Get Complete Project Material File(s) Now! »



In vitro procedures that mimicked pig digestion were used to evaluate Roxazyme® G2 (RX) and Viscozyme L ® V2010 (VZ) activities, and the fermentation characteristics of DF in feed ingredients and in fibrous, complete maize-soybean growing pig diets. Maize (Zea mays) grain, its hominy chop and dehulled soybean (Glycine max) were used as the basal ingredients in a standard (141 g TDF g-1 DM), and in five high fibre (246 g TDF g-1 DM) growing pig diets, each enriched in DF using maize cobs, soy hulls, brewer’s (barley; Hordeum vulgare L) grains, lucerne (Medicago sativa) hay or wheat (Triticum aestivum) bran. Among the feed ingredients, dissimilar, source-dependent (p≤0.001) activities on DF were observed between RX (0.02 to 0.12) and VZ (0.04-0.33). The lowest (p≤0.05) RX activity was observed on the maize and soybean fibres. The source of fibre influenced (p≤0.0001) fermentation gas (51.8-299.4 mL g-1 DM) and SCFA (2.3-6.0 mMol g-1 DM) production. Dehulled soy bean, maize meal and hominy chop fibre extracts were the most fermentable, while maize cob and brewers’ grain fibre were poorly fermented (p≤0.05). Among the fibrous complete diets, the source of DF influenced (p≤0.001) the enzyme activities, with the activity of RX (0.03-0.06) substantially lower (p≤0.05) than that of VZ (0.16-0.22). The DF source influenced (p≤0.001) fermentation gas (126.6-187.6 mL g-1 DM) similarly (p≤0.05) affected the SCFA (4.1-5.4 mMol g-1 DM) production. On the STD DF, the activity of RX was less (p≤0.05) (0.03) than that of VZ (0.25), with higher (p≤0.05) (205.3 mL g-1 DM) gas, but similar (p>0.05) (5.0-mMol g-1 DM) SCFA production compared to the fibrous diets. The leguminous fibres produced higher percent Ace and with less But (p≤0.05), and, except for the dehulled soybean meal fibre, less (p≤0.05) Pro. Except for MC, the cereal fibres produced higher (p≤0.05) percent Pro and less (p≤0.05) Ace. Maize meal and hominy chop fibres produced relatively high percent But (p≤0.05). Compared to the standard diet, fibre extracted from the high fibre diets produced higher (p≤0.05) percent Ace, and less (p≤0.05) Pro and But. Among the diets, correlation was strong (r = 0.99, p≤ 0.001) between the partial degradability of VZ and the SCFA production, justifying its status as the standard enzyme for in vitro, pig digestibility studies. The source-depended variation in the fermentability of DF among the high fibre diets suggested substantial scope to increase DF fibre fermentability by screening the ingredients on fibre fermentability. The results suggested RX may not be effective in pigs fed high DF maize-soybean diets in which the DF is dominated by maize co-products.
Keywords; growing pigs, fibre, non-starch polysaccharides, enzymes, fermentability


