Impact of kefir yeasts on Fusarium graminearum growth and production of deoxynivalenol

doi:10.21203/rs.3.rs-4707402/v1

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Impact of kefir yeasts on Fusarium graminearum growth and production of deoxynivalenol

https://doi.org/10.21203/rs.3.rs-4707402/v1

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The kefir granules are a microbial consortium with numerous properties, among which their antifungal action has been reported. The aim of this research was to evaluate the antifungal capacity of yeasts isolated from kefir against Fusarium graminearum for its possible application as biocontrol agents. The inhibition of fungal growth was analyzed, according to dual tests, and the production of volatile organic compounds. The inhibition of the production of the mycotoxin deoxynivalenol was also analized. The six isolates studied belonged to the species Geotrichum candidum (2), Saccharomyces cerevisiae (1), Pichia kudriavzevii (2) and Pichia membranifaciens (1). They were isolated from kefir and identified in a previous work. The yeasts tested produced a reduction in the growth of F. graminearum in all assays. In particular, three of the isolates, 1 belonging to the species S. cerevisiae and 2 to the species P. kudriavzevii, generated volatile organic compounds that significantly inhibited the growth of the fungus in a 49.61, 51.46 and 49.61% respectively. The yeasts produced an inhibition of the mycotoxin deoxynivalenol correlated with a reduction in the growth of the fungus. The isolates of Geotrichum candidum (Gc30) and Pichia kudriavzevii (Pk27, PK20) significantly reduced the deoxynivalenol content of F graminearum by 86, 89 and 88%, and the range of growth inhibition in the assay was 16 to 62%. The data obtained about yeasts of novel origin are promising for their possible use as fungal biocontrol agents in foods as an alternative to agrochemicals

Kefr yeasts

Fusarium graminearum

Antifungal capacity

Growth inhibition

Deoxynivalenol mycotoxin

The Fusarium genus is of global relevance since it contaminates a wide range of agricultural products used for human and animal consumption, producing economic losses and food safety threats due to the production of mycotoxins (Dumitru et al. 2020; Hoppe et al. 2024). Fusarium, Aspergillus and Penicillum are the main genera of mycotoxigenic fungi associated with the contamination of cereals (Deligeorgakis et al. 2023; Habschied et al. 2021). Fungal contamination can occur both in the field before harvest, which is closely related to environmental factors, and in the post-harvest due to inadequate storage or processing (Sadik et al. 2023). Different species of Fusarium are phytopathogens distributed in the crop soil, so their infection is associated with pre-harvest (Pitt and Hocking 2022; Del Palacio et al. 2023). Fusarium spp causes epidemics with high yield loss and reduced quality of cereals mainly due to contamination of grains with mycotoxins (Matny et al. 2015). The predominant species is Fusarium graminearum in most regions of the world, suggesting that it is more adapted to environmental variability (Osborne and Stein 2007). The mycotoxins most associated with F. graminearum are deoxynivalenol (DON) and nivalenol (NIV), which have several adverse effects on animal and human health, such as gastrointestinal disorders and severe depression of the immune system, promoting the development of secondary infections (Del Palacio et al. 2023).

The biological control of fungi is an alternative and complementary technique to the use of agrochemicals, which are harmful to humans, animals and ecosystems when their use is excessive and prolonged (Nguyen et al. 2017; Chulze 2023). Among the microorganisms evaluated as biological control agents, there are yeasts, bacteria, and filamentous fungi (Nešić et al. 2021; Podgórska-Kryszczuk 2023). The use of yeasts has advantages over other microorganisms, since their nutritional requirements are simple, they allow the colonization of a wide range of substrates and at the same time they do not produce allergenic spores, mycotoxins or antibiotics (Freimoser et al. 2019; Hernandez-Montiel et al. 2021).The main approach in mycotoxin control is the elimination or reduction of the inoculum of the mycotoxigenic fungus in the previous and subsequent stages of harvest (Neme and Mohammed 2017). Another strategy consists of the mycotoxin decontamination in the matrix, by physical, chemical or biological methods which eliminate, destroy or modify the structure of mycotoxins (Taheur et al. 2019; CAST 2003).

