EFFECT OF DIFFERENT HEAT TREATMENTS ON THE
STANDARDIZED ILEAL DIGESTIBILITY OF AMINO
ACIDS IN SOYBEAN MEAL FED TO GROWING PIGS
Ulrike MESSERSCHMIDT1, Meike EKLUND2, Vanessa T.S.RIST1, Pia
ROSENFELDER1, Hanna K.SPINDLER1, John K.HTOO3, Rainer MOSENTHIN4
ABSTRACT
The effect of heat treatment (mild, medium and strong) of soybean meal (SBM) on standardized
ileal digestibility (SID) values of crude protein (CP) and amino acids (AA) was determined in
growing barrows. The pigs were surgically fitted with simple ileal T-cannulas. The 3 SBM
cornstarch-based assay diets were fed to 6 pigs according to a replicated 3 × 3 Latin square design.
The basal ileal endogenous losses of CP and AA were determined using a nitrogen-free diet. The
SID of CP was 39, 72 and 49 % for the mild, medium und strong heated SBM, respectively. The
SID of AA in the mild heated SBM ranged from 29 % for cystine to 55 % for arginine. In the
medium heated SBM, SID of AA ranged from 59 % for cystine to 82 % for arginine. In the strong
heated SBM, SID of AA ranged from 38 % for cystine to 62 % for arginine. Standardized ileal
digestibility of CP and all AA was higher in medium heated SBM compared to mild and strong
heated SBM (P < 0.001). The SID of all AA in mild heated SBM was lower (P < 0.001) than in
medium und strong heated SBM. The large differences in SID values between SBM obtained in
the present study indicate considerable impact of different heat treatment conditions on protein
quality of SBM for growing pigs.
Keywords: standardized ileal digestibility, soybean meal, heat treatment, growing pig
1
INTRODUCTION
Soybeans (SB) play a major role worldwide as a plant protein component of diets for nonruminant livestock (Clarke and Wiseman, 2000). Raw SB or soybean meal (SBM), which
has been inadequately processed during solvent extraction (Baker, 2000), are known to
have a negative influence on feeding value when fed to non-ruminant animals (CervantesPham and Stein, 2010). This is mainly due to the presence of various anti-nutritional
factors (ANF), which may exert negative effects on digestion and utilization of nutrients
(Liener, 1989). The activity of trypsin inhibitors (TI), the most deleterious ANF in raw
SB, is higher in SB than in other legume seeds (Leterme et al., 1988; Jezierny et al., 2010).
Therefore, it is necessary to remove these ANF before SB or its by-products are included
in the diet of non-ruminants. If the ANF are inactivated by proper thermal processing,
SBM is an excellent source of plant protein source. Due the content of crude protein (CP)
and amino acids (AA) in SBM, which complements that of cereal grains, this feedstuff has
been the predominant protein source used to supplement swine diets (Dilger et al., 2004).
However, the effects of processing on final product quality may vary considerably as
influenced by different processing procedures or conditions (Qin et al., 1996). An ideal
thermal treatment procedure aims to sufficiently inactivate ANF while simultaneously
maintaining the bioavailability of essential AA in the product (Van Barneveld, 1993).
1
Dipl.-Agr.Biol., Institute of Animal Nutrition, University of Hohenheim, 70593 Stuttgart, Germany
Dr. sc. agr., Institute of Animal Nutrition, University of Hohenheim, 70593 Stuttgart, Germany
3
Dr. sc. agr., Evonik Industries AG, Rodenbacher Chaussee 4, 63457 Hanau-Wolfgang, Germany
4
Prof. Dr. Dr. h.c., Institute of Animal Nutrition, University of Hohenheim, 70593 Stuttgart, Germany
2
Under-toasting during processing of SBM may result in incomplete removal of some ANF
(Araba and Dale, 1990) which, in turn, may decrease AA digestibilities in these SBM.
