EFFECT OF DIETARY Agaricus bisporus AND Auricularia auricula CRUDE EXTRACT SUPLEMENTATION ON CARCASS QUALITY OF BROILER

This research was conducted to examine Agaricus bisporus and Auricularia auricula crude extract using different solvents (water, ethanol, and methanol) on infrared spectroscopy absorbance during extraction and the impact on broiler carcass. Agaricus bisporus and Auricularia auricula crude extracts were scanned using fourier transform infrared (FT-IR) spectroscopy. Each mushroom crude extract was chosen and applied into broiler diets as feed additive at 0%, 0.4%, 0.8%, 1.2%, and compared zinc bacitracin inclusion. Variable measured were final live weight, carcass yield, breast meat yield, and abdominal fat yield of broiler. Two hundred and forty day-old chicks were randomly allocated into eight dietary treatments, each treatment was replicated three times with ten chicks for each pen. Diets and water were offered ad libitum . Methanolic extract showed monosaccharide absorption peak in fingerprint region at wavelength 890 cm -1 , 930 cm -1 , 1050 cm -1 , 1150 cm -1 which indicates alpha and beta linkage than the others solvent. Even so, dietary inclusion of methanolic extracts of Agaricus bisporus and Auricularia auricula did not show any effect on final live weight and the yiled of carcass, breast meat and abdominal fat of broiler. In conclusion, methanolic extraction is effective to extract monosaccharides with α-and β-linkages from Agaricus bisporus and Auricularia auricula , while the dietary inclusion of methanolic extracts of both edible mushroom and zinc bacitracin has no effect on carcass quality of broilerin broiler diets did not show differences between treatments as well as zinc bacitracin group.


INTRODUCTION
The use of antibiotic growth promoter (AGP) in poultry and cattle industry in Indonesia has been banned since early 2018. Common AGP used in poultry industry was zinc bacitracin which is used to optimize broiler growth. Zinc bacitracin usage in long term in poultry diets may induce the deposition of the antibiotic residue to poultry products such as meat and egg. Antibiotics resdidue in poultry products may impact on human immune system against antimicrobial-resistance bacteria. There has been much research evaluating some alternative ways to replace AGP in poultry production. In this study, glucans from the mushroom play a role as prebiotic.
Agaricus bisporus and Auricularia auricula are two kinds of edible mushroom which contain many therapeutic polysaccharides. Most polysaccharide in Agaricus bisporus and Auricularia auricula are in the form of glucan. β-glucan from Agaricus bisporus and Auricularia auricula activate macrophages cell and may improve host immunity (Minato and Abe, 2013). Conventional extraction is usually performed in long time period of time (more than 2 h) at reflux temperature 90 °C. While microwave assisted extraction may increase yield extract at short time and reduce solvent usage (Proestos and Komaitis, 2008). Water usage as solvent will absorb more microwave energy, therefore solvent polarity is very important to be considered. Common polar solvents showed better result in polysaccharides extraction by using microwave assisted extraction than nonpolar solvents (Wang and Weller, 2006).
Agaricus bisporus and Auricularia auricula are two kinds of mushroom which cultivate in subtropical climate such as Indonesia. Liu et. al. (2013) reported that ethanolic extract of Agaricus bisporus showed better antoxidant activity against various radical scavengers, and Agaricus bisporus ethanolic extract could be natural antioxidant and therapeutic agents. Infrared spectra of Auricularia auricula extracted using microwave assisted extraction showed stretching vibration of -OH and C-H at about 3600-3200 cm -1 and 300-2800 cm -1 , then the absorption at 903 cm -1 indicates βglycosidic linkages as β-glucan and the peak at 1145, 1075, and 1042 cm -1 showed sugar form as pyranose. Microwave assisted extraction could influence the molecular weight of the polysaccharide and the molecular weight changes could affect the bioactivity of the polysaccharide (Zeng et. al., 2012;Feng et. al., 2010). The combination between β-glucan and antibiotic addition into weanling pigs diets could increase dry matter, gross energy, crude protein, and crude fat digestibility (Hahn et. al., 2006).
Agaricus bisporus is known to play a role as immunomodulatory agent and growth performance of broiler and turkey (Guimarães et al., 2014;Giannenas et al., 2011). Agaricus bisporus additional in laying hen diets showed 2% residual stem from white button mushroom improve laying hens performance and increase nutrient utilization (Yang et. al., 2020). Another study based on Shamsi et. al. (2015) reported that the inclusion of 0.2% powdered Agaricus bisporus into broiler diets significantly reduced daily feed intake and feed conversion ratio. Auricularia auricula crude extract structure changes during extraction process using microwave assisted extraction (MAE) with different solvents by infrared spectroscopy and the impact of the inclusion of the mushroom extracts in diets on the carcass quality of broiler.

