Proceedings of the 10 th International Conference on Environmental Science and Technology Kos island, Greece, 5 7 September 2007

Transcription

1 Proceedings of the 10 th International Conference on Environmental Science and Technology Kos island, Greece, 5 7 September 2007 ANIMAL FEED PRODUCTION BY SOLID STATE FERMENTATION OF BREWER S SPENT GRAINS AND MALT SPENT ROOTLETS WITH ASPERGILLUS AWAMORI, A. ORYZAE AND PHANEROCHAETE CHRYSOSPORIUM A. BEKATOROU 1, M. KANELLAKI 1, I.M. BANAT 2 and P. NIGAM 2 1 Food Biotechnology Group, Department of Chemistry, University of Patras, 26500, Patras, Greece 2 School of Biomedical Sciences, University of Ulster at Coleraine, BT52 1SA, N. Ireland, UK ampe@chemistry.upatras.gr EXTENDED ABSTRACT Brewer s spent grains (BSG), malt spent rootlets (MSR) and their mixtures (BSG-MSR), with/without addition of molasses, orange and potato pulp, as additional carbon sources, were treated with the filamentous fungi A. awamori, A. oryzae, and the ligninolytic whiterot fungus Phanerochaete chrysosporium, for the production of protein enriched animal feeds. Various types of solid state fermentation (SSF) processes were carried out at 30 o C. The fermented substrates had different initial moisture contents (60-80%) and different initial sugar concentrations. SSFs were carried out under aseptic conditions, in 3 types of bioreactors. All experiments were carried out without addition of extra nutrients. In the first case, SSFs of BSG, MSR and BSG-MSR, with A. awamori or A. oryzae were carried out with & without addition of a 10 o Be solution of molasses (initial sugar concentration ~0.3g per 20g of substrate). SSFs were carried out in conical flasks (a) and in petri dishes (b). Both fungi performed well in all the tested systems, with high biomass yields within 3-4 days. SSFs were also carried out at larger scale, in glass-tower reactors (c). Each fermentation system contained 100g BSG, suitable amount of Aspergillus spore suspension and various amounts of molasses (10 o Be), orange pulp (undiluted), potato pulp (1:1 with water) and finally mixtures of molasses, orange and potato pulps. Experiments were also carried out without addition of the above carbohydrate materials. The average real protein increase obtained in the above SSF systems after treatment with Aspergillus spores was about 35%. Fermentation times were lower in the case of bioreactor types (a) and (b), although the (c) type systems may allow larger scale applications, better moisture control and homogenous fungal growth within the fermented material bulk. In the second case, SSFs of BSG, MSR and BSG-MSR, with the white-rot, fungus P. chrysosporium, were carried out in petri dishes (b) with and without the addition of various amounts of molasses and orange pulp. Various initial moisture contents (40-80%) and initial sugar concentrations were evaluated. Fungal growth was also fast in all cases, with good protein increase levels as in the previous cases. Nevertheless, in all the studied systems, the fungi were unable to grow efficiently in the absence of extra fermentable sugars, as excepted. Fungal growth was higher in BSG compared to MSR, since MSR contain lower amounts of fermentable sugars or starch. All samples were readily accepted by chicken. Keywords: Brewer s spent grains, Malt spent rootlets, Aspergillus oryzae, A. awamori, Phanerochaete chrysosporium, Amylolytic, Ligninolytic, Protein enrichment, Animal feed B-60

