extfieldsofstudy: Biology Medicine

text:
Modulation of colonic hydrogen sulfide production by diet and mesalazine utilizing a novel gas-profiling technology

ABSTRACT Excessive hydrogen sulfide (H2S) production from gut microbial metabolism may have clinically important relevance in the pathogenesis of gut disorders, including ulcerative colitis. However, little is known regarding factors that alter its production. Using a newly-designed in vitro gas-profiling technology, the study aimed to verify real-time H2S measurement reproducibility and thereafter, assess its production following exposure to dietary factors and 5-aminosalicylate acid (5-ASA). Measurements of H2S, carbon dioxide, hydrogen and methane measurements were compared between gas-profiling systems. Homogenized slurries were prepared from freshly-passed healthy human feces. Fifty ml slurries were aliquoted into separate fermentation chambers and substrates added including 1 g highly fermentable fructo-oligosaccharides (FOS) or resistant starch Hi-Maize (RS), or minimally fermentable psyllium or sterculia, 1 g cysteine, 0.9 g sodium sulfate or 1.2 mL of 1 M 5-ASA alone or in combinations. H2S release was sampled every 5 mins over 4-h and expressed relative to unspiked controls. RS suppressed H2S production by a mean 89.0 (SEM 4.8)% and FOS by 82.2 (6.2)% compared to <35 (17)% by psyllium and sterculia (p<0.001, two-way ANOVA). Cysteine stimulated H2S production by 1557 (532)%. The addition of FOS to slurries containing cysteine significantly suppressed H2S by 90 (2)% over the addition of 5-ASA (0.3 (2)%, p<0.001). Sulfate and 5-ASA had minimal overall effects. In conclusion, the H2S-profiling technology is a reproducible tool. Production of H2S is greatly enhanced by sulfur-amino acids but not inorganic sulfate, and is effectively suppressed by readily fermentable fibers. These findings inform potential designs of dietary therapies to reduce H2S production in vivo.

Introduction
Alterations in colonic microbial metabolism of gaseous end-products have been implicated in the development of ulcerative colitis (UC). Evidence for such observations include elevated luminal hydrogen sulfide (H 2 S) concentrations driven either by increased metabolism of sulfur-containing substrates or impaired detoxification mechanisms. 1 Prolonged exposure of colonocytes to excessive luminal H 2 S and nitric oxide, another toxic gas, produce biochemical lesions in the mucosal lining described in UC. 2 Indeed, one of the possible mechanisms of 5-aminosalicylic acid (5-ASA), a first-line drug therapy, in inducing remission in UC, is the inhibition of H 2 S production. 3 .
It is well known that microbial fermentation of undigested carbohydrates yield hydrogen (H 2 ), methane (CH 4 ) and carbon dioxide (CO 2 ). 4 On the contrary, H 2 S production reflects either fermentation of sulfur-amino acids or the reduction of inorganic sulfate. 5 The relative contributions of each microbial pathway towards luminal H 2 S production is not well documented. Additionally, the contribution of other dietary factors to H 2 S production require further investigation. Limited data suggest that readily fermentable fibers, such as resistant starch and fructo-oligosaccharides (FOS) may influence H 2 S production by shifting fermentation away from sulfur-amino acids, 6,7 Since H 2 S may be pathogenic in UC and its microbial pathways are potentially modulable by diet, this gas may be a valuable biomarker to assess the success of dietary interventions in correcting functional dysbiosis in UC. Evidence that such a dietary strategy may be efficacious is limited to a single report in which patients with active UC had marked clinical and biochemical improvement after dietary restriction of sulfuramino acids and inorganic sulfate. 8 Whilst this dietary approach was targeted at modulating the involvement of H 2 S in pathogenic mechanisms of UC, a strategy was needed for assessing whether target end-points were achieved.
A key limitation in the design of new dietary therapies that target luminal H 2 S is the ability to measure its production and, perhaps more importantly, changes associated with such interventions. Existing measurement techniques in vivo and in vitro are either limited by volatility of H 2 S and, therefore, poorly reliable faecal assays. 9 or direct and unpleasant rectal sampling procedures. 10 In order to address these research gaps, we have developed a novel in vitro gas-profiling technology that enables accurate measurement of multiple gases in real time. The gas profiler, shown in Figure 1, is composed of three different compartmentsa fermentation chamber, an array of gas sensors capable of simultaneous measurements of CO 2 , CH 4 , H 2 and H 2 S, and a pressure sensor that assesses total gas production. The gas profiler has advantages over previous in vitro fermentation techniques where simultaneous measurements of individual gas production has been difficult, and dynamics and kinetics of gas production cannot be obtained. 11 The other significant benefit of the system is the ability to monitor oxygen levels in the gas chamber's headspace using an oxygen sensor to achieve an nearly oxygen-deplete anaerobic environment (>98% oxygen-free gas). As such, the new technology allows for qualitative prediction of the kinetics of microbial activity in a rapid, convenient and well-controlled environment.
We hypothesize that, with the invention of this novel gas-profiling technology, gas profiles generated are accurate, reproducible and comparable to existing data from the literature for carbohydrate fermentation; and that the technology can be applied to assess changes in the metabolism of colonic microbiota in faecal samples when their microenvironment is modulated by relevant dietary substrates and drugs. Specifically, sulfur-containing amino acids such as cysteine, and inorganic sulfate promote colonic H 2 S production, while its production will be suppressed by fermentation of readily fermentable fibers, FOS and resistant starch, over poorly fermentable fibers, psyllium and sterculia, and exposure to 5-ASA. Thus, the aims of the present study are first to validate measurements of the in vitro gas-profiling system for overall and individual gas constituents (H 2 , CH 4 , CO 2 and H 2 S) in faecal samples; secondly, to determine its utility to provide kinetic data on gas production; thirdly, to examine its experimental utility in identifying factors in the diet that might modulate H 2 S production; and fourthly, to use 5-ASA as a model drug for its ability to modulate ex-vivo production of H 2 S. Figure 2 shows the inter-variability of three gasprofiling modules using the unspiked faecal sample from one donor. Variations in individual gas measurements between modules were minimal over the 2.5 h period. The coefficient variations for individual sensors at 2.5 h were 2% for H 2 , 5.7% for CO 2 and 1.6% for H 2 S. Figure 3 shows the reproducibility of gas measurements across three replicate experiments with resistant starch. Measurements between experiments were consistent for total gas, H 2 , CO 2 production and H 2 S suppression over 4 h (coefficients of variation: total gas 2.5%; CO 2 1.9%; H 2 4.6%; H 2 S suppression 5.2%).

Subjects
Seven healthy controls (3 males, 4 females) were enrolled in the study. The mean age of these volunteers was 34 [range: 23 -56] years old. Median 4-h H 2 S production from unspiked faecal slurries of all seven volunteers was documented to be 64.6 [11.3-202.9] mL.

Fermentation profiles of the fibers
Four replicate experiments were completed for each fiber, each replicate representing pooled faecal slurries from three healthy volunteers. Figure 4 shows the extent and rate of gas production with different fibers over 4 h of incubation. The volume of each gas produced at the end of the 4-h period in response to varying fibers is also shown in Table 1. Mean total gas production was the greatest for FOS, moderate for resistant starch and minimal for both psyllium and sterculia. The production of CO 2 and H 2 had a rapid onset and was highest in magnitude for FOS followed by resistant starch, but occurred passively and minimally for psyllium or sterculia ( Figure 4A-D). The production of CO 2 for all indigestible carbohydrates was about four times higher than that of H 2 during the 4-h fermentation period (Table 1). Compared to H 2 , much smaller amounts of CH 4 were produced by all volunteers.

Modulation of faecal H 2 S production
Four-hour continuous gas profiling for H 2 S following the addition of various fiber types is also shown in Figure 4(G & H). The magnitude of H 2 S suppression throughout the 4-h experimental period was much greater with resistant starch and FOS (p<0.001; twoway ANOVA) than the effects seen for sterculia or psyllium. The kinetics featured in Figure 4G shows two mechanisms of suppressionpassive and active. Sterculia and psyllium featured low suppression rates whereas FOS and resistance starch are featured with high suppression rates in the initial 2 h period (p<0.001; two-way ANOVA).
In addition, a total of four replicates from four different subjects were also completed for each of the following substrates. Mean Figure 5(A & B) shows the degree and rate of H 2 S production in response to the addition of cysteine and sulfate relative to an unspiked sample. Stimulation of H 2 S occurred in a linear fashion for cysteine and, at the end of the 4-h period, mean H 2 S production had increased by 1557 (532)%. In contrast, the degree of stimulation by sulfate was small (10 (40)%) at the end of the 4-h period.
The addition of 5-ASA to faecal slurries produced an initial suppressive effect in H 2 S production that tapered off as the experiment progressed. At the end of 4 h, the degree of H 2 S suppression was minimal (-7 (30) %).

