Phosphate rather than surfactant accounts for the
main contribution to enhanced ®bre digestibility
resulting from treatment with boiling
neutral detergent
P.M. Kennedy, J.B. Lowry
*, L.L. Conlan
CSIRO Tropical Agriculture, PMB 3, Indooroopilly, Qld, Australia 4068
Received 6 December 1999; received in revised form 31 March 2000; accepted 8 June 2000
Abstract
It is known that extraction of some grasses with neutral detergent (ND) increases the in vitro digestibility [Kennedy, P.M., Lowry, J.B., Conlan, L.L., 1999. J. Sci. Food Agric. 79, 544]. Here, we report experiments which de®ned the contribution of ND components to digestibility increases. Substrates were prepared from spear grass (Heteropogon contortus) by boiling at neutral pH in solutions of 0.05 M disodium EDTA, 0.018 M sodium borate, and 0.03 M sodium phosphate, and a mixture of the three solutions. Phosphate was the most effective single component in increasing ND ®bre (NDF) digestibility in vitro, after 120 h of fermentation, from 472 to 522 g/kg NDF, equivalent to 68% of the increase found with boiling ND. NDF digestibility of bagasse at 120 h increased from 162 to 186, 230 and 277 g/kg NDF after boiling for 1 h in water, neutral phosphate and ND, respectively.
Phosphate treatment of bagasse produced a 44% increase in NDF digestibility, compared to increases of 5, 8, 14, and 16 % for rhodes grass (Chloris gayana), spear grass, angleton grass (Dicanthium aristatum) and carpet grass (Axonopus af®nis), respectively. Increases of cumulative gas production against incubation time indicated that most of the increased fermentation had occurred by 24 h of fermentation for the grasses, whereas 72 h was needed for bagasse. There were also improvements in NDF digestion with neutral phosphate treatment of spear grass at temperatures of 75, 85 or 958C, with prolonged treatment times required at lower temperatures. These treatments reduced the content of acid insoluble ash in NDF and increased the rate of production of gas during fermentation. Removal of minerals from the cell wall matrix appeared to be responsible for the increase in NDF digestibility caused by phosphate treatment. The possible commercial use of phosphate on-farm to upgrade nutritional quality of straws is discussed.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Pretreatment; Rumen microbes; Digestibility; Neutral detergent; Phosphate 86 (2000) 177±190
*Corresponding author. Tel.:61-7-32142840; fax:61-7-32142882. E-mail address: brian.lowry@tag.csiro.au (J.B. Lowry).
1. Introduction
Increasing digestibility of plant ®bre for use by ruminants is desirable both in European countries, where excess crop residues are a disposal problem, and in tropical countries where there is dry-season loss of pasture quality and optimal utilisation of ®brous residues is essential for animal production. Much research has been directed towards chemical treatment of crop residues and improvements have been achieved by treatment with alkali or urea or oxidative methods (e.g. alkaline hydrogen peroxide) which increase accessibility to cell wall polysaccharides by microbial enzymes. However, alkali and peroxide treatments have attendant safety and environmental concerns and treatment with urea solution requires several weeks for the evolved ammonia to react with the roughage.
In contrast to the partial solubilisation of hemicellulose that occurs during alkali treatment, boiling of feedstuffs in neutral detergent (ND) was developed to provide a laboratory assessment of ®bre by removing cell solubles (Van Soest et al., 1991). Kennedy et al. (1999) demonstrated that treatment of some mature grasses with boiling ND solution for 1 h increased extent and rate of digestion by rumen microbes measured in vitro. This enhancement of microbial digestion was substantial for spear grass (Heteropogon contortus), with digestibility of ND ®bre (NDF) increasing by 50% after 48 h fermentation by rumen microbes. In those experiments, we did not assess whether a milder treatment requiring fewer chemicals or lower temperatures would produce a similar improvement in digestion.