Mushroom preparation and extraction
Agaricus bisporus and Auricularia auricula were purchased from local mushroom farmer in Singosari sub district, Malang District, East Java, Indonesia. The fresh mushroom were sliced to reduce the size, then soaked using three different solvents (water, ethanol 96%, and methanol 70%) for 24 h. Extraction was performed using a modified microwave (50 Hz, 399 watt) (Purwanto et. al., 2010) with additional equipment vacum pump. MAE of the edible mushroom was done at 50°C for 15 min. The crude extract was then filtered using cheese cloth and its extraction schematics is shown in Fig. 1. The filtrate was evaporated using MAE for second time for 15 min to get crude extract from A. bisporus and A. auricula. Crude mushroom extract was stored into freezer at -20°C for in-vivo trial. Both mushroom crude extracts were centrifuged under 3500 rpm at about 15 min, then separate between supernatant and solid fraction. Solid fraction after centrifuged were dried using microwave under 50°C for 10 min. Then 3 mg of powdered Agaricus bisporus and Auricularia auricula crude extract mix with 97 mg of KBr (Merck KGaA, 54271 Darmstadt, Germany) then pressed by using KBr press holder. Finally the spectra was scanned in wavelength range 500-4000 cm -1 with FT-IR spectrofotometer. FT-IR analysis was performed at central laboratory of State University of Malang (Experiment 1).

Animals and experimental design
Two hundred and forty day-old chicks (unsex) were randomly allocated into 8 experimental groups with average body weight of 37,6±2,90 g/chick. Three replication were done for each treatment with ten chicks in each pen. The dimensions of the pen were 1 x 1,2 x 0,8 m 3 and equipped by hanging feeder and drinker. The feed and water given ad libitum. During ten days, all chicks got brooding phase with controlled room temperature at 26-35°C and humidity 65%-75%. The experiment was done in 35 d. Basal diet BR1 was given at the first 21 d (starter period), and BR2 was given later until 35 d (finisher period), basal diets purcashed from PT. Japfa Comfeed, Indonesia. Chemical composition of the basal diet is shown in Table 1.

Broiler carcass measurements
The spectra of Agaricus bisporus and Auricularia auricula crude extract using FT-IR spectroscopy to investigate the absorption banding from polysaccharides of both edible mushroom crude extracts. Meanwhile, in experiment 2 on day 35, the broilers were weighed and sampling was performed by marking a broiler with average weight for every pen. The samples were then slaughtered. The yield of the carcass, breast meat and abdominal fat was determined according to the following formula: Breast meat (%) = Filleted breast meat (g) Carcass weight (g) × 100 abdominal fat (%) = Abdominal fat (g) Live weight (g) × 100

Statistical analysis
The spectrum data from FT-IR spectroscopy analysis (experiment 1) were analyzed descriptively. Meanwhile, the data from experiment 2 were analyzed using a one-way ANOVA with a completely randomized design (CRD). The orthogonal contrast tests were performed later for all variables using Minitab ® 18.1 (Minitab Inc, USA).

