Biological treatment for enhancement of the antioxidant capacity in agro-biomass as animal feed

Rice straw was fermented by two strains of Aspergillus terreus (ATCC 74135 and ATCC 20542) to enhance the antioxidant content of this agro-biomass as ruminant feed. After 8 days of fermentation, antioxidant capacity of samples was determined using Ferric reducing antioxidant power (FRAP), 2,2′azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods. Results of all three methods showed that treatment by both strains of A. terreus significantly (P<0.01) improved the antioxidant capacity of rice straw with higher enhancement for A. terreus ATCC 74135 than A. terreus ATCC 20542. In vitro gas production method was used to examine the stability of antioxidant components of the fermented samples under rumen microbial influence. Our results showed that rumen liquid sample containing fermented rice straw have higher antioxidant capacity than the control (untreated rice straw) after 48 h incubation, indicating that antioxidant capacity of the fermented rice straw remains stable during ruminal fermentation. These results show that biological treatment using A. terreus can be a practical method for production of high quality agro-biomass as ruminant feed with natural antioxidant capacity.


INTRODUCTION
Fat content and its quality are among the most important factors determining the quality of meat in the market.Free radical oxidation of fatty acid content in animal fat is the primary factor that causes the deterioration of the quality of stored meat and meat products (Gokalp et al., 1983;Karami, 2010).Antioxidants are compounds or substances that are able to inhibit or interfere with the free-radical oxidation reaction fundamental to glyceride oxidation (Sherwin, 1990).Vitamin E is the major lipidsoluble antioxidant in animal tissues which delay oxidative deterioration of meat.Asghar et al. (1991) reported that vitamin E not only improves the oxidative stability of pork lipids, it also maintains the redness of the muscle and decreases drip loss.Addition of vitamin E to feed increases α-tocopherol concentration in sarcosomes and thus, significantly increased the stability of the lipids against oxidation and improved the quality of farm animal products (Armstrong, 2002;Pokorny et al., 2001) Lipid oxidation can also be reduced by supplementing antioxidants to the diet of the animals (Coronado et al., 2002;Karimi et al., 2010).Natural antioxidants are found in almost all plants, microorganisms, fungi and even in animal tissues (Akarpat et al., 2008).Although some synthetic antioxidants are available, toxicity and carcinogenicity of these components (Juntachote et al., 2006) and their low water solubility (Bekhit et al., 2003) reduce the usage of these antioxidants and thus natural antioxidants are preferred.Furthermore, consumers generally perceive natural antioxidants which are better than their synthetic counterparts.
Studies have shown that polysaccharides of fungal mycelia have high antioxidant capacity and different polysaccharides and polysaccharide-proteins have been isolated from fungi with high antioxidant capacity (Li and Tsim, 2003;Leung et al., 2009).In addition, biological treatment is a well-known method for enhancement of the quality of agro-biomass as animal feed.However, there is limited published research concerning the use of A. terreus to enhance antioxidant content of agro-biomass, including rice straw (RS).The present study focuses on the effect of biological treatment on enhancement of antioxidant capacity of RS using two strains of A. terreus (ATCC 74135 and ATCC 20542) and the stability of antioxidant capacity of the fermented rice straw (FRS) to rumen microbial degradation.

Chemicals
Unless otherwise stated, all chemicals used in this study were obtained from Nacalai Tesque, Kyoto, Japan.

Fungi, Substrate and Fermentation
Rice straw (RS) collected locally were dried at 60°C for 24 h and ground to uniform size (mesh size 6) and the two strains of A. terrus (ATCC 20542 and ATCC 74135) were obtained from American Type Culture Collection (ATCC).Spore suspensions of the fungal strains were prepared by washing 7 day old culture slants with sterilized 0.1% Tween-80 solution.Spores were counted whit a hemocytometer (Biirker-Tiirk;Schreck, Hofheim, Germany), adjusted to approximately 10 7 spores/ml, and were used as inocula in the study.Solid state fermentation of RS was carried out in 500 ml Erlenmeyer flasks.Twenty grams of each substrate was placed in individual flasks and moisture content was adjusted to 50% by addition of 20 ml water.The flasks were plugged with cotton and autoclaved at 121°C for 15 min.Each flask was inoculated with 10% (V/W) inoculums.The flasks were incubated at 25°C for 8 days in triplicate for each treatment.At the end of fermentation, flasks were removed from the incubator and the fermented rice straw samples were dried and stored at -20°C for antioxidant capacity study and in vitro gas production study.

