Nutritional Enrichment of Apple Pomace by Fungal Fermentations

This study tested effects of optimised fungal fermentation conditions on nutritional enrichments of apple pomace. Three fermentation experiments were conducted by using 2.5x106 spores of Pleurotus ostreatus (P. ostreatus), 2.5x106 spores of Phanerochaete chrysosporium (P. chrysosporium) and without microorganisms per g substrate, respectively, for 0, 7, 14 and 21 days using a pilot bioreactor. At the end of each incubation period, 3 steril sample were analysed to determine microbial growth, pH and nutrient contents levels. The results indicated significantly increased crude ash and protein contents (P<0.05) as well as decreased crude fiber and reducing sugar contents of apple pomace by both fungal microorganisms (P<0.05). Crude fat content increased by P. ostreatus fermentation while P. chrysosporium fermentation reduced crude fat content (P<0.05). Tannin content reduced at all fermentation periods by P. ostreatus while P. chrysosporium fermentation increased tannin content (P<0.05). Pectin increased by both fungal fermentations, but the effect of P. ostreatus was greater (P<0.05). It was concluded that fungal fermentations caused to remarkable improvements of apple pomace in nutritional properties, which could of high importance in animal nutrition. Research Article Article History Received : 04.10.2019 Accepted : 06.02.2020


INTRODUCTION
There is an increasing trend in adding value to or utilizing from ligno-cellulotic by-products for the production of several functional bio-molecules including enzymes, antioxidant and organic acids by the means of microbial fermentation. Apple pomace is a ligno-cellulotic by-product of fruit juice manufacturing sector, which annually processes approximately 20-40% of a total 83 million tonnes apple produced in 2017 (FAO 2018). Apple pomace composed of peel, nut and pulp, which contained about 36% insoluble and 15% soluble fractional fibres on a dried matter basis (Sudha et al., 2007).
Apple pomace of food and feed materials are cheap and rich source of carbohydrate, pectin, crude fibre and minerals in nutritional content (Kara and Doymaz, 2015). It can also provide an economic contribution to livestock production as a means of recycling as an industrial by-product that can cause environmental pollution (Waldbauer et al., 2017;Ulger et al., 2018;Ricci et al., 2019;Matejova et al., 2019). On the other hand, apple pomace can be added to the rations in ruminant animals, in fresh, dried or silage, in poultry as to dried form in amounts that will not have negative effect on animal health (Sudha et al., 2007;Ulger et al., 2018;Yasar and Tosun, 2019a). However, apple pomace has a high-water content of having difficulty for storage, has been seasonally produced Yasar and Tosun, 2019a). These can limit the use of apple pomace in livestock as a source of feed. On the other hand, some antinutritional factors are present in the apple pomace (Ajila et al., 2015). Although rumen fermentation can overcome the antinutritional factors of apple pomace. But the performance of monogastric young animals could be lowered by the use of apple pomace. Therefore, the antinutritional contents of apple pomace should be lowered and its nutritional content must be increased before fed to monogastric animals. One of the treatments to improve nutritional qualities of apple pomace is solid state fermentation (Joshi and Devender, 2006;Mukherjee et al., 2016).
Fungal fermentations of agricultural waste products in solid state fermentation (SSF) resulted in remarkable nutritional and chemical benefits in respect to adding value to these products (Kurt and Buyukalaca, 2010). Several fungal microorganisms were successfully used in various conditions of SSF using apple pomace for the enrichment of nutrients (Villas-Boas et al., 2003;Albuquerque et al., 2006;Vendruscolo et al., 2008;Ajila et al., 2015;Madrera et al., 2017), for improvements of antioxidant enzyme activities (Zheng and Shetty, 2000;Joshi and Devender, 2006;Ajila et al., 2011) and aromatic compounds (Ricci et al., 2019).
When fermented with S. cerevisiae the nitrogen and fat content of apple pomace have been reported to be increased (Joshi and Devender, 2006). Apple pomace fermented with 3 yeast strains (S. cerevisiae, ref: 32; S. bayanus, ref: C6; and H. uvarum, ref: 62) for 7 days has increased crude protein, fat and dietary fibre content, but depleted the sugars content (Madrera et al., 2017). As a result of fermentation with Saccharomyces cerevisiae AXAZ-1 and Kluyveromyces marxianus IMB3, some industrial by-products had significantly increased crude fat and protein contents, and Kluyveromyces marxianus was found an ideal microorganism for increasing crude protein and fat content (Aggelopoulos et al., 2014).
According to the results, when the apple pomace was fermented with white-rot fungal there was an increase in the content of free sugar, a decrease in the content of crude fibre, ADF and NDF contents (Zhong-Tao et al., 2009;Yasar and Tosun, 2018a). On the other hand, the study conducted by Yasar and Tosun (2019b) showed that the crude ash, ether extract and starch contents of apple pomace was increased by the fermentation of apple pomace with K. marxianus, wehereas the crude protein, total reducing sugar, crude fibre, ADF, NDF and lignin contents were reduced. Furthermore, apple pomace has been used to produce organic acid and lactic acid (Dhillon et al., 2011;Dhillon et al., 2012;Yasar and Tosun, 2019b). There were sporadic effects of SSF on the changes in the tannin and pectin by microbial SSF (Dhillon et al., 2012;Yasar and Tosun, 2019b).
In this study, the optimum conditions of pH, fermentation periods, stirring rate and moisture content of substrate selected from the above studies were fixed, optimised and controlled by a modern bioreactor and used to ferment apple pomace by two fungal microorganisms, Pleurotus ostreatus (Jacquin: Fries) Kummer. teleomorph (ATCC® 34673™) and Phanerochaete chrysosporium Burdsall. teleomorph (ATCC® 24725™). The objective of this study was to determine the effects of optimised fermentation conditions selected for two fungal microorganisms on the nutrient fortification of apple pomace, a wasteproduct of apple juice production.

