Effect on growth performance and digestibility in broiler chickens

The improvement of growth performance by feeding A. awamori appears to result from an increase in the feed efficiency of broiler chickens and metabolisable energy (ME) from the diet. The reason for this increase in ME could be due to the digestion of either raw starches or soluble non-starch polysaccharides. A. awamori possesses the ability to digest raw starches (Amsal et al., Reference AMSAL, TAKIGAMI and ITO1999) and to produce cellulase and xylanase, which are required for the digestion of soluble non-starch polysaccharides (Bhat and Hazlewood, Reference BHAT and HAZLEWOOD2001). In addition, Aspergillus could improve the nutritional quality of soybean meal because the trypsin inhibitor contained in unprocessed soybean is degraded by Aspergillus spp. (Hong et al., Reference HONG, LEE and KIM2004). Indeed, supplementation with exogenous enzymes (cellulase, hemicellulose and pectinase) has improved the growth performance of broiler chickens (Saleh et al., Reference SALEH, TAHIR, OHTSUKA and HAYASHI2005). It has been demonstrated that carcass weight increased in broiler chickens fed a diet containing 0.05% A. awamori due to the increased ME of the diet (Yamamoto et al., Reference YAMAMOTO, SALEH, TAHIR, OHTSUKA and HAYASHI2007). Similarly, Saleh et al. (Reference SALEH, EID, EBEID, KAMIZONO, OHTSUKA and HAYASHI2011) found that body weight gain and breast muscle weight increased, while feed intake and abdominal fat weight decreased when feeding broilers on a diet containing 0.05% A. awamori. Furthermore, feeding the Shōchū distillery by-product (SDBP) improved the skeletal muscle growth of broiler chickens. SDBP contains some growth promoters for the muscles of broiler chickens, i.e. butoxybutyl alcohol and dibutoxybutane (Kamizono et al., Reference KAMIZONO, OHTSUKA and HAYASHI2013). Since A. awamori is essential for producing Shōchū, it might also have the ability to produce these growth promoters in the enteric canal of broiler chickens fed a diet containing A. awamori.

Effect on protein metabolism in broiler chickens

The ubiquitin-proteasome system (UPS) is thought to play a major role in the degradation of most sarcoplasmic proteins and calpain might play a significant part in initiating the degradation of cellular structural protein by releasing protein fragments for proteolysis in the UPS. Also, atrogin-1 has been identified as a muscle-specific E3 that was highly expressed in muscle atrophy (Bodine et al., Reference BODINE, LATRES, BAUMHUETER, LAI, NUNEZ, CLARKE, POUEYMIROU, PANARO, NA, DHARMARAJAN, PAN, VALENZUELA, DECHIARA, STITT, YANCOPOULOS and GLASS2001; Gomes et al., Reference GOMES, LECKER, JAGOE, NAVON and GOLDBERG2001; Ohtsuka et al., Reference OHTSUKA, KAWATOMI, NAKASHIMA, ARAKI and HAYASHI2011). Calpains are a family of intracellular Ca²⁺-dependent cysteine proteases that are ubiquitously expressed in many cells and tissues (Suzuki et al., Reference SUZUKI, SORIMACHI, YOSHIZAWA, KINBARA and ISHIURA1995). Two major forms of calpain (m-calpain and μ-calpain) are activated in the presence of Ca²⁺ at micromolar and millimolar levels, respectively (Goll et al., Reference GOLL, THOMPSON, LI, WEI and CONG2003). These are heterodimers composed of a large catalytic subunit (80 kDa) and a small regulatory subunit (30 kDa). Smith and Dodd (Reference SMITH and DODD2007) have reported that calpain acts upstream of the ubiquitin-proteasome system and m-calpain mRNA is scarcely expressed in the muscle and liver in chicken. Saleh et al. (Reference SALEH, EID, OHTSUKA and HAYASHI2012a) found that the mRNAs of ubiquitin, proteasome and m-calpain significantly decreased when feeding A. awamori and those of atrogin-1 and m-calpain tended to decrease (Saleh et al., Reference SALEH, OHTSUKA, YAMAMOTO and HAYASHI2013a) These results are consistent with those of Kamizono et al. (Reference KAMIZONO, OHTSUKA and HAYASHI2010), who found that SDBP reduced the gene expression of the enzymes responsible for skeletal muscle protein breakdown. However, mRNA expressions of beta-actin, myosin and pax-7, which are related to the growth and development of muscle fibre (Somi et al., Reference SOMI, ANDRE and KLEIN2006; Chang et al., Reference CHANG, LIU, LIAO, CHEN, LUAN, ZHANG, DAI, MA, ZHOU, WANG and CHEN2011), were all increased but only slightly by feeding A. awamori as reported by Saleh et al.(2012a). Therefore, these results suggest the idea that the promotion of muscle growth by feeding A. awamori is due to the suppression of skeletal muscle protein degradation rather than the enhancement of muscle protein synthesis.