Overheating, both in terms of excessive temperature and (or) exposure time to heat
treatment may depress digestibilities of AA, in particular lysine (Van der Poel et al.,
1990). Therefore, the present study was designed to investigate the effects of different
thermal processing conditions in terms of temperature variation and duration of heat
treatment (mild, medium and strong) on standardized ileal digestibilities (SID) of CP and
AA in SBM for growing pigs.
2
MATERIALS AND METHODS
2.1
Animals and housing
The experiment was conducted with 6 barrows from the University of Hohenheim
Research Station (German Landrace × Piétrain), with an average initial and final body
weight (BW) of 23 ± 0.7 kg and 54 ± 2.8 kg, respectively. The pigs were kept in metabolic
crates (0.80 m × 1.50 m). The temperature in the research unit, which was equipped with
an automated temperature control system, was 20 ± 2 °C. Each crate was equipped with an
infrared heating lamp and a low pressure drinking nipple. Until the beginning of the
experiment, the pigs were fed a commercial starter diet (Porcigold®SMA 134, RKW Süd,
Germany) containing 170 g CP/kg (as-fed).
2.2
Surgical procedure
The pigs were fitted with a simple T cannula at the distal ileum according to the principles
described by Li et al. (1993). The cannulas were prepared from high molecular weight
polyethylene. The internal diameter of the barrel of the cannulas was 17 mm, the length of
the barrel was 80 mm and each of the 2 curved flanges was 55 mm in length. The washer
hat 70 mm in diameter and screw caps were used to seal the cannulas. During the
experiment, the skin around the cannula was cleaned with lukewarm water several times
daily, dried and covered with a skin protection paste (Stomahesive® Paste, Convatec,
Princeton, USA). Additionally, a sterile pad (Rondopad®, DEWECO Dr. Wüsthoff & Co.,
Wermelskirchen, Germany) was adjusted between the retaining ring and the skin to absorb
leaking digesta to prevent erythema. The pigs were allowed a recovery period of 14 days
(d). During this time period, the feed allowance was gradually increased, starting with 50
g (as-is)/d the day after surgery until 35 g (as-is)/kg average BW were consumed. The
research protocol was approved by the German Ethical Commission for Animal Welfare.
Care of the animals used in this experiment was in accordance with the guidelines issued
by German Regulation for Care and Treatment of Animals (Lorz and Metzger, 1999).
2.3
Soybean meal processing
Initially 500 kg of raw SB from the same batch (Audecoop cooperative, France) was
processed to produce differently heat-treated SBM according to the established method at
the research facility of Creol located in Pessac, France. After cracking, dehulling and
flaking, SB was defatted by extracting the oil with hexan heated at 50 °C in a batch
extractor before being toasted. The defatted SBM was divided into 3 groups to be toasted
under different conditions. Toasting was conducted in a pilot desolventizer (Desmet
Schumacher type), which has 1 m diameter and a capacity for indirect steam treatment
pressures from 1 to 9 bars to heat the trays of the desolventizer. Several holes in the tray
allowed for direct steam treatment. The exit orifice was closed, and the meals were filled
manually into the bottom tray.
Mild SBM received indirect steam pressure at 1.5 bar. As soon as meal temperature
reached 80 °C, the direct steam valve was opened until 105 °C were measured in the meal
(approximately 34 min). The meal remained at this temperature level in the desolventizer
for 20 min.
Medium SBM received indirect steam pressure at 5 bar. As soon as meal temperature
reached 80 °C, the direct steam valve was opened until 105 °C were measured in the meal
(approximately 45 min). The temperature was further increased to a maximum of 112 °C.
Thereafter, the meal remained at this temperature level in the desolventizer for 20 min.
Strong SBM received indirect steam pressure at 8.5 bar. As soon as meal temperature
reached 80 °C, the direct steam valve was opened until 105 °C were measured in the meal
(approximately 7 min). The temperature was further increased to a maximum of 139 °C.
Thereafter, the meal remained at this temperature level in the desolventizer for 30 min.