MATERIALS and METHODS
Apple pomace were purchased from a local provider dried and ground to pass a sieve with 3 mm and supplemented with additional nutrients (Table 1) and were further autoclaved at 120 o C for 15 min. Two fungal microorganisms, Pleurotus ostreatus (P. ostreatus) and Phanerochaete chrysosporium (P. chrysosporium) were obtained from DSM were cultivated according to the supplier instruction to collect sufficient amount of spores for inoculation. Optimum fermentation conditions selected from the literature (Ajila et al., 2011;Pathania et al., 2017;Yasar and Tosun, 2019b) were fixed in the study (see Table 1) and optimized by using a laboratory bioreactor of 2-3 L working capacity, LabforEtOH 5 (Infors Ltd., KSU J. Agric Nat 23 (3) Janssen (1989). Pectin Wang and Zhang (1999) and tannin Chemesova and Chizhikov (2004) were spectrophotometrically analysed. All chemical analysis results were expressed as percentage of dry matter.

RESULTS and DISCUSSION
The use of a modern bioreactor to optimise all the fermentation conditions was very successful. It was observed that the stirring, aeration and pH were excellently managed without any fluctuations from the fixed values.
As compared to experiment III, where no microbial development was seen, the fungal growth of P. ostreatus and P. chrysosporium in experiment I and II reached to a maximum level by 4 log increase at 14 and by 2 log increase at 7 days of fermentation, respectively (Table 2).  Table 3 contained all analytical data of the apple pomace fermented for 0, 7, 14 and 21 days without fungal inoculants (Experiment III). It can be seen that there were no statistically significant changes in any parameters over the periods of fermentation (days). The effects of fungal inoculants on each of parameters were presented in Figure 1, 2 and 3 as percentage decrease or increase from the blank fermentation at each fermentation period; the analysed values of blank samples were set as "0".
The fungal fermentations remarkably utilised from readily available carbohydrates as the microorganisms of P. ostreatus (Figure 1.A and 1.B) and P. chrysosporium (Figure 2.A and 2.B) significantly (P<0.05) consumed total reducing sugar of apple pomace at all fermentation periods, in comparison with blank fermentation experiment (Figure 1 and 2).
Overall, the fermentations with P. ostreatus ( Figure  1.A) and P. chrysosporium (Figure 2.A) significantly (P<0.05) increased crude ash and crude protein of apple pomace, expect there was a significant decrease of ash content of apple pomace in 21 days of P. ostreatus fermentation. Total increase in crude ash and protein was significantly (P<0.05) higher in P. chrysosporium (Figure 2.A) than those in P. ostreatus (Figure 1.A) fermentation.
However, there was a significant interaction between the fungal fermentations and crude fat contents. A remarkable increase in the content of crude fat, by about 22 folds (from 0.40% to 9.0%), of apple pomace by P. ostreatus fermentation, whereas the fermentation of apple pomace with P. chrysosporium Results in this research were completely agreed with the results of many studies reporting an increase in crude ash and protein content as a result of the fermentation of industrial by-products with various microorganisms including P. ostreatus and P. chrysosporium (Lateef et al., 2008;Ajila et al., 2015;Madrera et al., 2017;Yasar and Tosun, 2018b). In our study, the rate of increased crude ash and protein content by P. ostreatus and P. chrysosporium fermentation was comparably higher than these results in the above studies. The reason for the increase in the amount of crude protein in this study is thought to be due to the addition of ammonium sulphate to the medium. Because, Villas-Boas et al. (2003) has been reported that the addition of ammonium sulphate to the fermentation medium has a stimulating effect on fungal and yeast fermentations and increases crude protein production in fermentation. In this study, the reason for the increase in crude protein content more than other studies is thought to be due to the addition of ammonium sulphate to the medium. Altop et al. (2018) reported that in fungal fermentation microorganisms