Shuaibing Zhang*,Minghui Zheng,Huanchen Zhai,Ping'an Ma,Yangyong Lyu,Yuansen Hu,Jingping Cai*

College of Biological Engineering,Henan University of Technology,Zhengzhou 450001,China

ABSTRACT The prevention of fungal spoilage is an essential consideration in wheat storage.Recent studies have revealed that volatile organic compounds(VOCs), possibly with natural fungicidal properties,could be produced from stored wheat grains.In this study,the antifungal effect of hexanal,a main component of VOCs from stored wheat, against spoilage fungi on agar plate and in high-moisture wheat grains were investigated via the gas fumigation method.And the impact of hexanal fumigation on grain quality was evaluated through analysis of the malondialdehyde content,fatty acid values,germination percentages and vigor of 16%and 18%moisture wheat grains fumigated with 1.66,2.49,and 3.31 mmol/L hexanal vapor.The results of in vitro antifungal experiments on agar plates revealed that the minimum inhibitory concentration and minimum fungicidal (fatal) concentration of hexanal against the five fungi were 4-14 folds and 4-7 folds lower than those of propionic acid, respectively.The fungal spoilage of high-moisture wheat grains inoculated with pure fungal spores and with naturally occurring fungi could be completely inhibited by 1.66 mmol/L hexanal vapor.During 5-week storage of high-moisture wheat grains fumigated with 1.66, 2.49, and 3.31 mmol/L hexanal vapor,the malondialdehyde content in high-moisture wheat grains did not change significantly in all samples, and fatty acid values were slightly higher in 18% moisture wheat than in 16% moisture wheat.The germination percentages and vigor of wheat samples decreased with increased hexanal vapor concentrations and moisture content.These results indicated that hexanal fumigation could be used as an alternative chemical control method to prevent the fungal spoilage of postharvest wheat.

Keywords:Hexanal Chemical control Spoilage fungi Wheat storage Grain quality

1.Introduction

Wheat is a staple cereal grain cultivated and consumed worldwide [1].Wheat is usually stored for one or more years after harvesting to supply domestic cereal industries and satisfy import/export demands.One of the primary management challenges associated with grain storage is the prevention of fungal growth in wheat bulk [2].The growth of storage fungi,particularly Aspergillus and Penicillium species,in wheat bulk can seriously deteriorate grain quality and quantity and produce toxigenic or carcinogenic metabolites[3-5].Therefore,managing the growth of such fungi in stored wheat grains is essential for food safety.

Several physical and chemical approaches to prevent the fungal spoilage of wheat grains have been extensively studied.Controlling the temperature and moisture content of stored grain is still the main approach to prevent fungal growth in wheat bulk [2,6].The moisture content of wheat grains must be strictly controlled when loading into large warehouses for long-term storage.In China,regulations stipulate that the moisture content of stored wheat grains should remain below 12.5%(Chinese standard GB 1351-2008).Nevertheless, incidental fungal spoilage in stored wheat also occurs owing to water condensation due to the “cold wall contact” effect and moisture transfer resulting from temperature differences in grain bulk [7].Chemical fumigation has been studied as an alternative approach for preventing the fungal spoilage of stored grains.The ability of several chemical fumigants, such as ozone gas,phosphine,propionic acid,and essential oils,to control fungal growth in stored grains has been tested[8-12].However,these fumigants have not been widely used in the prevention and control of fungal spoilage in grain storage due to residual toxicity,limited antifungal effect,or high cost.Therefore, green, safe and efficient antifungal fumigants are still demanded for safe grain storage.

In recent years,the inhibitory effect of natural plant-or microorganism-produced volatile organic compounds(VOCs)on the fungal spoilage of agro-products has attracted increasing attention owing to their natural origin,low toxicity,and high potency[13].Stored wheat is a living,albeit dormant organism.VOCs produced through the lipoxygenase(LOX)pathway in wheat grains are mainly volatile aldehyde,alcohol,and acid chemicals,which may be responsible for the natural antifungal properties of wheat grains[14-17].Hexanal has been identified as one of the main components of VOCs produced by stored wheat grains[14,16-18].As a natural product originating from wheat grains and a food additive authorized by the U.S.Food and Drug Administration and the National Health and Family Planning Committee of China (Chinese Standards for Food Additives GB 2760-2014),hexanal is considered to be a natural metabolizable fungicide [19-21].The effectiveness of hexanal in preventing the growth of postharvest pathogens in fruits and vegetables is well known[22-24].Spores of spoilage fungi in peach,such as Botrytis cinerea,Monilinia fructicola,Sclerotiniasclerotiorum,Alternariaalternata,andColletotrichum gloeosporioides in fruits and vegetables could be completely inhibited or killed after exposure to 900 μL/L of hexanal vapor at 20°C for 12 h[25],which demonstrated the higher antifungal potency of hexanal.However,the effectiveness of the antifungal properties of hexanal in preventing spoilage in stored wheat grains has not been reported.