Inhibition by antagonistic microorganisms includes different antifungal mechanisms, the predominant one being the emission of volatile organic compounds (VOCs), others are competition for nutrients and space, parasitism, antibiotics, induction of host resistance and biofilm formation (Zhang et al. 2020; Zhao et al. 2022). VOCs released by yeast had the capacity to reduce the germination of spores, the growth of mycelium and the production of mycotoxins, which can be used in preventive food safety strategies. Each antagonist can produce a wide variety of VOCs. VOCs can also modulate the expression of genes involved in the biosynthesis of mycotoxins, so evaluating their effect on the level of gene expression is key in the biosynthetic pathway, as was studied for ochratoxin (Chang et al. 2015). The main VOCs emitted by yeasts used in biological control are alcohols (ethanol, 3-methylbutan-1-ol, and 2-phenylethanol) and esters (ethyl acetate and 3-methylbutylacetate) (Farbo et al. 2018; Hua et al. 2014).

Kefir granules are a microbial consortia with numerous properties, among which the antifungal action has been reported. The granules are composed of lactic acid bacteria (LAB), acetic acid bacteria and yeast (Moure et al. 2023). The antimicrobial properties of the granules result from the action of the microorganisms synergistically or individually. Among the yeasts isolated from kefir, there are species belonging to the genera Candida, Hanseniaspora, Issatchenkia, Kazachstania, Kluveromyces, Lanchancea, Pichia, Saccharomyces, Torulospora, Yarrowia, Zygosacharomyces among others (González-Orozco et al. 2022; Güzel-Seydim et al. 2021; Lynch et al. 2021; Moretti et al. 2022). Different investigations have reported high inhibitions of Fusarium sp. and Aspergillus spp. versus fermented kefir granules (Ismaiel et al., 2011; Tenorio-Salgado et al., 2021; Kawtharani et al. 2020). Zhimo et al. (2020) observed variable inhibition of Penicillum spp on fruits when confronted with different yeasts from kefir. Several authors determined that co-cultivating Fusarium graminearum with yeasts had an effect on growth and DON production (Kawtharani et al. 2020).

Although various authors have focused on the study of antifungal properties of kefir yeasts, the antecedents are still scarce. The objective of this work was to analyze the potential antifungal effect of kefir yeasts against Fusarium graminearum growth and DON production as a contribution to the search of a more environmentally friendly alternative to agrochemicals.

Biological material and inocula

The inocula of F.graminearum were prepared from 6-mm plugs cut out from the margin of a 7-day-old colony growing on Petri dishes with PDA growing medium (2% potato-agar) at 26ºC. The fungus was maintained as mycelium in tubes with 2% potato agar under a layer of mineral oil at 4ºC, until use. The yeasts used in this research were selected previously for their high effect in reducing conidial germination as an indicator of fungal inhibition according to Moure et al. (2023). The inocula of 6 yeast isolates (2 belonging to Geotrichum candidum: Gc; 1 to Saccharomyces cerevisiae: Sc; 2 to Pichia kudriavzevii: Pk and 1 to Pichia membranifaciens: Pm) were obtained by raising from the − 80°C strain to YPD broth (1% yeast extract-2% peptone-2% D-glucose) and incubating for 48 h at 28°C. Additional successive raising were carried out and the third broth was used as yeast inoculum, called fresh culture. The viability of cells was corroborated by growth in plate. Inocula were prepared by adjusting the suspensions to 10⁴conidia/ml using a Neubauer chamber.

Dual culture assay

The yeast inhibitory effect on F. graminearum was carried out in dual cultures, which consisted of the simultaneous growth of both in 90 mm diameter Petri dishes with PDA culture medium. The assay was carried out in 90-mm diameter Petri dishes with PDA, inoculating 10 µl of a 10⁴conidial/ml suspension of F.graminearum 2 cm away from the border of the plate and a 2-day-old yeast culture was streaked 5 cm from the fungal inoculum. Controls consisted only of the fungal inoculum. The assay was carried out in two independent repetitions, and each treatment was carried out in triplicate. Incubation was at 30°C in the dark and the fungal growth was periodically observed for 14 days (Pantelides et al. 2015).

Volatile organic compounds

The production of antifungal volatile organic compounds (VOCs) was determined with the double plate method according to Vero et al. (2013). The assay consisted of facing two Petri dishes − 5 cm diameter containing PDA- to form a chamber. The upper plate was surface inoculated with the corresponding yeast and the bottom plate was spotted with 10 µl of a 10⁴conidial/ml suspension of the fungus. The plates were sealed together with three turns of Parafilm®, incubated at 30°C in the dark, and the fungal diameters were periodically determined. The control consisted only of the fungal inoculum. The assay was carried out in two independent repetitions, and each treatment was carried out in triplicate.

