Academic Editor: Baohong Zhang
Asparagine is one of the precursors of acrylamide and toxic fungal secondary
metabolites, both carcinogenic compounds. In the present study, the optimal
conditions to deplete asparagine by Aureobasidium pullulans (L1 and L8)
from potato and wheat flour matrices were investigated. Through a colorimetric
plate-assay with phenol red as indicator dye, both strains demonstrated to be
able to produce L-asparaginase from 20
L-asparaginase is an amid-hydrolase that catalyzes L-asparagine to L-aspartate and L-ammonia [1]. Recently, its important applications in the pharmaceutical industry as cancers treatment determined a great interest from different international industrial sectors [2]. L-asparaginase enzyme is also used in the food industry for acrylamide prevention formation in foods processed with high temperatures [3], especially for baked and fried foods. It is known that plants, animals, and microorganisms can produce asparaginase enzyme [4, 5]. Among yeasts, L-asparaginase or L-glutaminase occurred frequently in Hansenula spp., Cryptococcus spp., and Rhodotorula spp. [6]. More recently, Saccharomyces cerevisiae stands up between filamentous fungi with a high asparaginase activity [7]. Much effort has been invested in the study of asparagine, related to acrylamide levels mitigation in food to reduce its exposure to the consumers. Acrylamide is a carcinogenic molecule to humans, resulting from the Maillard reaction that involves asparagine, reducing sugars, and high temperatures [8]. The yeast Aureobasidium pullulans can assimilate asparagine and determine a reduction of acrylamide content in fried potatoes [9, 10, 11]. Application of L-asparaginase in food process represent an alternative method to reduce acrylamide content in fried, baked, and toasted products. At the same time, the activity of this enzyme can improve the antagonistic capability of a biocontrol agent (BCA) against the development of some fungal pathogens [12]. It is known that a specific nutritional composition can enhance BCAs activity but inhibit the growth of the pathogens [13]. The amino acids involved in the studied food systems such as potato and wheat flour are mainly based on asparagine and aspartic acid. Both amino acids were studied because the first one can be utilized readily by the antagonists and poorly by the pathogens, and the second one showed an inhibiting effect on the growth of some molds [9]. About potatoes, Rhizoctonia solani represents an economically important disease [14] causing both quantitative and qualitative damages. In the case of wheat, Fusarium graminearum can cause severe economic losses for cereal farmers due to significant reductions in grain yield and quality, especially for mycotoxins production [15].
In this regard, the goals of the research were to (i) verify A. pullulans strains extracellular L-asparaginase enzyme activity at different growth conditions (temperatures and times) through in vitro assays and (ii) to confirm their ability to reduce asparagine content in presence of different vegetable matrices, such as potato and wheat flour, at different co-culturing conditions. (iii) The last aim was to verify how amino acids content influence R. solani and F. graminearum mycelial growth.
Aureobasidium pullulans L1 and L8 strains [16] were grown on nutrient
yeast dextrose agar (Oxoid, UK) in 1 L of distilled water, and stored at 4
Potatoes (“Primura”) from experimental fields (Bologna, Italy) were stored at
8
Asparaginase enzymatic activity was tested by a rapid plate assay made by
suspending: glucose 2 g, L-asparagine (Sigma Aldrich, Saint Louis, MO, USA) 10 g,
KH
Samples of homogenized potato (1 g) and wheat flour (1 g) were amended with L1
and L8 suspension concentrated 10
Asparagine and aspartic acid effect on fungal growth were assayed on PDA plates.
Agar medium was amended with different concentrations of the above-reported amino
acids (0 mg/L, 15 mg/L, 40 mg/L, 80 mg/L, and 120 mg/L) (Sigma Aldrich, MO, USA).
Subsequently, substrates were inoculated with a mycelial pathogen agar plug (6
mm, Ø) and incubated at 20
Statistical analysis was conducted by using Statgraphics software (version
centurion 15.0, The Plains, VA, USA). One-way ANOVA was used to analyze data and
the least significant differences test (LSD) to separate differences among the
means (p
To screen the asparaginase activity of the two yeasts a plate assay was set up.