Accordingly, the aim of the present investigation was to de®ne which component(s) (i.e. sodium dodecyl sulphate, sodium phosphate, sodium EDTA or sodium borate) of the ND salts mixture was responsible for the increased digestibility, and whether similar effects could be achieved using non-ionic detergents and treatment temperatures lower than 1008C. Initially, we suspected that the effect was due to surfactant dislodging a lignin fraction, as indeed occurs to a much larger extent during acid detergent treatment (Lowry et al., 1994). Alternatively, EDTA, by chelation and removal of calcium ions from the pectic components of the cell wall matrix, or borate, because of its well known ability to complex with hydroxyl groups in certain con®gurations (Sinner et al., 1975), might contribute to the activity shown by the complete ND. A subsidiary aim of this study was to determine whether less rigorous conditions that are more applicable for farm-scale applications, would result in economically signi®cant increases in ®bre digestion.
2. Materials and methods
2.1. Substrates and experiments
2.1.1. Experiment 1
Spear grass, ground through a 1.5 mm screen, was boiled under re¯ux for 1 h, or shaken at 208C for 1 h at 20 g/l, in the solutions in Table 1. These included the buffer mixture used in the ND solution, or 0.1 M bis-Tris. Individual components of the ND solution (i.e. 0.05 M Na2 EDTA, 0.018 M Na2B4O7, 0.015 M Na2HPO4 plus 0.015 M NaH2PO4) were adjusted to pH 7.0 by addition of HCl or NaOH. Detergents used were sodium dodecyl sulphate (SDS, BDH 44244, 30 g/l), and the non-ionic detergents Triton X-100 (Calbiochem 6484620, 30 g/l), and Tween 20 (polyoxyethylene-sorbitan monolaurate, Sigma P-1379, 30 g/l). Residues were recovered after treatment, on polyester cloth with a 25mm aperture and washed 11 times with 500 ml water at 95±1008C (substrates 2±12) or at 208C (substrate 13) and dried at 508C. Absorbance of each extract was measured at 280 nm against the appropriate extractant blank. A sample was subjected to centrifugal ultra®ltration through a 3000 Da membrane (Amicon Centriprep 3) and the polymeric contribution calculated from the absorbance of ultra®ltrate and retentate.
The NDF content (g/kg DM) and OM content (g/kg NDF) of substrates prepared from the same batch of spear grass, are in Table 1.
Substrates were fermented for 120 h in vitro, as described below. Gas pressure was manually monitored at intervals of 6±24 h, and NDF was recovered at 120 h as described below.
Table 1
Content in substrates prepared from the same batch of spear grass, of neutral detergent ®bre (NDF) and organic matter (OM), resulting from treatment by various surfactants and salts, together with the increase in NDF digestibility after 120 h of in vitro fermentation
Substrate NDF content (g/kg DM)
OM (g/kg NDF)
Temperature Surfactant Saltsa Increase in digestibility (g/kg)b
1 750 947 0
2 928 982 boiling SDS 32
3 943 977 boiling SDS P, B, E 73
4 938 971 boiling SDS bis-Tris 41
5 924 977 boiling Triton X-100 P, B, E ÿ5
6 908 968 boiling Triton X-100 bis-Tris ÿ19
7 896 968 boiling Tween 20 34
8 912 975 boiling Tween 20 bis-Tris 36
9 930 982 boiling P,B,E 46
10 929 970 boiling E 30
11 910 979 boiling B 31
12 922 986 boiling P 50
13 902 962 208C SDS P, B, E 11
aE: 0.05 M Na
2EDTA; B: 0.018 M Na2B4O7; P: 0.015 M NaH2PO40.015 M Na2HPO4, all adjusted to
pH 7.0.
bIncrease in NDF digestibility compared to treatment 1. All differences from treatment 1 were signi®cant
(P<0.001, MS error, 0.353) with the exceptions of treatments 5 and 13. NDF digestibility (120 h) of treatment 1 was 472 g/kg.
2.1.2. Experiment 2
Substrates were 1, 9 and 12 from experiment 1, and spear grass boiled for 1 h with 0.03 M PO43
ÿ
and 0.05 M Na2EDTA adjusted to pH 7.0. The latter treatment yielded a substrate comprising 924 g NDF/kg DM and 954 g OM/kg NDF. NDF was recovered after in vitro digestion for 24, 48 72, 96 and 216 h.