Cholesterol-lowering effect in broiler chickens

Several studies have concluded that diets containing probiotic bacteria with or without fungi have a negative effect on plasma total cholesterol, low-density lipoprotein cholesterol (LDL-C) and triglycerides but a positive effect on plasma high-density lipoprotein cholesterol (HDL-C) in chickens (Mohan et al., Reference MOHAN, KADIRVEL, BHASKARAN and NATARAJAN1995; Reference MOHAN, KADIRVEL, NATARAJAN and BHASKARAN1996; Jin et al., Reference JIN, HO and ABDULLAH1997; Kim et al., Reference KIM, PARK and YU2003). Mohan et al. (Reference MOHAN, KADIRVEL, BHASKARAN and NATARAJAN1995) reported that the supplementation of feed with probiotic bacteria resulted in lowering the serum total cholesterol level in white leghorn layers from 176.5 to 114.3 mg/dl. Further work by Mohan et al. (Reference MOHAN, KADIRVEL, NATARAJAN and BHASKARAN1996) reported that chickens receiving diets containing 75, 100 and 125 mg probiotic/kg had a lower serum total cholesterol concentration (93.3 mg/dl) compared with the control birds (132.2 mg/dl). Jin et al. (Reference JIN, HO and ABDULLAH1997) found that dietary supplementation (0.05 or 0.1%) with A. oryzae decreased the plasma total cholesterol in broiler chickens. Similar lipid-lowering effects were obtained with chickens fed a diet containing A. oryzae (Kim et al., Reference KIM, PARK and YU2003; Madhavi et al., Reference MADHAVI, SINGHAL and KULKARNI2005; Panda et al., Reference PANDA, RAMA RAO, RAJU and SHARMA2006; Safalaoh, Reference SAFALAOH2006; Sallh and Al Hussary, Reference SALLH and AL HUSSARY2009) and A. niger (Yoon et al., Reference YOON, NA, PARK, HAN, NAM and KWON2004; Al-Kassie et al., Reference AL-KASSIE, AL-JUMAA and JAMEEL2008). On the other hand, others have reported that feeding probiotics (with or without fungi) did not affect plasma total cholesterol levels in birds. Sahin et al. (Reference SAHIN, KAYA, UNAL and ELMALI2008) reported that plasma total cholesterol was not affected when growing quails were fed diets supplemented with 0.5, 1 and 1.5 g/kg probiotic fungi. Piray and Kermanshahi (Reference PIRAY and KERMANSHAHI2008) found no significant differences in plasma total cholesterol, but increased HDL content when broilers were fed diets with 0.15% A. oryzae.

Hajjaj et al. (Reference HAJJAJ, DUBOC, FAY, ZBINDEN, MACÉ and NIEDERBERGER2005) and Saleh et al. (Reference SALEH, EID, EBEID, KAMIZONO, OHTSUKA and HAYASHI2011) reported that the mechanism underlying the cholesterol-lowering effect of Aspergillus could be due to the inhibition of 3-hydroxyl-3-methylglutaryl-coenzyme (HMG-CoA) reductase. Statin is a well-known HMG-CoA reductase inhibitor extracted from a fungus, Penicillium citrinum (Endo, Reference ENDO1985). It has been recognised as safe and has been widely used to treat patients with hypercholesterolemia (Serruys et al., Reference SERRUYS, DE FEYTER and MACAYA2002). A HMG-CoA reductase inhibitor produced by A. awamori might be responsible for the decrease in carcass fat deposition. In addition, Aspergillus might affect fat deposition by influencing the activities of hormone-sensitive lipase and malate dehydrogenase enzyme in adipose tissues (Mersmann, Reference MERSMANN1998; Shen et al., Reference SHEN, WANG and ZHAO1991).