In growing pigs, the objective to control gut fermentation is motivated by its profound influence on the pig’s metabolism and digestive physiology. Examples include prebiotic (Williams et al., 2001), bactericidal (Verstegen and Williams, 2002) and enterotrophic (Tonel et al., 2010) effects of the SCFA products. Short chain fatty acids are also known to control satiety (Sleeth et al., 2010) and are involved in glucose (Theil et al., 2011) and lipid (Fushimi et al., 2006) homeostasis. Short chain fatty acids provided in excess of 10% of the total DE, equivalent to 17.6% of the total energy supply of the growing pig (Anguita et al., 2006).
Fibre fermentability is a concept increasingly considered critical in a broader strategy toward the productive use of low cost, typically fibrous grain processing co-product feed ingredients (Chen et al, 2013). To control fermentation, the fermentation kinetics of DF in the feed ingredients need to be quantified, followed by the application of the parameters in feed formulation. Non-starch polysaccharide degrading enzymes may provide additional controls to influence fermentation (Bindelle et al., 2011). A range of commercial NSPase cocktails are available on the market. There is uncertainty on their efficacy in pigs, particularly on maize-soybean based diets (Bedford and Cowieson, 2012).
Effective characterisation of the fermentation kinetics of DF and the matching of enzymes to the NSP composition are still constrained by methodology. In vitro assays are preferred if they can predict the hydrolytic and fermentative degradation of NSP in the gut with less difficulty, low cost and in less time compared to in situ and in vivo studies. Bindelle et al. (2007) adapted the in vitro fermentation gas production technique originally described for ruminants (Menke et al., 1979), to application in pig nutrition. The method conveniently uses faeces as inoculum to ferment fibre residues remaining after simulated gastric-ileal digestion by porcine pepsin + pancreatin. The procedure is a variation of the three-step in vitro method of Boisen and Fernandez (1997), which uses Viscozyme L ® V2010 (VZ) to predict the lower gut degradation of fibre. Viscozyme is a non-specific, multi-enzyme complex that contains a wide range of carbohydrases including arabinase, cellulase, β-glucanase, hemicellulose, pectinase, and xylanase (Cone et al., 2005).
The aim of the study was to examine the influence of high levels of different unconventional feed ingredients on the fermentation characteristics, and the activity of Roxazyme® G2 (RX) on DF in complete, maize-soybean growing pig diets.

Materials and methods

Experimental feeds

Maize (Zea Mays) grain (MM) was obtained from the Animal Production Institute of the Agricultural Research Council (ARC). Maize hominy chop (HC) and cobs (MC), dehulled soybean (Glycine max) (dSBM) and soybean hulls (SH), brewer’s (barley; Hordeum vulgare L) grains (BG), lucerne (Medicago sativa) hay (LH), wheat (Triticum aestivum) bran (WB) from OPTI FEEDS PVT LTD, Lichtenburg. The nutrient and fibre composition of the feed ingredients are described in Table 3.1. From these ingredients, six growing pig diets (Table 3.2 and Table 3.3) were formulated; a standard, low fibre (141 g total dietary fibre (TDF) kg-1 dry matter (DM)) growing pig diet and five similarly nutritionally balanced (NRC, 1998) but high fibre (246 g TDF kg-1 DM) diets. The high level of DF was achieved by substituting variable proportions of maize and dehulled soybean meals in the STD diet with WB, MC, BG, SH or LH.
Samples of feedstuffs and diets were milled through a 1 mm screen using an Ika® analytical mill and were subsequently oven-dried for 18 hours in a forced draught oven at 100 ºC to determine DM content. The samples were cooled for 30 minutes in a desiccator. Ankom® F57 filter bags (ANKOM Technology, Macedon, New York, USA) were rinsed in 99% acetone, air-dried and labelled using a solvent resistant marker. Feed samples (0.5 g ± 0.001) were weighed into the bags, and heat-sealed using a 200 mm AIE 60 Hz impulse sealer.

Chemical analyses

Feeds were analysed using AOAC (2000) Official method 934.01 for moisture, AOAC (2006) Official method 965.17 for phosphorus and AOAC (1999) methods 990.03 for crude protein (CP), 968.08, 942.05 and 920.39, for calcium, ash and fat (EE), respectively. Dietary fibre was analysed as soluble plus insoluble fibre, using the AOAC (2007) Official method 991.43. Crude fibre (CF), Acid Detergent Fibre (ADF) and Neutral Detergent Fibre (NDF) were analysed according to van Soest et al. (1991). Gross energy (GE) was determined using a DDS isothermal CP500 bomb calorimeter.

