Effect of Fermented Rice Bran and Cassava Waste on Growth Performance and Meat Quality of Crossbred Pigs

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2.2.2. Assay of Cellulase Activity

Cellulase assay was performed according to the method of ^[14] using carboxy-methyl cellulose as substrate. In brief, 250 µl of 1% CMC in 0.05 M citrate buffer (pH 4.8) was placed a test tubes and 250 µl of culture filtrate was added. The reaction mixture was incubated at 50^oC for 30 minutes. The reaction was terminated by adding 500 µl 3,5-dimitrosalisylic acid (DNSA) reagent. The tubes were heated at 100°C in boiling water for 5 minutes, and then cooled at room temperature. The absorbance was read at 540 nm. Enzyme activity was defined as one µmol glucose released per min.

2.2.3. Assay of Protease Activity

The protease activity was assayed by the method of ^[15] using casein as substrate. In brief, 175 µl culture filtrate was added to 350 µl casein in 2% phosphate buffer (0.05 M, pH 7.0) and was shaked at 500 rpm/min during 10 minutes. The reaction was stopped by adding 875 µl of 5% trichloracetic (TCA) and incubated at room temperature. After 20 minutes, culture filtrate was centrifuged at 12,000 rpm/min at 4°C for 10 minutes to remove the precipitate of casein. The supernatant of 200 µl added with 800 µl 6% of Na₂CO₃, well shaking. Then 200 µl of 0.2 N Folin reagent was added. The absorbance of supernatant was read with spectrophotometer at 750 nm. Enzyme activity was defined as one µmol tyrosine released per min.

2.2.4. Assay of Amylase Activity

The assay mixture consisted of 250 µl of enzyme solution and 250 µl of 1% starch in 0.1 M phosphate buffer (pH 7.0) incubated at 50°C for 10 min. The reaction was stopped by adding 500 µl of 1% dinitrosalicylic acid. The tube containing enzyme solution was heated at 100^oC in boiling water for 5 minutes, and then cooled at room temperature. Determine maltose released by spectrophotometer at 540 nm. Amylase activity was defined as the amount of enzyme that releases one µmol reducing sugar equivalent to maltose per min ^[16].

2.2.5. Preparation of Fermented By-Products

Fresh cassava waste was collected directly from a cassava starch processing factory in Hue. The cassava waste (CW) was sprayed directly on a clean concrete floor and turned occasionally to reduce moisture content. For preparation of fermented CW (CWF), the air-dried CW was thoroughly mixed with rice bran and maize (ratio 75: 15: 10), after which 2 mL per kg of each of Trichoderma longibrachiatum (1 x 10⁷ spores per mL), Aspergillus niger (1 x 10⁷ spores per mL), Pichia kudriavzevii (1 x 10⁶ CFU per mL) and Lactobacillus buchneri (1 x 10⁶ CFU per mL) were added together with 2.6 g urea/kg and once more thoroughly mixed as described by ^[17]. The cassava waste mixture was put into nylon bags for fermentation. The dry matter (DM) of the final product was around 35%.

For preparation of fermented rice bran (RBF), the rice bran (RB) was thoroughly mixed with maize (ratio 90: 10), after which 2 mL per kg of each of Trichoderma longibrachiatum (1 x 10⁷ spores per mL), Aspergillus niger (1 x 10⁷spores per mL), Pichia kudriavzevii (1 x 10⁶ CFU per mL) and Lactobacillus buchneri (1 x 10⁶ CFU per mL) were added and once more thoroughly mixed. Then, water (40 L per 100 kg feed) containing 4.8 g urea/kg was added and thoroughly mixed with the rice bran mixture. The rice bran mixture was put into nylon bags for fermentation. The DM of the final product was around 64%.

The impacts of the fermentation treatments on the characteristic of the fermented by-products are presented in Table 2. In general, there were increases in the content of crude protein (CP) and decreases in NDF and ADF of fermented rice bran and cassava waste as compared with the un-fermented CW and RB. After 21 days of fermentation, HCN content had been reduced from 306 to 26 mg/kg DM.

Table 2. Dry Matter (DM) Content (%), Chemical Composition (% of DM) and Hydrogen Cyanide (HCN) Content (Mg/Kg Dm) of Un-Fermented and Fermented Rice Bran and Cassava Waste ^{1, 2}

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2.2.6. Experimental Diets and Feeding

The seven experimental diets comprised: CO, control diet without fermented by-products; RBF1, diet with 15% and 30% fermented rice bran in the growing and finishing period, respectively; RBF2, diet with 20% and 35% fermented rice bran in the growing and finishing period, respectively; RBF3, diet with 25% and 40% fermented rice bran in the growing and finishing period, respectively; CWF1, diet with 15% and 30% fermented cassava waste in the growing and finishing period, respectively; CWF2, diet with 20% and 35% fermented cassava waste in the growing and finishing period, respectively; and CWF3, diet with 25% and 40% fermented cassava waste in the growing and finishing period, respectively. The ingredients and chemical composition of the experimental diets are presented in Table 3 and Table 4.