2 1. INTRODUCTION Agro-industrial wastes containing lignocellulose, starch and fermentable carbohydrates, such as cereals and their spent grains, citrus and potato residues, molasses, etc., have been used as animal feeds with or without treatment with microorganisms for protein enrichment [1-3]. The main solid by-product of the brewing industry, brewer s spent grains (BSG), are produced after the mashing stage in beer production and consist of about (% w/w on dry matter) cellulose, lignin, apparent starch (glucose, maltodextrins and residual starch), crude protein, crude fibre, 6-10 digestible fibre, 6-10 lipids and 3-5 total ash. Their composition depends on the barley variety as well as the malting and mashing programme. Due to their high fibre and protein content, including all essential amino acids, BSG are widely used as animal feed. They have also been found good substrates for the cultivation of mushrooms leading to increase of their nutritional value [2-7]. During the past 2 years, efforts funded by ESF, are being made to upgrade BSG, including the production of biocatalysts based on immobilized cells for alcoholic beverage production, and hydrolysis of their residual starch using fungi, in order to evaluate the potential use of the treated material as carbohydrate substrate for brewing yeast production, or alternatively, as protein enriched animal feed. The production of beer using cells immobilized on BSG is a very attractive perspective, because BSG are a natural, abundant and easily available natural cellulosic material, fully compatible with beer. Cells immobilized on BSG have been applied successfully in various brewing processes [8]. BSG and malt spent rootlets (MSR) are rich in fibre, starch and fermentable carbohydrates, and can therefore be treated by amylolytic (such as Aspergillus sp.) or ligninolytic microorganisms (such as white-rot fungi), or their combinations, to produce protein enriched animal feeds, leading at the same time to creation of added value and reduction of the pollution caused by their disposal. BSG residual starch has been evaluated as sole carbon source for the synthesis of enzymes by filamentous fungi such as A. oryzae and A. awamori [2,9]. Aspergillus sp. (A. oryzae, A. niger and A. awamori), are extensively used for industrial enzyme production (mainly a-amylases and glucoamylases) due to their superior ability to secrete enzymes [10,11]. A. oryzae is widely used for commercial a-amylase production, which is typically used in baking, brewing, in dietary supplements, in alcohol production and other food grade applications. It has also been evaluated as probiotic in animal feed production [12,13]. At research level, the depolymerization of starch of various substrates using A. oryzae and A. awamori strains for biomass or enzyme production, waste organic load reduction, animal feed production, etc., in submerged (SmF) and solid state fermentation (SSF) processes, has been reported [11,14,15]. White-rot fungi on the other hand, are used to convert lignocellulosic materials to more digestible feeds for ruminants. Phanerochaete chrysosporium for example is a widely used white-rot fungus, known for its high rates of lignin degradation through various enzyme activities including lignin peroxidases, laccases etc., [15]. Therefore, since lignocellulosic crop residues are low in protein content, they can be enriched through fermentation by white-rot fungi, for potential animal feed sources. This study explores ways to upgrade BSG and MSR, and their combinations with other by-products of the food industry such as molasses, citrus and potato residues, through treatment with A. awamori and A. oryzae or P. chrysosporium for animal feed production. B-61

3 2. MATERIALS AND METHODS 2.1. Microorganisms and media The Aspergillus sp. used in this study were the strains A. oryzae IMI (CABI- International Mycological Institute, Oxfordshire, UK) and A. awamori Nakazawa DSM63272 (DSMZ, Braunschweig, Germany). They were grown on potato dextrose agar (PDA) (20 g/l glucose) at 25 o C, and spores were collected using 0.1% Tween-80 solution under aseptic conditions. The white-rot fungus P. chrysosporium, obtained by the School of Biological Sciences of the University of Ulster, was grown at 30 o C under shaking in medium containing 5 g/l glucose, g CaCl 2 H2O, g/l MgSO 4, 0.5 g/l NH 4 H 2 PO 4, g/l FeSO 4, 0.02 g/l CuSO 4, 0.01 g/l yeast extract, g/l MnSO 4 (ph 6.0 buffered with KH 2 PO 4 / K 2 HPO 4 ). BSG and MSR were obtained from the Athenian Brewery S.A. BSG contained about 82% moisture, 20% dry matter, 2.5 o P remaining extract (washable), 21.6% crude protein, 4.7% ash, 6.8% fat, 5.05 and 13.6 mg/g dry material total phenolics expressed as gallic acid equivalents in organic and in aqueous extract, respectively, total antioxidant capacity equal with 8.51 mg /ml of extract, and had ph 6.0. Molasses, orange and potato pulp, and their mixtures were used, after dilution with water at suitable densities. All media were sterilized at 120 o C for 15 min Solid state fermentations Solid state fermentations were carried out at 30 o C, under aseptic conditions, in three types of bioreactors: (a) in conical flasks with occasional rotation and stirring of their content (slow rotating drum-type solid state fermentation reactors) (Figure 1,a), (b) in petri dishes (shallow tray-type reactors) (Figure 1,b), and (c) in 250 ml glass bioreactors (tower-type reactors) (Figure 1,c), connected with cylindrical glass vessels that contained sterilized water, through which air was supplied to the system in order to retain the moisture of the fermenting material. All experiments were carried out without addition of extra nutrients. Air A C B Figure 1: Solid state fermentation (SSF) reactors used in the present study: (a) conical flasks with occasional rotation and stirring (slow rotating drum-type), (b) petri dishes (shallow tray-type) and (c) glass bioreactors (tower-type) B-62