In vitro digestion of feeds

Feed ingredients were digested using a modification of the three step procedure described by Boisen and Fernandez (1997). Pepsin (porcine, 200 FIP-U/g, Merck No, 7190), pancreatin (porcine, grade IV, Sigma No P-1750) and Viscozyme L ® V2010 (VZ) (Viscozyme 120 L, 120 fungal β-glucanase U/g; mixture of microbial carbohydrases, including β-glucanase, xylanase, arabinase, cellulase, Novo-Nordisk, Bagsvaerd, Denmark) were used in the same dosages used in the original procedure. In the third step, Viscozyme was compared to Roxazyme® G2 (Novo DSM, from T. Longibrachitum, with endo -1,4-β-glucanase (8000 U/g), endo-1,3(4)-β-glucanase (18 000 U/g) and endo-1,3(4)-β- xylanase (26000 U/g). To determine the minimum effective dose of RX, calibration started from the feed dose (100 mg/kg), by calculating the dose in proportion to the mass of the sample used in the setup and increasing the dose until the maximum degradability was achieved for all the substrates.

Digestion by pepsin

Each digestion jar of the ANKOM DaisyII Incubator used in the digestion accommodated a maximum of 24 samples. Accordingly, in each run of the digestion, 24 sample bags containing the 8 feed ingredients with 3 replicates, or 6 diets with 4 replicates were placed in one of 4 digestion jars on the incubator. One empty filter bag was included in each jar as a blank. To each jar, 600 mL of phosphate buffer solution (0.1 M, pH 6.0) and 240 mL HCl (0.2 M) were added. The pH was adjusted to 2.0 using a 1 M HCl or a 1 M NaOH solution. The jars were placed on a rotating rack in the incubator. Temperature was calibrated to 39 oC. Pepsin (0.6 g) and 12 mL of chloramphenicol (5 g, Sigma No. C-0378, per 100 mL ethanol) solution were added to the digestion medium in each of the jars and mixed gently. The samples were digested for 2 hours at a temperature of 39 ± 0.5o C.
Digestion by pancreatin
After digestion by pepsin, 240 mL of phosphate buffer solution (0.2 M, pH 6.8) and 120 mL of 0.6 M NaOH solution were added to the medium in each of the jars. The pH was adjusted to 6.8 using 1 M HCl or NaOH solutions. Pancreatin (2.4 g, porcine, grade IV, Sigma No P-1750) was added to each jar. The samples were digested for 5 h at a temperature of 39 ± 0.5o C. After the digestion, the bags were rinsed in warm tap water with minimum agitation to prevent the escape of the particulates from the bags. The bags were further rinsed for 5 minutes in 95% ethanol, and for a further 5 minutes in 99% acetone. The samples were then oven-dried at 85o C for 18 hours in a forced draught oven and were cooled in a desiccator for 30 minutes.

READ  Plants used traditionally to treat age-related/neurological disorders

Digestion by Viscozyme and Roxazyme

At the termination of pancreatin digestion, the media in the jars were completely discarded. To each jar, 750 mL of a freshly prepared phosphate buffer (0.1 M, pH 4.8) was added. Temperature was calibrated to 39 ± 0.5 oC. To each jar, 12 mL of VZ or 0.017 g of RX was added. The samples were incubated for 24 hours at a temperature of 39 ± 0.5 oC. At the completion of the fermentation process, the bags were washed, rinsed and dried as described section above.

In vitro fermentation

Preparation of substrates and the experimental scheme

Fermentation tests were conducted on the filtered, washed pooled residues after several runs of pepsin-pancreatin (PP) digestion of feedstuffs and diets. The gas production setup accommodated 10 samples at a time. Accordingly, each fermentation run included the 8 feed ingredients + 2 blanks or the 6 diets + 2 blanks. The blanks contained the inoculum only.

Preparation of buffer and inoculum

A phosphate-bicarbonate buffer was prepared according to Marten and Barnes (1980). The temperature of the buffer was calibrated and maintained at 39 °C in a water bath.
The inoculum was prepared following the procedures described by Bindelle et al. (2007). Fresh faeces were collected directly from the rectum of three mature dry sows from the herd at the Agricultural Research Council, Animal Production Institute, Irene (Pretoria, South Africa). The sows were on an antibiotic-free, dry sow ration. The faeces were immediately placed into a sealable plastic bag filled with CO2 and placed in a pre-warmed thermos flask. The flask was flushed with CO2, and was closed tightly. The inoculum was prepared in 2 batches in stomacher bags, each batch containing 60 g of the pooled faeces suspended in 400 mL of the buffer at 39 oC. The faeces and buffer in each stomacher bag were subjected to 60 seconds of mechanical pummelling in a stomacher blender. The blended mixtures were filtered through 4 layers of mutton cloth into a pre-heated (39 °C) 2 litre flat bottomed flask, which was immediately flushed with CO2 and closed with a stopper. The inoculum was kept tightly closed and submerged in a water bath at 39 oC.