Effect on muscle lipid metabolism in broiler chickens

The total fat content in the breast muscle (Musculus pectoralis superficialis) increased in chickens fed a diet containing A. awamori (Saleh et al., Reference SALEH, EID, EBEID, OHTSUKA and HAYASHI2012b). Acetyl-CoA carboxylase (ACC) and fatty acid synthesis (FAS) are considered to play a major role in fatty acid synthesis and carnitine palmitoyltransferase 1A (CPT1A) is essential for fatty acid oxidation. Gene expressions of FAS and ACC increased concomitantly with the increase in the total fat content in the breast muscle of broiler chickens fed diets containing A. awamori (Saleh et al., Reference SALEH, EID, EBEID, OHTSUKA and HAYASHI2012b; Reference SALEH, HAYASHI and OHTSUKA2013b). Although fatty acid synthesis in chicken mainly occurs in the liver, these data suggest that FAS and ACC increased at least transcriptional level in skeletal muscle tissues, suggesting that fatty acid synthesis might increase in the skeletal muscles.

Polyunsaturated fatty acids (PUFA) play an important role in humans as they help to reduce the incidence of such life-style diseases such as coronary artery diseases, hypertension and diabetes, as well as certain inflammatory diseases such as arthritis and dermatitis (Simopoulos, Reference SIMOPOULOS2000). These diseases are an increasing problem all over the world due to the dominance of animal fats and partially-hydrogenated vegetable oils in the human diet. Betti et al. (Reference BETTI, PEREZ, ZUIDHOF and RENEMA2009) reported that consumers are becoming more aware of the effect of foods enriched with PUFA. Because of its high α-linolenic acid (LNA) content, dietary fermentation using A. awamori is a good method for increasing PUFA in poultry meat (Betti et al., Reference BETTI, PEREZ, ZUIDHOF and RENEMA2009). Several studies have shown that oleic and linoleic acids are the most common unsaturated fatty acids produced by Aspergillus probiotic and that linoleic acid is the major constituent of fungal lipid (Mazur et al., Reference MAZUR, NAKANISHI and EL-ZAYAT1991; Calvo et al., Reference CALVO, GARDNER and KELLER2001; Richard et al., Reference RICHARD, WILSON and CALVO2004; Tsitigiannis et al., Reference TSITSIGIANNIS, ZARNOWSKI and KELLER2004). Thus, the increase in oleic, linoleic and linolenic acids in the muscle is probably due to the intestinal activities of A. awamori and A. niger. Srianta et al. (Reference SRIANTA, NUGERAHANI and KUSUMAWATI2010) have reported that Aspergillus terreus (A. terreus) produces linolenic acid. Furthermore, Aspergillus has an ability to produce desaturase, which changes saturated fatty acids to unsaturated fatty acids (Richard et al., Reference RICHARD, WILSON and CALVO2004). Furthermore, delta 6-fatty acid desaturase (D6DES) plays an important role in the synthesis of PUFAs from microorganisms in higher animals. Interestingly, A. awamori also increased mRNA expression of D6DES in the breast muscle of broiler chickens (Saleh et al., Reference SALEH, EID, EBEID, OHTSUKA and HAYASHI2012b).