Discussion
The profile of gases produced in the colon depend not only on the functional capabilities of the unique microbiota of the individual, but also by the nature of the substrates to which the bacteria are exposed. Specific gases such as H 2 S can influence the physiology and pathology of the colon. 12 For example, at lower luminal concentrations, H 2 S has been seen to promote resolution of inflammation in rat studies 13 but at higher concentrations, activates pro-inflammatory pathways leading to the development of colitis. 14 Thus, the quantification of its production in vivo and in response to changing substrate availability provide potentially important clinical information. However, difficulties in accessing luminal gaseous content and production rate in vivo have limited this line of enquiry. The novel gas-profiling technology utilized in this study was developed to enable simultaneous, realtime measurement of a spectrum of gases produced by the faecal bacteria to improve current understanding of the interactions of substrates with end-products of likely pathogenic significance. Indeed, our pilot experimentation has demonstrated the potential of the system to be a novel tool in studying H 2 S production and its modulation in the colon, a previously almost inaccessible target.
The H 2 S-sensing technique in the current study was able to overcome existing challenges in sampling and measuring H 2 S. First, despite the volatile nature of H 2 S, its release was measured in a strictly anaerobic environment with minimal variability between the three gas-profiling modules or across experiments. The sensitivity and specificity of measurements by the gas-sensing technology has already been demonstrated in an earlier study. 15 Furthermore, faecal H 2 S measurements in unspiked samples of volunteers are comparable to other reported measurements in faecal slurries in healthy controls. 16 Secondly, the system was able to measure free H 2 S released into the gaseous headspace, a crucial ability as adsorption by sampling materials can result in reduced H 2 S recovery. 17 Exposure to air can also reduce H 2 S levels appreciably. 16 and this necessitates the rapid determination of H 2 S under anaerobic conditions when using gas chromatography or high-performance liquid chromatography. Thirdly, we have demonstrated the experimental utility of the gas-profiling technology by describing gas production kinetics when faecal microbiota were exposed to different common fiber supplements as a model with physiological relevance.
The two major pathways of H 2 S production by the colonic microbiota comprise fermentation of amino acid or dissimilatory sulfate reduction utilizing hydrogen as an electron donor. 18 The current study found that the sulfur-amino acid, cysteine, was a more powerful and reliable stimulator of H 2 S production than inorganic sulfur. This information could be used to speculate that, quantitatively, sulfate reduction in the Values shown represent mean (SEM) of four replicate experiments using pooled faecal slurries. 1 Differences between fibers were evaluated using one-way ANOVA. Significant differences (p < 0.05; post-hoc Tukey's multiple comparisons test) between fibers are highlighted with the use of different superscripts. 2 The first timepoint at which gas production was significantly higher (p < 0.05 compared to baseline, where gas production is zero) as determined using one sample t-tests.
large bowel by sulfate-reducing bacteria may not be the major pathway of generating free luminal H 2 S, although further experiments may be needed to examine factors that increase its activity. It is possible that a larger quantity of sulfate is needed to stimulate a larger response of H 2 S in our study. Lewis et al. 19 observed an almost 2-fold increase in faecal sulfide concentrations following a 14-day dietary supplementation with 20 mmol/day of sodium sulfate in a small cohort of healthy volunteers. 20 but their findings likely reflected an increase in both free and bound H 2 S levels. In contrast, our findings are in agreement with those of two other studies. Levine et al. 16 observed a 300 -fold stimulation of free faecal H 2 S production by cysteine relative to that of an unspiked faecal slurry and this was much greater than the two fold stimulation with sodium sulfate at the end of the 24-h experiment. 16 Similarly, Florin et al. 21 found sulfate to be a poor stimulant of H 2 S production in vitro compared to cysteine, or another sulfur-containing substrate, mucin. One of the reasons for the ineffective  production of H 2 S by sulfate could be related to the greater presence of protein-fermenting bacterial species within the colonic microbiota, whereas the process of sulfate-reduction is highly specialized and limited to the less abundant sulfate-reducing bacteria. Hence, it may be prudent for dietary strategies to reduce excessive H 2 S production in the large bowel to target a reduction in the fermentation of sulfur-amino acids rather than sulfate-reduction. The administration of 5-ASA has previously been postulated to inhibit the genesis of luminal H 2 S. Initial observations were based on the lower content of total faecal sulfide (both bound and unbound) in patients with active UC and who were taking 5-ASA than those not on 5-ASA therapy. The inhibitory effects of 5-ASA appear to be mediated by interfering with both the rate of dissimilatory sulfate reduction and, to a lesser extent, the fermentation of sulfur-amino acids. This has been demonstrated in two studies in vitro with a reduction of 30-60% in H 2 S production being observed after 18-h incubation periods with 20 mmol/ L 5-ASA. 3,22 one with faecal cultures of amino-acid fermenting bacteria. 22 and the other measuring dissimilatory sulfate-reduction rates. Somewhat in contrast, we did not see such potent inhibitory effects at the end of our 4-h experiments whether with the incubations of faecal slurries was with 5-ASA alone or in combination with cysteine. Reasons for this discrepancy could potentially lie in the differences in experimental time courses or the methodology employed for H 2 S measurement. A longer incubation period could have provided more time for adsorption of H 2 S by 5-ASA compared to our experiments. Alternatively, 5-ASA may exert its effects mostly on bound H 2 S as seen in the earlier studies, 3,22 which may be less relevant to its pathogenicity. This suggests that, in patients with UC, the efficacy of 5-ASA therapy is less likely to be via reduction in metabolically active H 2 S in the large bowel.
Of great interest, and potential therapeutic importance, are the findings that resistant starch and, to a lesser extent, FOS, modulate H 2 S production in vitro. Only a few studies have observed such effects of these fermentable fibers on reducing bacterial H 2 S production. In in vitro fermentation experiments using colonic content of pigs as the inoculum. 23 both inulin and FOS added to cecal content (as the 'control' substrate) reduced H 2 S levels by 15% and 5% respectively at the end of 24 h. Lewis and co-workers. 19 also observed a reduction in faecal sulfide concentrations following a 12-day supplementation with oligofructose, a type of FOS, in healthy controls. No changes in concentrations of faecal sulfate, populations of sulfatereducing bacteria or sulfate-reducing rates were observed in their study. It is possible that the mechanism of action for oligofructose was via reducing fermentation of sulfur-amino acids. This was demonstrated in a study in ruminants where increasing overall carbohydrate fermentation via a decrease in colonic pH effectively decreased ruminant production of H 2 S. 24 This is highly likely as resistant starch and FOS have previously been shown to reduce biomarkers of colonic protein fermentation, including faecal ammonia, phenols and branched-chain fatty acids in animal studies, 25,26 and several human interventional studies, [27][28][29] An increase in nitrogen disposal. 26 or utilization of nitrogen to support increased bacteria mass following increased carbohydrate fermentation shifts the colonic microbiota away from utilizing protein as substrates for fermentation, 27,29 The reduction in protein fermentation by supplying readily fermentable carbohydrate to increase carbohydrate fermentation and thus, divert 'attention' of bacteria to protein as a fermentative substrate most likely explains the current findings that, following the addition of FOS to cysteine, the production of H 2 S is actively and markedly reduced (by 90%) in comparison to cysteine alone. The shift to carbohydrate fermentation was confirmed by a concurrent 12-to 16-fold increase in both CO 2 and H 2 production during the experiment following the addition of FOS to cysteine compared to cysteine alone. The corollary of this finding is that effective suppression of H 2 S production by faecal microbiota requires active carbohydrate fermentation with concurrent reduction in protein fermentative activity.
A small effect on H 2 S suppression was also seen with slowly or poorly fermentable fibers despite minimal participation in bacterial fermentation. As poorly fermentable but highly soluble and viscous carbohydrates, both sterculia and psyllium can exert other gelforming effects. 30 It is quite possible that these carbohydrates 'mop up' H 2 S produced spontaneously by faecal microbiota as they attract water to form a viscous gel within the faecal slurries. This would explain the passive H 2 S suppression by these fibers. No other data are available to corroborate these observations.
There are several limitations of this study. First, the use of faecal samples provides a representation of fermentative events in the distal colon and will not necessarily reflect events in the proximal colon. 31 Secondly, interpretation of the in vitro findings must be interpreted in light of the fate of dietary carbohydrates as they traverse the colon. Orally-ingested FOS and resistant starch, when delivered as a single supplement, are completely fermented in the proximal colon. 30 Translating these findings into the in vivo situation will require a strategy that will carry the fermentation of these fibers to the distal colon. Indeed, addition of wheat bran to the same resistant starch used in the present study carries the fermentation distally in rats, pigs and humans, [32][33][34] Thirdly, as the in vitro fermentation method is a closed system, it is unable to simulate the absorptive and detoxification systems occurring in vivo. It is for this reason that the fermentation period was limited to 4 h as pH dropped below 5 following this timeframe. As most metabolic activities of the colonic bacteria change (mostly inhibited) when the pH is 5 or lower. 35 the drop in pH below 5 would have significantly affected the metabolic ability of the faecal bacteria in our study.
In conclusion, this simple, proof-of-concept study has demonstrated the development of a novel faecal gas-profiling technology that provides reliable and simultaneous measurements of the highly volatile H 2 S gas with results comparable to the published literature where available. More importantly, this pilot study has also demonstrated the utility of this gas-profiling system as an assessment tool for predicting factors that modulate H 2 S production in vivo. Readily fermentable fibers, resistant starch and fructans, were shown to effectively suppress H 2 S, even in the presence of excessive H 2 S stimulation with cysteine, compared to poorly fermentable fibers. In addition, not all sulfur-containing substrates stimulate H 2 S to a similar extent, with fermentation of a sulfur-containing amino acid generating H 2 S more vigorously than dissimilatory sulfate reduction. On the other hand, 5-ASA exerted minimal inhibitory effects, disproving our initial hypothesis. In this way, the gas profiling system provides a means of assessing individual characteristics and might be very important for studies in which therapeutic strategies to modulate gas production (such as H 2 S in patients with UC) are being evaluated. It would also be worthwhile utilizing clinical samples from patients with active or quiescent UC to demonstrate the repeatability of these findings. These findings also have significant implications for the design of dietary strategies for the reduction of luminal H 2 S concentrations as a therapy for ulcerative colitis and even malodorous rectal gas. They suggest the importance of incorporating dietary strategies that increase fermentable fiber intake whilst concurrently reducing consumption foods rich in sulfur-amino acid, but not the restriction of inorganic sulfate or the use of 5ASA. Future studies should investigate faecal H 2 S responses in patients with UC following dietary manipulation with varying substrates to support the design of therapies that reduce the involvement of H 2 S in the pathogenesis of UC.

