Improving Nutritional Qualities of Tomato Pomace by Pleurotus ostreatus and Phanerochaete chrysosporium Fermentation

In this study, it was aimed to improve nutrient quality by fermenting tomato pomace with Pleurotus ostreatus (P. ostreatus) and Phanerochaete chrysosporium (P. chrysosporium). Tomato pomace was incubated for 21 days at optimized conditions of pH (3.50-5.50), temperature (24-28 oC), moisture content (68% w w-1), aeration (0,25 L min-1) and stirring rates (10 rpm). Three samples taken at each incubation time were chemically analyzed. The results indicated that fermentation with P. ostreatus and P. chrysosporium significanly increased ash content by 25 and 21%, crude protein content by 16 and 30%, respectively (P<0.05). Fermentation with P. ostreatus decreased ether extract content from 7.22% to 0.29% at 21th day (P<0.05). However, there was an increase of ether extract content with P. chrysosporium fermentation (from 7.22 to 11.62% at 21 day) (P<0.05). Crude fiber of tomato pomace with P. chrysosporium were reduced by 64% (P<0.05). Both fungal fermentations reduced total reducing sugar content by about 30% (P<0.05). Fermentation with P. ostreatus and P. chrysosporium significantly changed tannin and pectin levels (P<0.05). As a result, fungal fermentation caused to nutritionally enriched tomato pomace with added active compounds, and could be used as functional feed in animal nutrition. Research Article Article History Received : 25.05.2019 Accepted : 31.10.2019


INTRODUCTION
Recently, there has been an increasing demand on the utilisation of agro-industrial waste products causing enviromental pollution to a greater extend. Tomato pomace is a by-product of tomato processing industry, and composed of tomato skin, seed and pulp. The world's annual tomato waste production reaches up to 11 million ton per year including 4 million tons of tomato pomace (FAO, 2016). Tomato pomace contains appreciable amount of proteins, lipids, carbohydrates, amino acids, carotenoids and minerals (Frexio et al., 2012;Liu et al., 2013;Ergun and Urek, 2017;Waldbauer et al., 2017;Ulker et al., 2018). It has mainly been used as feed material or soil fertilizer (Knoblich et al., 2015;Bennamoun et al., 2016). Tomato pomace can directly be fed to ruminant animals as fresh or dried forms and as a part of silage at appreciable amounts. The ruminant animals can easily utilize from the nutrients of tomato pomace as a result of microbial digestion of the rumen (Weiss et al., 1997;Mirzaei-Aghsaghali and Maheri-Sis, 2008;Ziaei and Molaei, 2010;Abdollahzadeh et al., 2010). In contrary, poultry species can not completely utilize from tomato pomace so as to ruminant animals since the digestive tract of most of the poultry species, especially young growing birds, do not sufficiently secret specific enzymes degrading the nutrients such as crude fibre (CF), tannin and pectin which are mostly considered as antinutritional factors (ANFs) (King and Zeidler, 2004;Al-Betawi, 2005;Wadhwa and Bakshi 2016;Yasar and Tosun, 2019). In addition, tomato pomace is a seasonal product and not available throughout the entire year and difficult for conversation due to its high moisture content of 75%. Moreover, drying tomato pomace at commercial scale to produce animal feed has been found not economically feasible (Weiss et al., 1997), but there are novel processing treatments including drying to produce bioactive compounds such as antioxidants, lycopene, oils and protein as food and feed ingredients (Lu et al., 2019).
The fungals of Rhizopus stolonifer LAU 07, Candida utilis, Trichoderma viride, Aspergillus niger, Fusarium, P. ostreatus and P. chrysosporium (Villas-Boas et al., 2003;Lateef et al., 2008;Yasar and Tosun, 2018), the yeast of Saccharomyces cervisiae and bacteria of Bacillus subtilis (Azza et al., 2013) have been used in SSF processes of agricultural by-products for crude protein enrichment. In addition, fungal microorganisms was reported to break down cellulose, hemicellulose and other complex polysaccharides in industrial by-products (Rashad et al., 2009;Díaz-Godínez et al., 2012). Previously studies reported that fermenting tomato pomace with several microorganisms increased the amount of ash and crude protein (CP) and decreased the levels of CF and hemicellulose (Assi and King, 2008;Azza et al., 2013;Roja et al., 2017;Yasar and Tosun, 2019).
Having evaluated all the above stated results, P. ostreatus and P. chrysosporium could ideally act as best fungal microorganisms to nutritional enrichment of tomato pomace at optimum conditions of 4.0-5.0 of pH, 25-35 o C of temperature and low stirring rates at occational intervals, which were selected from the literature and optimized and controlled throughout the study using a modern bioreactor. Therefore, the objective of this study was to test the effect of two fungal microorganisms used in SSF on the changes in nutritional composition of pomace.

