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Manganese Amendment and Pleurotus ostreatus Treatment to Convert Tomato Pomace for Inclusion in Poultry Feed

Department of Animal Science, University of California, Davis, One Shields Avenue, Davis 95616-8521

¹ Corresponding author: ajking{at}ucdavis.edu

	ABSTRACT

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If freshly dried tomato pomace could be fed to poultry, its naturally occurring -tocopherol would retard lipid oxidation during further processing, long-term frozen storage, and heating of poultry meat; however, the high fiber content in this agricultural by-product adversely affects its use. Experiments were conducted to investigate the chemical composition and in vitro true digestibility of amended (without and with 487 µmol of manganese/g) tomato pomace substrate after treatment with the white-rot fungus Pleurotus ostreatus. In treated pomace without manganese, protein content was improved by 3.1%, cellulose and hemicellulose decreased over time, but lignin degradation was not detected. In addition, treated pomace without manganese showed a significant reduction of in vitro true digestibility. Manganese in pomace inhibited fungal growth and did not enhance lignin degradation. Under the conditions of the experiment, P. ostreatus improved the nutritional composition of tomato pomace; however, it did not reduce lignin. It is possible that manganese amendment at the level used affected gaseous conditions (O₂ consumption and CO₂ evolution rates), important factors that must be considered when attempting to enhance accelerated lignin degradation by P. ostreatus.

Key Words: digestibility • manganese amendment • Pleurotus ostreatus • poultry feed • tomato pomace

	INTRODUCTION

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Investigators are constantly searching for ways to improve the worldwide use of agricultural residues by including them in animal feed. Special attention has been given to the microbial bioconversion of accumulated agricultural wastes. The potential use of white-rot fungi (Pleurotus spp.) for the biodegradation of lignin has been discussed (Kirk and Farrell, 1987; Kamra and Zadrazil, 1988; Singh et al., 1996; Fadel, 1999; Villas-Boas et al., 2002). Reviews have comprehensively described the applicability of white-rot fungi in several biotechnological and environmental applications. Authors have specified the role of extracellular ligninolytic enzymes in lignin degradation for several fungal species (Hatakka, 2001; Cohen et al., 2002a).

California produces more than 90% of the processed tomatoes in the United States (USDA, 2007). Tomato pomace is a by-product of tomatoes processed into various products, and consists of peels, cores, culls, trimmings, seeds, liquor, and unprocessed green tomatoes picked by harvest machinery. As with most agricultural by-products, the fiber content of tomato pomace affects its nutritional value. The major fiber components—cellulose (CELL), hemicellulose (HEMC), and lignin—are difficult for animals to digest because the polydispersed physical and chemical structure imposed by lignin polymers prevents free access of hydrolytic CELL- and HEMC-degrading enzymes from using plant polysaccharides (Cohen et al., 2002a).

In the first of a series of investigations on the use of tomato pomace as a possible feed for poultry, King and Zeidler (2003) included it in broiler diets to determine whether the -tocopherol in pomace could retard lipid oxidation during subsequent long-term frozen storage or heating of poultry meat. Although investigators concluded that -tocopherol from pomace fed to poultry could retard lipid deterioration in postmortem tissue, they noted that the high fiber in diets caused by adding pomace adversely affected feed conversion, thus raising the question of the cost-effectiveness of the by-product. One possible way to enhance the use of pomace, primarily through delignification, was to treat it with the white-rot fungus Pleurotus ostreatus during solid-state fermentation (SSF). The major concerns associated with this process were the quantity of retained -tocopherol and the conversion of CELL, HEMC, and lignin into digestible nutritional components.

Assi and King (2007) treated tomato pomace with P. ostreatus and determined the quantity of selected antioxidants (-tocopherol, lycopene, and β-carotene) remaining in the substrate after fungal treatment under SSF. Results of the study revealed that the carotenoid content was negligible, whereas more than 50% of the -tocopherol remained up to 104 d after treatment when mushrooms were produced. Thus, it seemed that freshly prepared and properly stored tomato pomace could be treated with P. ostreatus without destroying the major antioxidant of interest.

A few species of microorganisms (bacteria, protozoa, and fungi) are able to degrade the complex lignocellulosic components of fiber (Kirk and Farrell, 1987; Hatakka, 2001). As mentioned above, Pleurotus spp. are significant because they are purported to degrade lignin preferentially under SSF. Additionally, fungal bioconversion of agricultural by-products is an environmentally friendly biotechnological process (Hadar et al., 1992; Cohen et al., 2002a).