RESULTS AND DISCUSSION
Infrared spectrum of Agaricus bisporus crude extract are shown in Fig. 2 and Table 2. Agaricus bisporus crude extract showed peak at 930 cm -1 stretching vibration which indicates the presence of β-1,4-glucan. Then peak at 850 cm -1 in KAA, KAE, and KAM which is indicates the existence of alpha bound linkages in fingerprint region. KAE showed strongest alpha banding of monosaccharides that is alpha peak absorption at 893,0 cm -1 while KAA and KAM alpha peak absorption at 848,7 and 852,5 cm -1 . It indicates that solvent usage may dilute α-glucan from Agaricus bisporus. El-Batal et. al. (2008) stated that absorption on fingerprint region at 890 cm -1 indicates 1,3-β-glucan banding, while absorption at 930 cm -1 indicates β(1,4) polysaccharide.
Based on analysis of variance (ANOVA) there are no significant differences between treatments on carcass quality of broiler. The non significant differences caused of Agaricus bisporus and Auricularia auricula crude extract addition of 1.2% in broiler diets did not changes the nutrient utilization. In previous study Kavyani et. al. (2012) reported that 1% dry Agaricus bisporus inclusion in broiler diets significant increase broiler body weight at 21 days of rearing compared with antibiotics group (flavophospholipol). Gut health could improve broiler performance, which balancing intestinal microflora could improve efficiency of utilization and digestibility of feed. Based on IR spectroscopy analysis, both mushroom crude extract contained β-glucan which play antioxidant and therapeutic agents in poultry diets. β-glucan contained in Agaricus bisporus and Auricularia auricula may improve broiler live weight. Dietary βglucan could reduce digesta viscosity in small intestine, so nutrient absorption will increase with reduce organism fermentation time by endogenous enzyme (Huyghebaert et. al., 2011). Major polysaccharides in Agaricus bisporus and Auricularia auricula are β-D-glucan, which induces immune response to stimulate macrophage proliferation.
Carcass yield in Table 2 showed that Agaricus bisporus and Auricularia auricula crude extract addition in average 70.6 -73.5%, which is broiler carcass yield at about 70 -75%. In study of 3% yeast cell wall inclusion into broiler diets did not show significant differences compared with oxytetracycline group (Ahmed et al., 2015). Kavyani et al. (2012) also reported that 3% Agaricus bisporus inclusion in broiler diet showed 73.3% carcass yield with no significant differences between treatments. Another study showed 6% inclusion of white button mushroom simultaneous increase broiler carcass yield (Mazaheri et al., 2014). Breast meat yield in average of 29.1 -31.0%, there were no significant differences among the treatment, which indicates Agaricus bisporus and Auricularia auricula crude extract did not affect on broiler breast meat conformation. Meanwhile the lowest abdominal fat percentage showed in Agaricus bisporus crude extract 1.2% inclusion with 1.19% of body weight. In addition of edible mushroom crude extract contained β-gulcan could stimulate macrophages and dendritic cell which needed more energy transferred into peyer's patches to improve host imunity by increasing CD4 and TCR 1 cells (Yalcin et. al., 2014;Chae et. al., 2006), then balancing energy storages in whole body as fat. Carcass dressing in this study ranged from 70-73%, whereas similar study by Ahmed et. al. (2015) reported carcass dressing percentage in the range of 70-73% without showed significant differences between treatments using Saccharomyces cerevisiae inclusion. Saccharomyces cerevisiae cell wall is rich in β-1,3-glucan which is important for modulating immune response and nutrient utilization in the small intestine (Nie et. al., 2018).

CONCLUSION
In conclusion, absorption characteristics of the glycosidic bands in αor βtype of monosaccharides of A. bisporus and A. auricula crude methanolic extracts were identified using FT-IR spectroscopy. Meanwhile, both edible mushroom crude extracts did not change the carcass quality of broiler.