Potential role of A. awamori as an antioxidant

Lipid oxidation is one of the most important mechanisms causing quality loss and directly affects the consumers’ acceptability of meat and meat products (Morrissey et al., Reference MORRISSEY, BRANDON, BUCKLEY, SHEEHY and FRIGG1997). Lipid peroxidation is a process of vital importance to the food processing industry (Khan, Reference KHAN2011; Mohamed et al., Reference MOHAMED, HUSEEN and JAWAD2011) as it adversely affects the flavour, taste, nutritional value and the sensory quality of food (Botsoglou et al., Reference BOTSOGLOU, YANNAKOPOULOS, FLETOURIS, TSERVENI-GOUSSI and FORTOMARIS1997). Vitamin E (VE, α-tocopherol), is one of nature's most effective lipid-soluble antioxidants as it protects cell membranes from oxidative damage (Brigelius-Flohé and Traber, Reference BRIGELIUS-FLOHÉ and TRABER1999). Many of the benefits related to high VE content have been credited to the improved antioxidant status of animals, but the exact mechanisms involved are far from clear. Many studies have reported the antioxidant effect of Aspergillus probiotics: according to Saleh et al. (Reference SALEH, EID, EBEID, KAMIZONO, OHTSUKA and HAYASHI2011), the addition of A. awamori (0.1%) or A. niger (0.05%) to the diet decreased the thiobarbituric acid reactive substances (TBARS) value while α-tocopherol content increased in the broiler breast muscle. Furthermore, Saleh et al. (Reference SALEH, EID, EBEID, OHTSUKA and HAYASHI2012b) reported that muscle TBARS decreased and muscle α-tocopherol content increased by feeding A. awamori (0.05%), indicating that the fungus has antioxidative properties. Maraschiello et al. (Reference MARASCHIELLO and GARCÍA REGUEIRO1999) found a negative correlation between muscle TBARS and α -tocopherol content in chickens. These results indicate that A. awamori produces antioxidative substances. Indeed, an increase in TBARS levels in the breast muscle of broiler chickens induced by heat stress was negated by feeding a diet containing A. awamori (El-Deep et al., Reference EL-DEEP, IJIRI, EID, YAMANAKA and OHTSUKA2014). In addition, feeding diets containing A. awamori increased the mRNA expressions of antioxidant enzymes (i.e., glutathione peroxidase, catalase and superoxide dismutase). These results suggest that A. awamori can act as an antioxidant when fed to broiler chickens.

A. awamori as a supplement in broiler nutrition

Some feeding experiments with broilers have found that there are synergistic effects when using A. awamori with another source of probiotics. Saleh et al. (Reference SALEH, HAYASHI and OHTSUKA2013b) reported that feeding using a combination of A. awamori and S. cerevisiae synergistically promoted broiler growth performance. The improvement in weight gain and feed efficiency caused by this combination may be partially caused by an increase in the metabolisable energy of the feed (Saleh et al., Reference SALEH, EID, OHTSUKA and HAYASHI2012a). Alternatively, it may be related to the more balanced microbial population in the gastrointestinal tract of the broilers resulting from S. cerevisiae, which has an important role in health and performance (Thongsong et al., Reference THONGSONG, KALANDAKANOND and CHAVANANIKUL2008). However, the combination of A. awamori and S. cerevisiae changed the plasma lipid profile by decreasing total cholesterol, triglyceride and LDL-C and increasing HDL-C (Saleh et al., Reference SALEH, HAYASHI and OHTSUKA2013b). Saleh et al. (Reference SALEH, HAYASHI and OHTSUKA2013b) also found that muscle TBARS values decreased while the mRNA expression of glutathione peroxidase increased through feeding A. awamori and S. cerevisiae. These data supported the potential of A. awamori as an antioxidant. Antioxidative substances have a special role in maintaining animal health as well as productive and reproductive performance. This is largely because of their effects in eliminating free radicals and the toxic products from the various metabolic processes which reduce the physiological response to stress in animals, in increasing the protection of tissues against lipid peroxidation and in improving meat quality during storage (Okada, Reference OKADA1996; Ohtsuka et al., Reference OHTSUKA, OHTANI and HAYASHI1998). The mRNAs of ubiquitin, proteasome and m-calpain were significantly reduced by the combination of A. awamori and S. cerevisiae (Saleh et al., Reference SALEH, HAYASHI and OHTSUKA2013b). These results support the idea that the promotion of muscle growth by probiotics is caused by the suppression of skeletal muscle protein degradation. Saleh et al. (Reference SALEH, HAYASHI and OHTSUKA2013b) reported that the mRNA of delta 6-fatty acid desaturase (D6DES) in the muscle was increased by A. awamori and S. cerevisiae, which might relate or contribute towards the increases in n-6 fatty acids and D6DES plays a key role in the synthesis of PUFAs (Garcia et al., Reference GARCIA, GARRIDO and RODRÍGUEZ2002).