Effect on growth and the production of deoxynivalenol

The effect of yeast on DON production by F. graminearum was analyzed in Petri dishes with PDA culture medium in triplicate for each isolate. The yeasts extended on the surface and F. graminearum in the center of the plate. The plates were incubated at 28°C for 14 days, measuring the diameter daily with two measurements perpendicular to each other for each colony and at the end of the incubation time were stored at -20°C until their analysis (Vero et al. 2013).

Deoxynivalenol extraction

DON extraction was carried out using the total cultivation medium of Petri dishes which was placed in an Erlenmeyer flask with 125 mL of acetonitrile:acetylacetate:water (50:41:9) with agitation for 1 h at 300 rpm. The clean-up of de samples was performed with a column packed with charcoal:alumina:celite (0.7,0.5:0.3) and dried in Rotavap®. 100 µl of trifluoroacetic anhydride (TFAA) was added to the samples for derivatization, and 30 mg of sodium bicarbonate (NaHCO3) to destroy remains of the derivatizer that had not reacted. The samples were vortexed for 10 s, placed in a thermostatic bath at 80°C for 30 min, and placed at room temperature. The residue obtained was diluted in 100 µl of a solution made up of 2-amino-5- chlorobenzophenone (ACBP) in toluene at a concentration of 10 µg/ml, which was used as an internal standard. The samples were stirred for 10 seconds and 1000 µl of water were added to remove excess reactants and the aqueous and organic phases were separated. The toluene layer was transferred to a vial with anhydrous sodium sulfate to eliminate the water (Alvarez et al. 2009).

Deoxynivalenol quantification

DON detection was carried out with a Shimadzu GC17 gas-liquid chromatograph with an electron capture detector (ECD), with an RX-5MS capillary column (30 m; 0.25 mm x 0.25 µm). The injector temperature was 250°C and the detector temperature was 300°C. A temperature program that consisted of 2 min at 90°C, and then increased from 90 to 275°C (2 min) at 23°C per minute and from 275 to 290°C (5 min) at 30°C per minute was used. Nitrogen was used as auxiliary gas and carrier. The injection volume was 2 µl and the running time per sample was 27 min. For data analysis, a calibration curve was used between the standard toxin retention time (Sigma-Aldrich, USA) and the internal standard. The concentration range measured was between 50 and 400 pg. The calibration curve was constructed by graphing the relative area (AR) (relationship of the toxin area and the internal standard, ACBP) against the concentration at each point and the linearity range was determined using a regression curve. The results obtained were multiplied by the corresponding extraction and dilution factors of the sample and were expressed in µg/g (Alvarez et al. 2009).

Statistical analysis

The statistical analysis of the data (ANOVA test) was carried out employing STATISTICA 7 software (StatSoft).

Dual culture assay

When evaluating the inhibitory behavior of the yeasts on F. graminearum growing in the test of dual culture assays in Petri dishes, inhibition of the fungus was observed when both microorganisms were close, due to the weakening of the fungal mycelium. Measurements were carried out daily during the 14 days of assay, and towards the end of the test all yeasts maintained their inhibition capacity, except for the isolate P .membranifaciens − 9A, which was completely covered by F. graminearum on day 12.Various authors observed a similar behavior for Aspergillus tubigensis against Candida zeylanoides, C. sake, Cryptococcus magnusy, Aureobasidium pullulans and for Botrytis cinerea against Pichia membranifaciens (Masih and Paul 2002; Pantelides et al. 2015).The antifungal capacity of the isolates of G. candidum and P. Kudriavzevii has been previously reported. Kumar et al. (2017) detected a clear zone of inhibition in A. parasiticus NRRL299 when was cultivated with G. candidum on cereals, while Choińska et al. (2020) found that isolates of P. kudriavzeviiais in rye grains could inhibit the growth of Fusarim sp., Mucor sp., Botrytis cinerea, Penicillium sp. and A. fumigatus. The Fig. 1 shows the co-culture of F graminearum with Pichia kudriavzevii as representative of the assay.

Volatile organic compounds

When analyzing the generation of VOCs released in chambers created between the two facing Petri dishes by coincubation of yeasts and F. graminearum without contact with each other, it was observed that the growth rate of F. graminearum was significantly reduced by all isolates in a 24–51% range (Table 1). In particular, three of the isolates, 1 belonging to the species S. cerevisiae and 2 to the species P. kudriavzevii, generated volatile organic compounds that significantly inhibited the growth of the fungus by 49.61, 51.46 and 49.61% respectively.