MATERIALS and METHODS
Tomato pomace optained from a local provider dried and ground to pass a sieve with 3 mm were supplemented with nutrients as shown in Table 1 and was further autoclaved at 120 ℃ for 15 min. Two fungal microorganisms, Pleurotus ostreatus (P. ostreatus) and Phanerochaete chrysosporium (P. chrysosporium) obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany) were cultivated according to the supplier instruction to collect inoculating spores. Optimum fermentation conditions fixed in the study (Table 1) were selected from the literature and optimized by using a laboratory bioreactor of 2.5-3 L working capacity, LabforEtOlH 5 (Infors Ltd., Switzerland). A blank fermentation experiment was conducted with no fungal inoculation. The fixed pH values were well optimized by peristaltic pumps using buffer solutions of 0.1 M sodium acetate (pH=1.5) and 0.1 M sodium bicarbonate (pH=9.75).
At each sampling period (days), three independent samples were taken from each of fermentation experiments (Table 1) were consequently analysed three times as replicates for the determinations of nutritional and antinutritional factors parameters. *Added nutrients in experiment I, II and III were 20 g (NH4)2SO4, 10 g NH4Cl, 10 g CH4N2O and 60 g Molasses, and there was a constant rate of stirring rate (10 rpm for 2 min at every 12 h). **Blank fermentation, a non-pH optimised experiment (initial pH of 4.00 did not change throughout the fermentation period) under sterile fermentation conditions, the same as in experiments I and II Thus, 9 independent replicates per treatment were obtained, and data was analysed according to a general linear model (GLM) of variance analysis, where the differences between the treatments were separated at 0.05 significance level using SPSS software (IBM SPSS Statistics 22.0 for Windows). Fungal growth was determined by the method of TS ISO 21527-2: 2008 and the contents of dry matter (DM, %), ash %, crude protein (CP, %), crude fiber (CF, %), ether extract (EE, %) and reducing sugar (RS, %) by the methods specifically expressed in AOAC (2005). Pectin (Wang and Zhang, 1999) and tannin (Chemesova and Chizhikov, 2004) were spectrophotometrically analysed.