Assessment of repeatability of gas measurements
Gas-profiling modules were pre-calibrated using an external setup that includes an oxygen-free pressured chamber and a set of factory-calibrated gas-tanks with known concentration of the gases of interest. Each individual gas sensor was calibrated with a fixed gas concentration value and tested against typical concentrations of all common gases found in the colon to ensure specificity of measurements.
The inter-variability of the three gas-profiling modules was also assessed by measuring baseline gas production with unspiked fresh faecal inoculum from one healthy donor, incubated under anaerobic conditions at 37 C for 2.5 h. Intra-variability in measurements between three replicate experiments were also investigated by spiking pooled faecal slurries from three healthy donors with resistant starch under the same anaerobic conditions. Based on validation tests, the upper and lower limits of detection for each gas were determined and expressed as the volume of gas measured relative to the total headspace of the bottle or '% vol'.

Subjects
Faecal samples were collected from healthy volunteers with no known gastrointestinal diseases. Volunteers were excluded if they had taken any antibiotics, laxatives or fiber supplements in the preceding one month or probiotics in the three months prior to collection.
Volunteers were instructed not to change their diets during the collection period. All subjects provided written informed consent prior to collection of faecal samples. Ethical approval was obtained from Monash University Human Research and Ethics Committee (CF15/2454) for this study.

Sulfur-containing substrates
Two forms of sulfur-containing substrates were utilized to reflect differences in bacterial metabolism of sulfur-containing amino acid and inorganic sulfate. These comprised of 1.0 g cysteine (Sigma-Aldrich Co. LLC, NSW, Australia), a sulfur-amino acid, and 0.9 g sodium sulfate (Sigma-Aldrich Co. LLC, NSW, Australia), an inorganic sulfate (equivalent to 6.5 mmol). The dose of each substrate was calculated from the amount likely to be delivered to the colon from dietary intake in ileostomy studies, 36,37 .

5-ASA
A dose of 20 mmol/L of 5-ASA (MW 389.4, Sigma-Aldrich Co. LLC, NSW, Australia) was utilized to reflect the concentration of 5-ASA delivered to the large bowel during maintenance therapy. 38 .

In vitro fermentation procedure
The in vitro fermentation protocol was adapted from that of Edwards et al. 35 Fresh feces were collected into a sealed container, immediately gassed with oxygenfree nitrogen (N 2 ), and kept at 37 C in a waterbath. Samples from individual donors were used within 1.5 h of passage to ensure that colonic microbiota maintained viability. The inoculum was then prepared by mixing fresh feces with the sodium phosphate buffer to make up a 16% slurry (wt/vol). In the first set of experiments (assessing fermentation profiles of indigestible carbohydrates), the inoculum was initially made of faecal samples from three healthy donors and several replicates with the same fibre substrate was performed. Whilst this is standard procedure for in vitro fermentation experiments. 39 subsequent experiments (second, third and fourth sets) were repeated with faecal slurries from individual donors to capture potential individual variations in faecal bacterial metabolism and if necessary, exclude for outliers.
The mixture was homogenized in a household blender for 2 min under a stream of oxygen-free N 2 and filtered through four layers of muslin cloth to remove particulate materials. Aliquots (50 mL) of slurry were transferred to 200 mL bottles containing 10 mL of sodium phosphate buffer alone as a blank control or with test substrate. The headspace of the bottles were gassed with oxygen-free N 2 before incubation and then sealed. Each bottle was incubated with a shaking water bath at 37 C and at 50 shakes/ minutes.
Gaseous response to dietary substrates or 5-ASA Gaseous release from unspiked faecal slurries were measured via automated headspace sampling every 5 min over a 4-h period. In vitro experiments were conducted in four separate sets according to the postulated effects on H 2 S production. The first experimental set comprised spiking with various fiber substrates, the second with sulfur-containing substrates and third set with 5-ASA. Finally, samples were spiked with cysteine to stimulate excessive H 2 S production as well as with a H 2 S inhibiting substrate, either FOS or 5-ASA.
Total gas production was measured via a pressure sensor (ANKOM RF Gas Production System, ANKOM technology, Macedon NY) at 5-min intervals over 4 h. The data generated were then converted into volume in mL using the 'ideal-gas' and Avogadro's equations. The individual gases (CO 2 , H 2 S, H 2 and CH 4 ) were also sampled at a similar frequency and duration. Simultaneous measurements of CO 2 , H 2 and CH 4 response were used to assess the extent of bacterial fermentation.
Gas production of different gas species was calculated by multiplying the gas concentration at each timepoint with the headspace volume and expressed as volume (mL/g of fiber) after adjustment for any gas production originating from the unspiked faecal inoculum. Cumulative gas production at the end of the 4-h experimental period was used to assess the extent of fermentation. The rate of gas production was calculated using the derivative of the cumulative gas production volume as follows: gas production rate t ð Þ D d dt y t ð Þ where y t ð Þ stands for the cumulative gas production as a function of time.
Percentage of H 2 S suppression by each fiber was calculated using the equation below where H 2 S b indicates H 2 S production from a spiked sample whereas H 2 S g indicates production from an unspiked faecal inoculum

Statistical analysis
Statistical analyses were performed using SPSS v. 22 and MATLAB. Data were presented as mean (SEM) of cumulative gas volume per gram of fiber at different time points. Differences in the kinetics of gas production between substrates over the 4-h period were analyzed using two-way ANOVA. Differences in the percentage of change (whether stimulation or suppression) in gas production at the end of 4-h relative to an unspiked sample were analyzed using one-way ANOVA and, where significant differences identified between substrates, Tukey's multiple comparison analysis was performed.

Declaration of conflict of interest
All authors declare no competing interests in relation to the study.


s2fieldsofstudy:
Biology
Agricultural And Food Sciences

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Induction and recovery of copy number variation in banana through gamma irradiation and low‐coverage whole‐genome sequencing

Summary Traditional breeding methods are hindered in bananas due to the fact that major cultivars are sterile, parthenocarpic, triploid and thus clonally propagated. This has resulted in a narrow genetic base and limited resilience to biotic and abiotic stresses. Mutagenesis of in vitro propagated bananas is one method to introduce novel alleles and broaden genetic diversity. We previously established a method for the induction and recovery of single nucleotide mutations generated with the chemical mutagen EMS. However, officially released mutant banana varieties have been created using gamma rays, a mutagen that can produce large genomic insertions and deletions (indels). Such dosage mutations may be important for generating observable phenotypes in polyploids. In this study, we establish a low‐coverage whole‐genome sequencing approach in triploid bananas to recover large genomic indels caused by treatment with gamma irradiation. We first evaluated the commercially released mutant cultivar ‘Novaria’ and found that it harbours multiple predicted deletions, ranging from 0.3 to 3.8 million base pairs (Mbp). In total, predicted deletions span 189 coding regions. To evaluate the feasibility of generating and maintaining new mutations, we developed a pipeline for mutagenesis and screening for copy number variation in Cavendish bananas using the cultivar ‘Williams’. Putative mutations were recovered in 70% of lines treated with 20 Gy and 60% of the lines treated with 40 Gy. While deletion events predominate, insertions were identified in 20 Gy‐treated material. Based on these results, we believe this approach can be scaled up to support large breeding projects.