Table 1

Effect of volatile organic compounds generated by yeasts on the growth of *Fusarium graminearum*
Treatment	Growth rate % Growth
Treatment	(mm/h)	inhibition
Control	0,754^a
Gc 30	0,568^b	24,67
Gc 31	0,397^c	47,35
Sc 9B	0,380^c	49,61
Pm 9A	0,504^b,c	34
Pk 20	0,380^c	49,61
Pk 27	0,366^c	51,46
Control: Fusarium graminearum; Gc: Geotrichum candidum; Sc: Saccharomyces cerevisiae; Pk: Pichia kudriavzevii; Pm: Pichia membranifaciens. Different letters (per column) indicate significant differences determined by ANOVA and LSD p < 0.05

HERE Table 1

Our results showed that the tested yeasts generated volatile organic compounds that inhibited the growth of F. graminearum in a wide range. The results obtained for G. candidum were similar to what was observed by Kawtharani et al. (2020), who associated the ability to reduce growth in Fusarium species by this yeast to the release of phenylacetic acid (PLA) to the medium. Choinska et al. (2020) studied the VOCs released by an isolate of P. kudriavzevi with the capacity to inhibit the genera Penicillium, Fusarium, Aspergillus and Mucor, and among the compounds detected they found 2-phenylethanol and its acetates, which had demonstrated antifungal capacity (Hua et al. 2014; Huang et al. 2020; Liu et al. 2014). Other authors, when analyzing the volatiles generated by an isolate of G. candidum with high inhibitory capacity on the phytopathogen Rhizoctonia solani, detected 2-phenylethanol, isoamyl acetate and ethyl isovalerate as the predominant volatiles (Mookherjee et al. 2017). Abdel-Kareem et al. (2019) related the inhibitory capacity of S. cerevisiaea against A. flavus, with the compounds 4-hydroxyphenylethanol, 4,4-dimethyloxazole and dioctyl ester of 1,2-benzenedicarboxylic acid released by the yeast. Fiori et al. (2014) detected the inhibition of Aspergillus carbonarius sporulation by VOCs generated by Candida spp. and Lachancea spp.

Given that the inhibitory effect of yeasts by emission of volatiles on the growth or sporulation of filamentous fungi, particularly under conditions of oxygen limitation as occurs in grain storage, the results observed could represent a valuable tool for postharvest biocontrol (Huang et al. 2011). Yeast VOCs represent a promising tool for containing the growth and development of mycotoxigenic fungi, and a valuable aid in ensuring food safety and quality. Likewise, yeast VOCs can also modulate the expression of genes involved in OTA biosynthesis (Chang et al. 2015). Therefore, VOCs released by selected yeasts deserve attention for their ability to reduce spore germination, mycelium growth, and mycotoxin production in preventive food safety strategies.

Effect on growth and on deoxynivalenol production

From the cultures in Petri dishes of F. graminearum co-cultured with each yeast, periodic measurements of the diameter of the colonies were made, and the growth rate was calculated. Once the incubation time was over, the mycotoxin was extracted from the whole cultures for subsequent quantification by GC-ECD.

F.graminearum reduced its growth when confronted with all the yeast isolates tested (Gc30, Gc31, Sc 9B, Pm9A, Pk27, Pk20) in a range of 34 to 62%. As seen in the Table 2, the isolates G. candidum 30, P. kudriavzevii 20 and 27 significantly reduced DON in a 86, 88 and 89% respectively in relation to the control, therefore an inhibitory effect on the production of the toxin is inferred. For the rest of the isolates (S. cerevisiae 9B, P. membranifaciens 9A and G.candidum 31 no significant differences in DON content were detected, even though the growth rate of F. graminearum was reduced. The isolate of G. candidum 31 increased the DON production in a 29% but inhibited the fungal growth (28%). The isolate G. candidum 30 showed a high DON inhibition (86,6%) but the growth inhibition was of 34,2%. The isolates of P. kudriavzevii showed the more efficient performance reducing 89% of DON production and 61% fungal growth (Pk27), and 88% DON production and 48% fungal growth (Pk20). These isolates also showed the more efficient performance in growth inhibition in the volatile compound assay.