4 In the first case, solid state fermentations of BSG, MSR and BSG-MSR, with A. awamori or A. oryzae and were carried out at 30 o C, with and without the addition of a suitable amount of a 10 o Be solution of molasses (to obtain an initial sugar concentration of about 0.3 g fermentable sugar per 20 g of substrate). Fermentations were carried out in conical flasks (a) and in petri dishes (b). Fermentations were also carried out at larger scale, in glass-tower reactors (c). Each fermentation system contained 100 g BSG, suitable amount of Aspergillus spore suspension and various amounts of molasses (10 o Be), orange pulp (undiluted), potato pulp (1:1 with water) and finally mixtures of molasses, orange and potato pulps. Experiments were also carried out without addition of the above carbohydrate materials. In the second case, solid state fermentations of BSG, MSR and BSG-MSR, with the white-rot, ligninolytic fungus P. chrysosporium, were carried out at 30 o C, with and without the addition of various amounts of molasses (10 o Be) and water-orange pulp slurries (1:1) (to obtain different initial fermentable sugar concentrations). Fermentations were carried out in petri dishes (b). Various initial moisture contents (60-80%) and initial sugar concentrations were evaluated Assays Samples were collected, homogenized and analyzed for protein and moisture content, or other parameters such as residual fermentable sugar, ash, fat, crude protein, real protein increase, total phenolics and antioxidant capacity. Crude protein (total nitrogen) was determined according to the AOAC Official Method (Kjeldahl method). Experiments were performed multiple times in order to standardize the sampling for protein analysis using the Kjeldahl method since the produced samples contained different amounts of moisture. Moisture was determined by drying initially at 60 o C for 18 h and then at 105 o C for 5 h. The real protein increase (RPI) was calculated using the formulas of Iluyemi et al. Fat was determined by the Soxhlet method using diethyl ether as solvent. Total phenolics were determined after alkaline extraction with NaOH, followed by acidification with HCl and extraction with a mixture of diethyl ether/ethyl acetate (1:1). The organic phase was evaporated and the solid residue was diluted in methanol containing anhydrous Na 2 SO 4, and analyzed by the Folin-Ciocalteu method. The results were expressed as mg of gallic acid per g of dry material. The antioxidant capacity (DPPH scavenging capacity) was determined according to the method of Brand- Williams. Residual sugars were determined by RI-HPLC. For comparison reasons samples containing all the above combinations of materials, without treatment with fungi were also analyzed. Samples were supplied to chicken to rate their acceptability as feed. 2. RESULTS In this study, BSG and MSR, with or without addition of other by-products of the food industry such as molasses, orange and potato pulps, were treated with the filamentous A. awamori and A. oryzae or the ligninolytic white-rot fungus P. chrysosporium for potential protein enriched animal feed production. The work focused mainly on the technological feasibility and proximate analysis of the proposed animal feeds, as well as palatability of the treated materials for chicken. The Aspergillus species performed well in all the tested systems containing BSG, with vigorous growth and high biomass yields within 3-4 days. The real protein increase obtained in the above solid state fermentation systems after treatment with Aspergillus spores was above 30 % w/w (on dry weight basis) (Table 1). In systems containing MSR only, especially those without addition of a fermentable carbohydrate source, lower growth and therefore protein increase (20-25% w/w), was observed since filamentous fungi cannot utilize lignocellulosic materials directly. B-63

5 Generally, fungal growth at 30 o C was affected by the availability of fermentable sugars (including starch) in the substrates and not by the moisture contents in the range 40-80%. No significant changes on total phenolics (expressed as mg of gallic acid per g of dry material) and antioxidant capacity (expressed as DPPH scavenging capacity, mg/ml of extract) were observed after the solid fermentation of the substrates, compared with the non treated materials (Table 1). Table 1 shows the values of moisture, crude protein, protein increase, total phenolics and antioxidant capacity before and after the solid state fermentations of BSG, MSR, BSG/MSR(1:1), with/without addition of molasses, orange or potato pulp (added fermentable sugar ~1,5%w/w and initial moisture ~70-75%), at 30 o C, with A. oryzae, A. awamori or P. chrysosporium. These parameters are given as average values plus standard deviations for all the studied systems. Table 1: Parameters of the solid state fermentations of BSG, MSR, BSG/MSR(1:1), with/without addition of molasses, orange or potato pulp, at 30 o C, with A. oryzae, A. awamori or P. chrysosporium Substrate / Microorganism Assays BSG MSR BSG/MSR BSG MSR BSG/MSR BSG MSR BSG/MSR A. oryzae A. awamori P. chrysosporium SSF time (d) Moisture before SSF (%w/w) Moisture after SSF (%w/w) Protein before SSF (%w/w dry) Protein after SSF (%w/w dry) Protein increase (%w/w dry) Total phenolics aqueous (mg/g) Total phenolics organic (mg/g) Antioxidant capacity (mg/ml) 75.2± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.2 In the same manner, P. chrysosporium growth was also fast in all cases containing BSG and fermentable carbohydrates, with good protein increase levels as in the previous cases (~34%). Nevertheless, the fungus was unable to grow efficiently in the absence of extra fermentable sugars, as excepted, and in substrates containing only MSR, the growth rates were lower, since P. chrysosporium needed to go through ligninolytic activity in order to obtain the necessary fermentable sugars for growth (Table 1). In all the studied systems, fermentation times were lower in the case of bioreactor types a (slow rotating drum-type solid state fermentation reactors) and b (shallow tray-type B-64