2.1.3. Experiment 3
Spear grass, ground through a 1.5 mm screen, was boiled in ND salts for 5, 15, 30 and 60 min. In addition, spear grass was boiled under re¯ux for 60 min in ND salts at half and double normal concentration, or in water, 2% (w/v) NaCl, 2% (w/v) Na2SO4or 0.03 M PO43ÿ, all adjusted to pH 7.0. The 10 prepared substrates, plus the spear grass from which they were derived, were subjected to in vitro incubation for 120 h.
2.1.4. Experiment 4
Substrates of spear grass and of bagasse, with an original NDF content of 740 and 908 g/kg DM respectively, were prepared by boiling under re¯ux in water, 0.03 M PO43
ÿ
or ND solution for 60 min as described above and fermented for 120 h in vitro. Gas pressures were monitored at 12 h intervals, and NDF was recovered after 120 h.
2.1.5. Experiment 5
Mature carpet grass (Axonopus af®nis), angleton grass (Dicanthium aristatum), rhodes grass (Chloris gayana) and spear grass, ground to pass a 1 mm screen, were boiled under re¯ux in 0.03 M PO43ÿ(pH 7.0) for 0, 10, 20 30 and 60 min. For spear grass, additional re¯uxing times of 1, 3, and 6 min were used. Substrates were simultaneously incubated with rumen ¯uid for 120 h. Gas pressures were recorded at 3, 9, 18, 24, 32, 40 and 48 h and data, excluding 3 h, was ®tted to the equation:yab(1ÿeÿct
), whereyis the gas pressure andtis the sampling time. Time required for evolution of 50% of the 48 h value was calculated as an index of fermentation kinetics not confused by gas derived from microbial lysis.
2.1.6. Experiment 6
Spear grass, ground to pass 1 mm screen, was boiled (20 g/l of 0.03 M PO43 ÿ
, pH 7.0) for periods of 1±30 min under re¯ux or maintained in a water bath at 758C (1±480 min), 858C (1±300 min), or 958C (1±120 min)), with N2gas bubbling to simulate agitation at boiling. Substrates were recovered on 25mm polyester cloth and extensively washed (eight times with 100 ml water) at room temperature before being dried at 558C. The content of AIA in NDF (y) was ®tted to the equation: yabeÿct
, where t is the preparation time in PO43ÿsolution. Gas pressures were recorded at 3, 9, 18, 24, 32, 40, and 48 h during the 120 h fermentation, and were mathematically assessed as in experiment 5.
2.1.7. Experiment 7
differences in chemical composition of the ®nal pulverised and non-pulverised materials, this material was extensively washed on polyester cloth of 50mm aperture to allow passage of ®ne particles. A sample recovered on the cloth was subjected to pulverising in a Tema ring mill for 3 min to produce a ®ne powder which was stirred in water for 10 min and recovered on 10mm polyester cloth. Screens of 150 and 90mm passed 98.4 and 92.4% of this material, respectively. Samples of both substrates were boiled in 0.03 M PO43
ÿ
(pH 7.0) for 60 min, washed as in experiment 6, and recovered on 10mm polyester cloth. A portion of the phosphate-treated 1 mm substrate was pulverised and subjected to a second boiling in 0.03 M PO43
ÿ
for 60 min. Substrates were fermented in vitro for 72 h, while gas pressures were automatically logged at 15 min intervals.
2.2. Fibre digestion and gas production
All in vitro studies used about 100 mg DM of substrate incubated in 50 ml serum bottles following the protocol of Pell and Scho®eld (1993). NDF content of substrate and residues post-incubation were determined as described by Pell and Scho®eld (1993). In some experiments, gas pressure in the triplicate bottles was measured at intervals by puncturing the butyl rubber stopper by a needle attached to a pressure transducer. Corrections were made for changes caused by gas loss due to sampling and for atmospheric pressure variations, and for volume variations between bottles. In experiment 7, the inoculum size was reduced from 3.0 to 0.5 ml of rumen ¯uid and the gas pressure was automatically logged at intervals of 30 min (Pell and Scho®eld, 1993).
Acid-insoluble ash (AIA) was analyzed by the method of Van Keulen and Young (1977).