secrete phytase enzyme and this enzyme breaks down the phosphorus in complex form and as a result, the crude ash content of the substrate increases. In this study, the crude ash content was increased, however, the reason for the increase is not known precisely because no mineral substance analysis was performed. As a result of fermentation of industrial by-products with bacteria, yeast and fungi, it has been determined that the content of crude fat increases by 20-50% (Joshi and Devender, 2006;Madrera et al., 2017;Altop et al., 2018). Similar results were obtained from previous studies of P. ostreatus fermentation. In contrast to these studies, the crude fat content of apple pomace was increased by 2252% at the end of fermentation in our study. Unlike the studies in the literature, the crude fat content of apple pomace by P. chrysosporium fermentation decreased significantly. This is a first ever scientific outcome from the apple pomace fermentation by P. chrysosporium. Therefore, it is highly possible that P. chrysosporium needs more fatty KSU J. Agric Nat 23 (3): 754-761, 2020 Araştırma Makalesi Research Article acids for its microbial growth than P. ostreatus on apple pomace.
There are many studies reporting decreased crude fibre and its fractions by the solid-state fermentation (Lateef et al., 2008;Yasar and Tosun, 2018c;Karakurt et al., 2019). Altop et al. (2018) reported that the starch and sugar content of industrial by-products were reduced as a result of fungal fermentation. In our study, P. ostreatus and P. chrysosporium microorganism fermentation significantly decreased crude fibre, ADF, NDF and reducing sugar, similar to previous studies. In the fermentation study conducted by Aderemi and Nworgu (2007), it was reported that fungal microorganisms break down structural and non-structural carbohydrates by the enzymes they secrete and as a result of this degradation, crude fibre and its fractions and sugar contents decrease. Krishna (2005), Aderemi and Nworgu, (2007) and Altop et al. (2018) report that microorganisms secrete enzymes to break down structural and non-structural carbohydrates, thereby breaking down carbohydrates to meet their carbon needs. In this study, it is concluded that microorganisms meet the carbon need for growth and development by breaking down crude fibre, ADF, NDF and reducing sugars and consequently, these contents are reduced.
Fermentation with P. ostreatus (Figure 2.A) significantly (P<0.05) reduced the tannin contents and meanwhile increased the pectin contents of apple pomace. However, the changes in tannin and pectin contents throughout the fermentation periods were sporadic, not consistent by P. ostreatus and P. chrysosporium (Figure 2.B).  Degradation of tannin and pectin in apple pomace by fungal microorganisms were also reported earlier (Villas-Boas et al., 2003;Ruiz-Aguilar et al., 2004;Zhong-Tao et al., 2009). In contrary, the magnitude of tannin degradation in our study was larger with the fermentation of P. ostreatus, while no degradation of pectin, even led to significant increase in pectin content. Moreover, the tannin content tended to increase by the P. chrysosporium fermentation to a significant extend, where the pectin levels had a tendency of decrease towards to the end of fermentation. Such differences in the pectin content of apple pomace were due to the types of fungal microorganisms differing in the production of pectin esterase enzyme Zhong-Tao et al., 2009;Dhillon et al., 2012;Yasar and Tosun, 2019b).

CONCLUSION
It can be concluded that the studied fixed fermentation conditions selected from the literature for P. ostreatus and P. chrysosporium were well suited for the purposes of nutrient fortification of apple pomace in terms of KSU J. Agric Nat 23 (3): 754-761, 2020

Araştırma Makalesi
Research Article increased ash and crude protein as well decreased dietary fibre. This result is of significant importance in animal nutrition.

Acknowledgement
Many thanks to TÜBİTAK, Turkey VHAG 1001 (2140629 project number) for finacial support to conduct these experiments.

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

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