6 reactors), compared to the c type systems (tower-type reactors) which may allow larger scale applications, better moisture control and homogenous fungal growth within the fermented material bulk. On the other hand, such bioreactors may have higher construction and operation costs. Finally, all samples were readily accepted by chicken. Further experiments on animals were not conducted, and should be the aim of future research, studying the suitability of the proposed feeds from a nutritional-clinical point of view. Moreover, the possibility of producing protein enriched feeds with probiotic properties should be evaluated, since grains and grain fractions contain high amounts of fibre that can act as excellent probiotic sources to support growth of probiotic bacteria. ACKNOWLEDGEMENT We thank European Social Fund (ESF), Operational Program for Educational and Vocational Training II (EPEAEK II) and particularly the Program PYTHAGORAS, for funding the above work. REFERENCES 1. Bisaria R., Madan M., Vasudevan P. (1997) Utilization of agro-residues as animal feed through bioconversion, Biores. Technol., 59, Mussato S.I., Dragone G. and Roberto I.C. (2006) Brewer s spent grain: generation, characteristics and potential applications, J. Cereal Sci., 43, Santos M., Jimenez J.J., Bartolome B., Gomez-Cordoves C. and del Nozal M.J. (2003) Variability of brewer s spent grain within a brewery, Food Chem., 80, Hough J.S. (1985) The biotechnology of malting and brewing, Cambridge University Press, Cambridge, pp Wang D., Sakoda A., Suzuki M. (2001) Biological efficiency and nutritional value of Pleurotus ostreatus cultivated on spent beer grain, Biores. Technol., 78, Sabbioni A., Superchi P. and Bonomi A. (1995) Assessment of nutritive value of dry brewer's spent grain using the growth response of lambs, Riv. Sci. Alim., 24, Kanauchi O. and Agata K. (1997) Protein, and dietary fiber-rich new foodstuff from brewer s spent grain increased excretion of faeces and jejunum mucosal protein content in rats, Biosci Biotechnol. Biochem., 62, Branyik, T., Vicente, A.A., Dostalek, P., & Teixeira, J.A. (2005) Continuous beer fermentation using immobilized yeast cell bioreactor systems, Biotechnol. Prog., 21, Francis F., Sabu A., Nampoothiri K.M., Szakacs G. and Pandey A. (2002) Synthesis of a- amylase by Aspergillus oryzae in solid-state fermentation, J. Basic Microbiol. 42, Pandey A., Nigam P., Soccol C.R., Soccol V.T., Singh D. and Mohan R. (2000) Advances in microbial amylases, Biotechnol. Appl. Biochem., 31, Jin Q., Van Leeuwen J.H. and Patel B. (1999) Mycelial morphology and fungal protein production from starch processing wastewater in submerged cultures of Aspergillus oryzae, Process Biochem., 34, Barbesgaard P., Heldthansen H.P. and Diderichsen B. (1992) On the safety of Aspergillus oryzae-a review Appl. Microbiol. Biotechnol., 36, Lee K.W., Lee S.K. and Lee B.D. (2006) Aspergillus oryzae as probiotic in poultry-a Review, Int. J. Poultry Sci., 5, Singh H. and Soni S.K. (2001) Production of starch-gel digesting amyloglucosidase by Aspergillus oryzae Hs-3 in solid state fermentation, Process Biochem., 37, Chantasartrasamee K., Na Ayuthaya D.I., Intarareugsorn S. and Dharmsthiti S. (2005) Phytase activity from Aspergillus oryzae AK9 cultivated on solid state soybean meal medium, Process Biochem., 40, Basu S., Gaur R., Gomes J., Sreekrishnan T.R. and Bisaria V.S. (2002) Effect of seed culture on solid- state bioconversion of wheat straw by Phanerochaete chrysosporium for animal feed production J. Biosci. Bioeng., 93, B-65