2.3. Statistical analysis
Differences attributable to treatment at a particular time after inoculation were assessed by analysis of variance. Differences between multiple means, were tested by the Student±Neuman±Kuels range test (Steel and Torrie, 1980). Curve ®tting and associated statistics were completed with Table Curve1 2D (Jandel Scienti®c, San Rafael, CA).
3. Results
3.1. Experiment 1
NDF digestibility increased by 73 g/kg NDF due to treatment with boiling ND solution (i.e. SDS plus ND salts), but at 208C digestibility increased by only 11 g/kg NDF (P>0.05, Table 1). Among those treatments involving single components of the ND solution, 0.03 M PO43
ÿ
(treatment 12) was the most effective, resulting in increased digestibility of 50 g/kg NDF, equivalent to 68% of the increase obtained with the boiling ND solution (difference between treatments 3 and 12,P<0.05). The digestibility increases with SDS or Tween 20 alone, Na2B4O7or Na2EDTA were similar (30±34 g/kg
NDF). Use of Triton X-100 depressed (P<0.001) digestibility, whereas use of bis-Tris did not improve NDF digestibility. NDF digestibility was positively related to OM content of the substrate with the exception of those substrates prepared with Triton X-100 (Fig. 1) and also to the gas production between 3 and 19 h post inocula-tion (Fig. 2).
The highest concentration of solubilised lignin, based on absorbance at 280 nm and the proportion which was polymeric, was obtained with SDS plus ND salts. SDS alone extracted 54% of that maximum amount and the ND salts extracted 61%. The proportions extracted by the individual ionic components (EDTA2ÿ
, B4O7 2ÿ
, PO4 3ÿ
) were 52, 46 and 62%, respectively.
Fig. 1. Experiment 1. Relationship of NDF digestibility to OM content in spear grass subjected to treatments described in Table 1. Relationship, excluding treatments 5 and 6:yÿ1390.689x, S.E. 12.7,P<0.01.
3.2. Experiment 2
The increase (110 g/kg NDF ) in NDF digestibility when spear grass was treated with PO4
3ÿ
was evident after 24 h and was not increased further by addition of Na2EDTA or by Na2EDTA plus Na2B4O7(Fig. 3).
3.3. Experiment 3
There was a curvilinear relationship between NDF digestibility at 120 h, and time of boiling in ND salts during substrate preparation (Fig. 4). NDF digestibility for untreated spear grass (489 g/kg NDF), was progressively increased by boiling for 60 min in water, Fig. 3. Experiment 2. Time course of NDF digestion of untreated spear grass (*) or spear grass boiled for 60 min with ND salts (&) with PO43
ÿ
plus Na2EDTA (^) or PO43
ÿ
(&), S.E. 0.436. Three observations per mean.
Fig. 4. Experiment 3. Effect on spear grass of increasing time of boiling in ND salts on OM content (*), S.E. 1.1, NDF content (&), S.E. 1.5, and NDF digestibility after 120 h of fermentation (^), S.E. 1.54. Values are means of 2, 2, and 3 data, respectively.
2% NaCl, 2% Na2SO4, double ND salts, 0.03 M PO43 ÿ
, half ND salts and ND salts; values were 518, 536, 543, 572, 575, 577 and 586 g/kg NDF, respectively (S.E. of differences, 3.56). The data comprised four groups within which there was similar digestibility, but between which digestibility differed (P<0.05). These groups comprised (a) untreated spear grass, (b) grass boiled with water, (c) NaCl and Na2SO4treatments, (d) substrates prepared using all solutions containing PO43
ÿ
. Substrate treated with PO43 ÿ
alone was less digestible (P<0.05) than ND salts; relative to spear grass treated with boiling water, these treatments were 85% as effective as ND salts in increasing digestibility.
3.4. Experiment 4
For both spear grass and bagasse which had been boiled in water, treatment with PO43ÿ increased NDF digestibility by about 50% of the increase seen with ND (Fig. 5), but the absolute responses were greater in bagasse than for spear grass. NDF digestibility of bagasse boiled in water, neutral PO43
ÿ
or ND, was 186, 230 and 277 after 120 h, compared with 162 g/kg NDF for untreated bagasse. NDF digestibility of spear grass boiled in water, neutral PO43
ÿ
or ND, was 499, 522 and 545, compared with 472 g/kg NDF for untreated grass. Cumulative gas production (data not shown) showed that the increased gas production due to treatment by PO43
ÿ
or ND had developed after 24 h of fermentation for spear grass, whereas about 72 h was required for bagasse.