In vitro gas production
In vitro gas production method as described by Menke and Steingass (1988) was used for studying the effect of microbial fermentation of feed on antioxidant capacity of rumen liquid and stability of antioxidant capacity of feed during the microbial fermentation in the rumen.Five hundred milligrams of each treated sample (RS and FRS) were transferred into a 100 ml syringes.Buffered and mineral solution (Menke and Steingass, 1988) was prepared and placed in a 39°C water-bath under continuous flushing of CO2.
Rumen fluid was collected before the morning feeding from two ruminally fistulated male local Kedah-Kelantan cattle that were fed with an equal weight rations (approximately 4 kg dry matter) containing 40% concentrates and 60% grass hay, twice daily at 08:00 and 18:00.Rumen fluid, collected using a manually operated vacuum pump, was transferred into a pre-warmed bottle and immediately transported back to the laboratory, filtered through eight layers of cheesecloth and flushed with CO2.Rumen fluid was added to the buffered mineral solution with constant stirring, while maintaining the temperature in a water bath at 39°C.About 30 ml of buffered rumen fluid was dispensed into individual syringes, each containing the 500 mg of treated sample and the syringes were incubated at 39°C for 48 h.Incubation was performed in triplicate within each of the two runs.At the end of the incubation, liquid layer of each syringe was collected for subsequent antioxidant capacity analysis.

Sample Preparation for Determination of Antioxidant Capacity
For determination of antioxidant capacity of the RS and FRS, 10 ml of methanol were added to 1 g of the dried sample in 50 ml tube.The mixture was shaken continuously on an orbital shaker for 1 h.The extract was then centrifuged at 2500 ×g for 10 min using ultracentrifuge (Beckman Instruments Inc., Palo Alto, CA) and stored at -20°C for further analysis.The liquid component from the in vitro gas production study (previous section) was centrifuged at 2500 ×g for 10 min and the supernatant was used for determination of antioxidant capacity in rumen liquor.

DPPH Radical Scavenging Activity
Antioxidant capacity of the samples was measured on the basis of scavenging activities of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (Tagashira and Ohtake, 1998).In a test tube containing 150 µl of samples, 5 ml of DPPH methanol solution (100 mg/l) was added.After 30 min of incubation in the dark at room temperature, the absorbance was measured against a blank (methanol) at 517 nm using spectrophotometer (Barnstead Turner SM110255, USA).Inhibition of DPPH radical was calculated as a percentage (%) using the formula: Where, Acontrol is the absorbance of the control reaction (containing all reagents except test compound), and A sample is the absorbance of the test compound.α-tocopherol was used as standard (Figure 1).

Ferric reducing antioxidant power (FRAP) assay
In the FRAP method, the yellow Fe 3+ -TPTZ complex is reduced to the blue Fe 2+ -TPTZ complex by electron-donating substances under acidic conditions (pH less than 7).Any electron donating substance with a half reaction of lower redox potential than Fe 3+ /Fe 2+ -TPTZ will drive the reaction, resulting in the formation of the blue complex.To prepare the FRAP reagent, a mixture of 300 mmol/l acetate buffer pH 3.6 (6.4 ml 2 M sodium acetate solution and 93.6 ml 2 M acetic acid solution (1 l)), 10 mmol/l TPTZ (in 40 mmol/l HCl) and 20 mmol/l ferric chloride (10:1:1, v:v:v) was made.One hundred and fifty microliters of sample was mixed with 4.5 ml of FRAP reagent.After 5 min, the absorbance was read at 593 nm using a spectrophotometer (Barnstead, USA).The FRAP reagent was used as blank and the final absorbance of each sample was compared with those obtained from the standard curve made from 0 to 1000 µmol/l ferric sulphate (FeSO4-7H2O) (Figure 2).The results  were expressed in µmol Fe2+/l sample (Szollosi and Szollosi Varga, 2002).

ABTS Assay
For ABTS assay, the procedure described by Arnao et al. (2001) was used with some modifications.The stock solutions contained 7.4 mM ABTS •+ solution and 2.6 mM potassium persulfate solution.The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in the dark.The solution was then diluted by mixing ABTS •+ solution with methanol to obtain an absorbance of 1.1±0.02units at 734 nm using the spectrophotometer.Fresh ABTS •+ solution was prepared for each assay.Each sample (150 µl) was allowed to react with 2850 µl of the ABTS •+ solution for 20 min in the dark condition before the absorbance was read at 734 nm.The capacity to scavenge the ABTS •+ radical was calculated using the following equation: Where, A Control is the initial absorption of the ABTS •+ and A Sample is absorbance of ABTS •+ in the presence of samples.α-tocopherol was used as standard (Figure 3).

Statistical analysis
Data were analysed using the general linear model (GLM) procedure of SAS version 9.1 (2008).All multiple comparisons among means were performed using Duncan's new multiple range test.Data were expressed as mean ± standard deviation.

