CHAPTER 6 Effects of roasting, extrusion and enzymes on black-eyed beans (Vigna
6.2 Materials and methods
Black- eyed beans (Vigna unguiculata) purchased in Missouri, USA extruded at Wenger Manufacturing, Inc. (Sabetha, Kansas, USA) and were used in the current study.
6.2.2 Processing of black-eyed beans
The raw black- eyed beans (RBEB) were first ground (Restch- saw mill; SM 2000; Serial:
127211118J) through a 2 cm x 2 cm screen, followed by a 1cm x 1cm screen to obtain a particle size of 1.5 mm mesh.The raw black-eyed beans were roasted (ROBEB) using a hotbox oven at 120°C for 45 min. The seeds were removed from the oven and cooled, then ground. A second batch of the raw black-eyed beans were also extruded at 120°C for 3 min, cooled, and ground through a 2 cm x 2 cm screen then ground again through a 1cm x 1cm screen to obtain a particle size of 1.5 mm mesh. In addition, a third batch of raw black- eyed bean were ground as described previously, and extruded at 111°C, then cooled to room temperature. The expanded, extruded pellets were then ground through a 2 cm x 2 cm screen.
119 6.2.3 Birds and housing
A total of 336 day-old male broilers (Ross 708) chicks were purchased from a commercial hatchery, weighed, wing banded, and assigned to stainless steel chick batteries. All pens had the same initial body weight (about 41 g/bird). A completely randomized design was used with seven replicate pens of eight chicks assigned to each of the six dietary treatments. The chicks were fed starter diets from day 0 to 14. Lighting comprised 24 hours of light (approximately 2 foot candles) from days 1 to 9, followed by 20 hours of light (approximately 2 foot candles) and 4 hours of darkness from Days 10 to 14. The temperature in the house ranged between 35 and 36.1oC at the beginning of the experiment and between 30 and 31oC towards the end of the experiment. The health status of the birds was assessed daily. All abnormalities were recorded, including body weights of dead chicks and culls.
6.2.4 Treatments and experimental design
Six dietary treatments were used as shown in Table 6.1. Tables 6.1 and 6.2 have shown the ingredient and nutritional composition of the diets, respectively. Treatment 1 was the control maize-soya meal diet, while treatment 2 was a diet containing the raw black-eyed beans (RBEB). Treatment 3 was a diet that contained roasted black-eyed beans (ROBEB) and Treatment 4 contained extruded black-eyed beans (EBEB). Treatment 5 was a diet that contained raw black-eyed beans supplemented with a mixture of enzymes. Treatment 6 was a diet that contained extruded black-eye beans supplemented with a mixture of enzymes. The enzyme mixture included a combination of a commercial phytase (Ronozyme), Cibenza DP100 (protease) and Cibenza CSM (xylanase, α-galactosidase and β-glucanas) were used. Sixty g of each commercial enzyme was mixed with 5.82 kg of ground maize to make a premix. A randomized complete design was used, and included 6 treatments with 7 replicate pens of 8 birds per pen. A maize-soybean meal-based basal diet (mash form) formulated to meet the nutritional requirements of chicks (1-14 d post-hatch), as recommended by the National Research Council (NRC, 1994), was used. The raw black- eyed beans, treated black- eyed beans and treated black- eyed beans plus enzymes were included at 400 g/kg inclusion.