3.5. Experiment 5
The predicted asymptote in NDF digestibility after boiling in PO43ÿwas equivalent to 115.6, 113.5, 108.1 and 104.9 % of the digestibility of untreated carpet, angleton, spear and rhodes grasses, respectively (Fig. 6).
The relationship between NDF digestibility and content of AIA in NDF differed markedly between diets (Fig. 7a). With the exception of angleton grass, NDF digestibility Fig. 5. Experiment 4. Effect of boiling for 60 min in water, 0.03 M PO43
ÿ
PO42
ÿ
for angleton (*), rhodes (&), carpet (^) and spear (~) grasses. S.E. 0.420, three observations per mean.
Fig. 7. (a) Experiment 5. Relationship of NDF digestibility at 120 h with acid insoluble ash content of NDF for PO43
ÿ
increased with a decrease in AIA content of NDF. Furthermore, within grasses, content of AIA in NDF was positively related to time required for evolution of 50% of the 48 gas value (Fig. 7b). For angleton and rhodes grass, the relationship was not seen until substrates had been boiled for 20 and 10 min, respectively.
3.6. Experiment 6
Untreated grass contained 48 g AIA/kg NDF. The effects of preparation temperature on the content of AIA content in NDF were approximated by ®tting mono-exponential relationships (Fig. 8). Increasing temperature decreased they-intercepts and increased the rate of decline with time of AIA/NDF. At 758C, 480 min of treatment resulted in an increase in digestibility to 585 g/kg NDF; this digestibility was achieved in 180 min at 858C, within 45 min at 958C, and in 3 min for boiled substrate. NDF digestibility was inversely related to the content of AIA in NDF, but there was a discontinuity in the relationship, with data from substrates subjected to higher temperatures (958C, times greater than 30 min, boiling longer than 6 min) having higher digestibilities at a given AIA content than the other substrates (Fig. 9a). The time required for evolution of 50% of observed gas at 48 h was positively related to the AIA content of NDF (Fig. 9b).
3.7. Experiment 7
Reduction of particle size of spear grass increased NDF digestibility at 72 h from 400 to 552 g/kg. The effect of boiling with 0.03 M PO43ÿfor 60 min was similar for both particle sizes and resulted in increased NDF digestion of 81±96 g/kg NDF. A second boiling with PO43
ÿ
after grinding of PO43 ÿ
-treated grass, did not further increase digestibility.
Fig. 8. Experiment 6. Relationship of acid-insoluble ash with preparation time in 0.03 M PO43
ÿ
at 758C (*), 858C (&), 958C (^) and boiling (~) temperatures. 758C;y14.434.0 exp(ÿ0.00580x), S.E. 2.557,P<0.001. 858C; y13.433.5 exp(ÿ0.0214x), S.E. 1.60, P<0.001. 958C; y9.6421.3 exp(ÿ0.0307x), S.E. 0.466,
4. Discussion
The ®nding that most of the increase in ®bre digestibility in boiling ND solution was attributable to PO43
ÿ
, with much less attributable to the surfactant (SDS), was surprising. Although effects of SDS and Tween 20 surfactants were similar, the depression of ®bre digestibility with Triton X-100 may have been caused by microbial inhibition by some surfactant binding to the ®bre despite exhaustive washing. The effects of the relatively mild treatments in the experiments reported here are small compared with those caused by more severe conditions. For example, the 42% increase in ®bre digestibility observed when bagasse was boiled with PO43ÿis smaller than 100% increase with lime treatment (10% for 1±2 h at 1008C) or phosphoric acid (3% at 1978C/13.5 atmospheres pressure) Fig. 9. (a) Experiment 6. Relationship of NDF digestibility at 120 h with acid insoluble ash content of NDF for PO43
ÿ
-treated spear grass prepared at 758C (*), 858C (&), 958C (^) and boiling (~) temperatures. Upper line:y634ÿ1.8375x, S.E. 0.388,P<0.001. Lower line:y614ÿ1.638x, S.E. 4.096,P<0.001. (b) Experiment 6. Relationship of estimated time (h) for 50% of 48 h gas evolution with acid insoluble ash content of NDF of PO43
ÿ
-treated spear grass prepared at 758C (*) 858C (&), 958C (^) and boiling (~) temperatures.
y19.3750.1062x, S.E. 0.5672,P<0.001.