Introduction
The continual improvement of crops is necessary to mitigate expanding pressures on food security such as increasing population, yield reduction due to climate change and changing food preferences (Ronald, 2011). Accessible genetic variation is a fundamental component of plant breeding as it provides a repository of alleles that can be exploited for developing novel traits for crop improvement and adaptation. Traditional breeding methods in seed propagated crops utilize natural nucleotide and chromosome structure differences within or between species to create new allelic combinations and alter heritable traits. Where sufficient variation is not available or accessible, mutations have been induced by treatment of plant cells with chemical mutagens or ionizing radiation . The use of mutagenesis to facilitate breeding has been termed 'mutation breeding', and to date, there are over 3275 officially registered mutant varieties with an estimated global economic impact in the billions of dollars (Ahloowalia et al., 2004;Kharkwal and Shu, 2009).
Mutation-based approaches can be especially useful in species with a narrow genetic base or those that are recalcitrant to traditional breeding methods. For example, major bottlenecks exist for obligate and facultative vegetatively (clonally) propagated species where meiosis is rare or nonexistent. Additionally, mutagens that cause dominant or hemizygous (dosage based) phenotypes can increase the efficiency of generating novel traits in polyploids as the expression of phenotypes arising from recessive mutations requires the combination of mutations from homeologous sequences (Krasileva et al., 2017). Gamma irradiation has been used widely as a mutagenizing agent for breeding programmes for many crops. The Joint FAO/IAEA Mutant Variety Database (MVD) lists 2519 officially released mutant crops produced by treatment with physical mutagens from which 1614 produced by gamma irradiation (FAO-IAEA, 2017). Advances in genome sequencing technologies have allowed more precise evaluation of the effect of gamma irradiation on plant genomes Shirasawa et al., 2016). At higher dosages, the creation and repair of double-strand breaks result in genomic insertions and deletions. For example, using exome capture sequencing in maize, Jia reported the recovery of a 6203bp deletion linked to opaque kernel phenotype (Jia et al., 2016). In poplar, treatment of pollen with gamma irradiation resulted in indels varying between whole chromosomes to small fragments . This work further showed that large genomic indels could be effectively recovered using low-coverage whole-genome sequencing (LC WGS), making mutation discovery more cost-effective and data analyses more streamlined.
Large insertions and deletions lead to variation in copy number of many loci that can have profound effects on phenotypes of the organism. Copy number variations especially affect haploinsufficient genes for which a single functional copy of a gene is not sufficient for normal function (Birchler and Veitia, 2012;Casanova-S aez et al., 2014). Haploinsufficiency has been demonstrated to be induced by various stresses such as drugs, salt and high pH, and therefore may be important loci for improving crop resiliency to changing climates (Deutschbauer et al., 2005;Giaever et al., 2004;Lum et al., 2004). In addition, species that propagate asexually lack the mechanisms of meiosis and independent assortment to expunge deleterious alleles that arise in their genomes via spontaneous mutation. This phenomenon, known as Muller's ratchet, results in a high accumulation of heterozygous and potentially deleterious alleles as observed in species such as banana and potato (Jankowicz-Cieslak et al., 2012;Muller, 1932;Potato Genome Sequencing et al., 2011). Single-copy mutations, therefore, can potentially knock out the function of genes where only one functional copy is being maintained. Lack of meiosis also means that newly induced mutations will be more likely maintained in comparison with sexually propagated species. For example, in Arabidopsis thaliana (L.) Heynh. the majority of large deletions spanning millions of base pairs that are induced by treatment of pollen with gamma rays or carbon ion irradiation were not transmissible (Naito et al., 2005).
Bananas of the genus Musa L. are the fourth most important food crop and among the top ten food commodities for South-East Asia, Africa and Latin America (FAO, 2018a). The majority of the edible cultivars of bananas are selected parthenocarpic diploid and triploid clones from the inter-and intraspecific hybridization of diploid seeded Musa acuminata Colla and M. balbisiana Colla (Perrier et al., 2011;Simmonds and Shepherd, 1955). However, at present only a few of the selected clones are having consumerpreferred traits and dominate the global market. This genetic bottleneck has been perpetuated by tissue culture-based mass clonal production, propagation and maintenance systems developed since the 1970s (Gowen, 1995). The most common export banana is the triploid M. acuminata. Banana plantations are comprised of nearly genetically identical cloned plants. The lack of genetic diversity poses the threat of mass devastation in the event of a pathogen attack, as occurred in the late 1890s and early 1900s with the outbreak of Panama disease, caused by soilborne fungus Fusarium oxysporum f.sp. cubense (Foc) Race 1. This led to a widespread epidemic and resulted in collapse of the cultivar 'Gros Michel' from the global market (Ploetz, 2005). Resistant Cavendish bananas maintained as specimens in the botanical gardens in the United Kingdom and in the United Fruit Company in Honduras were identified and used to replace 'Gros Michel' (Ploetz, 2006). However, Panama disease by Foc Tropical Race 4 (TR4) affecting these Cavendish cultivars emerged in late 1960s in Taiwan and has been spreading across borders. Reports of its spread outside of South-East Asia to Jordon, Pakistan, Lebanon, Oman and Mozambique by 2013 have raised serious alarms as no commercial replacement for Cavendish bananas resistant to TR4 has been found yet (FAO, 2018b;Garc ıa-Bastidas et al., 2013;Molina et al., 2009). TR4 is potent, affects several local banana cultivars in addition to Cavendish types and can remain in soil for decades. Use of traditional fungicides to control TR4 has been largely unsuccessful. Its spread thus threatens not only the global export trade but also the local market and therefore can have a negative impact both on commercial economies and farmer's food security.
Inducing mutations in triploid M. acuminata provides an approach for generating genetic and phenotypic variation that can be clonally propagated. Previously, we established a system for inducing and maintaining SNP mutations induced by chemical mutagenesis. Treating shoot apical meristems of tissue cultured bananas with the chemical mutagen ethyl methanesulphonate (EMS) introduced a high density of GC-AT transition mutations (Jankowicz-Cieslak et al., 2012). We further showed that genotypic heterogeneity (also known as mosaicism or chimerism) caused by accumulation of different mutations in different cells of the plant propagule could be rapidly removed via isolation of shoot apical meristems and subsequent longitudinal bisection. We sought to establish a similar system for inducing and maintaining insertions and deletions. According to the MVD, only three mutant banana cultivars have been officially released. All three were produced by treatment with gamma irradiation. The cultivar 'Al-Beely' showing 30% higher yield was released in 2007 in Sudan, while 'Klue Hom Thong KU1' with larger bundle was approved in 1985 for release in Thailand. 'Novaria', demonstrating early flowering, and improved fruit quality, was released in Malaysia in 1995 and is reported as the most commercially successful (Mak et al., 1996;Nov ak et al., 1990).
Screening for putative mutant candidates is an intensive step in any mutation breeding programme. We therefore aimed to develop an efficient pipeline for the generation and recovery of large copy number variations (CNV) in gamma-irradiated Cavendish banana cultivars, employing tissue culture, low-coverage whole-genome sequencing (LC WGS) and chromosome dosage analysis. We chose a chromosomal dosage analysis that was previously successful in detecting aneuploidy, insertions and deletions in Arabidopsis, rice and poplar (Tan et al., 2015(Tan et al., , 2016. To establish a pipeline for banana, we first adapted sequencing and dosage analysis for the previously released mutant banana cultivar 'Novaria'. Large genomic deletions of up to 3.8 Mbps were recovered. We next developed a newly mutagenized banana population and tested two different irradiation dosages to establish that new genetic variation can be induced and maintained in vitro. This work suggests that a large-scale mutagenesis pipeline can be created for routine production of mutant populations suitable for glasshouse and field evaluations.

Establishing LC WGS dosage screening in banana mutant cultivar 'Novaria'
To establish recovery of large genomic indels induced by gamma irradiation, we used the officially released mutant cultivar 'Novaria'. This triploid is derived from gamma irradiation of the cultivar 'Grand Naine' (AAA). In vitro plant material of both was acquired from the Bioversity International Musa Germplasm Transit Centre (ITC) and propagated to produce sufficient material for pilot studies. Low-coverage whole-genome sequencing was performed on four leaves from both cultivars using 2x300PE sequencing with an Illumina MiSeq. Between 4.8 and 7.4 million reads were produced for samples, generating between 2.20-and 3.47-fold coverage (Table S1). Reads were aligned to the Musa acuminata reference sequence (D'Hont et al., 2012). A relative read coverage approach was used to discover variations in copy number in mutant samples whereby one nontreated 'Grande Naine' sample was chosen for all comparisons. Read coverage was normalized for a ploidy of three in each sample and then averaged over 100-kb bins along the 11 chromosomes. Putative mutations were subsequently scored as deviations from copy number in nontreated 'Grande Naine' when variation was present in mutant 'Novaria' replicates (Figures 1, S1 and S2). Visual analysis of coverage graphs revealed putative deletions in 'Novaria' ranging between 0.3 and 3.8 million base pairs (Mbp). Analysis further suggested that all events were single copy with dosage changing from three to two. We next sought to evaluate mutation scoring via direct analysis of frequency coverage tables that are the output of the bioinformatics analysis. Ten putative deletions were called when using a filter whereby indels were called only if coverage deviated over three consecutive 100-kb bins ( Table 1). As expected with this approach, the smallest deletions recovered were 0.3 Mbp in size.

Validation of genomic deletions in 'Novaria'
To test the accuracy of mutation scoring by visual analysis and high stringency filtering approaches, we chose the largest and smallest deletions identified to evaluate copy number by quantitative PCR. Primers were designed in the boundary regions flanking the putative deletion events where copy number was predicted to be unaffected, and also within the region of the predicted deletion. In both chromosomes, an approximate 33% reduction in yield was observed in the affected region, suggesting the presence of single-copy deletions as predicted by LC WGS assay ( Figure 2; Table S2). We next investigated genes predicted to be present in the deleted regions. One hundred eighty-nine genes fall within the affected areas (Tables 1 and S3). Gene Ontology (GO) analysis was performed to further evaluate affected regions ( Figure 3; Table S4). This revealed genes involved in diverse processes such as gene expression, cellular biogenesis, protein phosphorylation, protein and DNA binding, and membrane components.

Establishment of a pipeline for routine induction and screening of large indels in Cavendish bananas
Based on the results obtained with the previously released mutant 'Novaria', we developed a system for rapid mutation induction and screening of banana meristematic cultures using gamma irradiation that can be incorporated into a breeding programme ( Figure 4). Briefly, triploid bananas of the cultivar 'Williams' were clonally propagated through bisection of shoot apical meristems in liquid culture. Two gamma dosages were applied towards the banana populations, 20 Gy and 40 Gy. Genetic mosaicism resulting from mutagenizing multicellular tissues was removed through repeated rounds of longitudinal bisection of the shoot apical meristem as previously described (Jankowicz-Cieslak and . In order to quickly establish if a dosage treatment resulted in accumulation of novel mutations, we chose to evaluate a single plantlet per line. Leaves were isolated from 10 lines from each chosen treatment (20 Gy and 40 Gy). Whole-genome sequencing and copy number variation detection analysis were applied using the graphical approach established for 'Novaria' (Table S5). Mutations in 7/10 lines treated with 20 Gy and 6/10 lines with 40 Gy could be quickly visually identified, suggesting both dosages are suitable for use in forward-genetic screens ( Figure S3). Dosage graphs also indicate the presence of putative single-copy insertion events. In some lines, more than one mutation event could be visually detected. In total, we observe 10 events in 20-Gy-treated samples and nine events in 40-Gy samples. Biological replicates were sequenced to further evaluate accumulation of mutations in gamma-irradiated cultivar 'Williams'. This controls for potential false positive signals resulting from endoreplication events. Mutant line 40 Gy w-9 was propagated for four in vitro subcultures, and two clones were selected for LC WGS. All identified mutations were found in both replicates, indicating the gamma-induced lesions are stably mitotically inherited ( Figure 5). Analysis of frequency coverage output revealed 18 putative deletions accumulating in this line ranging from 0.3 to 6.8 Mbp. This covers a region containing 2762 predicted coding regions (Table 2).