Gas production measurements

Gas production was measured using an ANKOMRF gas production system (ANKOM® Technology). Residues of PP digestion were dried to constant weight at 50o C and stored in a desiccator prior to weighing 0.75 g ± 0.001 g samples into a 200 mL glass bottle. To each bottle of the gas measurement module, 100 mL of buffer at 39 oC and 50 mL of inoculum were added to make up 150 mL of fermentation medium containing 0.05 g fresh faeces per mL of buffer. The gas bottle headspace was flushed with CO2 before closing tightly and incubation in a warm bath at 39 °C. Gas production measurements were taken over 64 hours, at a recording interval of 5 minutes.

Analyses for short chain fatty acids in the fermentation residues

Acetic (Ace), propionic (Pro), n-butyric (But), iso-butyric (Iso-But) and n-valeric acids were determined using a modification of the procedures described by Webb (1994). Residues of fermentation were filtered through Cameo 30 (0.45 µm) filters before analyses for SCFA in a Varian 3300 FID Detector Gas Chromatograph, using a CP Wax 58 (FFAP) CB Cat no 7654 column (25 m, 0.53 mm, 2.0 m), with Helium as the carrier gas. Ace, Pro, But, Iso-But and n-Valeric were used as standards. The temperature program started at initial column temperature of 50 C for 2 minutes, which was increased at 15 C per minute to a final temperature of 190 C, for 5 minutes.

Statistical analyses

The PROC NLIN procedures of Statistical Analysis Systems (SAS) Institute software, version 9.3 (SAS, 2010) were used to estimate gas production kinetics using the monophasic model described by Groot et al. (1996);
Analysis of variance was conducted on the partial in vitro digestible dry matter (IVDDM) coefficients and on fermentation parameters using the PROC MIXED procedures of SAS software, version 9.3 (SAS, 2010). The IVDMD dry matter by PP (IVDDMPP) and the fermentation parameters of the residues of the digesta were analysed using model I;
Where: µ is the overall mean, αi the effect of the ith source of fibre and ei the random error.
Partial IVDMD by RX (IVDMDRX) and VZ (IVDMDVZ) were analysed using model II; ( ) Where: µ is the overall mean, αi the effect of ith source of fibre, βj the effect of the jth enzyme cocktail, (αβ)ij the interaction of fibre source and enzyme cocktail and eij the random error.
Comparison of means was performed using the Bonferroni t-test at the α-level of 0.05.


Degradability by Roxazyme and Viscozyme

The IVDMDPP, IVDMDRX and IVDMDVZ of the feed ingredients and of the diets are indicated in Table 3.4 and Table 3.5, respectively. IVDMDPP; IVDMDRX and IVDMDVZ differed among the feed ingredients and among the diets. Compared to other feedstuffs, the activity of RX was lower (p≤0.05) on fibre extracted from dehulled soybean meal and from maize and its co-products. Interaction was also significant (p≤0.001) among the diets, whereby the activity of VZ was highest on fibre extracted from the STD and SH diets, whereas RX had among the least activity on the same diets.