2.4
Experimental design and dietary treatments
The experiment was arranged as a replicated 3 × 3 Latin square design. The 3 SBM
containing diets were formulated to meet or exceed the dietary threshold levels for CP and
AA according to Fan et al. (1994), and the NRC (1998) nutrient recommendations for
growing pigs in a BW range from 20 to 50 kg. A nitrogen-free diet was used to determine
the basal ileal endogenous loss of CP and AA (Stein et al., 2007). The pigs received their
assay diets at a daily level of 35 g (as-fed)/kg of their average BW, corresponding to 3
times the energy requirement for maintenance (i.e., 106 kcal metabolizable energy ×
BW0.75; NRC, 1998). The BW of the pig was determined at the beginning of each
experimental period. The formulation of the assay diets was based on the analyzed AA
composition of the SBM (Tab. 1). Each assay feed ingredient was added to a cornstarchbased basal diet (Tab. 2).The assay diets were fed in a mash form mixed with water (1/1
w/v) twice daily in 2 equal meals at 06.00 and 18.00 h.
Table 1. Analyzed chemical composition of the assay feed ingredients (g/kg dry matter)
Treatment
Dry matter
Crude protein
Neutral detergent fiber
Acid detergent fiber
Ash
Ether extracts
TIA1
Indispensable amino acids
Arginine
Histidine
Isoleucine
Leucine
Lysine (total/reactive)
Methionine
Phenylalanine
Threonine
Tryptophan
Valine
Dispensable amino acids
Alanine
Aspartic acid
Cystine
Glutamic acid
Glycine
Proline
Serine
Mild
920
559
155
33
79
16
17.6
Soybean meal
Medium
930
561
213
30
79
16
4.4
Strong
947
559
284
37
78
18
17.1
41.8
15.3
25.4
43.3
33.1/30.4
8.0
28.3
21.9
7.8
26.7
41.2
15.0
26.1
43.7
32.3/28.3
8.0
28.3
22.4
7.7
27.4
40.6
14.4
25.8
43.4
29.9/25.0
7.9
27.8
22.3
7.6
27.0
24.6
65.4
8.6
103.9
24.0
25.5
28.4
24.6
66.6
8.6
104.3
24.1
24.2
28.2
24.3
65.7
8.4
103.3
23.8
24.3
28.0
1
TIA = trypsin inhibitor activity in mg/g CP (88% DM).
Table 2. Formulation of the assay diets (g/kg, as fed)
Treatment
Soybean meal1
Cornstarch2
Dextrose2
Cellulose3
Oil4
Vitamin and mineral premix5
Monocalcium phosphate
Calcium carbonate
Titanium dioxide
1
Soybean meal assay diets
400.0
412.0
100.0
30.0
30.0
16.0
2.5
4.5
5.0
Nitrogen-free
–
812.0
100.0
30.0
30.0
16.0
2.5
4.5
5.0
Mild, medium or strong heated.
Roquette, GmbH, Frankfurt, Germany.
3
Arbocell®, J. Rettenmaier & Söhne GmbH + Co.KG, Rosenberg, Germany.
4
Blend of 750 g/kg rapeseed oil and 250 g/kg soybean oil.
5
Vilomin 18950, Deutsche Vilomix Tierernährung GmbH, Neuenkirchen-Vörden, Germany.
2
2.5
Experimental procedure
Each experimental period included 5 d for adaptation to the assay diets and 2 d for digesta
collection. Ileal digesta were collected for a total of 24 h on d 6 from 06.00 to 18.00 h and
on d 7 from 18.00 to 06.00 h. The collection procedure was adapted from Li et al. (1993)
using plastic tubing attached to the barrel of the cannula by elastic bands. The bags were
changed at least every 20 min and immediately frozen at -30 °C. During collection, 4 ml
of 2.5 M formic acid were added to the sampling bags to minimize further bacterial
fermentation. Samples of digesta were pooled within each animal and period, freeze-dried
and ground to 0.5 mm.
2.6
Analytical procedure
Determination of dry matter (DM) and proximate nutrients (except for crude fiber, CP and
AA) in the SBM samples were performed as outlined by Naumann and Bassler (1997).
Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined as
described by Van Soest (2006). The nitrogen contents in the SBM, diets and ileal digesta
samples were analyzed using a gas combustion method according to the official method
990.03 of the Association of Official Analytical Chemists (AOAC, 2000; FP-2000, Leco
Corp., St. Joseph, MI, USA). Ethylenediaminetetraacetic acid was used as a reference
standard before and after all nitrogen analyses. The AA contents in the SBM, diets and
ileal digesta samples were determined by using ion-exchange chromatography with
postcolumn derivatization with ninhydrin. The AA were oxidized with performic acid
which was neutralized with sodium metabisulfite (Llames and Fontaine, 1994;
Commission Directive, 1998), and they were hydrolyzed by means of 6 N HCl for 24 h at
110 °C. Afterwards, they were quantified with the internal standard method by measuring
the absorption of reaction products with ninhydrin at 570 nm. Tryptophan was determined
by HPLC with fluorescence detection (extinction 280 nm, emission 356 nm), after alkaline
hydrolysis with barium hydroxide octahydrate for 20 h at 110 °C (Commission Directive,
2000). Tyrosine was not determined. Reactive Lys contents in the SBM batches were
determined according to Fontaine et al. (2007). Titanium dioxide concentrations in the
assay diets and ileal digesta samples were analyzed according to the method described by
Brandt and Allam (1987).
2.7
Calculations and statistical analysis
The apparent ileal digestibilities (AID) of CP and AA in the assay diets were calculated
according to the following equation:
AIDD = 100% - [((ID / IF) × (AD / AF)) × 100%]
where
AIDD = apparent ileal digestibility of CP or AA in the assay diet (%)
ID = marker concentration in the assay diet (g/kg DM)
AF = nutrient concentration in ileal digesta (g/kg DM)
AD = nutrient concentration in the assay diet (g/kg DM)
IF = marker concentration in ileal digesta (g/kg DM).
The basal ileal endogenous loss (IAALB) of CP and AA were determined according to the
following equation:
IAALB = ((AAD × (ID / IF))
where
IAALB = the basal endogenous loss of CP or AA (g/kg dry matter intake (DMI))
AAD = CP or AA content in the assay diet (g/kg DMI)
ID = marker concentration in the assay diet (g/kg DM)
IF = marker concentration in ileal digesta (g/kg DM).
The SID of CP and AA in the assay feed ingredients were calculated by correcting AID of
CP and AA in the assay diets for basal ileal endogenous losses of CP and AA expressed as
g/kg DMI. The SID of CP and AA in the assay feed ingredients were obtained according
to the following equation:
SIDD = AIDD + ((IAALB / CPD or AAD) × 100%)
where
SIDD = SID (%) of CP or AA in the assay feed ingredients
AIDD = AID of CP and AA in the assay diets (%)
IAALB = basal endogenous loss of CP or AA (g/kg DMI)
CPD or AAD = CP or AA (g/kg DM) content in the assay feed ingredients
The data were subjected to mixed model analysis using the MIXED procedure of SAS
(2008). The linear model included the fixed effects of SBM treatment and animal. Periods
and animals within a square were considered as random effects assuming a compound
symmetry variance-covariance structure. Homogeneity of variances and normal
distribution of the data were confirmed by analysis of the residuals using the
UNIVARIATE procedure of SAS (2008). All results are reported as least square means
(LSmeans). Multiple comparisons among SBM treatments were performed using a t-test
with degrees of freedom determined by the Kenward-Roger-method (Kenward and Roger,
1997). The significance level for all pair-wise t-tests was α ≤ 0.05. Significant differences
between SBM treatments were indicated by different superscript letters using the
algorithm for letter-based representation of all pair-wise comparisons according to Piepho
(2004). Basal ileal endogenous CP and AA losses were calculated as means ± standard
deviation using the GLM procedure of SAS (2008).
3
RESULTS AND DISCUSSION
3.1
General observations
All animals were healthy throughout the experiment and readily consumed their feed
allowances.