Discussion
An important step in any programme employing induced mutations for functional genomics or trait development is the establishment of optimized parameters for the routine induction and recovery of mutations that can be applied to a large scale. This is especially important for clonally propagated crops such as banana where labour-intensive in vitro culture is needed and phenotypic evaluation requires large greenhouse and field resources. Decreases in sequencing costs coupled with advances in next-generation sequencing (NGS) offer a new avenue for improving the efficiency of banana trait development through induced mutations. Large insertions and deletions provide several advantages when considering methods for improving polyploid banana. Simultaneously affecting the copy number of many genes can potentially lead to dosage-dependent phenotypes and also reveal haploinsufficient loci. In the pilot screen, we recovered singlecopy genomic deletions in the commercially released 'Novaria' cultivar. More than 180 genes are predicted to be affected in 10 predicted deletion events. GO term analysis revealed genes involved in diverse processes such as gene expression and cellular biogenesis that may contribute to the phenotype of the cultivar. Further study is required to evaluate the effect of CNV on the expression of these genes and to estimate which targets may be causative for improved fruit quality and early maturity. While lack of meiosis and segregation makes establishing causality of mutations difficult, clonal propagation ensures mutations are  genetically fixed and phenotypes are stable; two major advantages. Another advantage to targeting large indels is that they can be efficiently discovered using LC WGS. In the pilot study of 'Novaria', we recovered deletions with approximately threefold coverage per sample. Such low-coverage whole-genome sequencing was previously shown to be an effective means of recovering CNV in poplar, rice and Arabidopsis and has been applied for evaluation of human clinical samples Kader et al., 2016;Tan et al., 2016). In our pilot, we chose to evaluate four leaves from the 'Novaria' cultivar and four nontreated 'Grande Naine' samples. Biological replicates were included to control for presence of CNV arising from endoreplication events. Endoreplication is a phenomenon in which cell genomes can replicate without a subsequent cellular division and chromosome reduction (Gutierrez, 2016). This has been observed in both plants and animals, can be stimulated by environmental conditions, and is controlled by genetic and epigenetic mechanisms (Breuer et al., 2014;H ribov a et al., 2016;Lee et al., 2009). The result of endoreplication events are cells that can have altered copy number of loci, thus affecting the ability to discover CNV induced by gamma irradiation. It is possible to differentiate between endoreplication events and true mutations by sampling different tissues. Endoreplication events are expected to be cell linage specific and may be random in different cells with respect to chromosomal region. This means that CNVs resulting from endoreplication are not expected to occur in all tissues of a plant. Such variation is therefore not expected in multiple progeny resulting from clonal propagation. In contrast, after plants are propagated to remove chimeric sectors, all detected mutation events are expected in all tissues and propagates. In our pilot screen, we found no example of endoreplication as putative mutation events were identified in all clonally replicated material. Further, no evidence of endoreplication was found in biological replicates of a newly mutagenized cultivar 'Williams'. Together, this suggests that interrogation of a single DNA sample (e.g. from a leaf) per mutant plant line is a suitable approach in banana.
To identify mutations in 'Novaria', we applied a dosage analysis method previously used for recovery of gamma-induced indels in poplar, rice and Arabidopsis Tan et al., 2015Tan et al., , 2016. We used a graphical approach and found that binning coverage into 100-kb bins provided a rapid visual method for finding large indels in the banana genome. To evaluate the accuracy of this approach, we chose the largest (3.8 Mbp) and the smallest (0.3 Mbp) deletions and confirmed these to be single-copy deletion events by qPCR. Single-copy events are expected for a gamma-induced deletion in mitotically propagated material. Validation of deletions by qPCR further shows the efficacy of using real-time PCR for the development of dosagebased molecular markers. In addition to being rapid, the analysis method is further advantageous in that direct comparisons are made between treated and untreated material and not to a published reference sequence. This is especially useful in cases where cultivars diverge significantly in sequence or structure from the published reference.
Owing to the fact that we did not observe endoreplication in 'Novaria', we chose to streamline our screening by sequencing only one sample per line in newly mutagenized material. This allowed seven mutants and one nontreated control to be sequenced on a single MiSeq run with a coverage of~3.59, similar to that used for screening 'Novaria'. Sample number and throughput can be increased using a higher throughput Illumina sequencing platform. Based on our results, we estimate 30 banana plants can be sequenced per lane on a HiSeq 3000/4000 for a coverage of 59. Sequencing biological replicates facilitates mutation calling. As sequencing costs drop, early identification of plants harbouring mutations will allow preselection of material for field evaluations based on affected genomic regions and/or specific coding sequences.
Induced mutations were relatively frequent in cultivar 'Williams' and were found in 70% of lines treated with 20 Gy and 60% of lines treated with 40 Gy. The observed rate of mutation accumulation in lines is slightly higher than the 50% recovery of mutations reported in gamma-irradiated poplar . Interspecific comparisons are difficult as variations in the density of induced mutations are expected due to differential responses in mismatch repair and programmed cell death pathways (Fulcher and Sablowski, 2009;Yoshiyama et al., 2009). Higher mutation rates, however, are expected for polyploids, in part due to the buffering effect of additional nontreated 'Grande Naine' genomes (Birchler, 2012;Stadler, 1929). Further, lack of meiosis in vegetatively propagated bananas means that most lesions will be maintained.
Ten lines produced by treatment with 20 Gy and ten lines treated with 40 Gy were evaluated in this study. Visual detection resulted in 10 mutation events observed in 20 Gy-treated material and nine events in 40 Gy-treated material. While the sample size is too small to be significant, this trend is comparable to that reported for gamma-irradiated rice where similar mutation rates were reported for seeds irradiated with 165 and 246 Gy . However, an effect of dosage on mutation accumulation was reported for poplar and observed at higher dosages in rice Li et al., 2016). Interestingly, putative insertion events were observed in two lines treated with 20 Gy, but none in material treated with 40 Gy. In addition, no insertion events were identified in 'Novaria'. Further studies are required to determine whether irradiation dosage or genotype affects the accumulation of insertions or has an affect on the size of insertions or deletions.
When considering the large field resources needed to screen bananas, the ability to sequence a small number of individuals in order to fine-tune mutagenesis should reduce costs and improve success rates. However, sequencing only one DNA sample per line likely results in an underrepresentation of the number of mutation events. For example, our analysis of two clones of a 40-Gy-irradiated 'Williams' revealed 18 putative deletion events covering approximately 20.5 Mbp of sequence. The inclusion of a biological replicate allowed the filtration of dosage fluctuations and enabled the scoring of more putative mutation events. This streamlines the analysis step as filtering rules can be applied to the output dosage table. In poplar, three consecutive bins with dosage variation were considered as true events . In banana, we tested this with the 'Novaria' data and found that deletions identified by graphical interface had at least  Figure 4 Schematic diagram of banana mutagenesis and recovery of mutations via LC WGS. Shoot tips isolated from banana plantlets propagated and maintained by shoot apical meristem culture were subjected to 20 or 40 Gy of gamma irradiation in bulk. Propagules were then recovered in groups of five and allowed to grow for approximately 4 weeks. Each plantlet at this stage is taken as the starting point of a mutant line to ensure unique mutation events in selected plantlets. The shoot tip of each plantlet is bisected and maintained in culture until growth of two leaves. The cycle is repeated for a minimum of three generations to remove genotypic heterogeneity resulting from mutagenesis of multicellular tissues. Leaves of plantlets from unique lines are then used for DNA extraction and subsequent whole-genome sequencing. Chromosomal dosage analysis is carried out to detect indels. Once suitable mutagenesis conditions are established, the procedure can be scaled up for large-scale plant phenotyping. Lines identified with desired phenotypes can be sequenced to develop dosage-based molecular markers to enable sample tracking and validation in multilocation field trials. three consecutive bins of similar dosage variation in the output dosage table for indel calling. We further set the filter value of ploidy in the dosage output tables to 3 (normal triploid state) and those in mutants to +/À 1, +/À 2 (corresponding to single-or double-copy indel events) for identical mutation calling events in all samples. When using low-coverage sequencing of biological replicates, however, false negative errors may be unavoidable. While it has been reported that larger indels predominate in   gamma-irradiated poplar and maize, an abundance of SNP and small indel variation has been observed in seed-irradiated rice Li et al., 2016;Yuan et al., 2014). Cataloguing all nucleotide variation requires higher coverage sequencing. Currently, this is cost-prohibitive for evaluation of many samples.
In summary, we have established a pipeline for routine induction and recovery of genomic indels in polyploid banana that can be scaled up for large mutation breeding projects. Efficient pipelines for mutagenesis and recovery of deletion mutations will remain an important tool, especially in vegetatively propagated crops where dosage effects may be key for generating phenotypes such as Foc TR4 resistance. Mutagenized bananas are currently being evaluated for variations in Foc TR4 response . Based on the results presented here, we propose a banana mutation breeding pipeline using gamma irradiation (Figure 4). We expect that further improvements and cost reductions in genome sequencing will make routine recovery of smaller indels and SNP mutations feasible in the near future, thus further expanding the resources for banana improvement.