Results from many studies have suggested that to achieve significant bioconversion with elevated lignin degradation in an array of agricultural residues, various conditions should be optimized. Reported conditions have included primarily the fungal strain and growth parameters—temperature, gaseous phase (O₂ and CO₂ concentration), particle size, and substrate porosity (Kamra and Zadrazil, 1988; Hadar et al., 1992; Stamets, 1993; Zadrazil and Puniya, 1995; Bisaria et al., 1997; Curvetto et al., 2002). Moreover, amending the substrate with manganese, as an active mediator, regulated and enhanced the activity level of manganese peroxidase, improved colonization conditions, and increased lignin degradation (Kerem and Hadar, 1993; Kerem and Hadar, 1995; Cohen et al., 2001, Cohen et al., 2002a,b; Curvetto et al., 2002). When manganese was added, manganese peroxidase production was enhanced in wood poplar and fir culture sawdust substrates (Giardina et al., 2000).

Tomato pomace could be a valuable feed ingredient for poultry if the conditions for preferential delignification could be identified. Thus, the objective of the work reported here was to investigate further changes in the chemical composition and in vitro true digestibility (IVTD) of tomato pomace substrate to determine the effectiveness of manganese amendment, followed by treatment with P. ostreatus.

	MATERIALS AND METHODS

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Substrate Preparation and Inoculation

Tomato pomace (70% moisture) was obtained from the Processed Foods Research Unit (Western Regional Research Center, USDA, Agricultural Research Service, Albany, CA). Pomace was divided into 2 containers. Manganese sulfate (MnSO₄, stock no. M7634, lot no. 074K0139, Sigma-Aldrich, St. Louis, MO) was added as 487 µmol/g to one container. This level of MnSO₄ was intermediate to the lowest and highest levels used by other investigators (Kerem and Hadar, 1993). After thoroughly mixing the substrates in each container, pomace was placed in Erlenmeyer flasks (250 mL) for experiment 1 and in flasks and autoclavable bags (2 L) for experiment 2. Flask or bags with pomace and without manganese were designated POT, whereas those with pomace and manganese were designated POT x Mn.

Experiment 1

The effect of P. ostreatus over time was compared with that at 0 d (control). Flasks with POT were sterilized in an autoclave (Model SV-120 Sterilizer, Steris Corporation, Mentor, OH) at 121°C and 193 kPa for 1 h, then cooled (25 ± 1°C) and inoculated with 5% sterile sawdust spawn of the white-rot fungus P. ostreatus (Fungi Perfecti LLC, Olympia, WA) under a laminar flow hood (model 201–430, NuAire, Plymouth, MN). These sterilized and inoculated flasks were stored at room temperature (25 ± 1°C) to initiate the mycelial colonization and the fungal fermentation process. They were maintained at room temperature throughout the experiment. Duplicate samples of the control (sterilized, untreated pomace) for 0 d and POT for d 14, 35, 42, 49, 56, and 63 were analyzed for chemical composition and IVTD (see below). The experiment was repeated.

For further evaluation of the effect of manganese on fungal growth, triplicates flasks were prepared with pomace containing either POT or 0.5 (POT x 0.5Mn) or 1.5 times (POT x 1.5Mn) the initial concentration of added MnSO₄. These flasks were inoculated, cooled, and stored as described above. Visual comparison of fungal growth was made from 0 to 63 d. This procedure was repeated.

Experiment 2

The effects P. ostreatus across treatments within a sampling period and over time were evaluated. Duplicate bags of the control, POT, and POT x Mn were sterilized, inoculated, and cooled as described above, then stored (25 ± 1°C) for 49 d of incubation. Mycelial growth of P. ostreatus was visually assessed and documented during incubation, full colonization, and formation of the aggregated mass of mycelia. The bags were then placed in a refrigerator (6 to 10°C) for 24 h to induce cold shock before transferring them to a fully controlled environmental growth chamber with a temperature of 18°C, ventilation, RH of 98%, and 4 h of light/d (using four 60-W incandescent light bulbs) to initiate the oyster mushroom fruiting stage. To determine the fate of lignin in pomace after mushroom growth, samples from the spent residue (residue of tomato pomace substrate from bags after mushroom cultivation) at 104 d were chemically analyzed with other samples, as explained below.

Triplicate flasks of the control, POT, and POT x Mn were prepared and stored as described above. Chemical and IVTD analyses were conducted on triplicate samples at d 11, 18, 21, and 26 and on duplicate samples of the spent residue at d 104. The experiment was repeated.