3.2
Chemical composition of soybean meal and assay diets
The analyzed chemical composition of the assay feed ingredients and the assay diets are
presented in Tables 1 and 2, respectively. The CP content of the differently heated SBM
averaged 560 g CP/kg DM. The analyzed chemical composition in the SBM containing
assay diets (Tab. 3) is in good agreement with that calculated from the single ingredients,
and met the dietary threshold levels for CP and AA in pigs (Fan et al., 1994). The values
of all AA with the exception of lysine were higher than average values for SBM (480 g
CP/kg (as is)) reported by (AminoDat® 4.0). The values for total lysine and reactive lysine
decreased from mild to strong heated SBM indicating a steady increase in lysine damage
due to more intensified heat treatment (Fontaine et al., 2007). The content of NDF also
increased by increasing the intensity of heat treatment to the SBM.
Table 3. Analyzed crude protein and amino acid composition of the assay diets (g/kg dry matter)
Treatment
Dry matter
Crude protein
Indispensable amino acids
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Valine
Dispensable amino acids
Alanine
Aspartic acid
Cystine
Glutamic acid
Glycine
Proline
Serine
Mild
908
207
Soybean meal
Medium
916
245
Strong
922
230
15.7
5.6
9.7
16.6
13.0
3.0
11.0
8.6
3.0
10.2
16.4
5.8
10.3
17.5
13.2
3.1
11.5
9.0
3.2
10.7
16.4
5.9
10.5
17.7
12.6
3.0
11.6
9.1
3.1
11.1
9.4
24.9
3.2
39.5
9.1
11.0
11.0
9.8
26.4
3.5
41.7
9.6
11.5
11.7
10.0
26.7
3.5
42.1
9.8
11.3
11.6
The IAALB, determined by means of the nitrogen-free method, amounted to 12.2 g/kg
DMI for CP, and ranged from 0.1 g/kg DMI for methionine to 1.1 g/kg DMI for glutamic
acid (Tab. 4). The IAALB values of CP and AA determined in the present study were
similar to mean values summarized by Jansman et al. (2002).
Table 4. Basal ileal endogenous losses of crude protein and amino acids (g/kg dry matter
intake, mean ± standard deviation)
Crude protein
Indispensable amino acids
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Valine
Dispensable amino acids
Alanine
Aspartic acid
Cystine
Glutamic acid
Glycine
Proline
Serine
3.3
Basal ileal endogenous losses
12.2 ± 2.62
0.5 ± 0.12
0.2 ± 0.05
0.4 ± 0.13
0.7 ± 0.20
0.5 ± 0.18
0.1 ± 0.05
0.4 ± 0.14
0.5 ± 0.13
0.2 ± 0.05
0.5 ± 0.16
0.6 ± 0.14
0.9 ± 0.28
0.2 ± 0.05
1.1 ± 0.34
0.9 ± 0.20
1.0 ± 0.62
0.5 ± 0.12
Effect of heat treatment on standardized ileal digestibility of crude protein and
amino acids
The SID of CP was 39, 72 and 49 % for the mild, medium und strong heated SBM,
respectively (Tab. 5). The SID of indispensable AA in the mild heated SBM ranged from
40 % for leucine to 55 % for arginine. In the medium heated SBM, the SID of
indispensable AA ranged from 69 % for threonine to 82 % for arginine. In the strong
heated SBM, SID of indispensable AA ranged from 47 % for tryptophan to 62 % for
arginine. The SID of AA in the mild heated SBM was up to 12 percentage units lower
compared to the strong heated SBM, with significant differences for arginine, isoleucine,
leucine, phenylalanine, valine, alanine, aspartic acid, glutamic acid and serine. The SID
values in the medium heated SBM were up to 35 percentage units higher compared to the
mild heated SBM with significant differences for CP and all indispensable and
dispensable AA.