RESULTS AND DISCUSSION
Effects of biological treatment of RS using two strains of A. terreus (ATCC 20542 and ATCC 74135) on its antioxidant capacity are shown in Table 1.The results showed that antioxidant capacity of FRS treated using the two strains of A. terreus significantly increased (P<0.01) the antioxidant capacity of the FRS in all the three antioxidant test procedures adopted in this study (FRAP, ABTS and DPPH).
In the FRAP essay, antioxidant capacity was measured as the ability of the antioxidant components in the test sample to reduce ferric (III) to ferrous (II) in a redox-linked colorimetric reaction (Li et al., 2006).Our results showed that biological treatment of rice straw significantly increased (P<0.01) its capability on reduction of Fe 3+ to Fe 2+ in the FRAP reagent (Table 1).ABTS scavenging capacity denotes the ability of antioxidant components in the test sample to remove the ABTS •+ radicals, which were generated from the oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS) by potassium persulphate (K 2 S 2 O 8 ).The ABTS scavenging activity of RS increased (P<0.01) from 32.90 to 76.33% and 65.37%, respectively, for FRS treated by ATCC 74135 and ATCC 20542 (Table 1).
Likewise, DPPH is a compound that contains a nitrogen free radical which is readily destroyed by a free radical scavenger.This assay is a test on the ability of the antioxidant compounds functioning as proton radical scavengers or hydrogen donors (Singh and Rajini, 2004).Our data (Table 1) indicated that treatment of RS with the two strains of A. terreus resulted in higher DPPH scavenging activity than that of untreated RS.The overall results of this study showed that treatment RS by ATCC 74135 resulted in higher enhancement in antioxidant capacity than that by ATCC 20542.
The higher antioxidant capacity recorded in the FRS is assumed to be due to the polysaccharides of mycelial biomass that were produced during the growth of fungi on this substrate as suggested by data from the following studies.Li and Tsim (2003) reported strong antioxidant capacity of a polysaccharide (with a molecular composition of 10 glucose: 60 mannose: 75 galactose) which they isolated from the mycelia biomass of a Cordyceps anamorph fungus.Similarly, Chang et al. (2007) reported that polysaccharides isolated from biomass of Phellinus baumii Pilat (basidiomycete fungus belonging to the genus Phellinus) also exhibited antioxidant capability.Natural antioxidants isolated from plants, fungi and marine algae have been suggested to be the most useful nutraceuticals and functional foods constituents in health and disease prevention (Gutteridge and Halliwell, 1994).Leung et al. (2006) reported that polysaccharides produced from biomass of fungi are potential natural antioxidants.It has been suggested that antioxidant capacity of polysaccharides is due to the hydroxyl group of its molecules (Lindsay, 1996).However, SH, COOH, C=O, -Sand -Ofunctional groups in some polysaccharides and most polysaccharide-protein complex molecules can also confer a radical scavenging effect (Leung et al., 2009).
It is important that the antioxidant ability of the FRS to be stable to ruminal microbial degradation, if it is to be of use as an antioxidant agent to the host animals.To examine the above property of the antioxidant components, RS and FRS samples were separately incubated for 48 h under in vitro condition by inoculating rumen fluid into the buffer and mineral solution.The results showed that FRS significantly improved the FRAP (P<0.05),DPPH (P<0.05) and ABTS (P<0.01)scavenging activities in the rumen fluid (Table 2).The above results thus showed that the antioxidant capacity of the FRS is stable to ruminal microbial activity and therefore suggesting that biological treatment of rice straw using A. terreus offers a practical method to enhance the nutritive value of this agrobiomass as feed for ruminant animals.

Conclusion
Results of the present study showed that solid state fermentation using A. terreus is a viable method to enhance feeding value of agro-biomass, such as rice straw, by increasing its antioxidant capacity.Our results further showed that the antioxidant components in the FRS were stable in the rumen ecosystem and not degraded by the microorganisms in the rumen.Since antioxidant is beneficial to the health of the animal and can also improve the quality of their products (meat and milk), antioxidant enriched FRS, produced using the present method, can be a high quality roughage source for ruminant production.

Figure 2 .
Figure 2. Standard carve of FeSO4 for used in FRAP method.
± SD. **, Significantly different at 1% level; a, b and c: indicating means within column differed significantly.

Table 1 .
Effect of biological treatment on antioxidant activity of rice straw (RS) and fermented rice straw (FRS).

Table 2 .
Antioxidant activity of rumen fluid after 48 h incubation of rice straw (RS) and fermented rice straw (FRS).