IJAMBR 3 (2015) 96-103 ISSN 2053-1818

Bacterial quality of postharvest Irvingia gabonensis (Aubry-Lecomte ex O’Rorke) fruit wastes

Ebimieowei Etebu* and Gloria Tungbulu

Department of Biological Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria.

Article History ABSTRACT Received 01 October, 2015 Irvingia gabonensis is an economically important fruit tree. Although the fungal Received in revised form 28 postharvest quality of its fruits have been studied, associated with its October, 2015 Accepted 03 November, 2015 decay are yet unknown. Hence in this research, the bacterial quality of Irvingia fruit wastes was studied during 0, 3, 6 and 9 days after harvest (DAH). The Keywords: results obtained show that postharvest I. gabonensis fruits decayed with time. Irvingia gabonensis, Fruit weight and pH were significantly (P=0.05) influenced by DAH. Mean fruit Bacterial weight at 0, 3, 6 and 9 DAH was 31.99, 29.46, 26.56 and 23.37 g respectively. Postharvest, Mean pH value decreased from 6.42 at 0 DAH to 6.31, 6.22 and 6.17 at 3, 6 and 9 Decay, DAH respectively. Phylogenetic analysis of 16S rRNA partial gene sequences Phylogenetic analysis. showed that bacterial species related to Bacillus, Enterobacter, Oceanobacillus and Staphylococcus were obtained from I. gabonensis fruits up to 3 DAH but not beyond. Whilst recommending proper washing of fresh Irvingia fruits to avoid Article Type: food poisoning, findings of this work offer the potential use of I. gabonensis fruit Full Length Research Article wastes as substrate for antibiotics production. ©2015 BluePen Journals Ltd. All rights reserved

INTRODUCTION

Irvingia gabonensis (Aubry-Lecomte ex O’Rorke) is a most known uses and is therefore considered the most highly, economically important fruit tree native to most valuable component of the fruit. Thus, once it is tropical forests in West and Central Africa (Harris, 1996; extracted, the mesocarp and other parts, which constitute Lowe et al., 2000). It ranks highest of all non-timber forest over 80% of the fruit, are discarded as waste and left to products being proposed for domestication (Agbogidi and rot in dumps, water bodies, pits or nearby bushes (Etebu, Okonta, 2003; Leakey et al., 2003; Ndoye et al., 1998) in 2012; Ladipo et al., 1996). West and Central Africa. As such I. gabonensis is However, some works have shown that the pericarp of prominent among the trees of choice in agroforestry Irvingia fruits just like the kernels contain several practices (Koyejo and Omokhua, 2001; Leakey, 1999; nutritionally beneficial substances such as carbohydrates, Okafor, 1985, 1991; Okafor et al., 1996). It is sometimes proteins, lipids, vitamins etc. (Onimawo et al., 2003). This called bush mango or African mango because the trees is in addition to various phytochemicals perceived to bear mango-like fruits (Matos et al., 2009; Ngondi et al., contribute to the wellbeing of locals who consume the 2005) of 4-7 cm long, green when unripe and yellow fleshy components (mesocarp) of the fruit (Etebu, 2012, when ripe with a fleshy mesocarp (Etebu, 2013). 2013). Furthermore, some researchers have reportedly Although, Irvingia sp. is widely recognized and exploited, used the mesocarp as feed for pigs, and in the production it has been observed that the interest of most local of fruit drinks, wine, jam and other syrups (Ayuk, 1999). harvesters is in the kernel or seed because it has the I. gabonensis fruit ‘wastes’ are beginning to attract the attention of scientific research owing to the afore- mentioned potential benefits and uses. Etebu (2012, 2013) recently studied the fleshy component of the fruit *Corresponding author. E-mail: [email protected]. Tel: +234 (mesocarp) with a view to understanding some conditions (0) 802-982-9015. under which Irvingia decays as well as the fungi

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associated with such decays. These studies showed that was measured with a pH meter, and 1 ml of the slurry mature fruits of I. gabonensis fruits are usually completely was plated onto Nutrient agar (Oxoid Ltd, Hampshire, green at harvest with very few patchy specks of dark UK) previously impregnated with a fungicide, Itraconazole colourations without rot, but these fruits turn yellow as (4 µgml-1) according to Cutsem (1989). The plates were they ripened after about three days of harvest and then incubated at 28°C for 48 h. Different distinct colonies develops brownish-black fruit rot disease symptoms were repeatedly sub-cultured after every 48 h onto newly which increases in severity with the passage of time. prepared Nutrient agar plates until pure cultures were Aspergillus, Penicillium, Fusarium and Botrytis have been obtained. The entire process was repeated for fruit implicated with postharvest fruit rot of Irvingia (Etebu, wastes on 3, 6 and 9 DAH. 2012, 2013; Joseph and Aworh, 1991). Although these pioneering works on the pathology and fungal quality of postharvest Irvingia sp. fruits are DNA extraction and sequencing appreciated, there is still need to study the potential role of bacteria in the postharvest quality of the fruits, being Pure cultures of bacterial cells obtained, as described very important determinant of postharvest spoilage of above, from fermenting postharvest Irvingia fruit wastes fruits, especially juicy and succulent fruits. were washed three times in 1 ml of ultra-pure water and Hence, this research was aimed at identifying the spun by centrifuging at 12,000 rpm for 5 min. Thereafter bacteria associated with decaying postharvest Irvingia sp. DNA was extracted and purified using ZR fruit waste. Findings from this work would serve to Fungal/Bacterial DNA MiniPrep™50 Preps. Model D6005 forewarn locals on the potential health risk or benefits (Zymo Research, California, USA) according to the and implications associated with consumption of Manufacturer’s protocol. Briefly, 50-100 mg of bacterial harvested fruits. cells were re-suspended in 200 μl of deionized sterile water and transferred into a ZR BashingBead™ Lysis Tube. Exactly 750 μl Lysis solution was added and MATERIALS AND METHODS bacterial cells were lysed by bead beating for 5 min at maximum speed prior to centrifugation at 10,000 × g for 1 Samples collection min (Zymo Research, California, USA). Using a pipette, 400 μl of the upper aqueous phase Irvingia fruits were harvested from a natural forest containing nucleic acids was then transferred into a situated in Amassoma town (Latitude 4° 58′ 09″ N and Zymo-Spin™ IV Spin Filter collection tube and Longitude 6° 06′ 34″ E) of Bayelsa State, Nigeria. A total centrifuged at 7,000 × g for 1 min. The filtrate was then of 900 fresh and green fruits were randomly selected and mixed with 1.2 ml of Fungal/Bacterial DNA binding buffer split with a machete to extract the kernel, and the and 800 μl of the mixture was thereafter transferred into a pericarp which is usually considered waste by locals were Zymo-Spin™ IIC column in a collection tube and thereafter separated into three replicates (300 fruits per centrifuge at 10,000 × g for 1 min. The flow through was replicate). A quadrant measuring about 3 m × 1 m having discarded from the collection tube and the process was three equal compartments (representing three replicates) repeated. DNA was thereafter washed with 200 μl DNA of 1 m × 1 m was constructed and fruits wastes from pre-wash buffer and centrifuge at 10,000 × g for another each replicate were spread in the three equal 1 min. The DNA was finally washed with 500 μl of the compartments of the quadrant respectively. The quadrant Fungal/Bacterial DNA Wash Buffer provided into the was barricaded at the sides with nets to exclude reptiles Zymo-Spin™ IIC column and centrifuged at 10,000 × g and the fruits were left to decay for 9 days after harvest for 1 min and eluted with 100 μl of DNA Elution Buffer (DAH). into a clean and sterile 1.5 ml Eppendorf tube by centrifugation at 10,000 × g for 30 s. The DNA samples were thereafter sent to Inqaba Measure of weight, pH and isolation of bacteria Biotechnology Pretoria South Africa for polymerase chain reaction (PCR) and sequencing according to Weisburg et The same day the fruits were harvested, soon after the al. (1991). The 16S rRNA partial gene sequence were experiment was set up (DAH = 0), 10 split Irvingia fruits targeted and amplified through PCR using primers (27-F were randomly selected from each replicate and and 1492-R) with sequences being 5'-AGA GTT TGA assessed for weight and brownish-black rot disease TYM TGG CTC AG-3' and 5'-TAC CTT GTT AYG ACT T- according to Etebu (2012). The average fruity weight for 3' respectively (Martin and Collen, 1998). each replicate was recorded for further statistical analysis. After assessing for weight, the mesocarps of the fruit wastes were sliced in equal amount (w/v) of sterile Data analyses water and blended with a household blender for 30 s under aseptic conditions. The pH of the resultant slurry Data on fruit weight and pH were subjected to ANOVA

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Table 1. Effect of days after harvest on pH, fruit weight and bacterial quality of Irvingia sp. fruit wastes.

Days after harvest Fruit weight pH Bacterial isolate code 0 31.99b 6.42c BA1, BA2 3 29.46ab 6.31bc BB1, BB2, BB3 6 26.56ab 6.22ab None 9 23.37a 6.17a None

using Generalized Linear Model of SPSS, Version 16.0 amounts of acid which decreases as the fruits ripen. The statistical software. Mean data of the fruit weight and pH acid content of fresh fruits decreases in content during were further subjected to Turkey’s mean separation test. ripening because they are utilized or converted to sugars DNA sequencing results were subjected to BLAST during respiration (Kader and Barrett, 2005). Although the analysis on the National Centre for Biotechnology acid content of the Irvingia fruit wastes was not assessed Information platform on the web in this work, results showed that the pH steadily reduced (http://blast.ncbi.nlm.nih.gov), and bacterial isolates were as the days after harvest increased (Table 1). A steady identified based on the resultant top hits with maximum decrease in pH signified an increase in acidity, different percentage identity, minimum gaps and minimum E-score from what obtains with majority of fruits as reported by (Altschul et al., 1990). Furthermore, the first top 10 hits Kader and Barrett (2005). Previous works have shown for each bacterial test isolate were subjected to that fungal species belonging to taxonomic genera such phylogenetic analysis using the Molecular Evolutionary as Aspergillus, Penicillium, Botrytis, Mucor, etc, constitute Genetics Analysis software version 6 according to Hall the fungal biota of postharvest I. gabonensis fruits (2013) and Tamura et al. (2013). (Etebu, 2012, 2013; Joseph and Aworh, 1991). Fungi are generally acidophilic, as opposed to bacteria (Talibi et al., 2014) and being a significant part of the microbiota of RESULTS AND DISCUSSION postharvest I. gabonensis fruits, they probably utilized the carbohydrate and sugar contents of the fruits to produce Postharvest I. gabonensis fruit wastes were observed to acids which could have been responsible for the decay after harvest and this in turn affected the weight downward pH values recorded in this work as the days and pH of its fruit (Table 1). Postharvest I. gabonensis after harvest of the fruits increased. A decrease in pH fruits were differentially, significantly (P≤0.05) influenced with increase in days of storage of the fruits apparently by the number of days fruit wastes were left in the open translates to increase in acid content. field. The fruit weight and pH generally showed a decline Locals who harvest the fruits from the forest often eat as the number of DAH increased. Mean fruit weight at 0, the fleshy mesocarp while splitting the fruits to harvest 3, 6 and 9 DAH was 31.99, 29.46, 26.56 and 23.37 g, their valuable kernels (Etebu, 2012; Ladipo et al., 1996). respectively. Mean pH value decreased from 6.42 at 0 In addition to the overall aim of assessing bacteria DAH to 6.31, 6.22 and 6.17 at 3, 6 and 9 DAH, associated with decaying I. gabonensis fruits, this study respectively (Table 1). Although the fruit weight and pH was also aimed at accounting for the potential bacteria separately declined with the number of days after locals consume alongside the fruits. Most locals who harvest, the differences between the successive days consume the fruits whilst harvesting the kernels from the were not always significant at P=0.05. In particular, the fruits in the field simply pick the fruits from the soil, having reduction in mean fruit weight (31.99 g) on DAH (0) was fallen from the trees, wipes off the dirt with their palm or significant (P=0.05) in comparison to 23.37 g recorded on on their clothes and ingest straightaway. For this reason, DAH (9), but the reduction in fruit weight from 31.99 g at Irvingia fruit assessed in this study were not sterilized or 0 DAH to 29.46 g at 3 DAH was not significant (P=0.05). even washed with water, but were simply shaken to Similar to fruit weight, the reduction in pH of postharvest dislodge soil particles and assessed for its bacterial fruit wastes from 6.42 at 0 DAH to 6.31 on 3 DAH was quality. also not significant (P=0.05) either. Whilst the mean fruit Five bacterial isolates coded as BA1, BA2, BB1, BB2 weight on 0 DAH was 31.99 g and its weight on 6 DAH and BB3 were isolated from the postharvest I. was 26.56 g. The difference between these two gabonensis fruits which were left to ferment in the open measurements was not statistically significant at P=0.05. field. BA1 and BA2 were isolated from freshly harvested But unlike fruit weight, the mean pH (6.42) at 0 DAH was Irvingia fruits (DAH=0) while BB1, BB2 and BB3 were significantly (P=0.05) different from 6.22 recorded as pH isolated from postharvest Irvingia fruits left in the open for for the fruits after 6 DAH. days 3 after harvest. Molecular analyses were used to Most fresh fruits are known to have considerable identify and or establish their phylogenetic relationships

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using 16S rRNA partial gene sequence. Bacterial (Mezzatesta et al., 2012). Postharvest I. gabonensis fruits has been revolutionized by the application of usually ripen after 3 days (Etebu, 2012) and become molecular biological techniques and, in particular, more appealing to consume. Although clinical cases analysis of 16S rRNA gene sequences to determine arising from consumption of Irvingia fruits are not yet phylogenetic relationships. This approach is being known, it would be foolhardy to assume that it is safe to considered much more robust as opposed to traditional consume postharvest Irvingia fruits without first washing cultural techniques and is now routinely used for them properly. Apart from Enterobacter cloacae, two classification and identification of soil isolates, including more bacterial isolates, Oceanobacillus spp. and S. (Mandic-Mulec and Prosser, 2011). BLAST and cohnii were also isolated from the fruits at 3 DAH (Figure phylogenetic analyses of partial 16S rRNA gene 1, Table 2). sequences showed that BA1, BA2, BB1, BB2 and BB3 The discovery of the genus Oceanobacillus is relatively shared ≥ 99% DNA sequence similarity with Bacillus recent. The genus belongs to the bacterial family spp., Enterobacter spp., Oceanobacillus profundus, . The first known member, Oceanobacillus Enterobacter cloacae and Staphylococcus cohnii, iheyensis was first proposed only in 2001 (Lu et al., respectively (Figure 1 and Table 2). 2001). Members are Gram-positive, motile, rod shaped, It is instructive to note that virtually all the bacteria produces ellipsoidal spores within a swollen sporangia. isolated and identified in this work have been variously They are generally halotolerant, aerobic or facultatively reported to associate with soil in one way or the other. In alkaliphilic (Kim et al., 2007; Lee et al., 2006; Lu et al., particular, Bacillus spp. are aerobic, endospore-forming 2001, 2002; Yu et al., 2014). So far, about 15 species often viewed as saprophytic organisms that live naturally isolated from marine environments and fermented food in the soil (Vilain et al., 2006). sauces have been described and recognized (Hezayen Spore forming bacteria, such as Bacillus, are known to and Younis, 2010; Kim et al., 2007; Lu et al., 2001; Whon remain dormant in soil but are able to resume growth et al., 2010). All references on this genus, to the best of when appropriate conditions such as high moisture our knowledge are centered on discoveries from Asia and content of soil easily decomposable organic matter Middle Eastern countries. This is probably the first work becomes available (Nicholson et al., 2000). The easily in Nigeria wherein a bacterium whose 16S rRNA partial decomposable nature of I. gabonensis fruits apparently gene sequence shares a high 99% sequence similarity contributed to the activation and subsequent growth of with members of the genus is isolated. Given the Bacillus spp. on synthetic media. It is instructive to note beneficial use of halotolerant and hypersensitive that a number of Bacillus spp. are known to be either microorganisms (Hezayen et al., 2000; Ventosa and opportunist pathogens or outright pathogens on plants Nieto, 1995), it would be interesting to further study this and/or animals, including humans (Chattopadhyay et al., apparently halotolerant bacterial isolate obtained from 2004; Ivanova et al., 2003; Schoeni and Wong, 2005). It decaying I. gabonensis fruit wastes (deposited as BB1 in would be worthy to mention at this point that I. the Microbiology Unit of the Department of Biological gabonensis like other forms of water-soluble dietary fibre, Sciences, Niger Delta University, Wilberforce Island, has a laxative effect on consumers (Ngondi et al., 2005). Nigeria). Although, such experiences have not been scientifically BB3 was also isolated from postharvest I. gabonensis linked to bacterial infection, the bacterial types isolated in fruit wastes that were left in the open field to this study may be responsible for the laxative effects. The ferment/decay naturally. This isolate was isolated from 16S RNA gene sequences of two bacterial isolates the fruits at 3 DAH, alongside BB1 and BB2 (Tables 1 obtained from this work had 99% identity with and 2). BLAST and CLUWSTAL alignment analysis of its Enterobacter spp. (Table 2). Enterobacter spp. are 16S rRNA partial gene sequence (834 nucleotide bp) commonly Gram-negative rods, non-sporing, facultative obtained through PCR showed that it shared a 100% anaerobic bacteria belonging to the family sequence identity with S. cohnii without a single gap Enterobacteriaceae (Mezzatesta et al., 2012). Members (Data not shown). S. cohnii was first described by of this genus are not only ubiquitous in nature but a good Schleifer and Kloos (1975) as Gram-positive, coccal number of them are pathogenic on a wide array of plants shaped, coagulase negative and catalase positive. The and humans (Davin-Regli and Pagès, 2015; Humann et potential involvement of the bacterium in various human al., 2011; Ramesh et al., 2014). The 16S rRNA partial infections including brain abscess, pneumonia, urinary gene sequence of one of the bacterial isolates found to tract infection, endocarditis, arthritis, etc, has been voiced be associated with I. gabonensis fruit wastes on 3 DAH by several Scientists (Hu et al., 2014; Soldera et al., had 99% sequence identity, specifically with Enterobacter 2013). Like Oceanobacillus spp., this high sequence cloacae (Table 2). This bacterium has been shown very similarity of BB3 with S. cohnii was least expected recently to have taken on clinical significance as because the latter is usually known to be more frequently opportunistic human pathogen, especially with respect to isolated from hospital than non-hospital intensive care patients who are on mechanical ventilation environments(Soldera et al., 2013). Given the high

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Figure 1. Phylogenetic analysis based on 16S rRNA gene sequences of test isolates and related bacterial sequences obtained from NCBI database using the software package MEGA version 6. Bootstrap values based on 1000 replications are listed as percentages at the branching points. Bar, 0.05 nucleotide substitutions per site.

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Table 2. Molecular identification of bacteria isolated from postharvest Irvingia gabonensis at different days after harvest.

Day after Bacterial Query Topmost hit of bacteria with Accesion Percentage E-value harvest isolate code length closest related sequence number identity 0 BA1 807 Bacillus spp. KC884677.1 99 0 BA2 738 Enterobacter spp. KP278235.1 99 0

3 BB1 810 Oceanobacillus spp. JN602242.1 99 0 BB2 828 Enterobacter cloacae CP011650.1 99 0

BB3 834 S. cohnii LN774330.1 100 0

6 No bacterium isolated 9 No bacterium isolated

percentage similarity of the 16S rRNA partial gene Apart from potential antibiotics produced by fungal sequence with that of S. cohnii, the indiscriminate species previously shown to colonize naturally fermenting consumption of I. gabonensis fruits without due attention postharvest I. gabonensis fruits, endospore-forming to hygienic practices such as washing fruits picked from bacteria, some of which are species of Bacillus, produce the soil is worrisome; there is therefore the need for a wide range of antiviral, antibacterial and antifungal public enlightenment among locals that harvest the fruit. compounds. Antimicrobial compounds produced by soil Interestingly, all bacteria isolated from this study were inhabiting endospore-forming bacteria obviously dictate obtained from freshly harvested I. gabonensis fruits (day the interactions of the latter with other soil 0) and those left to rot for 3 DAH. Contrary to microorganisms (Mandic-Mulec and Prosser, 2011). expectations, bacteria were not isolated from the fruits as Although, BA1 could not be identified up to the species the days after harvest increased up to 6 days and level molecular analyses showed that its 16S rRNA beyond, notwithstanding the fact that they were left to rot partial gene sequence shared a high 99% sequence on soil in an open field. A couple of reasons could be similarity with gene sequences of Bacillus spp. deposited advanced for this observation. Firstly, I. gabonensis fruits in the NCBI gene database. BA1 identified as Bacillus during decay probably produced chemical substances spp. may have produced antibiotics that inhibited the that inhibit bacterial growth. Although phytochemicals growth of all other bacteria on the 6 DAH. Furthermore, were not assessed in this work, earlier works showed that being an endospore former the bacterium may have I. gabonensis fruit wastes produce and release saponins formed spores that did not grow within 48 h of culture in a as they decay on soil (Etebu, 2012), and these are known synthetic media. These assumptions would need to be to inhibit microbial growth (Mert-Türk, 2006). The lack of proven through proper investigation, and calls for further growth of bacteria on the fruits as from the 6 DAH work. onwards may be attributable to saponins produced by the Apart from Bacillus spp., BA1 was observed to be latter. Similar to saponins, previous works have also closely related to members of the genus fingered tannins as possessing microbial inhibitory Chryseomicrobium and Planococcus (Figure 1). capacity (Kraus et al., 2004; Nierop et al., 2006). It would Chryseomicrobium spp., in particular are aerobic, gram- be interesting to study the effect of phytochemicals positive non-motile, non-endospore-forming rods (Arora formed in postharvest I. gabonensis on bacteria. Another et al., 2011). Chryseomicrobium imtechense was the first possible explanation as to the lack of growth of bacteria known member of the genus. The bacterium was isolated on the fruits as from the 6 DAH harvest may be due to from a subsurface marine water sample, and named C. chemical substances produced by fungi. As earlier on imtechense after ascertaining through vigorous stated, fungal species belonging to four genera - characterization that it was a new species (Arora et al., Aspergillus, Penicillium, Botrytis and Mucor have been 2011). The second known species of the genus was shown to associate with decaying I. gabonensis fruits isolated from a semi-arid tropical soil from India and the (Etebu, 2012, 2013; Joseph and Aworh, 1992). Some of name C. amylolyticum was only proposed in a recent these fungal genera are known to constitute species that publication (Raj et al., 2013). Interestingly, this bacterium produce antibiotics inhibitory to bacteria (Kück et al., is reported to be moderately alkalophilic (optimum pH 7- 2014). It is therefore possible for this group of fungi to 8; range 6.5-11) (Raj et al., 2013). Whether or not it have colonized I. gabonensis fruits in this study to would survive a pH of less than 6.5 has not been produce antibiotics in course of their association with the reported. However, results obtained from this study show fruits; resulting to annihilation of bacteria as from the 6th that all five bacterial isolates were obtained from day onwards after harvest. postharvest I. gabonensis fruits on days 0 and 3 DAH

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with pH of 6.42 and 6.31, respectively. No bacterial antibiotic treatment. Fontiers Microbiol. 6:1-10. isolate was obtained from the fruits left after 6 and 9 days Etebu E. (2012). Postharvest pathology and phytochemicals of Irvingia gabonensis (Aubry- Lecomte ex O’Rorke) fruits and wastes. Agric. of harvest when the pH of the I. gabonensis fruits Sci. Res. J. 2(6):285-294. dropped to 6.22 and 6.17, respectively. Etebu E. (2013) Differences in fruit size, postharvest pathology and phytochemicals between Irvingia gabonensis and Irvingia wombolu. Sustain. Agric. Res. 2(1):52–61. Hall B. G. (2013). Building phylogenetic trees from molecular data with Conclusion MEGA. Mol. Biol. Evol. 30(5):1229-1235. doi:10.1093/molbev/mst012 Harris D. J. (1996). A revision of the Irvingiacea in Africa. Bulletin du Findings from this work showed that bacterial species Jardin Botanique National de Belgique. 65:143-196. related to Bacillus, Enterobacter, Oceanobacillus and Hezayen F. F. & Younis M. A. M. 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