Table 5. Standardized ileal crude protein and amino acid digestibility (%, LSmean ±
standard error of mean) in differently heat treated soybean meal
Treatment
Mild
Crude protein
39a
Indispensable amino acids
Arginine
55a
Histidine
47a
Isoleucine
41a
Leucine
40a
Lysine
45a
Methionine
48a
Phenylalanine
43a
Threonine
42a
Tryptophan
43a
Valine
42a
Dispensable amino acids
Alanine
43a
Aspartic acid
39a
Cystine
29a
Glutamtic acid
46a
Glycine
39a
Proline
41 ± 2.7a
Serine
42a
Medium
72c
Strong
49b
Pooled SEM
2.5
P-values
< 0.001
82c
74b
76c
75c
68b
80b
76c
69b
73b
73c
62b
52a
53b
51b
48a
56a
54b
49a
47a
52b
2.4
2.6
2.5
2.5
2.7
2.8
2.4
2.5
2.5
2.5
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
70c
68c
59b
72c
65b
75 ± 2.6b
74c
51b
48b
38a
54b
46a
47 ± 1.7a
50b
2.4
2.4
3.5
2.6
2.9
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
2.4
Within a row LSmean with a common superscript are not significantly different at α ≤ 0.05
SEM = standard error of mean
LSmean = last square mean
a,b,c
The SID of indispensable AA in the 3 SBM used in the present study were consistently
lower than SID values reported for commercially available SBM (e.g. NRC, 1998;
AmiPig, 2000; AminoDat® 4.0). The mild heated SBM was the most gently treated SBM,
whereas heat treatment for the medium heated SBM was intermediate and the strong
heated SBM was the most severe. It is well accepted that SBM contains several ANF, such
as TI (Grant, 1989). The primary mode of action of TI is inhibition of the proteolytic
pancreatic enzyme trypsin by forming stable inactive complexes with the enzyme (Liener
and Kakade, 1980; Lalles and Jansmann, 1998, Jezierny et al., 2010). Furthermore, as a
secondary effect, TI may increase pancreatic secretion of trypsin and chymotrypsin resulting
in enhanced loss of endogenous AA which, in turn, reduce SID of AA (Gatel, 1994; Jezierny
et al., 2010).
To better understand the additional effect of the ANF on the SID of AA, the SBM under
investigation were also analyzed for their TI concentration (Lab of Mouriscade, Spain).
The results showed that the TI activity values were 17.6, 4.4 and 17.1 mg/g for the mild,
medium and strong heated SBM, respectively (Tab. 1). Thus, the lowest SID of AA in the
mild heated SBM was due to the presence of TI (under-heating) coupled with toasting in
wet condition (personal communication). A markedly lower TI activity in the medium
heated SBM indicates that TI was inactivated, suggesting the processing condition was the
most optimal.
Excessive heat processing may lead to the formation of Maillard reaction products that are
biologically unavailable (Pahm et al., 2008). Maillard reactions involve binding of AA to
the carbonyl group of reducing sugars such as glucose. Lysine is the AA most affected by
the Maillard reactions because it has an ε-amino group that readily reacts with reducing
sugars (Fontaine et al., 2007). Heat damage in the strong heated SBM is confirmed by a
smaller proportion of reactive lysine to total lysine (84 vs. 92 % compared with mild
heated SBM). Therefore, low SID values in the strong heated SBM reflect considerable
heat damage (Van der Poel et al., 1990). Surprisingly, the TI activity in the strong heated
SBM was still similar to that of mild heated SBM. During the production of the strong
SBM, temperature rise was very fast while the duration of the direct stream contact was
too short (7 vs. 45 min compared with medium heated SBM). Thus, it is possible that the
stream injected into the strong SBM was not long enough to inactivate the TI activity
(personal communication).
4
CONCLUSIONS
The SID of CP and AA in medium heated SBM was significantly higher than values
obtained for mild and strong heated SBM. The SID of CP and most AA in mild heated
SBM were similar to that of strong heated SBM. In general, SID values of CP and AA
determined in differently heat treated SBM, used in this study, were consistently lower
than SID values for SBM previously reported (e.g. NRC, 1998; AmiPig, 2000;
AminoDat® 4.0). In conclusion, the heat treatment of the medium heated SBM would
appear to have efficiently reduced the ANF in the SBM without seriously affecting protein
quality. Thus, it is important to avoid under- or over-heating of the SBM to maximize the
digestibility of AA.
5
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