To evaluate the potential antifungal effect of hexanal on the fungal spoilage of stored wheat grains,the in vitro antifungal effects of hexanal vapor on five common storage fungi, Aspergillus glaucus, A.flavus, A.niger, A.ochraceus,and Penicillium expansum, were compared with those of propionic acid using agar plates.Further,differences in the antifungal effect of hexanal vapor on fungal spoilage in high moisture-content wheat grains were assessed.Meanwhile,the effect of hexanal fumigation on malondialdehyde(MDA)content,fatty-acid values(FAV),germination percentage, and vigor in wheat grains were tested to understand the impact of hexanal fumigation on storage quality of wheat grains.

2.Methods

2.1.Effects of hexanal and propionic acid fumigation on in vitro mycelial growth

The effect of fumigants on mycelial growth of A.glaucus,A.flavus,A.niger,A.ochraceus,and P.expansum was evaluated via the gas fumigation method.The five storage fungi were conserved in our laboratory after isolation from wheat grains and identification via morphological observations and internal transcribed spacer sequence analysis[26,27].The spore concentration was enumerated using a hemocytometer.For A.flavus, A.niger, A.ochraceus, and P.expansum,1 μL spore solution(2×106spores/mL)was inoculated in the center of 9 cm-diameter glass Petri dishes containing 20 mL of Czapek-Dox agar(3%sucrose, 0.1%K2HPO4, 0.3% NaNO3, 0.05% KCl, 0.05% MgSO4·7H2O,and 2% agar powder).For the xerophilic fungus A.glaucus,1 μL spore solution(2×106spores/mL)was inoculated in the center of 15 cm-diameter glass Petri dishes containing 100 mL of Czapek's agar (3% sucrose, 0.1%K2HPO4, 0.3% NaNO3, 0.05% KCl, 0.05% MgSO4·7H2O,and 2%agar powder).The antifungal effectiveness of hexanal was tested in comparison with propionic acid,which is usually used as fumigant in stored grains and commercially manufactured feed[11].Hexanal and propionic acid were purchased from Macklin Biochemical Co.,Ltd.(Shanghai,China).Different concentrations of hexanal(0,1,2,4,8,12,and 16 μL)and propionic acid(0,2,4,8,16,24,and 48 μL)were added to the Petri dish covers,and the culture dishes were inverted.

The concentration of fumigant vapor was calculated as:

where Mfis the moles of fumigant,and V is the empty volume in the glass Petri dishes.The dishes were sealed with Parafilm and incubated at 28°C.After incubation for 5 days,two perpendicular diameters of each colony zone were measured(cm)with a ruler and averaged to represent inhibitory efficacy.

The percentage of mycelial growth inhibition rate(MGI)was calculated according to the following formula:

where d0(cm)is the mean colony diameter for the control sets, and dt(cm) is the mean colony diameter for the treatment sets.The minimal inhibitory concentration(MIC) was determined as the lowest concentration that completely inhibited fungal growth after 5 days of incubation.Then,the fumigant in the glass Petri dishes in which fungal growth had been completely inhibited was replaced with sterile air and incubated for another 3 days at 28°C.The lowest concentration at which no growth occurred on the plates after the prolonged 3-day incubation was determined as the minimal fungicidal concentration (MFC).Each experiment was performed in triplicate.

2.2.Fumigation system design

The fumigation system was constructed using a mini vacuum pump (FAA8006-12 V, Xinweicheng, Chengdu,China), a chemical reagent bottle, and a grain container loaded with wheat grains,which were connected by soft silicon tubes.The flow-through system was designed to evaporate liquid fumigant by circulating air through the chemical reagent bottle.The concentration of fumigant was calculated as:

where Mfis the moles of fumigant,and V is the total volume of the flow-through system.