Mutagenesis
Shoot tips of in vitro 'Williams' plantlets were isolated in water. 10-15 shoot tips were bulked in a Petri dish and subjected to 20-Gy or 40-Gy gamma irradiation using a cobalt-60 source (2.144 Gy/sec, Seibersdorf, Austria). Groups of five mutagenized shoot tips were placed in individual culture flasks and kept for recovery. Each shoot tip was used as the starting material for subsequent cultures and referred to as a line as previously described (Jankowicz-Cieslak and Till, 2016;Jankowicz-Cieslak et al., 2012). A minimum of three rounds of meristematic isolation and bisection were carried out to ensure removal of mutation-induced genotypic heterogeneity (mosaicism) as was previously established (Jankowicz-Cieslak et al., 2012).

Library preparation and sequencing
DNA was extracted from leaf material using the DNeasy Plant Mini Kit (Qiagen, Cat Nr: 69106) according to the manufacturer's guidelines. Genomic DNA was assessed for quality and quantity using NanoDrop, agarose gel electrophoresis and Qubit fluorimeter as previously described (Duitama et al., 2017). Genomic libraries were prepared using the TruSeq â Nano DNA Library Prep Kit (version 15041110 Rev. D) following the recommended protocol for 550 bp fragment size. Dual indices were used, and the libraries were normalized to 4 nM prior to pooling. Seven libraries were pooled together for primary screening of 'Novaria' and mutated cultivar 'Williams', each pool containing a combination of mutant lines and nontreated controls. Nine libraries were pooled together for sequencing of 40 Gy-treated 'Williams' line 9 (40 Gy w-9) biological replicates. Sequencing was performed on an Illumina MiSeq using 2 9 300 Paired End version 3 chemistry.

Data analysis
Raw data from the MiSeq were evaluated for the presence of large insertions and deletions as previously described . Briefly, the reads were first interleaved to form paired fastq files. A custom Python script (bwadoall) was then executed with default parameters (Comai, 2012). Bwadoall filters for quality, trims, aligns the reads against a given reference genome and converts the.sai files to give.sam files as output. The reference genome Musa acuminata v1 (Macuminata_304_v1) was used. Resulting BAM files are available at the Sequence Read Archive hosted by NCBI (SRA accession: SRP130725). Dosage plots were generated by running a custom Python script (BinbySam, 2013). The resulting dosage tables were then used to prepare graphs that were inspected for the presence of indels. Different binning sizes were evaluated, and the binning size of 100 kb was selected. To detect CNV in 'Novaria', data were compared to the nontreated 'Grande Naine' (GN), using two different approaches. First, the mean percentage values of all the individuals were used as a control. Secondly, a single GN sample was selected and all comparisons made to it. Similar dosage variation patterns were observed in both the methods (not shown), and so using a single reference sample was chosen for all subsequent analyses. The analysis of newly mutagenized cultivar 'Williams' material was performed by direct comparison to a selected nonirradiated 'Williams' control. Except for the parameter -p for ploidy, which was set at 3, default parameters were used in all other cases. Relative coverage values less than 3.0 indicate deletion of one or more copies of a chromosome fragment while higher values (>3.0) indicate potential insertional events. A dosage variation was considered to be true if at least three consecutive bins exhibited low or high relative coverage values. Read coverage and mapping statistics were generated from raw Bam files using QualiMap v.2.2.1 (Okonechnikov et al., 2016).

qPCR evaluation of copy number
Selected CNV regions identified by sequencing were visualized using the integrative genomics viewer (IGV) in order to select regions falling within and outside the called predicted mutations (Robinson et al., 2011). Primers were designed using Primer3 with Tm between 65 and 72°C (Rozen and Skaletsky, 2000). Primer sequences are listed in Table S6. qPCR was carried out on Qiagen Rotor Gene Q (Qiagen GmbH, Hilden, Germany) using the KAPA Library Quantification kits for NGS according to the manufacturer's guidelines and recommended temperature cycle settings except that kit-supplied primers were substituted with designed primers. The average of the concentration of the qPCR product (copies per mL) in the unaffected regions of 'Grand Naine' and 'Novaria' was calculated. These averages were used for calculating the ratio of the concentration of the qPCR product for each sample at each region. These data were used for plotting graphs. Standard deviation was calculated for the ratios obtained from two to three replicates.

Gene ontology analysis
Gene Ontology analysis was performed using Blast2GOBasic (Conesa and G€ otz, 2008). Sequences for genes in the regions of predicted CNV were acquired from the banana genome hub (BGH, 2018). Blast2GOdefault parameters were used to make multilevel pie charts.

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article: Figure S1 Relative sequence read coverage (RSRC) plots showing mutation calls where dosage differences are detected in the mutant in at least three consecutive 100 kb bins. Figure S2 RSRC plots showing the presence of the 3.8 Mbp deletion identified in mutant 'Novaria' in 4 biological replicates. Figure S3 RSRC plots of newly mutagenized cultivar 'Williams'. Data from 10 lines mutagenized at 20 and 40 Gy. Table S1 Raw read coverage statistics for cultivar 'Novaria' and cultivar 'Grande Naine'.


extfieldsofstudy: Chemistry Medicine

text:
Novel Antifungal Compounds, Spermine-Like and Short Cyclic Polylactates, Produced by Lactobacillus harbinensis K.V9.3.1Np in Yogurt

Lactobacillus harbinensis K.V9.3.1Np was described as endowed with high antifungal activity. Most of the studies associated this activity to the produced organic acids, i.e., lactic acid, acetic acid, and hexanoic acid. The aim of this study was to purify and identify, other not yet described, antifungal molecules produced by L. harbinensis K.V9.3.1Np when used in yogurt fermentation. Active compounds were extracted through several extraction processes using organic solvents and protein precipitation. The fractions of interest were purified using flash chromatography and preparative HPLC for specific characterization. The bioactive compounds identification was performed using Nuclear Magnetic Resonance and Mass Spectrometry. Activity tests against Penicillium expansum and Yarrowia lipolytica showed that the active compounds from L. harbinensis K.V9.3.1Np are benzoic acid and a polyamine identified as a spermine analog, which has not been reported earlier. However, the highest activity was shown by a mixture of short (n = 2–5) polycyclic lactates. Our overall results demonstrate the efficiency of the proposed extraction/purification approach. The new compounds described here have promising antifungal activities but further studies are still needed to decipher their mode of action and production pathways. Even though, they present an interesting potential application in food, feed, as well as, in pharmaceutical industries and could serve as alternative to chemical additives.

INTRODUCTION
There is a growing concern on microbiological safety of dairy food as their contamination can cause considerable economic losses. More specifically, the contamination of food matrices with various mycotoxins or other undesirable fungal metabolites became a serious issue in food industry (Moretti et al., 2017). Molds' development in food may be accidental or related to environmental factors promoting fungal growth and metabolite secretion. The fungal contamination inducing physicochemical deterioration of dairy foods is usually concomitant to organoleptic damages which consist mainly in off-odors and flavors associated to visible color and texture defects. In fermented milk products, fungal growth induces a decrease in the acidity promoting subsequently the growth of some undesirable microorganisms (Leyva Salas et al., 2017). Thus, the need of preventing food from microbiological spoilage challenged scientists to develop appropriate preservative solutions. Avoiding microbial contamination is achieved through suitable treatment, and/or appropriate preservative agents or by preventing the growth and the activity of those that were not eliminated after treatment (Leyva Salas et al., 2017).
Legislations and consumers requirements have led food manufacturers to look for an alternative to chemical preservatives and additives. In this context, the use of bioprotective agents is considered as a suitable solution to alleviate fungal deterioration. Biopreservation is ensured by natural or controlled microbiota and/or their metabolites. Among the most suitable candidates as natural preservative agents, Gram-positive lactic acid bacteria (LAB) are widely used (Dalié et al., 2010;Voulgari et al., 2010;Crowley et al., 2013). Historically, four main "core genera" were defined being involved in food fermentation (Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus) (Wessels et al., 2004). However, a reclassification was achieved and several other genera were added to this original grouping, including Lactococcus, Micrococcus, Oenococcus, Aerococcus and others (Crowley et al., 2013). Homofermentative and heterofermentative LAB induce pH decrease and produce antimicrobial molecules generating naturally conserved fermented foods. The antimicrobial mechanisms associated to the LAB involve a wide range of metabolite production including organic acids, phenyllactic acid, reuterin, cyclic dipeptides, fatty acids as well as proteinaceous and miscellaneous antifungal compounds (Crowley et al., 2013). Moreover, LAB have been granted a GRAS (Generally Recognized As Safe) status and were also included to the "Qualified Presumption of Safety" (QPS) list in Europe (Crowley et al., 2013;Delavenne et al., 2013).
Recently, a new strain isolated from cow milk and identified as Lactobacillus harbinensis K.V9.3.1Np, demonstrated a strong antifungal activity against six species commonly involved in yogurt fungal spoilage, Debaryomyces hansenii, Rhodotorula mucilaginosa, Yarrowia lipolytica, Penicillium brevicompactum, Kluyveromyces lactis, and Kluyveromyces marxianus (Delavenne et al., 2015). In fermented yogurt, the antifungal activity was not affected through the storage and the technological processes indicating its adequacy as a substitute to chemical additives (Delavenne et al., 2013). In separate reports, Belguesmia et al. (2014) and Mieszkin et al. (2017) quantified and tested several organic acids produced by L. harbinensis K.V9.3.1Np, such as acetic, lactic, hexanoic, 2-hydroxybenzoic, and 2pyrrolidone-5-carboxylic acids. These compounds were shown to constitute part of the overall antifungal activity of the strain by acting in synergy with other, yet unidentified antimicrobial agents.
The aim of the current study was to identify, purify and characterize potential new compounds responsible for the antifungal activity of L. harbinensis K.V9.3.1Np. Our experimental design was based on a multi-step extraction and purification protocols (proteins and organic fractions) from 7 liters-yogurt culture. The identified biomolecules were then subjected to structural elucidation using nuclear magnetic resonance spectroscopy and mass spectrometry.

