Detection and control of off-flavour compound-producing streptomycetes on locally produced nuts using streptophages

Keywords: actinomycetes, bacteriophages, food taints, geosmin, nuts, Streptomyces, streptophage, volatile organic compounds.

Over a thousand microbial volatile organic compounds (VOCs)¹ have been identified from streptomycetes, many of which are acids, alcohols, aldehydes, alkenes, benzenoids, esters, ketones, pyrazines, and terpenes.² VOCs produced by Streptomyces species can benefit agriculture via the production of bioactive compounds, which can assist in bacterial and fungal growth inhibition, plant growth promotion or inhibition, and invoke resistance mechanisms.²

Geosmin (trans-1,10-dimethyl-trans-9-decalol), 2-methylisoborneol (2-MIB; 1,2,7,7-tetramethyl-exo-bicyclo-heptan-2-ol), and dimethyl disulfide are three of the major VOCs produced by Streptomyces species.^3–5 Geosmin and 2-MIB are semi-volatile and terpenoid secondary metabolites.⁶ Streptomycetes produce these compounds via the 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol 4-phosphate (DOXP/MEP) and melavonic (MVA) pathways.⁷ Likewise, dimethyl disulfide is a volatile sulfur compound produced via methionine degradation followed by methanethiol oxidation.^8,9

VOCs are also known as taste and odour compounds (T&Os) due to their detectability by humans. Humans can detect these T&Os-VOCs at concentrations of 4 ng/L, due to their olfactory sense.^4,10 Furthermore, geosmin synthase genes, which enables geosmin production, are broadly distributed within the members of the genus Streptomyces.^4,11 These compounds arise in food products, such as nuts and fish, via bioaccumulation from plant debris, soil, and water use.^6,12,13 Further accumulation of geosmin can occur in storage silos if left unmaintained due to continued growth of streptomycetes, not only resulting in strong odours but also giving rise to organic dust toxic syndrome.¹⁴ Geosmin has an earthy flavour,^15,16 yet there has been no successful technique to remove these VOCs due to the ineffectiveness and high cost of current methods.¹⁷ A recent study conducted at the University of the Sunshine Coast (USC) aimed to remove VOCs on locally-produced and openly sold nut samples using streptophages.

Bacteriophages have seen a rise in use as biocontrol agents in agriculture, bioprocessing, and healthcare.^18,19 Bacteriophages are regarded as safe for animals, humans, and plants, further promoting their use in the previously mentioned industries.^20–22 Chibani-Chennoufi et al.²³ reported that mice exposed to an oral four-phage cocktail did not experience a decline of their commensal E. coli biota. Bruttin and Brüssow²⁴ also reported that human volunteers orally exposed to phage T4 maintained their commensal E. coli population.

Eight streptomycetes were isolated from seven different locally produced and openly sold nut samples using two different isolation methods (air compaction using an air sampler²⁵ and conventional serial dilution²⁶) and incubation temperatures (28°C and 37°C) to maximise the chances of detection of these odorous species. Details of these isolates are given in Table 1.

Table 1.  Key characteristics of the Streptomyces isolates from nut samples.

Like bacteria, bacteriophages are present in agricultural environments where the host bacteria reside.²⁷ Usually bacteriophages are host specific, however, they also display polyvalency within the host’s taxonomic rank.²² The three major families of actinophages are Myoviridae, Siphoviridae, Podoviridae. Siphoviridae morphology is the most abundant one, particularly in soil among the actinophages.^28,29 This group of phages is mostly polyvalent within the Streptomycetaceae family to which the genus Streptomyces belongs. They are commonly known as streptophages³⁰ and morphologically consist of a long and flexible noncontractile tail. The siphoviridae heads contain portal protein at the vertices, which connects the head and tail segments, while the other vertices contain capsid proteins³¹ and the tails usually are 100–400 nm in length depending on the species.³¹

Bacteriophages target host populations via phenotype modification, predation, and lysogeny.²⁷ Soil is the major reservoir for actinophages, and they most commonly target actinomycete genera Streptomyces, Actinoplanes and Mycobacterium.²⁷

Nine different polyvalent streptophages from USC’s Microbial Library³² were selected and used to create a composite phage suspension (Fig. 1) at a concentration of 10⁸ pfu/mL. A composite streptomycete suspension was also created by mixing all eight streptomycete isolates at a concentration of 10⁴ cfu/mL. This concentration was selected as it represents unacceptable contamination value determined by the NSW Food Authority in their guidelines³³ for microbiological quality of ready to eat foods in Australia. Surface sterilised and UV irradiated nut samples using the methods by El-Tarabily³⁴ and Thomas and Puthur³⁵ were deliberately infected with this composite sample of streptomycetes. After 3 days of incubation streptophage composite sample was applied onto the streptomycete inoculated nuts with a host/phage ratio of 1:2. This ratio was selected due to past successful applications of phages onto hosts.³⁶ VOC production was examined using Headspace-Gas chromatography mass spectrometry (HS-GC/MS) throughout the 14 days of incubation in tightly capped bottles. A mixed standard of Geosmin and 2-MIB (Sigma-Aldrich) was used to detect Geosmin, which is known to be detected at 8.83 min.

Fig. 1.  TEM micrograph of the streptophages in the composite phage suspension displaying typical siphoviridae morphologies.

Fig. 2.  Decreased values of geosmin after phage application. Key: blue – geosmin production (control); orange – geosmin production with streptomycetes application; grey – geosmin production with streptomycetes application then streptophage application after day 7.

Findings indicated a sharp decrease immediately in geosmin production after the composite phage suspension application onto streptomycete infected nut samples (Fig. 2). After day 8, the geosmin levels were near zero and streptomycete cfu/mL came down to the acceptable levels by the NSW Food Authority (<10²).

2-MIB was not detected on the streptomycete or streptomycete plus phage treated nut samples at any stage of this study. Yanxia et al.¹⁷ reported a strong correlation between geosmin and 2-MIB concentration, indicating that 2-MIB may be dependent on geosmin production so the sharp decrease in geosmin might be the reason of its absence.

The continual rise in demand of agricultural products has resulted in an increase in preferences in the use of environmentally and human health friendly methods replacing other synthetic agents. Therefore, bacteriophage treatments gained attention to minimise product losses and nutritional properties from disease causing bacteria. Like the previous successful treatments of potatoes^37–39 and strawberries,⁴⁰ the observed success of streptophage treatment on nuts in this study may indicate similar positive outcomes might be possible. Therefore, information generated via the studies like the one presented here can contribute toward development of effective phage biocontrol methods targeting different problems in agriculture. Such methods might subsequently reduce the economic losses of the growers due to unmarketable product including the ones possessing earthy-musty smells.

The data that support this study will be shared upon reasonable request to the corresponding author.

The authors declare no conflicts of interest.

This research did not receive any specific funding.