2.3.Effects of hexanal fumigation on the growth of pure fungi in wheat grains

Wheat grains were immersed in 1%sodium hypochlorite solution for 4 min and washed three times with sterile water.The washed wheat was dried at 45°C for 6 h under aseptic conditions.The moisture contents of wheat grains were adjusted to 14%, 16%, and 18% by spraying calculated amounts of sterile distilled water over the wheat grains and maintaining under airtight aseptic conditions at 4°C for 24 h to allow the moisture to equilibrate.

Wheat grains inoculated with the spores of the xerophilic fungus A.glaucus were stored at 14% and 16% moisture content.Wheat grains inoculated with A.flavus, A.niger,A.ochraceus, and P.expansum spores were stored at 16%and 18%moisture content.One thousand grams of wheat grains containing 1 × 103fungal spores per gram were loaded into 1.5 L airtight grain containers.The wheat grains containing fungal spores from each species were fumigated with 0,0.41,0.83,1.66,and 3.32 mmol/L of hexanal and stored at 28°C to simulate typical grain storage conditions.The fungal development in fumigated wheat grains was analyzed every 7 days,as reported previously[6].

2.4.Effects of hexanal fumigation on the growth of natural fungi in wheat grains

To evaluate the antifungal effect of hexanal on natural fungi in wheat grains,wheat grains naturally contaminated with fungi were adjusted to 16%and 18%moisture content.One thousand grams of wheat grains were loaded into 1.5 L airtight grain containers and fumigated with 0,0.41,0.83,1.66, and 3.32 mmol/L of hexanal or 0, 3.38, 6.76, and 13.51 mmol/L of propionic acid.Grain storage quality and microbiological analysis of wheat grains were performed as described above.

2.5.Grain quality assessment

One thousand grams of wheat grains with 16%and 18%moisture contents were loaded into 1.5 L airtight grain containers and fumigated with hexanal vapor at concentrations of 1.66, 2.49, and 3.14 mmol/L.The fumigated wheat grains were stored at a constant 28°C to simulate typical storage conditions.The MDA content and FAVs of fumigated wheat grains were analyzed once a week,according to the methods of Tian et al.[28].

Bioassays to determine the level of germination within the wheat samples were performed before and after the 5-week storage period.One hundred wheat kernels were randomly withdrawn from the fumigated grain samples and placed on a filter paper(presoaked in sterile water)at the bottom of a Petri dish (9 cm diameter) and cultivated at 20 °C in an artificial climate chamber.The germination vigor and percentage of wheat kernels experiencing germination were calculated at 72 h and 120 h of incubation,respectively.Germination was considered to have occurred when the radicle protruded by 2 mm.All observations were performed in triplicate.

2.6.Statistical analysis

Analyses of variance and the correlation coefficients of different concentrations of fumigants (hexanal and propionic acid)and their inhibition rates against the five fungi were analyzed using SAS 9.0.Tukey's honestly significant difference test was used to compare the changes in inhibition rates corresponding to the different concentrations of fumigants.P values ＜0.05 were considered to represent significant differences between the results.

3.Results

3.1.In vitro inhibitory effects of hexanal on spoilage fungi

The inhibitory effects of hexanal on five spoilage fungi-A.glaucus,A.flavus,A.niger,A.ochraceus,and P.expansum,which are commonly associated with fungal spoilage in wheat with relatively high moisture content,were tested and compared with those of propionic acid.The results showed that hexanal at lower concentrations could significantly inhibit mycelium growth of the five fungi than propionic acid, particularly for the xerophilic fungi A.glaucus (Table 1).The MICs of hexanal for the five fungi were approximately 4-14 fold lower than those of propionic acid, and the MFCs of hexanal were approximately 4-7 fold lower.A regression analysis between theconcentrations of hexanal and propionic acid vapor and their inhibition rates against the five fungi yielded a correlation efficiency of over 0.97.

Table 1 In vitro inhibitory effect of hexanal and propionic acid vapor on the growth of storage fungi.