Chemical Analyses and IVTD

Tomato pomace from experiments 1 and 2 was sampled (in duplicate) on varying days and frozen at –80 C° until analyzed. Samples were placed under dry ice in cardboard boxes with double-sided styrofoam interiors and sent to Dairy One Forage Laboratory (Ithaca, NY 14850). The AOAC (1990) methods used included method 976.06 for CP and method 942.05 for ash. Acid detergent fiber (ADF) was quantified following Ankom Application Note 01–02 (Ankom Technology, 2002) and AOAC (1999) method 973.18. Nitrogen detergent fiber (NDF) and lignin were determined following the procedures of Van Soest et al. (1991) and AOAC (1999; method 973.18), respectively. In vitro true digestibility was analyzed by using Ankom Application Note 11-00 (Ankom Technology, 2000). Cellulose was calculated as the difference between ADF and lignin, and HEMC was calculated as the difference between NDF and ADF.

Statistical Analysis

Data were analyzed by using the general linear model procedure of SAS (SAS Institute Inc., Cary, NC). To allow for the possibility of correlation among the observations within a given replicate, a repeated measures ANOVA model was used to compare the mean responses of different time points across the replicate trials. In this setting, the repeated measures model was computationally equivalent to a blocked ANOVA, where the blocking was on the replicate trial. Post hoc comparisons among treatments were performed by using Tukey’s honestly significant differences method. Unless otherwise stated, differences were considered significant at P = 0.05.

	RESULTS

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For all experiments, visual observations revealed that tomato pomace was a suitable substrate for P. ostreatus; some mycelia prints were clearly visible by 48 to 72 h after inoculation. For experiments 1 and 2, P. ostreatus fully colonized and thoroughly permeated the pomace substrate between 18 and 22 d of inoculation, except for POT x Mn (experiment 2), for which full colonization occurred approximately 8 to 10 d later. The order of colonization of pomace substrates with varying concentrations of MnSO₄ was POT > POT x 0.5Mn > POT x Mn > POT x 1.5Mn.

Table 1 provides a range of measurements for treated tomato pomace compared with the control at 0 d. Ash content increased at 49 d and CP increased at 63 d of fungal incubation as compared with the control. Acid detergent fiber and NDF did not change over time, nor were the CELL and lignin fractions degraded. Results revealed sustained HEMC degradation at 42 d and thereafter. Pomace IVTD generally decreased over time and had decreased at 49 d with no further change at d 63.

View larger version (11K): [in this window] [in a new window]   Figure 1. Crude protein in tomato pomace without and with manganese during treatment with Pleurotus ostreatus. The x-axis is compressed between 26 and 104 d. Control = sterile, untreated tomato pomace; POT = sterile tomato pomace treated with 5% sterile sawdust spawn of P. ostreatus; POT x Mn = POT + MnSO4 (487 µmol/g of substrate). For POT, means (6 to 8 samples) were different for d 11 and 104 at P = 0.05. For POT x Mn, means (8 to 12 samples) were different for d 11, 26, and 104 at P = 0.05.

View larger version (12K): [in this window] [in a new window]   Figure 2. Cellulose in tomato pomace without and with manganese during treatment with Pleurotus ostreatus. The x-axis is compressed between 26 and 104 d. Control = sterile, untreated tomato pomace; POT = sterile tomato pomace treated with 5% sterile sawdust spawn of P. ostreatus; POT x Mn = POT + MnSO4 (487 µmol/g of substrate). For POT and POT x Mn, means (8 to 12 samples) were not generally different over the incubation time.

View larger version (11K): [in this window] [in a new window]   Figure 3. Hemicellulose in tomato pomace without and with manganese during treatment with Pleurotus ostreatus. The x-axis is compressed between 26 and 104 d. Control = sterile, untreated tomato pomace; POT = sterile tomato pomace treated with 5% sterile sawdust spawn of P. ostreatus; POT x Mn, POT + MnSO4 (487 µmol/g of substrate). For POT, means (8 to 12 samples) were different for d 11, 18, and 26 at P = 0.05. For POT x Mn, there were generally no differences among means.