Phenol red was added to the agar to reveal the pH alterations induced by the
release of ammonia ions by the L-asparaginase activity. The pH indicator is
normally yellow at acid conditions and turns pink at alkaline pH. L-asparaginase
activity by the yeasts was detected through a pink halo around the colony. By
culturing L1 and L8 on the screening plate amended with asparagine, a more
intensive pink halo was detected around the colony, starting from the two days of
incubation at 20
Incubation time | ||||||||
20 |
2 d | 4 d | 7 d | |||||
Colony diameter (mm) | Halo diameter (mm) | Colony diameter (mm) | Halo diameter (mm) | Colony diameter (mm) | Halo diameter (mm) | pH | ||
Control | 0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
6.2 | |
L1 | 9.7 |
21.5 |
13.7 |
32.2 |
16 |
39 |
7.2 | |
L8 | 11.7 |
18.0 |
17.5 |
18 |
23.5 |
18 |
6.9 | |
Incubation time | ||||||||
25 |
2 d | 4 d | 7 d | |||||
Colony diameter (mm) | Halo diameter (mm) | Colony diameter (mm) | Halo diameter (mm) | Colony diameter (mm) | Halo diameter (mm) | pH | ||
Control | 0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
6.2 | |
L1 | 11.7 |
21.5 |
17.7 |
37.7 |
20.5 |
56.2 |
7.2 | |
L8 | 15.7 |
0.0 |
22.7 |
0.0 |
29 |
0.0 |
6.4 | |
Incubation time | ||||||||
30 |
2 d | 4 d | 7 d | |||||
Colony diameter (mm) | Halo diameter (mm) | Colony diameter (mm) | Halo diameter (mm) | Colony diameter (mm) | Halo diameter (mm) | pH | ||
Control | 0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
6.2 | |
L1 | 9.5 |
17 |
10.2 |
29.7 |
12.5 |
29.7 |
6.8 | |
L8 | 11.2 |
0.0 |
16 |
0.0 |
20.2 |
0.0 |
6.2 | |
The halos were recorder after 2, 4, 7 days of incubation at 20 |
Yeast strains resulted both good consumers of asparagine contained in the target food matrices. At each tested temperature the asparagine amount increased over time in control samples. Especially at 60 min of incubation at each temperature, the increase of the asparagine content in potato homogenate and wheat flour was respectively of 83%, 84%, 66% and 66%, 48%, 96%, for the recorded values at 15 min.
In the potato matrix, L1 and L8 strains showed a great ability to completely
reduce asparagine content especially at 20
Asparagine amount in potato homogenate after 15, 30
and 60 min of incubation with Aureobasidium pullulans L1 and L8 strains
at 20
In wheat flour, the two strains showed different behavior. L1 showed faster asparagine consumption, with the best performances at 60 min of incubation at each tested temperature, almost completely reducing the target amino acid (Fig. 2).
Asparagine amount in wheat after 15, 30 and 60 min
of incubation with Aureobasidium pullulans L1 and L8 strains at 20
Also A. pullulans L8 strain reduced asparagine content in wheat flour
but as described above for potato homogenate, its efficacy (68.6%) concerning L1
(100%) was reduced by the highest temperature (30
Different concentrations of asparagine and aspartic acid (0, 15, 40, 80, 120 mg/L) were infused on MEA plates to evaluate their effect on Rs1 and F3 mycelial growth. The presence of the highest concentration of aspartic acid determined a significant reduction of both pathogens’ mycelial diameter with respect to the control, respectively by 8.2% and 12.0%. About the asparagine, the amino acid concentration of 120 mg/L resulted the most effective with respect to the control by significantly stimulating R. solani and F. graminearum growth by 4.4% and 18.9% respectively (Fig. 3a,b).