3.2.Inhibitory effect of hexanal vapor on fungal growth in wheat grains

To evaluate the effectiveness of hexanal vapor on inhibiting fungal growth in wheat grains,a fumigation system was designed to evaporate hexanal in chemical reagent bottles and fumigate wheat grains loaded in a grain container by circulating air via an airtight flow-through system(Fig.1).Hexanal-fumigated wheat grains inoculated with pure fungal spores showed remarkably inhibited fungal growth in high moisture-content wheat grains, and the growth of the five fungi was completely inhibited by fumigation with 1.66 mmol/L hexanal.The regression analysis between hexanal concentrations and fungal counts in wheat grains after the 5-week storage period yielded a correlation efficiency of over 0.95(Table 2).

Fig.1.Hexanal fumigation system for wheat grains:(A)vacuum pump,(B)chemical reagent bottle,(C)grain container.

3.3.Inhibitory effect of hexanal vapor on the growth of natural fungi in wheat grains

To further determine the inhibitory effectiveness of hexanal on natural fungi in wheat grains,the fungal development in 16% and 18% moisture-content wheat grains fumigated with hexanal vapor was compared with that in wheat grains fumigated with propionic acid.The results showed that fumigation with hexanal and propionic acid both remarkably reduced the fungal growth in stored wheat (Figs.2 and 3).However, 1.66 mmol/L hexanal vapor completely inhibited the fungal growth in wheat,while the same inhibitory effectiveness was only reached with 13.51 mmol/L propionic acid vapor,demonstrating the superior fungal inhibition performance of hexanal.

Table 2 Regression equations and correlation coefficients(r)between fungal counts(cfu/g)of wheat grains stored at different levels of moisture content(m.c.)and fumigated with hexanal after a 5-week storage period.

Fig.2.Fungicidal effects of hexanal fumigation on 16%(A)and 18%(B)moisture-content wheat grains stored at 28°C.

Fig.3.Fungicidal effects of propionic acid fumigation on 16%(A)and 18%(B)moisture-content wheat grains stored at 28°C.

3.4.Changes in grain quality of stored wheat fumigated by hexanal

To investigate the effect of hexanal fumigation on grain quality,wheat samples fumigated with higher concentrations of hexanal vapor (1.66, 2.49, and 3.14 mmol/L)were stored and changes in MDA, FAV, germination percentage, and vigor in hexanal-fumigated wheat were analyzed as indicators of grain deterioration during simulated storage.Fungal growth was completely inhibited in all wheat samples.The MDA content did not change significantly in 16%and 18%moisture-content wheat grains fumigated with hexanal after the 5-week storage period,although a slight decreasing tendency was observed during the first two weeks(Fig.4).FAVs were slightly higher in 18% moisture-content wheat than in 16% moisturecontent wheat,and a sharp increase in FAVs was observed in both groups of wheat grains during the final two weeks of storage(Fig.5).The germination percentage and germination vigor of wheat grains decreased with increasing concentrations of hexanal vapor and increasing moisture content(Fig.6).

Fig.4.Changes in malondialdehyde(MDA)content in 16%(solid line)and 18%(dashed line)moisture-content wheat grains fumigated with hexanal vapor during storage.

Fig.5.Changes in fatty-acid values(FAV)in 16%(solid line)and 18%(dashed line) moisture-content wheat grains fumigated with hexanal vapor during storage.

4.Discussion

Fig.6.Comparisons of the germination vigor(A)and percentages(B)of 16%and 18% moisture-content wheat grains fumigated with hexanal vapor before and after a 5-week storage period.

The use of natural products with fungistatic properties is an attractive approach for fungal spoilage prevention in wheat grains during storage.In this study,the antifungal effectiveness of hexanal vapor against five common storage fungi associated with wheat grain spoilage was evaluated.Hexanal vapor showed remarkably higher antifungal potency against the investigated storage fungi compared with that of propionic acid in agar plates and naturally contaminated wheat grains,indicating the potential of hexanal as an alternative chemical for the management of fungal spoilage of stored wheat grains.It was observed that hexanal vapor can reduce spore viability and hyphae growth of spoilage fungi on Czapek's agar plate and wheat grains in a concentration and time dependent manner,which is consistent with the antifungal effect of hexanal against pathogens on fruits reported previously [18,22,23].Hexanal vapor might be able to penetrate into the cell membrane of fungi and disturb the normal metabolism of fungal cells.To date,the antifungal mechanisms of hexanal against spoilage fungi have not been determined.It was reported that less than 3.0 μg/kg hexanal could be released from wheat grains [29].Although hexanal could be endogenously produced from wheat grains,it is still needed to be supplemented to completely control the spoilage fungi in stored wheat grains.Higher concentrations of hexanal and propionic acid were required to completely inhibit fungal growth in wheat grains than those required in agar plates;these results might have been caused by differences in the growth statuses of fungi on agar plates and in the stored grains or in the adsorption of fumigants by the wheat grains[30].In the fumigation system,the effectiveness of fumigants can be affected by several factors including the environmental conditions of temperature, humidity and atmospheric pressure.And adsorption of fumigants by cereal grains usually occurs,which can reduce the effectiveness of funmigant [31,32].In the successive study, the adsorption of hexanal and propionic acid by wheat grains and the fungal sensitivity of fumigants under different storage conditions of temperature and moisture content,as well as the fungicidal mechanisms of hexanal must be further investigated.

Hexanal fumigation did not cause a significant increase in MDA,indicating that the lipid peroxidation of cell membranes in wheat grains did not occur to a significant degree during storage.Hexanal has been reported to inhibit the enzyme activity of phospholipase,which is a key associated with the degradation of membrane phospholipids [33].Therefore,hexanal fumigation may be helpful in preventing the physiological deterioration of wheat grains in storage.The increased FAVs with prolonged storage time suggested that the fats in wheat were hydrolyzed during the metabolism of wheat kernels.The reduced germination percentage and vigor of wheat kernels might be attributable to the increased free fatty acids,which can inhibit seed germination[34].Further, increased concentrations of hexanal vapor were found to result in decreased germination vigor in wheat grains.These results align with those of Gardner et al.,who reported that hexanal could inhibit the germination and subsequent growth of soybeans[35].The effect of hexanal fumigation on processing quality of wheat grains and its final product still needs further study.

In this study, hexanal vapor was found to have potent fungicidal effects on wheat grains.The fungicidal effects of other VOC components associated with stored wheat on spoilage fungi must be further investigated in subsequent experiments.To date,several researchers have elucidated the defensive mechanisms of VOCs in many plants against pathogenic diseases [13,36].Recent studies have shown that the main components of VOCs from stored wheat are short-chain aldehydes and alcohols[14,16],which might be responsible for the naturally evolved defensive properties of wheat seeds against pathogenic diseases or pest.Recently,Germinara et al.[37]reported that volatile aliphatic aldehydes,individually and in various combinations,can effectively disrupt the ability of some insects to recognize and attack wheat grains,which showed that these bioactive compounds could be used as repellents for sustainable control in granaries.This suggests that the bioactive components of VOCs from wheat grains are promising alternatives to artificial chemicals for the control of spoilage fungi and pests in stored products.

5.Conclusion

Our study results demonstrated that hexanal has remarkably higher antifungal potency against spoilage fungi commonly existed in stored wheat grains in comparison with propionic acid.Over 1.66 mmol/L hexanal vapor could effectively control the growth of storage fungi in relatively high moisture-content wheat grains.Hexanal fumigation can inhibit the production of MDA in wheat grains, thereby preventing lipid peroxidation in wheat grain.Hexanal fumigation also slightly increased FAVs, and the germination percentage and vigor of wheat kernels decreased with increasing concentrations of hexanal vapor and grain moisture content.These results suggest that hexanal fumigation is a promising approach for controlling fungal spoilage in high moisture-content wheat grains.

Author Contributions

Shuaibing Zhang: Conceptualization, methodology,investigation,writing-original draft preparation,funding acquisition;Minghui Zheng:Investigation,software;Huanchen Zhai: Methodology, software; Ping'an Ma: Methodology ;Yangyong Lyu: Editing; Yuansen Hu: Writing, reviewing;Jingping Cai:Conceptualization,funding acquisition.

Conflicts of Interest

The authors declare that there are no conflicts of interests.

Acknowledgements

This work was supported by the National Natural Science Foundation of China(Project No.31772023),the National Key Research and Development Project of China(Project Nos.2017YFC1600903 and 2017YFD0401404), the National Key Research and Development Project of China(Project No.2019YFC1605303-04),and the Scientific Research foundation of Henan University of Technology(Project No.2018RCJH14).