6.2.5 Measurements
6.2.5.1 Gut morphologyAt day 12, two birds per pen were injected with 10 mg/kg of bromodeoxyuridine (BRDU) as a marker for gut epithelial growth. Two days later, the same birds were again injected with
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Table 6.1. Ingredient composition of experimental diet used in the study (g/kg)
TreatmentsIngredients 1 2 3 4 5 6
Maize 593.4 351.4 351.4 379.3 351.4 379.3
Soy bean 318.1 168.1 168.1 140.9 168.1 140.9
Raw BEB 0.0 400.0 0.0 0.0 400.0 0.0
Extruded beans 0.0 0.0 0.0 400.0 0.0 400.0
Roasted beans 0.0 0.0 400.0 0.0 0.0 0.0
Soy bean oil 10.0 33.3 33.3 10.0 33.3 10.0
Dicalcium - foods 18.0 18.6 18.6 18.0 18.6 18.0
Limestone 11.1 11.4 11.4 11.8 11.4 11.8
Salt 3.6 3.5 3.5 3.5 3.5 3.5
Sodium bicarbonate 2.6 2.8 2.8 2.7 2.8 2.7
Alimet 1.9 3.2 3.2 3.2 3.2 3.2
L-lysine hydrochloride 78 1.3 1.3 1.3 1.7 1.3 1.7
Threonine 0.7 1.4 1.4 1.3 1.4 1.3
Choline 60 0.8 1.9 1.9 2.0 1.9 2.0
Organic trace mineral mix 2.0 2.0 2.0 2.0 2.0 2.0
Foods vit mix 0.5 0.5 0.5 0.5 0.5 0.5
Mould guard 0.5 0.5 0.5 0.5 0.5 0.5
Santoquin-mix6 0.01 0.01 0.01 0.01 0.01 0.01
Sand 3.54 0.00 0.00 2.25 0.00 2.25
Total 100.0 100.0 100.0 100.0 100.0 100.0
Treatment 1: control maize-soya meal; Treatment 2: raw black- eyed beans (RBEB); Treatment 3: roasted black- eyed beans (ROBEB); Treatment 4: extruded black- eyed beans (EBEB);
Treatments 5: raw black-eyed beans plus enzymes; Treatment 6: extruded black- eyed beans plus enzymes
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Table 6.2. Calculated nutrient composition of the diets
Calculated nutrient composition 1 2 3 4 5 6
ME (kcal/kg) 2900 2900 2900 2900 2900 2900
Crude Protein (%) 20.44 19.66 19.66 20.12 19.66 20.12
Available phosphorus (%) 0.45 0.45 0.45 0.45 0.45 0.45
Calcium (%) 0.92 0.92 0.92 0.92 0.92 0.92
Sodium (%) 0.22 0.22 0.22 0.22 0.22 0.22
Choline (ppm) 1600 1600 1600 1600 1600 1600
Digestible lysine (%) 1.07 1.07 1.07 1.07 1.07 1.07
Digestible sulphur amino acid (%) 0.80 0.80 0.80 0.80 0.80 0.80 Digestible threonine (%) 0.72 0.72 0.72 0.72 0.72 0.72 Digestible tryptophan (%) 0.23 0.20 0.20 0.20 0.20 0.20 Treatment 1: control maize-soya meal; Treatment 2: raw black- eyed beans (RBEB); Treatment 3: roasted black- eyed beans (ROBEB); Treatment 4: extruded black- eyed beans (EBEB);
Treatments 5: raw black- eyed beans plus enzymes; Treatment 6: extruded black- eyed beans plus enzymes.
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BRDU, before they were weighed and euthanized. Samples of duodenum (~ 1 cm), midgut (~ 1 cm), ileo-cecal junction with 1 cm of ileum attached, and a cross section of mid cecal pouch were collected from each bird. The lumen was flushed gently with Notox and preserved in labeled bottles containing 10-20 × volume of Notox (samples were pooled by treatment).
A 1-cm piece of duodenum was collected from one bird per pen, flushed with Notox and fixed with 10-20× volume of Notox in a labeled bottle. The fixed tissues were processed and embedded in paraffin wax. Tissue blocks were then sectioned to 5 microns thickness, stained with haematoxylin and eosin (H&E) or immunostained with anti-BRDU, and examined using an Olympus light microscope. The H&E stained slides were used for examination of gut morphology and measurement of gut morphometry. The anti-BRDU stained slides were used for examination of cell proliferation. The height and width of 5 randomly selected villi, the depth of 5 randomly selected crypts, and the thickness of mucosa tissue at 5 different locations per sample were measured using an Olympus light microscope with a 10× eyepiece lens with a 10× objective lens given a magnification of 100×. The villi height, villi width, crypt depth, crypt depth/villi height (CV) ratio and mucosa tissue thickness (TMUC) were compared among treatments. Greater villi height and lower villi width indicate a greater absorption area. Lower crypt depth and crypt depth/villi height ratio indicates less need of cell proliferation to maintain gut integrity. The mean of villi heights, villi widths, crypt depths and TMUCs for each sample were used for statistical analyses.
6.2.5.2 Growth performance
The birds were weighed by pen on days 0, 7 and 14, and feed consumption was determined.
Mortality was checked twice daily and weights of dead birds were used to adjust for feed conversion. Body weight gain was estimated as: = (body weight at a weigh day) – (body weight at the previous weigh day). The feed: gain ratio corrected for dead birds was estimated as: (feed consumed) / (incremental pen weight + incremental dead bird weight).
The feed consumed was estimated as the difference between the total weight of feed offered and the residual for each week/pen divided by the number of birds per pen. The cumulative performance index (CPI) was calculated as: = ((Cumulative livability x ((body weight x 1000) /Day of study) x 10)/ (Cumulative feed: gain ratio corrected for dead bird weight)) were calculated.
123 6.2.6 Statistical analyses
Data were analyzed using the General Linear Models procedure of SAS® (SAS, Institute, Cary, NC, USA). When the null hypothesis was rejected, means were separated using Fisher’s protected least significant difference method.