Table 2

Inhibition of growth and DON production by *F. graminearum* against yeasts
Treatment	Growth rate (mm/h)	DON (µg/g)	% Growth inhibition	% DON inhibition
Control	0,892^a	0,8715^a
Gc 30	0,587^d	0,1163^c	34,2	86,66
Gc 31	0,638^d	1,1239^a	28,48	+ 28,96
Sc 9B	0,333^e	0,6684^a,b,c	62,67	23,31
Pm 9A	0,748^b	0,7622^a,b	16,15	12,55
Pk 27	0,343^e	0,0939^c	61,55	89,33
Pk 20	0,463^c	0,1017^b,c	48,10	88,34
Control: F. graminearum; Gc: Geotrichum candidum; Sc: Saccharomyces cerevisiae; Pk: Pichia kudriavzevii; Pm: Pichia membranifaciens. Different letters (per column) indicate significant differences, determined by ANOVA and LSD p < 0.05.

Armando et al. (2013) observed that the effect on DON production by F.graminearum co-cultured with S. cerevisiae depended on the isolate studied, and could be inhibited or enhanced. Other studies demonstrated that G. candidum, in addition to having the ability to reduce the growth of Fusarium spp., could reduce the production of associated toxins, which was related to the production of PLA (phenyllactic acid) by yeast (Kawtharani et al., 2020). Aspergillus spp have been studied more frequently than Fusarium spp with respect of yeast biological control. Among the yeasts tested for the control of Aspergillus spp, there are Saccharomyces spp. strains, S. cerevisiae (de Souza et al., 2021), Pichia anomala, Pichia kluyveri, and Hanseniaspora uvarum (Masoud et al. 2005), which inhibited both their growth and OTA production.

Killer yeast strains have the characteristic of secreting toxins lethal to other yeast cells and filamentous fungi. The production of killer toxins is one of the possible mechanisms by which yeasts control growth and toxin production in filamentous fungi. The killer phenotype was found for the first time in Saccharomyces cerevisiae but many other genera of yeasts also possess it. The killer yeasts have a wide field of application in pre- and post-harvest biocontrol of fungal plant pathogens. As an example A. carbonarius and A. niger are responsible for grape rot and for the accumulation of ochratoxin A, a potent nephrotoxic and carcinogenic compound in grape products. These fungal pathogens could be controlled by killer yeasts isolated from grapes in the southern Italy (Diaz et al. 2020).

HERE Table 2

In conclusion, the isolates selected for this study due to their antifungal capacity against Aspergillus spp. showed also an antifungal effect on F. graminearum. Our results demonstrate that the yeasts have an inhibitory

effect on the growth and the production of the mycotoxin DON. All yeasts isolates reduced mycelial growth in dual tests carried out and in the volatile compounds assays. Pichia kudriavzevii isolates showed the greatest effect on the inhibition of fungal growth and DON production. They were also the isolates that produced the highest growth inhibition in the volatiles assay. The possible use of these promising yeasts as biocontrol agents against fungi in foods could be a better alternative for the preservation of the environment.

ACBP 2-amino-5-chlorobenzophenone AR relative area

DON Deoxynivalenol

ECD electron capture detector

LAB lactic acid bacteria NaHCO3 sodium bicarbonate

NIV Nivalenol

PDA medium Potato dextrose agar medium

TFAA Trifluoroacetic anhydride

VOCs Volatile organic compounds

YPD broth Yeast extract-peptone-D-glucose broth

Author contribution Candela Moure, Diana Ramírez Alburquerque, Angela León Pelaez, Virginia Fernández Pinto and Teresa Alconada were involved in the design, data collection, data analysis, data interpretation, elaboration, and discussion of the manuscript.

Funding This work was supported by Consejo Nacional de Investigaciones Científcas y Técnicas (PIP: 0585),

Universidad Nacional de La Plata (11×838), Agencia Nacional de Promoción Científca y Tecnológica (PICT 2018 03702) and Programa Iberoamericano de Ciencia y Tecnología para el desarrollo (CYTED 121RT0110)

Data availability Please contact author for data requests.

Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication Not applicable.

Conflict of interest The authors declare no competing interests

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Impact of kefir yeasts on Fusarium graminearum growth and production of deoxynivalenol

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Biological material and inocula

Dual culture assay

Volatile organic compounds

Effect on growth and the production of deoxynivalenol

Deoxynivalenol extraction

Deoxynivalenol quantification

Statistical analysis

Results and discussion

Dual culture assay

Volatile organic compounds

HERE Table 1

Effect on growth and on deoxynivalenol production

HERE Table 2

Abbreviations

Declarations

References

Additional Declarations

Status:

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