Table 3. Ingredient Composition of The Experimental Diets (g/kg DM)

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The pigs were fed ad libitum and were given new feed three times per day (06.00, 11.00 and 18.00 h). The daily feed allowance was based on feed offered and refused the previous day. The pigs had free access to drinking water via automatic drinking nipples.

2.2. Data Recording

Feed refusals were recorded daily and were used for correction of the feed intake data. Pigs were weighed every month.

2.3. Carcass Traits

For evaluation of carcass traits, three pigs (2 females and 1 male) from each treatment were randomly selected at the end of experiment, starved of feed overnight, weighed and slaughtered at a commercial slaughterhouse. The back-fat thickness was measured at P2. The loin area was determined at the thirteenth and fourteenth rib. After slaughter, pH of the longissimus dorsi muscle (at the middle of the thirteenth and fourteenth rib) was measured with a pH-meter (Hanna Instruments 99163) by putting a pH probe into the muscle at 45 minutes (pH45) and at 24 hours (pH24). The iodine index of carcass fat was analyzed according to Wijs (method 993.20, ^[18]). Water-holding capacity of meat was determined as described by ^[19] using samples from the longissimus dorsi muscle at the middle of the thirteenth and fourteenth rib. In brief, 24 h after slaughter a muscle sample was collected and weighed (approximately 0.5 g) and put in a centrifuge tube with a filter paper (pore size 90 µm) at the bottom of the tube. The muscle sample was weighed after centrifugation at 30 x g for 1 h at 4^oC. The water-holding capacity was calculated from weight of samples before and after centrifugation. The muscle samples were analyzed for moisture, crude protein (CP), ether extracts (EE) and ash with standard ^[20] methods (for method number see below).

Table 4. Dry Matter (DM) Content (g/kg DM), Chemical Composition (g/kg DM) and Metabolizable Energy (ME) Value (kcal/kg DM) of Experimental Diets

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2.4. Chemical Analysis

The feed samples were analyzed for DM (method 930.15), ash (method 942.05), CP (method 984.13), EE (method 920.39), crude fibre (CF, method 978.10), acid detergent fiber (ADF, method 973.18) and neutral detergent fiber (NDF, method 4.6.03) according to standard AOAC methods ^{[18, 20]}(AOAC, 1990; AOAC, 1997). True protein content of feed mixtures was analyzed using precipitation with trichloroacetic acid (TCA) as described by ^{[20, 21]}. The HCN content of cassava waste was analyzed by the method of ^[22].

2.5. Statistical Analyses

The data were analyzed as a 2x4 factorial completely randomized design with diet type, level of inclusion and interaction (diet type by level of inclusion) as factors using the GLM procedure of Minitab Software, version 13.31 ^[23]. Treatment means which showed significant differences at the probability level of p<0.05 were compared using Tukey's pairwise comparison procedure.

3. Results

3.1. Performance

In the growing period, the average daily feed intake (ADFI) and feed conversion ratio (FCR) differed between the fermented feeds with lower ADFI and better FCR (p<0.01) on diet CWF than on diet RBF (Table 5). Inclusion of fermented feeds reduced the ADFI compared with the control diet (p<0.01). The FCR was improved with increasing inclusion of fermented feeds (p<0.01). There were no differences in average daily gain (ADG) between diet type or between inclusion levels of fermented feeds (p=0.43 and p=0.30, respectively).

In the finishing period, the ADFI, ADG and FCR differed between the fermented feeds with lower ADFI and ADG, and better FCR (p<0.01) on diet CWF than on diet RBF. Inclusion of fermented feeds reduced ADFI and ADG (p<0.01), while there was no clear effect of inclusion level on the FCR (p=0.066) compared with the control diet.

Overall, pigs fed diet CWF showed lower ADFI and ADG, and better FCR than pigs fed diet RBF (p<0.01). Inclusion of fermented feeds reduced ADFI and ADG (p<0.01), and improved FCR compared with the control diet (p<0.01).

3.2. Carcass Traits

Live weight at slaughter and hot carcass weight was lower on diet CWF than on diet RBF (p<0.01), while the dressing percentage was higher on diet RBF than on diet CWF (p<0.03) (Table 6). Carcass percentage, carcass length, loin muscle area and back fat thickness were unaffected by diet type. Increasing inclusion of fermented feeds resulted in lower live weight at slaughter, lower hot carcass weight and lower back fat thickness (p<0.01). The carcass percentage was affected by inclusion level of fermented feeds (p<0.01) but did not show any consistent pattern. The iodine index was lower on diet CWF than on diet RBF (p<0.01), but was unaffected (p>0.05) by inclusion level of fermented feeds. Dressing percentage, carcass length, loin muscle area and parameters measured on muscle samples (DM, CP, EE, ash, pH45, pH24 and water-holding capacity) were unaffected (p>0.05) by inclusion level of fermented feeds.

Table 5. Performance of Growing Pigs Fed Diets With Inclusion of Fermented Rice Bran (RBF) and Fermented Cassava Waste (CWF)

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Table 6. Carcass Traits in Growing Pigs Fed Diets With Inclusion of Fermented Rice Bran (RBF) and Fermented Cassava Waste (CWF)

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