Microorganisms and Culture Conditions
Lactobacillus harbinensis K.V9.3.1Np was previously isolated from cow milk (Delavenne et al., 2012). It showed strong antifungal activity against a panel of yeasts and molds in yogurt as an antifungal protective culture (Delavenne et al., 2013).
Yarrowia lipolytica UBOCC 211004 and Penicillium expansum UBOCC 108102, two fungi commonly encountered in dairy food and yogurt spoilage (Fleet, 1990;Cousin, 2002), and particularly resistant to organic acids (Delavenne et al., 2013), were chosen as indicators for antifungal assays. They came from the Culture Collection of Université de Bretagne Occidentale (UBOCC, Plouzané, France). P. expansum was cultivated on potato dextrose agar (PDA, Difco, Le Pont de Claix, France) slants for few days at 25 • C until spores were formed. Spores were then harvested with sterile distilled water supplemented with 0.1% Tween-80 and the suspension was spread on PDA in a Roux flask to increase spore production (Roy et al., 1996). Spores were harvested, enumerated using a Malassez cell and stock suspensions were standardized to a final concentration of 10 7 spores/ml before storage at −80 • C in glycerol (10%, v/v).
Yarrowia lipolytica was stored in yeast extract and malt based medium (YEMA) supplemented with glycerol (30%, v/v) at −80 • C, and cultivated aerobically at 25 • C on YEMA agar.

Yogurt Supernatant Recovering, Activity Control, and Protein Precipitation
Yogurts were produced with half-fat pasteurized milk supplemented with low-fat milk powder (4%) (Lait en poudre écrémé, Casino, France). After heating at 85 • C for 30 min, milk was rapidly cooled to 45 • C before being inoculated with the milk freeze-dried starter according to the manufacturer instruction.
Milk was then mixed and dispatched as 100 ml cultures into glass containers of 120 ml.
Preliminary control experiments and activity detection were performed on 1 L-cultures inoculated or not with L. harbinensis at a final concentration of 5.10 7 CFU/ml. Subsequent experiments aiming to separate and purify protein and organic fractions were then achieved with 7 L-culture inoculated with L. harbinensis. Fermentations were conducted at 42 • C for 6 h. Yogurts were stored for 2 weeks at for 10 • C after fermentation. Yogurts were centrifuged for 10 min at 14.000 g and supernatant was recovered by filtration on 0.45-µm nitrocellulose filter.
From the 7-L inoculated culture, protein precipitation was achieved using the following procedure (modified from Bhattacharyya and Babu (2009): (i) Ammonium sulfate precipitation (from 10% saturation to 100% saturation in 10 steps) by gradual incorporation of the salt and stirring for 2 h at 4 • C and centrifugation at 9000 g for 20 min for each step. The precipitate of each step was resuspended in water at 4 • C; (ii) From each recovered fraction (n = 10), soluble proteins with low molecular mass and peptides were separated by cell centricon (Amicon Millipore), with 10 kDa cut-off. A total of 20 FSP fractions were collected, lyophilized and tested for their antifungal activity against the indicator strains.

Solvent Extraction and Purification of Secondary Metabolites
Ethyl acetate extractions and activity tests were performed on the 1 L inoculated and non-inoculated control cultures and on the 7 L inoculated culture. From this later, protein-free supernatant was extracted with the same volume of ethyl acetate (three times) (Khan et al., 2016). The organic phases were combined and evaporated to dryness, under vacuum at 45 • C. The resulting DOA fraction (3.4 g) was solubilized in ethyl acetate dried with anhydrous MgSO 4 generating a major soluble fraction named FOP (3 g) and a minor insoluble fraction resuspended in bidistilled water FOE (0.4 g).
The first step of the purification of the organic ethyl acetate fraction (FOP) was assessed by flash chromatography on silica gel (Stevens and Hill, 2009). Elution was performed using dichloromethane (DCM)-methanol gradient (0 to 10% methanol for 18 min followed by an isocratic flow of 10% methanol for 10 min and finally isocratic gradient of 20% methanol-80% DCM for 20 min). The final purification was achieved by TLC with DCM-methanol (90:10, by vol.) and spot visualized in UV detector. A total of 18 fractions were evaporated under vacuum at 45 • C and stored at −20 • C until used for antimicrobial tests (Uzair et al., 2018).

Chromatography Purification
Active FOP and FOE fractions were analyzed by RP-HPLC on a XTerra C18 column (4.6 mm * 100 mm, 3.5 mm) using a Waters 2696 system equipped with a 600 PDA and Empower software. Solvents A and B were H 2 O and MeCN supplemented with 0.08% (v/v) TFA. Linear 0-60% gradients of B into A over 45 min, 60-95% B into A for1 min followedby 95% A isocratic gradient during 10 min were used for elution at 1 mL/min flow rate, with UV detection at 214, 268, 280, and 220 nm. For separation, preparative RP-HPLC was performed usingan XTerra C18 column (19 mm × 300 mm, 10 mm, Waters) with the solvents A and B, 0.1% TFA (v/v) in H 2 O and 0.08% TFA (v/v) in MeCN, respectively. For elution we used linear 0-60% gradients of B into A over 40 min, 60-95% B into A for1 min followedby an isocratic gradient during 10 min with 95% A and 5% B at 10 mL/min flow rate, with UV detection at 214 nm (Solecki et al., 2015).

Antifungal Activity of Fractions
The antifungal activity of each fraction was determined by the agar diffusion method (Delavenne et al., 2015). Yeast suspensions were prepared in sterile 0.1% peptone water by scraping colonies from the surface of YEMA agar after 2 days incubation at 25 • C. Spores suspensions were directly prepared from the stock suspensions. Cells and spores were enumerated with a Malassez cell. Appropriate amount of spores or yeasts cells suspension were added to YEMA agar (0.8%) to achieve a final concentration of 5.10 4 spores or cells/ml. The medium was mixed properly and poured in Petri dishes (90 mm). After solidification, wells of 9 mm diameter were made in the center of each agar plate. FSP fractions were dissolved in 200 µl water/methanol 80/20 and FOP fractions in 200 µl of DCM/methanol 80/20. Each well of both Y. lipolytica and P. expansum agar plates, were filled with 80 µl of the tested fractions. Water/methanol 80/20 and DCM/methanol 80/20 were both used as negative controls. Agar plates were then put at 10 • C for 2 h to allow products diffusion, and then incubated at 25 • C for 48 to 72 h. Upon incubation time, inhibition zones were measured.

NMR Analysis
1D NMR spectra were recorded using a Bruker Avance 500 spectrometer equipped with a BBO cryoprobe at the PRISM core facility (Rennes, France). Sample were analyzed in CDCl 3 solution or in water solution with 10% D 2 O using a sequence with presaturation.

ESI-TOF, ESI-MS/MS, and LC-ESI-Q-TOF ANALYSIS
All ms Spectra were recorded at on Waters Q-TOF 2 and Bruker MicrOTOF-Q II and Maxis 4G Mass Spectrometers the CRMPO core facility (Rennes, France).

RESULTS
Several studies were conducted using a targeted approach on the antifungal activity of L. harbinensis K.V9.3.1Np to assess the active biomolecules (Belguesmia et al., 2014;Mieszkin et al., 2017). The obtained results suggested that acetic, lactic, hexanoic 2-hydroxybenzoic, and 2-pyrrolidone-5-carboxylic acids, acted as antifungal compounds but other antifungal agents, not targeted in these studies, could be involved in the high antifungal potential. In the current work, using an untargeted approach, we focused on the identification, purification, and characterization of all non-acids antifungal compounds produced by L. harbinensis.

Screening of the Fractions Harboring Antifungal Activity
Antifungal susceptibility tests of the different extracts were carried out using the disk diffusion method. After the fermentation process with L. bulgaricus, S. thermophilus amended or not (control culture) with L. harbinensis K.V9.3.1Np, the yogurt culture supernatant was separated into different fractions using ammonium sulfate precipitation, ethyl acetate, and dichloromethane. Then the extracted fractions were tested using P. expansum and Y. lipolytica as reference organisms. Antifungal activity was retrieved only from the organic extracts of the L. harbinensis inoculated cultures (data not shown).
In a second step, the organic and aqueous extracts using ethyl acetate and dichloromethane were tested. As preliminary results shown in Figure 1, we observed a quite pronounced inhibition zone for the organic fraction of L. harbinensis K.V9.3.1Np supernatant extracted with ethyl acetate (FOP). In fact, after 48 h of incubation at 25 • C with the disks, clearing zones of 3.5 mm and 4 cm in diameter were observed in P. expansum and Y. lipolytica, respectively ( Figure 1A). The aqueous fraction (FOE) also exhibited an antifungal activity but of a relative weak intensity compared to the organic one ( Figure 1B). Based on these results, we focused on the ethyl acetate organic extracts for further characterization of L. harbinensis K.V9.3.1Np anti-fungal potential.

Identification of Bioactive Extracts
Ethyl acetate organic fraction (FOP) is a mixture of several compounds that may react in an antagonistic or synergistic way to exhibit the overall antifungal activity. Thus, we were interested in the identification of the bioactive compounds present in this fraction. To rapidly target the molecule responsible for this activity, a flash chromatography was performed. This technique was used to increase the compounds separation resolution and to collect individually purified molecules.
Eighteen fractions were collected (Figure 2) and then dried to be used for antifungal assays using P. expansum and Y. lipolytica indicator strains. Among the tested fractions, the active ones are presented in Figure 3. The inhibition zone varied with the different sub-extracts as well as with the two tested fungal strains. Six fractions, i.e., DOA4, DOA5, DOA7, DOA12, DOA13, and DOA14, showed an interesting inhibition zone ranging from 0.9 to 4 cm.