Fermentation properties

The fermentation gas production profiles of the fibre extracted from the ingredients and the diets are indicated in Figure 3.1 and Figure 3.2, respectively. Based on a regression model forced through the origin, the algorithm used to predict gas production achieved stronger prediction of the final gas (Y, at t = ∞) from the gas produced at t = 64 hours for the feed ingredients (Y=2.37X; R2=0.78), compared to the complete diets (Y = 1.89X; R² = 0.74).
Fermentation parameters of the fibre extracted from the ingredients and the diets are indicated in Table 3.6 and Table 3.7, respectively. Gas production differed by source of fibre among the feed ingredients and among the diets. There was greater variation in gas production among the feed ingredients, where maize and soybean fibres produced 3-4 times the gas produced by BG and maize cob fibres. The time taken to produce half the predicted gas volume was influenced by the source of fibre among the feed ingredients, but not among the diets. The source of fibre also influenced both the composition and the total (Ace, Pro, But, Iso-But, Val-a) SCFA concentration among the feedstuffs. Leguminous fibre tended to produce more percent Ace and less Pro and But. On the other hand, cereal fibre produced relatively high percent Pro and less Ace, and except for maize fibre, less But. Maize fibre had high Pro, low Ace and, except for maize cobs, high But.
Among the diets, SCFA production was not different, except for a tendency (p=0.069) towards higher SCFA in the LH diet, compared to the MC diet. Compared to the STD diet, the high fibre diets increased the proportions of Ace at the expense of Pro and But. Overall, due to the moderating effect of the highly fermentable maize and dehulled soybean fibres, there was less variation in fermentation parameters among the diets compared to the feed ingredients.
Short chain fatty acid production of the fibre extracted from the fibrous ingredients was correlated (r=0.89; p=0.045) to that of the diets in which they were used to increase DF. There was lower (r=0.550; p>0.337) correlation of the gas production. There was correlation (r=0.99; p <0.0001)) between SCFA and gas production among the feed ingredients, and not among the diets (r=0.67; p>0.05).
Different relationships were observed when the RX and VZ partial DM degradability were profiled in scatter plots against SCFA or gas production (Figure 3). Among the diets, correlation was significant (r = 0.99, p≤ 0.001) between the partial degradability of VZ and the SCFA production. The correlation was not significant for RX. Correlation of fibre degradability by enzymes to gas production was not significant for both the feed ingredients and the complete diets.

READ  Mathematical Fundamentals of MR Dampers


The dissimilar, source depended activities of VZ and RX on DF were attributed to differences in NSP specificities (Bedford and Cowieson, 2012). The low enzyme activities on maize fibre could be indicative of the presence of resistant starch (RS). Resistant starch is almost totally recovered in the filtered PP residues (Giuberti et al., 2013).
Similar large variation in the fermentation properties of DF among and within feeds was previously reported (Bindelle et al, 2011; Jha et al., 2011; Jha and Lerteme, 2012; Jonathan et al., 2012). The variation is attributed to differences in the NSP composition of DF (Bach Knudsen et al., 2001). For example, cereal fibre consists mainly of arabinoxylans, β-glucans and cellulose, whereas legume fibre contains mainly pectins, cellulose and xyloglucan (McDougall et al., 1996). Whereas NSP such as pectins and β-glucans are rapidly and practically totally fermented, insoluble cellulose and xylans are almost undigested (Noblet and Le Goff, 2001). In the present study, the low fermentability of brewer’s grains and wheat bran was attributed to the concentration of the husk and pericarp, which typically contain the insoluble, lignified fibre. Chemical analysis of maize cobs and lucerne hay also showed that the fibre was largely insoluble, and for maize cobs, highly lignified. On the other hand, the high fermentability of the soybean fibre was attributed to the high content of pectin (McDougall et al., 1996). Branching of the pectic polysaccharide chains provides numerous cleaving sites for enzymes (Jonathan et al., 2012). The hulls contain relatively more insoluble, relatively slowly fermentable cellulose and xylans (de Vries, et al., 2012) compared to the dehulled grain, hence were relatively less fermentable.
It is difficult to relate the observed profiles of fermentation SCFA to the NSP composition of DF, and to the likely gut microbial interactions. The general shift from Ace to Pro production in response to leguminous or high DF was previously associated with the presence of a diverse microbial population, in response to high substrate availability (Macfarlane and Macfarlane, 2003). Depending on the abundance of sNSP, a shift from Ace to But production by the maize meal and hominy chop fibres was previously associated with enhanced bacterial proliferation and a diverse microbial community (Jha et al., 2011). In the present study, the high But production by maize fibres could be attributed to RS, whose fermentation characteristically yields high But (Giuberti et al., 2013; Jonathan et al., 2013).
The fermentation test is premised on the assumption that in vitro gas and SCFA production predict in vivo degradation of of DF (Jonathan et al., 2012; Chen et al., 2013). Interstingly, in the present study, there was strong correlation between the two parameters among the feed ingredients, but not among the diets. In feed ingredients, Jonathan et al. (2012) reported significant correlation in the early stages of fermentation, and not by the end of fermentation. They attributed the poor correlation to fermentation pathways which differently partition carbon into SCFA or gases. For example, lactate, which is metabolised early during fermentation to yield Pro, But and the gases, was detected after 72 hours of fermentation of oat β-glucans and inulin, and not in other substrates. CO2 produced by bicarbonate buffering also depends on the quantitative and qualitative production of SCFA (Bindelle et al., 2011). It is therefore important to determine the gas composition, and the intermediate metabolites during the course of the fermentation (Jonathan et al., 2012).
Given strong prediction of the in vivo degradability of DF by the in vitro fermentation test (Chen et al., 2013), the positive correlation of VZ activity to the SCFA production validated its prediction of the in vivo degradation of DF. The large variation in the fermentation characteristics of DF from different sources, and its expression in high fibre diets create scope to control fermentation through diet formulation. The in vitro methods used in this experiment are largely currently restricted to the characterisation of fibre in feed ingredients. There is need for further, in vivo evaluation to test whether the inclusion of high levels of insoluble fibre-rich ingredients, enzyme supplements and screening fibre sources for fibre fermentability can be effective tools to control fermentation, sufficiently to induce beneficial the digestive metabolic and physiological functions.