(Deschamps et al., 1996; Gandi et al., 1997). More severe conditions such as alkali treatment combined with disruption of the cell wall matrix by steam explosion allows solubilisation of up to 70% of bagasse OM by Onozuka/pepsin (Playne, 1984). The effect on rate of digestion of cell wall constituents due to boiling with PO43
ÿ
is possibly related to increased surface area accessible to microbial cellulases (Weimer et al., 1990), although gross modi®cation of cell wall structure would not be expected (Akin et al., 1975). Alternatively, given that microbial adhesion is mediated by the chemistry of the surface layer (Russell et al., 1988), it might be due to more effective colonisation or earlier cellulase secretion by the rumen bacteria adhering to the plant surface. The results of experiment 7, in which an enhanced rate of digestion caused by PO43
ÿ
treatment of ground grass was observed even after pulverising spear grass, indicated that the PO4
3ÿ
effect is additional to that due to reduction of surface area constraints affecting microbial digestion.
There is evidence that mineral deposits in the cell wall provide a barrier to microbial digestion, which is supported by the relationships between ®bre digestibility and the content of AIA in NDF in Fig. 7a and Fig. 9a. In the case of cereal straws, silica bodies as well as cuticle and lignin cell wall in the outer layers, are not subjected to appreciable attack by rumen microbes whereas the ready digestion of alkali-treated straws was partially attributed to removal of silica bodies (Russell et al., 1988). ND solutions tend to remove chlorine and potassium (McManus et al., 1979). The other elements, predominantly silicon, phosphorus and calcium form a mineral crystalline intrusion throughout the structures of the cell wall, with silicon predominating in the case of grasses (McManus et al., 1979). McManus (1983) observed that the cell ash digestibility accounted for 75% of the variation of cell wall digestibility in 15 grasses, and concluded that the mineral component contributes to the `masking' of potentially digestible carbohydrate. Emanuele and Staples (1990) concluded that a large amount of Ca was associated with the cell wall, and that the rumen was the major site of its slow release. The effectiveness of PO43ÿ treatment in removing minerals from cell wall was not measured in our experiments, except as AIA content of cell wall, but clearly there was an increased rate, as well as extent, of ®bre digestion (Fig. 7b and Fig. 9b). In addition, absorbance results show that PO43ÿ extracts much less of the lignin fraction than is extractable with hot ND, and thus modi®cation of the bonds within the lignin-polysaccharide matrix would seem of minor importance to the increase in digestibility. Agitation of substrate during PO43
ÿ
treatment, as provided in the present experiments during boiling or by bubbling with nitrogen gas, appears to markedly accentuate the improvement in ®bre digestibility (Kennedy et al., unpublished results), presumably by facilitation of mineral removal.
At 958C and boiling temperatures with PO43 ÿ
production. For the four grasses studied, increased NDF digestibility in carpet and angleton grasses appeared to be mainly through mineral leaching and high temperature responses respectively, whereas both mechanisms appeared effective in rhodes and spear grasses.
5. Conclusion
Results demonstrate that temperatures lower than 1008C can be employed to increase ®bre digestibility provided prolonged exposure times are used. This poses the question as to whether a practical farm scale process could be developed and suggests that currently uneconomic pre-treatments, such as steam explosion of bagasse (Playne, 1984) or steaming or soaking of rice straw (Doyle et al., 1986), may be worth re-investigating with the inclusion of PO43ÿin the medium. In addition to increasing digestibility of NDF, the nutrition of ruminants may be improved through increased supply of sodium and phosphorus, particularly since tropical animals are often chronically sodium de®cient.
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