± 456 a82.6 ± 0.3 ab42.3 ± 1.4 ab47.5 ± 6.7 a80.9 ± 2.4 a
AFA—Apparent final attenuation, DP—Diastatic power, FAN—Free amino nitrogen, BG—Beta-glucans in wort, RE—Relative extract at 45 °C, KI—Kolbach index. a–c values: Values with different letters in columns are significantly different (p < 0.05).
Table
Table 6. Quality parameters of beers brewed from barley malt (BM) with and without P. oligandrum M1 (M1) oospores treatment added in the second steeping water (1 × 106 oospores M1/kg barley). The original extract of both beers was 13.1 ± 0.06%.
Table 6. Quality parameters of beers brewed from barley malt (BM) with and without P. oligandrum M1 (M1) oospores treatment added in the second steeping water (1 × 106 oospores M1/kg barley). The original extract of both beers was 13.1 ± 0.06%.
SampleColor (EBC)AE (%)RE (%)Alcohol (%)AA (%)RA (%)pH
BM10.6 ± 0.5 a2.60 ± 0.02 a4.62 ± 0.06 a4.40 ± 0.04 a80.1 ± 0.6 a64.7 ± 1.2 a4.47 ± 0.05 a
BM + M1 11.2 ± 0.5 b2.51 ± 0.02 b4.54 ± 0.06 a4.43 ± 0.04 a80.7 ± 0.6 a65.2 ± 1.2 a4.49 ± 0.05 a
AE—Apparent extract, RE—Real extract, AA—Apparent attenuation, RA—real attenuation. a–b values: Values with different letters in columns are significantly different (p < 0.05).
Table
Table 7. Deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) in beer prepared from barley malt (BM) with and without P. oligandrum M1 (M1) treatment added in the second steeping water (1 × 106 oospores M1/kg barley).
Table 7. Deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) in beer prepared from barley malt (BM) with and without P. oligandrum M1 (M1) treatment added in the second steeping water (1 × 106 oospores M1/kg barley).
SampleDON (μg/kg)D3G (μg/kg)
BM104 ± 19205 ± 40
BM + M1 77 ± 15168 ± 35
Note: Values in columns are not statistically different (p > 0.05).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Ng, C.A.; Pernica, M.; Litvanova, K.; Kolouchova, I.; Branyik, T. Biocontrol Using Pythium oligandrum during Malting of Fusarium-Contaminated Barley. Fermentation 2023, 9, 257. https://doi.org/10.3390/fermentation9030257

AMA Style

Ng CA, Pernica M, Litvanova K, Kolouchova I, Branyik T. Biocontrol Using Pythium oligandrum during Malting of Fusarium-Contaminated Barley. Fermentation. 2023; 9(3):257. https://doi.org/10.3390/fermentation9030257

Chicago/Turabian Style

Ng, Carlo Antonio, Marek Pernica, Katerina Litvanova, Irena Kolouchova, and Tomas Branyik. 2023. "Biocontrol Using Pythium oligandrum during Malting of Fusarium-Contaminated Barley" Fermentation 9, no. 3: 257. https://doi.org/10.3390/fermentation9030257

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop