Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae

Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e9, 2016 www.elsevier.com/locate/jbiosc Biosynthesis of eight-carbon volatiles from tomato ...

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Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e9, 2016 www.elsevier.com/locate/jbiosc

Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae Onur Günes¸er1 and Yonca Karagül Yüceer2, * Us¸ak University, Engineering Faculty, Department of Food Engineering, 64200 Us¸ak, Turkey1 and Çanakkale Onsekiz Mart University, Engineering Faculty, Department of Food Engineering, 17020 Çanakkale, Turkey2 Received 17 April 2016; accepted 29 November 2016 Available online xxx

The aim of this study was to investigate the possibility of using tomato and red pepper pomaces for the production eight-carbon volatiles by Trichoderma atroviride and Aspergillus sojae. The fermentation of tomato and pepper pomacebased media by both moulds was conducted in shake flasks and bioreactors. Microbial growth behaviours and fermentation abilities of T. atroviride and A. sojae under both fermentation conditions were followed by microbial counting. The production of flavours from tomato and pepper pomaces by fungal metabolism was determined by gas chromatographyeolfactometry, gas chromatographyemass spectrometry and sensory analysis. The results showed that T. atroviride grew faster than A. sojae, and the survival of T. atroviride in the tomato pomace was longer than that of A. sojae. However, T. atroviride grew slower than A. sojae in the pepper pomace. Eight-carbon flavour compounds, including (Z)-1,5-octadien-3-ol, 1-octen-3-ol, (E)-2-octenal and (E)-2-octenol, were produced by T. atroviride and A. sojae from the tomato and pepper pomaces. The highest production levels (265.55 ± 2.79 and 187.47 ± 0.92 mg kgL1) were observed for 1-octen-3-ol in the tomato fermentation by T. atroviride and A. sojae, respectively. The relationships between volatile compounds and their flavour characteristics in tomato and pepper pomaces were analysed using principal component analysis. Ó 2016, The Society for Biotechnology, Japan. All rights reserved. [Key words: Flavour; Fungal metabolism; Agro-waste; Microbial fermentation; Gas chromatography-olfactometry, Sensory analysis]

Only a few of bioresources have been studied for the biotechnological production in food industry so far (1,2). Use of agrowastes from the fruit and vegetable industries becomes very popular for the production of high-value products. Production of natural food colours, enzymes, some gums and flavours from agrowastes by biotechnological processes has been a focus for many studies (3e6). Tomato and red pepper pomaces, which contain seeds and peel residues, are the most abundant agro-wastes in the fruit and vegetable industries. Heuze et al. (7) have indicated that tomato waste constituted about 11 million tons, including a little more than 4 million tons of tomato pomace, in 2007. Unfortunately, we could not obtain accurate information on the volume of pepper pomace in the world. While tomato pomace has 15e24% of protein, 5e20% of fat, 28e51% of total sugar, and 3e6% of mineral substances on a dry basis, very limited compositional data are available for red pepper pomace. Pepper seeds contain 24%, 35%, 25% and 4% of protein, fibre, oil and mineral substances, respectively. The seeds can be excellent sources for biotechnological processes because of their rich nutritional content for microbial growth (7e9). Trichoderma and Aspergillus are filamentous fungi belonging to Deuteromycetes, which occur in a large variety of ecosystems. They are remarkable organisms due to their rapid growth, capability of

* Corresponding author. Tel.: þ90 286 218 00 18 x2272; fax: þ90 286 2180541. E-mail address: [email protected] (Y.K. Yüceer).

utilising diverse substrates and resistance to noxious chemicals. Trichoderma and Aspergillus contain some biotechnological workhorse species due to their high enzyme activities and easy in vitro culturing. Some species of Trichoderma produce 6-pentyl-a-pyrone associated with a characteristic sweet or coconut odour, whereas some other Trichoderma species are used as cellulolytic and hemicellulolytic enzyme producers for the food, textile, pulp and paper industries. Aspergillus species such as Aspergillus sojae and A. oryzae have been used for years for the production of traditional fermented foods such as soy sauce, Sake and Koji in Asian countries (10e14). Biotechnological approaches such as microbial fermentation have attracted increasing attention of researchers due to a possibility of valorisation of wastes and low-cost production steps using microorganisms (15,16). In the biotechnology of natural flavours, many studies were conducted on the production of flavour compounds using enzymatic approaches (17e19) and yeast metabolism in synthetic media or certain agro-wastes (20e22). Much of what is known about the synthesis of natural flavour compounds by biotechnological processes comes from the metabolism of yeasts and bacteria (4,15,23). However, there is still lack of information on the production of natural flavours from agro-wastes by fungal metabolism, except vanillin production (24e28). From this perspective, mushroom-like flavours such as 1-octen-3-ol, 1-octen3-one and octanol can be produced from tomato and pepper pomaces by primary and secondary metabolism of filamentous fungi such as Trichoderma and Aspergillus (5,29).

1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.11.013

Please cite this article in press as: Günes¸er, O., and Yüceer, Y. K., Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.013

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J. BIOSCI. BIOENG.,

The aim of this study was to investigate the possibility of using tomato and red pepper pomaces to produce certain aroma compounds and then comparing the flavour production behaviour of Trichoderma atroviride and A. sojae in the agro-wastes. MATERIALS AND METHODS Preparation of tomato and red pepper pomaces Tomato and red pepper pomaces were prepared according to Guneser et al. (30). The chemical compositions of the tomato and pepper pomaces are shown in Table 1. About 500 g of each pomace was ground using a knife mill (Restch GM 200, Haan, Germany), and 1 L of a 100 g L1 suspension was prepared with distilled water for shake flask fermentation, carried out in 250-mL Erlenmeyer flasks. Each pomace solution was homogenised at 24,000 rpm using ULTRA-TURRAX (IKA-Werke GmbH, Germany). The solutions were sterilised at 121  C for 15 min in an autoclave (Hirayama, Saitama, Japan). Cultivation and preparation of microbial cultures and suspensions The strains of T. atroviride (NRRL 31396) and A. sojae (NRRL 1988) were obtained from the ARS (NRRL) Culture Collection (Peoria, IL, USA). T. atroviride NRRL 31396 was cultured on malt extract agar (MEA), while A. sojae NRRL 1988 was cultured on Dichloran Rose Bengal Chloramphenicol Agar (DRBC). Both moulds were incubated at 30  C for seven days. The spores of T. atroviride and A. sojae were collected by washing the fungal growth on the surface of agar with Tween 80 (1 g kg1). The spore suspensions were filtered through two layers of sterilised cheese cloth to remove residual agar, then centrifuged at 1000 g for 5 min, and washed with a saline solution (8.5 g L1 of NaCl) to remove the Tween solution. Spore concentrations in the microbial suspensions were determined by counting with a Thoma slide using a light microscope (Olympus CX 31, Olympus, Philippines) (24,31). The concentrations of the spore suspensions obtained were 107e108 spores mL1. Fermentation experiments Fermentation experiments were conducted in 250-mL Erlenmeyer shake flasks with a 100-mL working volume. About 7 8 1 10 e10 spores mL were inoculated to the tomato and pepper pomace solutions, and the flasks were incubated at 30 C for 120 h in a shaking incubator at 120 rpm. The same procedure was applied to prepare a control group without the microorganisms. The shake-flask experiment was conducted in duplicate. Batch fermentation was performed at 30 C in a 5-L stirred tank bioreactor (STR) (Biostat A-plus, Sartorius, Melsungen, Germany) with a 4-L working volume. The initial pH values of the tomato and pepper pomace solutions were 4.43 and 3.93, respectively. The aeration rate was 0.325 vvm, and the agitation speed was 120 rpm. Batch fermentation conditions were determined based on the microbial growth and aroma production results from the shake-flask fermentation. The STR was equipped with two six-blade impellers, a pH probe (EasyFerm K8/325, Hamilton) and a PT 100 temperature sensor. Microbial growth Microbial counts of T. atroviride and A. sojae were monitored under both fermentation conditions to determine their growth behaviours and fermentation abilities on the agro-wastes. MEA was used for T. atroviride, and DRBC was used for A. sojae. Plates were incubated at 30 C for 5e7 days. Specific growth rates were calculated using growth curves to determine the characteristics of microbial growth in batch fermentation (32). Analysis of flavour compounds Flavour compounds in the fermented tomato and red pepper pomace solutions were determined by gas chromatographyeolfactometry (GCO) and gas chromatographyemass spectrometry (GCeMS). Extraction of flavour compounds Flavour compounds for the GCO and GCeMS analyses were isolated from the fermented tomato and red pepper solutions by solid-phase microextraction (SPME) (33). Three grams of the solutions were weighed in a 40-mL amber-coloured screw-top vial with a hole-cap polytetrafluoroethylene/silicon septum (Supelco, Bellefonte, PA, USA), and 1 g of NaCl was added to the vial. The vial was kept at 40 C in a water bath for 20 min to equilibrate volatiles in the headspace. Then, an SPME fiber (2 cm, 50/30 mm DVB/Carboxen/PDMS, Supelco) was inserted into the vial. The SPME fibre was exposed at a depth of 2 cm in the headspace of the vial. Then, the SPME needle was immediately injected into a GCO or GCeMS column. Gas chromatographyeolfactometry analysis GCO analysis was conducted using HP 6890 GC (Agilent Technologies, Wilmington, DE, USA) equipped with a flame ionisation detector (FID), sniffing port and splitless injection system. A nonpolar column (HP-5, 30 m length, 0.32 mm i.d., 0.25 mm film thickness; J&W Scientific, Folsom, CA, USA) was used for sniffing. Helium was used as a carrier gas. The inlet pressure was 48.74 kPa, and the flow rate was 1.2 mL min1. The GC oven

TABLE 1. Chemical compositions of tomato and pepper pomaces. Chemical properties pH Dry matter (g kg1) Total nitrogen (g kg1) Ash (g kg1)

Tomato pomace

Red pepper pomace

4.59 139.90 3.50 6.50

4.57 205.80 4.80 6.90

temperature was programmed from 40 C to 230 C at a rate of 10 C min1, with the initial and final hold times of 5 and 20 min, respectively. The FID and sniffing port were maintained at temperatures of 250 C and 200 C, respectively. The GCO procedure was duplicated by two sniffers. A post-peak intensity method was used for the determination of aroma intensity using a 10-point scale anchored to the left with ‘not’ and to the right with ‘very’ (34). The sniffers had 300 h of experience with the GCO technique, scale use and odour description. Aroma-active compounds were identified by comparing the retention indices and odour quality of unknowns with those of references analysed under the same experimental conditions by the sniffers during the GCO procedure. Retention indices were calculated using an n-alkane series (35). Gas chromatographyemass spectrometry analysis Volatile compounds were tentatively identified by GCeMS. A nonpolar HP5 MS column (30 m  0.25 mm i.d.  0.25-mm film thickness; J&W Scientific) was used for separation of flavour compounds. The GCeMS system consisted of an HP 6890 GC and 7895C massselective detector (MSD; Agilent Technologies, Wilmington, DE, USA). The GC oven temperature was programmed from 40 to 230 C at a rate of 10 C min1, with the initial and final hold times of 5 and 20 min, respectively. Helium was used as a carrier gas at a flow rate of 1.2 mL min1. The MSD conditions were as follows: capillary direct interface temperature, 280 C; ionisation energy, 70 eV; mass range, 35e350 amu; scan rate, 4.45 scans s1. The identification of flavour compounds was based on comparison of the mass spectra of unknown compounds with those in the databases of the National Institute of Standards and Technology and Wiley Registry of Mass Spectral Data. Flavour compounds were quantified based on their relative abundances. 2-methyl pentanoic acid (0.093 mg mL1) and 2-methyl-3-heptanone (0.486 mg mL1) were used as internal standards for acidic and neutralebasic compounds, respectively (36). All chemicals and reagents were of chromatographic grade and obtained from SigmaeAldrich Chemical Co. (St. Louis, MO, USA) and Merck KGaA (Darmstadt, Germany). Sensory analysis A roundtable discussion was conducted to determine descriptive sensory properties and changes in aroma profiles of fermented versus control samples (31). Seven trained panellists conducted sensory evaluation. The panellists were staff and graduate students at the Department of Food Engineering of Çanakkale Onsekiz Mart University; four were females and three were males; the age ranged from 24 to 45 years old. The panel received about 300 h of training on definition of descriptive terms for various kinds of products. The panellists quantified the attributes using a 15-point product-specific scale anchored to the left with ‘not’ and to the right with ‘very’ (37). Statistical analysis One-way analysis of variance (ANOVA) was conducted to determine the differences in intensities of flavour compounds among the fermented and unfermented solutions, obtained by GCeMS analysis for the shake-flask and bioreactor fermentations. Moreover, two-way ANOVA was conducted to evaluate the growth of the microorganisms in each pomace during the shake-flask fermentation. Welch’s test, a parametric alternative to ANOVA, was also used for the data that did not meet the prerequisites (homogeneity of variance and equality of variance) for ANOVA. Tukey’s honestly significant differences test was used for separating means (38). SPSS for Windows (version 15.0) was used for the ANOVA analyses. Principal component analysis (PCA) was performed using the XLSTAT statistical program (trial version, 2015, Addinsoft, Inc., New York, NY, USA) for interpreting the quantity of produced flavour compounds and their sensory impacts or perceptions.

RESULTS AND DISCUSSION Growth of T. atroviride and A. sojae in tomato and pepper pomaces It was determined that the microbial growth in the tomato and pepper pomaces depend on the mould type and fermentation time (P ¼ 0.01). The maximum count of A. sojae was observed at 72 and 48 h of fermentation in the tomato and pepper pomaces, respectively. A significant increase in the count of T. atroviride was determined at 96 and 72 h of fermentation (Table 2). When the increases in the counts of A. sojae and T. atroviride at the end of the shake-flask fermentations of both pomaces were compared, T. atroviride showed slightly higher growth in the tomato pomace; meanwhile, the growth (2.28 log colony-forming units mL1) of A. sojae was higher than that of T. atroviride in the pepper pomace. These results were confirmed by the growth behaviours for the batch fermentations. Using nutrients from agro-wastes for microbial growth promotion is quite challenging. Many agro-wastes from the fruit and vegetable industries have cellulose, hemicellulose and lignin-type polysaccharides. These polysaccharides cannot be directly utilised by many microorganisms, while free glucose, lactose and sucrose in synthetic media are more effectively used by microorganisms

Please cite this article in press as: Günes¸er, O., and Yüceer, Y. K., Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.013

VOL. xx, 2016

FLAVOUR PRODUCTION FROM POMACES BY FUNGI

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TABLE 2. Growths of A. sojae and T. atroviride in tomato and pepper pomaces during shake flask and batch fermentation (n ¼ 2). Shake flask fermentation

Batch fermentation

Microbial count (log cfu mL1  SE)

Microbial count (log cfu mL1  SE)

Fermentation time (h)

Tomato pomace A. sojae 0 24 48 72 96 120 Specific growth rate (h1) (for batch fermentation)

Pepper pomace

T. atroviride BCa

6.0  0.01 5.47  0.01Ca 7.0  0.01ABa 7.49  0.19Aa 7.0  0.01ABa 6.41  0.01ABCb e*

Ba

6.0  0.01 6.22  0.45Ba 5.85  0.37Bb 6.93  0.06ABa 7.56  0.12Aa 7.62  0.14Aa -

A. sojae

Tomato pomace

T. atroviride Ca

6.0  0.01 6.54  0.23Bca 6.54  0.23BCa 8.04  0.44Aa 7.0  0.01Bb 7.0  0.01Bb -

Ba

6.0  0.01 6. 19  0.10Ba 6. 19  0.10Ba 7. 58  0.01Aa 7. 80  0.03Aa 7. 73  0.03Aa e

A. sojae

Pepper pomace

T. atroviride BCb

4.97  0.02 4.95  0.04Cb 6.0  0.01Abb 5.48  0.02Bb 6.23  0.23Ab 6.15  0.15Ab 0.146

Da

5.69  0.01 6.88  0.06Ca 8.74  0.01A 8.38  0.10ABa 8.07  0.12Ba 7.92  0.10Ba 0.056

A. sojae

T. atroviride Db

4.79  0.31 4.51  0.21Db 5.93  0.010Cb 6.84  0.06BCa 8.04  0.10Aa 7.30  0.10ABa 0.109

6.15  0.10Ca 6.58  0.02BCa 7.15  0.15ABa 6.80  0.20ABa 7.42  0.42Aa 6.80  0.19ABa 0.028

SE: standard error; cfu: colony forming unit. Means followed by different lowercase letters represent significant differences between microorganisms in the same fermentation time. Means followed by different uppercase letters represent significant differences for the same microorganism during fermentation (P < 0. 05). * Not calculated. aeb

AeD

for growth (39,40). The physical and chemical properties of agricultural wastes, such as a C/N ratio and nutrient composition, as well as fermentation conditions, are also important factors for the growth of microorganisms in agro-wastes (16,40). These factors have effects on the production of secondary metabolites (e.g., volatile compounds) by microorganisms (41). The calculated maximum specific growth rates for T. atroviride and A. sojae were found to be 0.056 h1 and 0.146 h1, respectively, in the batch fermentation of tomato pomace. In the case of pepper pomace fermentation, specific growth rates of 0.028 h1 and 0.109 h1 were calculated for T. atroviride and A. sojae, respectively. There is limited information about the growth characteristics of Trichoderma and Aspergillus species. However, the microbial growth results obtained in this study were similar to previous findings obtained for different strains of Trichoderma and Aspergillus under different culture conditions (42e46). Kancelista et al. (43) calculated specific growth rates for Trichoderma virens TRS 107 and Trichoderma harzianum TRS 72 during solid-state fermentation of sugar cane bagasse, wheat bran and corn cobs,

which ranged between 0.045 h1 and 0.194 h1 depending on the strain and agro-waste material. In a study by Muthuvelayudham and Viruthagiri (42), it was found that Trichoderma reesei had a specific growth rate of 0.120 h1 in sugar cane bagasse, while a specific growth rate 0.10 h1 was observed for the same strain in rice straw fermentation. Chipeta et al. (46) observed specific growth rates of 0.198 h1 and 0.137 h1 for A. oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 in shake-flask cultures with spent sulphite liquor, a wastewater of the pulp and paper industry, as a carbon substrate. Consequently, it can be concluded that T. atroviride grew faster than A. sojae and the survival of T. atroviride was longer than that of A. sojae in tomato pomace fermentation. Conversely, T. atroviride grew slower than A. sojae in the pepper pomace. It was found that the counts of A. sojae increased during pepper pomace fermentation. Flavour production characteristics of T. atroviride and A. sojae in tomato and pepper pomaces Table 3 shows aroma-active compounds of the tomato pomace fermented with

TABLE 3. Aroma-active compounds of unfermented and fermented tomato pomace with T. atroviride and A. sojae (n ¼ 2). RIa

<500 582 647 761 795 850 854 863 876 891 901 916 961 969 974 1037 1049 1081 1121 1135 1166 1186 1230 1325 1373

Aroma compounds

Methanethiol Diacetyl Acetic acid Isoamyl alcohol Hexanal Isoamyl acetate Butyric acid Isovaleric acid Unknown 1 2-Heptanone Methional 2-Acetyl-1-pyrroline 1-Octen-3-ol 2-Octanol (Z)-1,5-Octadien-3-ol 2-Phenylacetaldehyde (E)-2-Octenal (E)-2-Octenol 2-Phenylethanol (E)-2-Nonenal 2,3-Diethyl-5-methyl-pyrazine Unknown 2 (E,E) 2,4-Nonadienal (E,E)-2,4-Decadienal Unknown 3

Aroma quality

Sulphur Butter Sour, vinegar Banana Green grass Fruity Cheesy Sour, fruity Popcorn Oxide, broth Boiled potato Popcorn Metallic, mushroom Mushroom Geranium Rose, flower Oxide nutty, mushroom Dust, cement, mushroom Rose Hay Green pepper Rose, sharp Fat, dirty Soap Lactone

Aroma intensity (mean  SE)

Identification methods

RI, O RI, MS, RI, MS, O RI, MS, O RI, MS, O RI, MS, O RI, O RI, O RI, O RI, MS, O RI, O RI, O RI, MS, O RI, O RI, MS, O RI, MS, O RI, MS O RI, MS, O RI, MS, O RI, MS, O RI, MS, O RI, O RI, MS, O RI, MS, O RI, O

Controlb

T. atroviridec

A. sojaec

ND 2.75  0.25 ND ND 1.15  0.35 ND. 2.0  2.0 2.75  0.25 ND 1.50  0.01 4.50  0.25 0.40  0.40 ND 6.0  0.01 ND 0.90  0.10 ND ND ND 1.50  0.01 3.0  0.01 ND 0.90  0.10 ND ND

0.25  0.25 1.25  1.25 0.50  0.50 1.15  1.05 1.60  1.60 1.0  1.0 2.90  2.10 1.50  1.50 ND ND 5.25  0.75 3.0  0.50 5.50  0.50 ND 7.0  0.01 1.0  0.25 2.0  0.01 5.0  0.01 0.5  0.01 1.75  1.75 4.50  0.50 ND 0.25  0.25 ND 0.25  0.25

ND 0.80  0.01 ND ND 0.25  0.25 0.40  0.40 ND ND 0.25  0.25 ND 5.75  0.25 6.75  0.75 4.25  0.75 4.25  0.75 5.50  1.50 1.65  0.85 3.0  2.0 3.75  1.25 ND 0.50  0.50 3.50  1.50 0.50  0.50 0.50  0.50 1.0  1.0 ND

SE: standard error; ND: not detected; MS: mass spectrometry; O: odor. Intensities of aroma compounds marked in bold increased in fermented tomato pomace. a RI: retention indices based on HP-5MS column. b Aroma intensity of unfermented pomace (120 h). c Aroma intensity of fermented pomace (120 h).

Please cite this article in press as: Günes¸er, O., and Yüceer, Y. K., Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.013

4

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J. BIOSCI. BIOENG.,

both moulds. Total 21 and 18 aroma-active compounds were identified in the tomato pomace fermented by T. atroviride and A. sojae, respectively. Acids, alcohols and aldehydes were the most abundant aroma-active compounds in the unfermented and fermented tomato pomace. Diacetyl (buttery), butyric acid (cheesy), isovaleric acid (sour, fruity) and (E,E)-2,4-nonadienal (fatty, dirty) were determined at high intensities in the unfermented tomato pomace. 1-octen-3-ol (mushroom), (Z)-1,5octadien-3-ol (geranium), (E)-2-octenal (oxide nutty) and (E)-2octenol (dust, cement) were detected at higher intensities in the tomato pomace fermented by both moulds. The tomato pomace fermented with T. atroviride had higher intensities of (Z)-1,5octadien-3-ol (geranium) and (E)-2-octenol (dust, cement) than that fermented with A. sojae. However, the intensity of (E)-2octenal was found to be higher in the tomato pomace fermented with A. sojae. The aroma-active compounds of the pepper pomace fermented with both moulds are shown in Table 4. Similar to the tomato pomace, the identified aroma-active compounds in the unfermented and fermented pepper pomace consisted mainly of acids, alcohols and aldehydes. 1-Octen-3-ol and (E)-2-octenol were detected at higher intensities in the fermented pepper pomace. The intensities of (Z)-1,5-octadien-3-ol, 3-octanone (mushroom) and (E)-2-octenal were higher in the pepper pomace fermented by A. sojae; meanwhile, only 1-octen-3-ol, (E)-2octenol, ethyl butyrate and 2-phenylethanol were found at high intensities in the pepper pomace fermented by T. atroviride (Table 4). When all GCO results were taken into consideration, it was concluded that (Z)-1,5-octadien-3-ol, 1-octen-3-ol, 3-octanone, (E)2-octenal, (E)-2-octenol and 3-octanol could be produced from pepper and tomato pomaces by fermentation with T. atroviride and A. sojae. The production of eight-carbon flavours such as 1-octen-3ol and 3-octanone was achieved via the enzyme-catalysed oxidation and cleavage of polyunsaturated fatty acids, especially by lipoxygenase and hydroperoxide lyase from the plant. The production of eight-carbon volatiles is largely unknown in fungal metabolism

and is related to the glucose and fatty acid metabolism of both fungi because these flavour compounds are end products of fungal fatty acid metabolism. Also, acetyl-CoA, which is a breakdown substance of glycolysis, can be used as a precursor for the production of these compounds (47). In the biosynthesis pathway, the oxidation step of polyunsaturated fatty acids was achieved by haem dioxygenase, which is different from lipoxygenase, and the cleavage stage of intermediate hydroperoxide compounds was performed by fungalspecific 10-hydroperoxide lyase (29). Stopphacer et al. (47) reported that 25 volatile compounds were produced by T. atroviride ATCC 74058 on potato dextrose agar. Among the volatiles, 3octanone, 1-octen-3-ol and 3-octanol were identified by the researchers as abundant eight-carbon volatiles, and concentrations of 1-octen 3-ol and 3-octanone were found at the highest level after 72 and 96 h of incubation, respectively, similar to our findings. In our previous study (48), we observed that similar mushroom alcohols and aldehydes could be produced at high concentrations from olive mill waste using T. atroviride. Moreover, Nemcovic et al. (49) concluded that the composition of volatile compounds is associated with conidiation in Trichoderma species. The researchers found that the fungi could produce 1-octen-3-ol and its analogues, 3-octanol and 3-octanone, and conidiation of the fungi was accompanied by an increase in these flavour compounds (49). Table 5 shows the concentrations of the flavour compounds produced from the tomato and pepper pomaces in shake-flask fermentation. Significant differences were observed among the concentrations of the flavour compounds in the unfermented and fermented tomato and pepper pomaces (P < 0.05). As seen in Table 5, 3-octanone and 3-octanol were not identified in the fermented tomato pomace by GCO, while these compounds were determined by GCeMS. Similar results were also observed for ethyl acetate (sweet), ethyl butyrate (fruity) and phenyl ethyl alcohol (rose) in the pepper pomace fermented with T. atroviride (data not shown). These differences may be attributed to the odour threshold, the odour recognition threshold, the effect of the matrix and sensitivities of

TABLE 4. Aroma-active compounds of unfermented and fermented pepper pomace with T. atroviride and A. sojae (n ¼ 2). a

RI

577 601 614 762 799 848 855 860 864 891 896 918 968 976 981 992 1049 1083 1099 1128 1141 1144 1156 1165 1201 1233 1374

Aroma compounds

Diacetyl 2-Butanone Acetic acid Isoamyl alcohol Hexanal Ethyl butyrate Butyric acid Unknown 1 Isovaleric acid 2-Heptanone Methional 2-Acetyl-1-pyrroline 1-Octen-3-ol 3-Octanone (Z)-1,5 -Octadien-3-ol Hexyl acetate (E)-2-Octenal (E)-2-Octenol 3,5-Octadien-2-one 2-Phenylethanol (Z)-2-Nonenal Unknown 2 (E,Z)-2,6-Nonadien-1-ol 2,3-Diethyl-5-methyl-pyrazine (E,E)-2,4-Nonadienal 3-Carvomenthenon Unknown 3

Aroma quality

Butter Sweet, camphor Sour, vinegar Banana Green grass Fruity Cheesy Dust Sour, fruity Oxide, broth Boiled potato Popcorn Mushroom Mushroom Geranium Cologne Oxide nutty, mushroom Dust, cement, mushroom Burnt oil Rose Cucumber Dust Hay, tobacco Green Pepper Fat, dirty Fishy Sweet

Aroma intensity (Mean  SE)

Identification methods

RI, O RI, MS, RI, MS, O RI, MS, O RI, MS, O RI, MS, O RI, O RI, O RI, MS, O RI, MS, O RI, O RI, O RI, MS, O RI, MS, O RI, MS, O RI, MS, O RI, MS, O RI, MS, O RI, O RI, O RI, O RI, O RI, MS, O RI, MS, O RI, MS, O RI, MS, O RI, O

Controlb

T. atroviridec

A. sojaec

3.0  0.01 1.75  0.25 1.0  1.0 ND 0.75  0.75 ND. 1.0  0.01 ND 1.0  0.01 1.75  0.25 ND 3.0  0.01 ND ND ND 0.5  0.5 ND ND 2.50  0.5 ND 2.25  0.75 1.0  0.01 1.75  0.25 3.50  0.50 ND 3.0  1.0 ND

1.0  0.5 ND ND 0.65  0.15 1.25  0.25 2.50  0.50 2.50  0.50 ND. 1.50  0.50 ND 1.75  0.25 4.50  0.50 7.0  0.01 ND ND ND ND 4.50  0.75 0.75  0.25 1.25  0.25 ND 1.0  0.01 0.75  0.75 2.0  0.01 0.8  0.01 0.40  0.4 ND

0.75  0.25 0.65  0.15 ND ND 1.90  1.10 ND ND 0.25  0.25 ND ND 1.25  0.75 4.0  0.01 5.25  1.25 5.75  0.25 3.0  3.0 0.75  0.75 3.0  1.0 3.0  1.0 ND ND ND ND 1.75  0.25 1.0  0.50 0.8  0.01 ND 0.40  0.40

SE: standard error; ND.: not detected, MS: mass spectrometry, O: odor. Intensities of aroma compounds marked in bold increased in fermented pepper pomace. a RI: retention indices based on HP-5MS column. b Aroma intensity of unfermented pomace (120 h). c Aroma intensity of fermented pomace (120 h).

Please cite this article in press as: Günes¸er, O., and Yüceer, Y. K., Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.013

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TABLE 5. Concentration of eight-carbon flavour compounds in fermented tomato and pepper pomaces in shake flask fermentation (n ¼ 2). Volatiles

(Z)1,5-Octadien-3-ol 1-Octen-3-ol 3-Octanone 3-Octanol (E)-2-Octenal (E)-2-Octenol

Aroma quality

Geranium Mushroom Mushroom Mushroom Mushroom Mushroom

Tomato pomace

Pepper pomace

Mean  SE (mg kg1 tomato pomace solution)a

Mean  SE (mg kg1 pepper pomace solution)a

Control

T. atroviride

ND ND ND ND ND ND

4.26  1.18A 297.46  46.64A ND 13.68  6.25 3.38  1.27A 31.55  0.33A

A. sojae 1.55 5.25 3.66 ND 2.48 4.59

 0.02A  1.60B  0.65  0.83A  2.60B

Control

T. atroviride

ND ND ND ND ND ND

ND 81.28  11.32A ND ND 3.71  2.13B 22.68  1.97A

A. sojae 40.97 53.88 32.96 ND 53.02 10.08

 1.24  8.85A  0.69  10.76A  0.59B

SE: Standard error; ND.: not detected. A, B Means followed by different superscript letter represent significant differences for the same compound in each pomace (P  0.05). a Relative abundance of aroma compound in pomace (120 h).

both GCO and GCeMS (50,51). The odour threshold is the lowest concentration of aroma compounds, which is perceived by the olfactory system of humans; the odour recognition threshold is the lowest concentration at which aroma quality of volatile compounds can be described (52). Therefore, to determine volatile compounds by GCO techniques, the concentration of volatile compounds in the matrix has to be higher than both threshold values (53). The quantities of 1-octen-3-ol, 3-octanol and (E)-2-octenol were higher in the tomato pomace fermented with T. atroviride than in that fermented with A. sojae and in unfermented tomato pomace samples. 3-octanone was only detected in the tomato pomace fermented by A. sojae. The tomato pomace fermented with T. atroviride had 297.46 mg kg1 of 1-octen-3-ol, 13.68 mg kg1 of 3-octanol and 31.55 mg kg1 of (E)-2-octenol, while the concentration of 3octanone was 3.66 mg kg1 in the tomato pomace fermented with A. sojae. In the pepper pomace fermentation, (Z)-1,5-octadien-3-ol, 3-octanone and (E)-2-octenal were produced by A. sojae at high concentrations. On the other hand, T. atroviride produced a higher concentration of (E)-2-octenol from the same pomace, while produced no (Z)-1,5-octadien-3-ol and 3-octanone (Table 5). Obviously, these differences can be attributed to the mould genus and the composition of the pomaces since synthesis of secondary metabolites by microorganisms, including flavour compounds, is greatly influenced by the concentration of nutrients in agro-wastes and by microbial metabolic pathways (41,54). For instance, De Araujo et al. (55) found that a higher amount of 6-pentyl-a-pyrone (3 mg g1 of dry substrate) was produced from sugarcane bagasse by an isolated Trichoderma sp. in solid-state fermentation. Bonnarme et al. (56) reported that Trichoderma viride TSP2 produced 6-pentyl-apyrone from castor, hazelnut and grape seed oils. In a study by Kalyani et al. (57), it was reported that T. harzianum produced 455 and 167 mg L1 of 6-pentyl-a-pyrone in potato dextrose broth under surface and submerged fermentation conditions, respectively. Although previous studies have shown that production of 6-pentyl-a-pyrone at a high concentration can be achieved by Trichoderma species, we did not find this compound in the tomato and pepper pomaces fermented with T. atroviride under both shakeflask and bioreactor fermentation conditions. However, we observed in our previous study (48) that 423.10 mg kg1 of 1-octen3-ol and 42.45 mg kg1 of (E)-2-octenol were produced by T. atroviride from olive mill waste. The findings of the present study are in good agreement with the previous data on the flavour production behaviour of Aspergillus species. Some Aspergillus species are responsible for the flavour formation in fermented Asian foods such as soy sauce and Koji. In a study by Kaminski et al. (58), it was determined that Aspergillus niger, A. ochraceus, Aspergillus oryzae, and Aspergillus parasiticus produced eight-carbon volatiles, including 3-octanone, 3-octanol, 1-octen-3-ol, 1-octanol and (E)-2-octenol on coarse wheat meal. Feng et al. (59) determined that A. oryzae HN 3.042 produced 1octen-3-ol, 3-octanol and (E)-2-octenal at high concentrations during fermentation of soy sauce. Moreover, Ito et al. (60) reported

that acetaldehyde, butyl aldehyde, acetone, 2-butanone, acetoin, diacetyl and ethyl acetate, as well as mushroom alcohols, were produced by A. oryzae during fermentation of rice Koji. The flavour compounds that had high concentrations were also considered for batch fermentation in the present study. The changes in the concentrations of flavour compounds are shown in Fig. 1. The fermentation time had a significant effect on the concentrations of these volatile compounds produced by both moulds from the tomato and pepper pomaces. It was determined that the formation of volatiles, including (Z)-1,5-octadien-3-ol, 1-octen-3ol, (E)-2-octenal and (E)-2-octenol, started after 24 or 48 h depending on the mould strain. The highest production was detected for 1-octen-3-ol at concentrations of 265.55 and 187.47 mg kg1 in the tomato pomace fermentation by T. atroviride and A. sojae, respectively (Fig. 1). During fermentation of the tomato pomace, the biosynthesis of (Z)-1,5-octadien-3-ol, (E)-2-octenal and 3-octanone by A. sojae was found to be higher than that shown by T. atroviride; meanwhile, T. atroviride produced 1-octen3-ol and (E)-2-octenol at higher concentrations than A. sojae. Moreover, it was observed that the concentrations of flavour compounds produced by A. sojae gradually increased during 120 h of fermentation, but no similar increase was observed in the fermentation with T. atroviride. On the contrary, a slight decrease was observed in the concentrations of flavour compounds produced by T. atroviride. The concentrations of flavour compounds reached the maximum level at 72 h of fermentation, after that the production of volatile compounds slowed down and their concentrations decreased. However, A. sojae continued to produce flavour compounds through the end of the fermentation. This may be related to the secondary metabolism of T. atroviride. It was thought that the secondary metabolite production by T. atroviride slowed down due to the reduction of usable nutrients in agro-waste and physicochemical changes of culture conditions after a certain period of fermentation (61,62). Moreover, process parameters such as agitation and the stripping effect might have also influenced the concentrations of the volatile compounds. The stripping effect is the most important parameter, known as the unavoidable discharge of flavour compounds in the bioreactor due to their volatilities and dissolving behaviours in culture media during the fermentation period (63,64). In the pepper pomace fermentation, (E)-2-octenal was produced by A. sojae at a high concentration at 48 h. However, the same volatile was produced by T. atroviride at a very low concentration. T. atroviride did not produce (Z)-1,5-octadien-3-ol and the flavour compounds produced by A. sojae did not gradually increase during fermentation (120 h). However, the concentrations of 1-octen-3-ol and (E)-2-octenol produced by T. atroviride gradually increased compared to those in the tomato pomace fermentation. The concentrations of (Z)-1,5-octadien-3-ol and (E)-2-octenal greatly decreased from 48 to 72 h in pepper pomace fermentation by A. Sojae (Fig. 1). This might be explained by the metabolic activities and numbers of the microorganisms in the media during

Please cite this article in press as: Günes¸er, O., and Yüceer, Y. K., Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.013

6

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J. BIOSCI. BIOENG.,

FIG. 1. Changes in some flavour compounds in tomato and pepper pomaces during batch fermentation.

fermentation. Fig. 1 shows concentration of volatile compounds in the fermentation condition. Until 48 h, both of these volatiles were not detected. The number of A. sojae increased in pepper pomace specifically after 48 h batch fermentation (Table 2). Based on the population, media may not have enough nutrients for high number of microorganisms and they may produce some other metabolites after certain period of fermentation. Therefore amount of these volatiles produced by A. sojae can decrease after 48 h. In addition, agitation and stripping effect during fermentation may affect the concentration of the volatiles. The results showed that the changes in the concentrations of the flavour compounds produced by T. atroviride and A. sojae in batch fermentation were in agreement with the findings of previous studies conducted with different yeasts and fungi to produce flavour compounds under bioreactor conditions (30,48,63). Yılmaztekin et al. (65) investigated the production of isoamyl

acetate from sugar beet molasses by Williopsis saturnus var. saturnus. They determined that the concentration of isoamyl acetate produced by W. saturnus NCYC 22 increased until approximately 140 h of fermentation, and after that, its concentration gradually decreased in bioreactor fermentation. In a study by Rocha-Valadez et al. (64), an extractive fermentation process was scaled up for 6pentyl-a-pyrone production by T. harzianum from a synthetic medium in a 10-L bioreactor. Similar to our results, the researchers observed 6-pentyl-a-pyrone depletion in the culture medium towards the end of fermentation. Sensory evaluation of tomato and pepper pomaces fermented with T. atroviride and A. sojae In the flavour research, it is required to interpret the relationship between the quantity of a flavour compound and its sensory impact or perception. Instrumental analysis should be performed together with

Please cite this article in press as: Günes¸er, O., and Yüceer, Y. K., Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.013

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FIG. 2. Principal Component Analysis (PCA) biplot of volatile compounds and sensory attributes of fermented and unfermented tomato (A) and pepper (B) pomaces.

Please cite this article in press as: Günes¸er, O., and Yüceer, Y. K., Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.013

8

GÜNES¸ER AND YÜCEER

sensory studies (37,66). Therefore, Spectrum Analysis was conducted in this study to determine the sensory perception of the produced flavour compounds and to confirm their sensory characteristics with sensory evaluation. The relationships between the sensory terms and the flavour compounds produced from the tomato and pepper pomaces are shown in Fig. 2. The panellists identified 10 sensory terms for the fermented tomato and pepper pomaces. Significant differences were observed between the fermented and unfermented tomato pomace in terms of their sensory attributes (P ¼ 0.01). Tomato pomace fermented with T. atroviride was characterised as boiled tomato, metallic, sweet aromatic and cheesy aromas, while tomato pomace fermented with A. sojae was characterised as nutty/dust and storage/mould flavours. Moreover, it was found that 3-octanone was associated with nutty/dusty aroma whereas 3-octanol, (E)-2-octenal, (Z)-1,5octadien-3-ol, and 1-octen-3-ol have a close relationship with earth, storage/mould, sweet aromatic and cheesy aromas in the tomato pomace (Fig. 2A). The pepper pomace fermented with T. atroviride was characterised by sweet aromatic, fermented vegetable, cooked and storage/mould aromas, while that fermented with A. sojae was characterised by pumpkin and rose/flower aromas. It was also observed that the unfermented pepper pomace was characterised by a high-level intensity of green pepper and herbal aromas. In the case of the pepper pomace, (E)-2-octenol and 1-octen-3-ol were associated with storage/mould and sweet aromatic aromas. Pumpkin and rose/flower aromas were found to be associated with 3-octanone, (Z)-1,5-octadien-3-ol, 3-octanol and (E)-2-octenal (Fig. 2B). These results confirmed that the changes in the flavour characteristics of the tomato and pepper pomaces occurred due to the production of volatile compounds by metabolisms of T. atroviride and A. sojae. Thus, eight-carbon volatiles, including 1octen-3-ol, 3-octanone, (Z)-1,5-octadien-3-ol and (E)-2-octenol, have particular flavours, from mushroom-like to sweet and fruity aromas (29). Observations similar to the sensory analysis findings of the present study were reported for different agro-wastes fermented with different moulds and yeasts in our previous studies (30,48). For instance, olive pomace fermented by T. atroviride had a mushroom aroma at a higher intensity with higher concentrations of 1octen-3-ol and (E)-2-octenol (48). Tomato pomace fermented by Debaryomyces hansenii had the most intense green bean aroma, while fermented vegetable and storage/yeast aromas were found at high intensities in pepper pomace fermented by D. hansenii and were shown to be associated with isoamyl acetate and phenyl ethyl acetate (30). In conclusion, the production of mushroom alcohols and aldehydes from tomato and pepper pomaces by fungal metabolism was investigated. It was determined that T. atroviride grew faster than A. sojae and the survival of T. atroviride was longer than that of A. sojae in the tomato pomace. However, T. atroviride grew slower than A. sojae in the pepper pomace. In contrast to previous studies, the present study reports the production of aliphatic alcohol- and aldehyde-type flavour compounds associated with mushroom and geranium-like aromas of fungal metabolism. (Z)-1,5-Octadien3-ol, 1-octen-3-ol, (E)-2-octenal, and (E)-2-octenol were produced from the tomato and pepper pomaces by T. atroviride and A. sojae. In particular, 1-octen-3-ol and (E)-2-octenol were produced at high concentrations from the tomato pomace using T. atroviride, and (Z)-1,5-octadien-3-ol and (E)-2-octenal were produced from the pepper pomace using A. sojae. Therefore, tomato and pepper pomaces can be considered appropriate raw materials in the production of biotechnological flavour compounds by fungal metabolism. Further studies should be conducted to optimise and evaluate the production of natural flavours on a large scale by fungal fermentation.

J. BIOSCI. BIOENG., ACKNOWLEDGMENTS This study was funded by The Scientific and Technological Council of Turkey (TUBITAK, Ankara Turkey; Project No. 110O903). The authors would also like to thank Bioflavor COST Action FA0907 for supporting this scientific work. The authors have no conflict of interest to declare.

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Please cite this article in press as: Günes¸er, O., and Yüceer, Y. K., Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.013