The influence of source of DF on enzyme activity and on its fermentation provides scope to formulate diets uniquely designed to induce desirable fermentation in the gut by screening the fibrous feed ingredients based on the fermentation kinetics of the fibre, and by correctly matching the NSP compostion to NSPases. The use of VZ as the standard enzyme for in vitro pig feed digestibility studies was supported by the strong correlation of its activity with the fermentability of DF. The low activity of RX on maize and soybean fibres may result in low efficacy in pigs fed maize-soybean based diets. If validated in an in vivo model, the in vitro approach used in the study could be a practical method to match the fibre in complete diets to different enzyme cocktails, and to test combinations of different cocktails for a specific diet to exploit possible additive or synergistic enzyme action.

1.1 Background
1.2 Problem statement
1.3 Hypotheses
1.4 Objectives
1.5 Delineation and limitations of the research
1.5.1 Scope of the research
1.5.2 Limitations of the research
2.1 Introduction
2.2 The definition of DF
2.3 The chemistry of fibre
2.4 Composition of DF in common pig feed ingredients
2.5 Methods of analysis for DF
2.6 Physiologically important physico-chemical properties of DF
2.7 Fermentation
2.8 Metabolic, physiological and environmental impacts of fibre
2.9 NSPases in pig nutrition
2.10 Evaluating pig feeds
2.11 Metabonomics
2.12 Summary
3.1 Introduction
3.2 Materials and methods
3.3 Results
3.4 Discussion
3.5 Conclusion
4.1 Introduction
4.2 Materials and methods
4.3 Results
4.4 Discussion
4.5 Conclusion
5.1 Introduction
5.2 Methods and Materials
5.3 Results
5.4 Discussion
5.5 Conclusion
6.1 Introduction
6.2 Methods and Materials
6.3 Results
6.4 Discussion
6.5 Conclusion
7.1 Introduction
7.2 Methods and Materials
7.3 Results
7.4 Discussion
7.5 Conclusion
8.1 Introduction
8.2 Methods and Materials
8.3 Results
8.2 Discussion
8.3 Conclusion
9.1 Overview of the current state of knowledge
9.2 Physico-chemical and fermentation properties of dietary fibre
9.3 Performance of pigs fed high fibre diets
9.4 Effect of high fibre on the histometry of the ileum
9.5 1H-NMRS and the biochemical influence of fibre in growing pigs
9.6 Effects of RX
9.7 Overall conclusions
9.8 Recommendations for future research

Related Posts