View larger version (12K): [in this window] [in a new window]   Figure 4. In vitro true digestibility of tomato pomace without and with Mn during treatment with Pleurotus ostreatus. The x-axis is compressed between 26 and 104 d. Control = sterile, untreated tomato pomace; POT = sterile tomato pomace treated with 5% sterile sawdust spawn of P. ostreatus; POT x Mn, POT + MnSO4 (487 µmol/g of substrate). For POT, means (8 to 12 samples) were different for d 11, 26, and 104 d at P = 0.05. For POT x Mn, means (8 to 12 samples) were different for 11 and 104 d at P = 0.05.

	DISCUSSION

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As reported previously, successful colonization of various substrates by P. ostreatus indicated that it may have the potential to degrade the lignocellulosic complex of fiber and increase the nutritional value of poultry feed (Kirk and Farrell, 1987; Kamra and Zadrazil, 1988; Hadar et al., 1992; Kerem and Hadar, 1995; Zadrazil and Puniya, 1995; Bisaria et al., 1997; Cohen et al., 2001, 2002a, b). In the present study, some, positive nutritional changes occurred. For example, the significantly higher concentration of ash for POT at 104 d indicated that there was organic matter degradation. However, results for ash in POT x Mn, as compared with those in POT and the control, were due to amendment with MnSO₄ and did not indicate significantly elevated organic matter degradation.

An increase for CP was achieved in experiments 1 and 2 (3.1% increase in the spent residue). It seems that the oyster mushroom P. ostreatus decomposed nonprotein nitrogen from substrate lignoprotein and synthesized the released nitrogen into fungal protein as suggested by Hadar et al. (1992), Bisaria et al. (1997), and Nigam and Singh (1996).

Overall results showed that P. ostreatus first attacked the HEMC and CELL. This was probably due to the complex structure of lignin as compared with those of CELL and HEMC. Observations of degradation in the present study seemed to corroborate findings that CELL and HEMC hydrolytic enzymes could be detected in the fungal biomass (Kirk and Farrell, 1987; Bisaria et al., 1997; Hatakka, 2001; Cohen et al., 2002a).

Differences in results for biodegradation of the lignocellulosic complex may have been caused by the use of different substrates and experimental conditions. For instance, substrates used in other studies were generally dried agricultural residues (e.g., wheat straw) containing a lower amount of HEMC than lignin. Thus, P. ostreatus in the residues with low HEMC would ultimately attack the lignocellulosic complex, resulting in lignin degradation. Conversely, tomato pomace contains CP and high amounts of HEMC that were probably more accessible to initial attack by P. ostreatus. Thus, our results contradicted findings for significant lignin degradation (Kamra and Zadrazil, 1988; Hadar et al., 1992; Kerem and Hadar, 1995; Zadrazil and Puniya, 1995; Bisaria et al., 1997; Cohen et al., 2001, 2002a, b) and supported the results of Singh et al. (1996) and Zhang et al., (2002), who noted that fungal fermentation did not improve the feeding value of substrates.

The significant reduction of IVTD observed was most likely due to degradation of the CELL and HEMC over the fungal treatment period, consequently providing reduced amounts of nutrients in the samples for digestion by anaerobic microorganisms during testing for IVTD. Contrary to the results reported here, many investigators have noted increased digestibility of byproducts treated with the oyster mushroom (Kamra and Zadrazil, 1988; Hadar et al., 1992; Bisaria et al., 1997). However, as noted above, Singh et al. (1996) reviewed studies in which wheat straw was treated with the oyster mushroom and found the digestibility of the spent residue to be lower than that of the control.

Several scientists have noted that continuously accumulated agricultural residues and agroindustrial byproducts could be converted by the white-rot fungus P. ostreatus into value-added products such as improved animal feed. The results of the work reported here revealed that treating tomato pomace with this fungal species enhanced the ash and CP contents and degraded CELL and HEMC, resulting in lower digestibility. Lignin degradation was not achieved. Adding manganese to the tomato pomace substrate inhibited the growth of P. ostreatus and negatively affected colonization. It is possible that manganese amendment at the level used affected gaseous conditions (O₂ consumption and CO₂ evolution rates), important factors that must be considered when attempting to enhance accelerated lignin degradation by P. ostreatus.

	ACKNOWLEDGMENTS

 
The skillful assistance of R. Michael Davis (Department of Plant Pathology, University of California, Davis) is gratefully acknowledged. The authors wish to thank David LeBauer, research assistant (Department of Plant Pathology, University of California, Davis), for his assistance during oyster substrate preparation and the inoculation process. The advice of Neil Willits and his assistance with statistical analyses is greatly appreciated.

Received for publication September 7, 2007. Accepted for publication March 9, 2008.

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