RESULT and DISCUSSION
The results of Table 2 that the growth rate of P. ostreatus in experiment I and P. chrysosporium in experiment II significantly increased by 3 log at the end of 21 days of fermentation (P<0.05). And the pH was well controlled in the pre-fixed ranges in both experiments (Table 1), indicating a successful fermentation of fungal microorganism on tomato pomace. Ash content of tomato pomace increased in both fungal fermentations. Dei et al. (2008) reported increased ash content of industrial by-products as a result of fungal fermentation. The reason for the inc rease in ash content was elaborated that during fermentation, microorganisms secrete enzymes which degrade complex minerals such as phosphorus in phytic acid form, and as a result of fermentation, the liberated minerals are released and thus the ash content increases. Many other studies found increased ash content of the fermenting substrates as a result of fermentation (Dei et al., 2008;Okpako et al., 2008;Aguilar et al., 2008;Altop et al., 2018). 00±0.20 a,b,c, d Different supercripts showed significant differences between the fermentation periods at each of the coloumn parameters. Lateef et al. (2008), clearly showed that fungal fermentation using the strain of Rhizopus stolonifer LAU 07 significantly improved nutritional qualities of some agro-wastes by increasing CP contents by 35-90% and reducing CF by 7.0 to 44% (P<0.05). Similar improvement rates were also reported by several other fungal species including P. ostreatus and P. chrysosporium in the study of Assi and King, (2008); Rashad et al. (2009 and2010);Díaz-Godínez, (2012) and Yasar and Tosun, (2018). In fungal fermentation fungal growth and reproduction of micelles formed as a result of the substrate used in fermentation has been reported to increase the CP content (Altop et al., 2018). On the other hand, Oboh and Akindahunsi (2003) claimed that the increased CP content was due to the increased enzymes which are in nitrogenous nature. It is thought that the increase of CP content in the fermentation of tomato pomace with P. ostreatus and P. chrysosporium is due to the above reasons.
However, in our study, the amount of increased CP content differed in the case of both microorganisms, showing that the type of fungal microorganisms may have affected the degree of increased CP. As overal the P. ostreatus and P. chrysosporium fermentations were found useful for increasing CP of tomato waste products.
The content of EE in tomato pomace was nearly consumed by the strain of P. ostreatus in our study. P. ostreatus was also previously shown to reduce the EE contents of oil-seed meal (Yasar and Tosun, 2018). The reduced lipid content could be due to the accumulation of lipids by some strains of fungal microorganisms which have lipase enzyme activity (Lateef et al., 2008;Agbo and Prah, 2014;Altop et al., 2019) since some fungal microorganisms should assimilate lipids from the fermenting substrates in order to produce other biomasses (Tinoco et al., 2011;Iandolo et al., 2011;Frexio et al., 2012;Jannathulla et al., 2018). In contrast P. chrysosporium significantly increased the EE content of tomato pomace. This increase in EE is thought to be due to the release of lipolytic enzymes that break down glycerol and fatty acids in some microorganisms. Because Onweluza and Nwabugwu (2009) reported that the EE increases when millet (Pennisetum americanum) and Pigeon pea (Cajanus cajan) fermented and as a reason for the secreted lipolytic enzymes during fermentation. However, in the previous studies, EE decreases; and lipolytic enzyme secretion and the increase in EE in fermentation are not sufficient in the literature.
Some microorganisms generally use easily soluble carbohydrates such as starch and sugar to meet carbon requirements during fermentation, and then prefer to use complex carbohydrates or other nutrient as carbon sources (Papagianni, 2007;Altop et al., 2019). And as well Xie et al. (2016) and Altop et al. (2019) reported that during solid state fermentation, fungals release enzymes such as cellulase, hemicellulose, which break down structural carbohydrates, and these enzymes break down structural carbohydrates. It was concluded that the microorganism used as a carbon source as a reason for the decrease in RS and HS content in fermentation of tomato pomace with P. chrysosporium. On the other hand, P. chrysosporium is a white-rotfungus, the release of enzymes that break down structural carbohydrates is thought to be high and therefore the HS content of tomato pomace may be reduced. In P. ostreatus fermentation, the HS content did not change, but the RS content decreased significantly, suggesting that this microorganism did not use either the carbon requirement of P. chrysosporium or the HS as a carbon source or does not release enough enzymes to break down structural carbohydrates There was no significant effect on the contents of ANFs (tannin and pectin) of tomato pomace of the fermentation carried out without fungal microorganisms in this study (P>0.05) (Table 4).
However, fermenting tomato pomace with P. ostreatus and P. chrysosporium significantly influenced these compounds (P<0.05). There was an average of 60% decrease in the tannin content at the 14 day of the fermentations carried out both fungal microorganisms (P<0.05). Fermentation of tomato pomace with P. ostreatus did not significantly change the pectin content (P>0.05). However, there was a significant sporadic effect of P. chrysosporium fermentation on the pectin content, which was increased (P<0.05) by 48% at 14 day and then decrease (P<0.05) by 28% at 21 day of in the pectin contents.
The pectin and tannin are usually available in the tomato peel tissue, and its degradation is very important to free up important biologically active compound such as phenolic compounds and lycopene with antioxidant property (Lavecchia and Zuorro, 2008;Rodríguez-Fernández et al., 2011;Saleh et al., 2018). Biz et al., (2016) reported that they produce pectinase enzyme by fermenting agricultural byproducts with Aspergillus oryzae. Enzyme production in the presence of pectin in the fermentation of microorganisms in the presence of pectin to break down the enzyme is connected to secrete. In our studythe pectin content of tomato peels was extracted and released into the matrix of fermentation substrate at the 14 day of fermentation with both fungal species, due to a possible increase in the activitiy of pectinase. Finally, the degradation of tomato peels could be said to be completed by fungal fermentations. This mechanism was more pronounced with the fermentation using P. chrysosporium. Thus, it could possible that some important phenolic compounds are released during the fungal fermentations and this lead to a possible increase of antioxidant capacity.
The tannin content of tomato pomace decreased in fermentation with both fungal microorganisms. In previous studies, it has been reported that in fermentation microorganisms break down tannin into smaller molecules such as gallic acid, catechin, glucose and gallicatechin (Rodriguez et al., 2008;Nazarni et al., 2016;Shang et al., 2019). In this study, it is thought that the decrease in tannin content secretes tannase enzyme and the tannin content is converted to gallic acid, catechin, glucose or gallicatechin.

CONCLUSION
As a result, the fermentation of tomato pomace by fungal microorganism yielded an enrichment of nutritional qualities and added some biologically functional compounds. The fermented tomato pomace with improved nutritional qualities is holding a great potential in farm animal nutrition.

ACKNOWLEDGEMENT
We gratefully acknowledged the TUBITAK, Turkey for their financial support of the study (VHAG: 214O629 -1001 project).

Statement of Conflict of Interest
Authors have declared no conflict of interest.

Author's Contributions
The contribution of the authors is equal.