Selection of Sub-Fractions of Interest From the Identified Bioactive Extracts
Further investigation was performed on the antifungal activity of these collected fractions. For that purpose, an additional fractionation step was performed using preparative chromatography. The results shown in Figure 4A clearly demonstrated that those fractions contained multiple compounds that may be responsible for this antifungal activity. Thus, a second activity test was achieved with the recovered sub-fractions. After this step, we could identify sub-fractions DO4-1, DO5-1, DO7-1, DO13-1, and DO14-1 as responsible for this antifungal activity ( Figure 4B).

Compounds Identification in the Sub-Fractions of Interest
The structural elucidation of the compounds present in subfractions DO4-1, DO5-1, DO7-1, DO13-1, and DO14-1 was a key    step of this study. Thus, mass spectrometry and nuclear magnetic resonance were used for that purpose. Structural elucidation of both compounds DOA4-1 and DOA5-1 was based on NMR spectral analysis. These compounds were identified as benzoic acid as clearly shown in Figure 5. In addition, Figure 6 shows their mass spectra obtained by ESI-TOF and confirmed their identification as benzoic acid.
The purified fraction DOA7-1 also exhibited an interesting antifungal activity. ESI-MS/MS was performed in order to elucidate the structure of this compound. Based on the fragment ions observed after collision induced dissociation associated to the molecular formula assigned to these ions using high resolution measurement, two structures were proposed for the compound DOA7-1 (Figure 7). The proposed structures were FIGURE 5 | NMR spectra in water of the actives compounds identified as benzoic acid in the DOA4 and DOA5 fractions. unsaturated polyamine named spermine analogous compounds that have not been reporter earlier.
It is worth noting that the attributions of these structures were initially based on the difference of 2 mass unit with spermine (precursor ion [MH] + m/z 203). Moreover, the molecular formula attributed to the DOA7-1 molecule with high resolution measurement was C 10 H 24 N 4 (experimental mass: 201.2082, theoretical mass: 201.2079, and relative error: 1 ppm). The spermine molecular formula being C 10 H 26 N 4 , it is irrevocable that a cyclization occurs or an instauration is added to form the new structure explaining the loss of the two hydrogen. Moreover, for all the amines, in MS/MS analysis, the loss of a neutral fragment of ammonia led to the formation of a fragment with an m/z signal that corresponded to the species [M+H-NH 3 ] + . According to literature for spermidine, the product ion was generated by the loss of 2 molecules of ammonia, giving m/z 112 [M+H-2NH 3 ] + (Sagratini et al., 2012). For spermine, the fragmentation of the precursor ion [MH] + m/z 203, produced an ion at m/z 112 [M+H-(CH 2 ) 3 N 2 H 4 -NH 3 ] + (Sagratini et al., 2012). These results are coherent with the proposed structures as we observed after MS/MS fragmentation of the precursor ion [MH] + m/z 201, a product ion m/z 184 [M+H-NH 3 ] + . The other characteristic product ions confirmed further the proposed structures as they corresponded to specific losses associated to this structure. In fact, the ions m/z 144, m/z 127, m/z 113 were identified as [M+H-(CH 2 ) 3 NH 3 ] + , [M+H-NH 3 -(CH 2 ) 3 NH 2 ] + , and [M+H-(CH 2 ) 3 NH 2 -(CH 2 )NH 3 ] + .
The analysis of the most active compound DOA13-1 using Maxis 4G II, LC-ESI-Q-TOF, revealed the presence of polyester compounds probably formed through the polycondensation of lactic acid molecule. The short polycondensation of lactic acid to form a short poly (lactic acid) is described in equation (1). In fact, as shown in Figure 8, the four detected peaks with m/z 401, m/z 329, m/z 257, and m/z 185 correspond to di, tri, tetra-, and penta-lactate compounds, respectively.
Eq. (1) The analysis of the compound DOA14-1 was performed using the Waters Q-TOF II Mass Spectrometer, an LC-MS/MS system that incorporates a quadrupole with the high efficiency of an orthogonal time-of-flight analyzer. Accurate masses of the product ions were measured by the Waters Q-TOF II ( Table 1). The spectra obtained through this analysis and the accurate mass measurements allowed the identification of the compound DOA14-1 also as a poly (lactic acid) (n = 1-4) (Figure 8).

DISCUSSION
Several studies reported the ability of lactic acid bacteria for production of proteins or proteinaceous compounds as antifungal agents (Magnusson and Schnürer, 2001;De Muynck et al., 2004;Falguni et al., 2010). For that purpose, the first extraction experiments from L. harbinensis K.V9.3.1Np-mediated fermented yogurt supernatant, consisted in protein precipitation and centrifugal filtration for protein, concentration. In order to minimize the mass effect of casein, we proceeded to a gradual precipitation with ammonium sulfate ranging from 10 to 100% saturation, followed by a separation with a centricon of a 10 kDa cutoff to obtain 20 final proteinaceous fractions. Those fractions were tested for their antifungal potential. Our results revealed the absence of antifungal compounds of proteinaceous nature.  The incorporation of weak acids in industrial food and beverage production for shelf-life increase is a common practice (Huang et al., 2010). Benzoic acid is one of the most commonly acid preservatives used in food for preventing microorganism growth. Benzoic acid produced by L. plantarum VTTE-78076 isolated from beer (Niku-Paavola et al., 1999) as well as from L. plantarum MiLAB 393 isolated from grass silage (Broberg et al., 2007) were also reported for their antifungal properties. The origin of benzoic acid is not fully elucidated. Lactic bacteria convert hippuric acid which is naturally present in milk into benzoic acid via hippurate hydrolase [EC:3.5.1.32] (phenylalanine degradation) (Sieber et al., 1995). Also, benzoic acid can be produced by auto-oxidation of benzaldehyde (Sieber et al., 1995;Urbienë and Leskauskaitë, 2006) and β-oxidation in the catabolism of fatty acids (Hertweck et al., 2001). Hence, analysis of the available genomes of the starter bacteria S. thermophilus JIM 8232 (SAMEA2272807, PRJEA68521, and GCF_000253395.1), L. plantarum strains WCFS1, and 5-2 (SAMEA3138345, PRJNA356, GCA_000203855.3, SAMN02953961, PRJNA257680, and GCA_001278015.1) and L. harbinensis DSM16991 (SAMN02440850, PRJNA188920, and GCA_000425885.1), showed the absence of several of the enzymes involved in these pathways (data not shown). Further genomic analyses will give us a better understanding of the genetic features and possible pathways responsible for the benzoic acid production or transformation by lactobacilli. Organic acids generated during L. harbinensis K.V9.3.1Np growth were previously reported to play an important role in antagonism toward molds (Belguesmia et al., 2014). The implication of weak organic acids in disruption of membrane organization and oxidative stress was supported in several studies (Hazan et al., 2004).
Spermidine and spermine are biogenic polyamine known as an essential polycationic compounds found in all living organisms (Kwon and Lu, 2006). Spermine, widely spread in human and animal tissues was historically reported for its antibacterial activity against a wide range of microorganisms (Fair and Wehner, 1971). However, it was previously reported that due to the predominance of spermidine in fungi, spermidine synthase inhibition or spermidine function alteration may cause fungal growth decrease (Mackintosh et al., 1997). Some spermidine analogs were proved to reduce infection of barley, bean and apple seedlings by a variety of powdery mildew fungus (Mackintosh et al., 1997). Further investigation should be performed to understand the mechanism of action of those new molecules as antifungal compounds but this is the first report on molecules from the spermine family acting as antifungal compounds and produced by bacteria.
Polylactic acid belonging to the class of aliphatic polyesters are known as a biodegradable polymer obtained through the condensation and polymerization of a monomer, lactic acid, that can be derived through the fermentation of carbohydrate feedstock. PLA is well known for its antimicrobial potential used in oligomers solution or associated to organic acids and other antimicrobial agents (Liu et al., 2007). PLA matrix is frequently used in addition to other compounds like pectin (Liu et al., 2007), nisin (Jin and Zhang, 2008), cellulose nanocrystals and silver nanoparticles (Fortunati et al., 2012) and many others for antimicrobial food packaging (Tawakkal et al., 2014).

CONCLUSION
In this study, new types of antifungal compounds produced by L. harbinensis K.V9.3.1Np were characterized. The ability of extracting, purifying and identifying those compounds was clearly demonstrated. Although the new compounds described in the current work have promising antifungal activities, deeper investigation should be performed to assess their production pathways, mode of action (either acting alone or in synergistic manner) and how they mediate the overall antifungal activity of L. harbinensis K.V9.3.1Np Of particular interest, short polylactates, present an interesting potential application in food, feed as well as pharmaceutical industries.

AUTHOR CONTRIBUTIONS
AM contributed to study conception, extraction, purification of compounds, data acquisition, analysis and interpretation, and manuscript drafting. ED contributed to data acquisition (antifungal test), analysis, and interpretation. YS contributed to MS spectra analysis and critical revision of the manuscript. MM performed bioinformatics and annotation analysis. PJ and NLY acquired the data. AC interpreted the results and critically revised the manuscript. AB contributed to NMR spectra acquisition and revision of the manuscript. JM conceived the study and revised the manuscript. MB-F contributed to study. GLB conceived and coordinated the study and revised the manuscript.

FUNDING
This study was conducted with the financial support of the French National Agency for Research (ANR): program FUNGINIB (ANR-09-ALIA-005-01.

ACKNOWLEDGMENTS
We are most grateful to the PRISM and CRMPO core facility (Rennes, France) for their technical support.