Asparagine and aspartic acid concentrations effect
on Rhizoctonia solani (a) and Fusarium graminearum (b) mycelial
growth (colony diameter, mm) after 7 days of incubation at 20
In a previous study, L1 and L8 displayed their capability to drastically alter the amino acids pattern of peach juice with a strong consumption of asparagine [9]; this activity was confirmed also in fried potato chips by 80% of reduction of acrylamide content [10]. The present study reports the asparaginase activity exerted by both yeast strains through the direct method of the plate assay, where both strains showed a different capability to produce L-asparaginase enzyme. This was more expressed by L1 with respect to L8 strain. However, the enzymatic activity exerted by microorganisms was strictly influenced by environmental conditions such as temperature and pH [19].
L1 strain displayed more intense halos at each temperature notably increasing
pH, with the halos diameters directly connected to the pH values. Conversely, L8
showed asparaginase halos only at 20
However, this activity is strictly connected to L1 and L8 strains mechanisms of action: competition for nutrients [9]. In fact, through the capability of the BCAs to compete for asparagine, pathogens’ aggressiveness was reduced, as showed also against Botrytis cinerea and Monilinia laxa [9, 12].
Results showed how each fungal growth was stimulated by the presence of asparagine. As reported by Giese et al. [20], asparagine was found to be a preferential nitrogen source for F. graminearum. About R. solani, its virulence was increased by organic sources of nitrogen such as asparagine [21].
Yeasts use asparagine as their nitrogen source through its deamination to aspartic acid [22]; this was the reason why the study provided the same assay for the evaluation of the aspartic acid effect on pathogens’ growth.
Amino acids are fundamental compounds, sometimes toxic if at high intracellular levels [23]. For example, bacterial cells growth can be inhibited by valine and phenylalanine, meanwhile causing the enzymes repression involved in isoleucine and tyrosine synthesis [24, 25].
However, amino acids such as aspartic acid, glutamic acid, glycine, serine, alanine, and leucine can reduce B. cinerea growth in vitro tests [12]. Nevertheless, amino acids were often used as antiseptic agents due to their low toxicity level [26, 27, 28, 29].
The application of yeasts or their enzymes in various technological processes may have also an inhibitory effect on certain toxins producers’ fungi such as F. graminearum. These microorganisms can accumulate mycotoxins from agricultural products decontaminating them [30].
Mycotoxigenic fungi are capable to grow on a broad range of substrates. However, not all of these substrates are equally good for mycotoxin production.
Bullerman et al. [31] asserted that the formation of aflatoxin is stimulated by the presence of certain amino acids, fatty acids, and zinc. Asparagine has been reported to be a good nitrogen source for aflatoxin production in stationary cultures [32].
Based on the obtained results, other experiments should be conducted for more details on the influence of the two yeasts on the asparagine content in food matrices and if they could represent an innovative, environment friendly, and effective method to mitigate toxic fungal secondary metabolites and acrylamide formation in cooking.
The present study reports the versatility use of BCAs, especially yeasts, such as mitigating agents of toxic substances in food and fungal pathogens antagonists. It is in fact known how some BCAs have the capacity to detoxify foods from toxic compounds and control fungal pathogens development ensuring food security and quality. The presented results could be added to the others with the aim to streamline procedures for the application and regulation of BCAs in agriculture and food technologies. However, all the food industry starting from the agricultural practices and storage management should be considered and improved to reduce pathogens’ development and toxic compounds formation. By the way, novel enzymes’ production by A. pullulans with other potential effects is still to be investigated and studied in-depth.
ADF conducted all the experiments, analysis and writing of the manuscript.
This study was developed on the base of the patent n. 102017000105265 “Ceppo di lievito utilizzabile per ridurre la quantità di acrilammide in un alimento trattato termicamente”. (Authors: Marta Mari, Alessandra Di Francesco and Luisa Ugolini; Alma Mater Studiorum- Bologna and CREA) (20.09.2017). The author the author would like to thank Marta Mari for her support during the research.
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This research received no external funding.
The author declares no conflict of interest.