Disassociate bound protein in tree leaves by trypsin hydrolysis and its effect on feed utilization, microbial protein and weight gain in sheep – an experimental study

B Srinivas, N Swain and N P Singh 

Division of Animal Nutrition, Central Sheep and Wool Research Institute,
Avikanagar-304501, Via. Jaipur, Rajasthan, India

bandla_srinivas@rediffmail.com

Abstract 

Twenty indigenous Malpura breed of sheep of average age 8.6+0.4 mo and body weight (BW) 15.9+0.7 kg were randomly divided into 2 equal groups. Animals were fed Alianthus excelsa tree leaves (200 g) as roughage at 08:00 h. Uneaten leaves removed at 14:00 h and experimental group (EG) fed feed mix consisted trypsin treated tree leaves (200g) and concentrate supplement (CS) (300g) at 14:00 h. Control group (CG) fed feed mix without trypsin treatment.  BW changes recorded at 5 d intervals for 45 d. After 25 d of preliminary period of feeding, a metabolism trial for 7 d was conducted.

Non protein nitrogen (NPN) fraction in feed mix increased (P< 0.05) after trypsin treatment. Dry matter intake (P<0.01) and digestibility were higher in EG than CG, yet there was 50% reduction in weight gain. CP digestibility reduced (P<0.05) after trypsin treatment and contrary was true for hemicelluloses (P<0.01) digestibility.  Fecal excretion of nitrogen was higher in EG than CG but vice versa was true in urinary excretion. Excretion of purine derivatives in urine was also 39 % lesser (P<0.05) in EG than CG. MBP absorbed (P<0.05) in CG and EG was 8.42 and 4.51 mM, d^-1, respectively. Residual trypsin appears to be still active in rumen environment and might have damaged the MBP production. Microbial N, g kg^-1 digestible organic matter intake (DOMI) was 21.48 and 10.36, respectively in CG and EG (P<0.05). Lower efficiency of MBP (g/kg DOMI) in EG was indicative of imbalance in protein and energy compared to CG. N retention was lesser besides inefficiency in the metabolizable N utlization in EG than CG (P< 0.05). Weight gain also affected because of lesser metabolizable N availability per MJ of ME and higher heat increment caused energy inefficiency. 

It is concluded that trypsin was effective in improving the solubility of bound protein fraction in tree leaves however, it had adverse affect on rumen microbial synthesis, protein digestibility and induced protein-energy imbalance. Its adverse impact on weight gains were observed after 30 d of feeding and hence, not suitable for its use in ruminants unlike in non-ruminants. Alternative source of proteolytic enzyme to disassociate bound protein in tree leaves without any adverse affect on rumen may need to be explored.

Keywords: derivatives, digestibility, enzyme, feed, microbial N, proteolysis, purine rumen, sheep, weight gain

Introduction

Tree-fodder is important feed resource in semi-arid regions to feed sheep round the year (Singh and Patnayak 1977, Kumawat and Chaudhary 2004). Although protein content of some of the tree leaves is higher than grasses, their degradation is not complete due to binding with cell wall polysaccharides particularly with maturity (Singh and Srinivas 1998, Karabulut et al 2006). Disassociating these bound proteins is necessary to improve protein availability from scanty feed resources in semi-arid regions. Trypsin is commonly used protease for site-specific hydrolysis of peptide fragments in metabolomics to derive bioactive peptides (Daniel 2003, Mota et al 2006). It is a very potent proteolytic enzyme catalyzing the breakdown of peptide bonds of protein involving lysine and/or arginine into smaller oligopeptides in the small intestine of either non-ruminants or ruminants (Alpers 1986). Proteolytic enzymes, used in the diets of non-ruminant animals to improve feed utilization efficiency, have been seldom tried in ruminant diet owing to high proteolytic activity in rumen (Bedford 1993, Kung 1996). Improving the hydrolysis of feed protein by trypsin to polypeptides or oligopeptides or free amino acids can optimize the growth of bacteria (Fadda et al 1999, Russell et al 1992), enhance nutrient uptake, digestibility and bioactivity in animal system (Wu 1998, Pintado et al 1999, Foregeding et al 2002). There are hardly any attempts in the past to disassociate cell wall bound protein by proteolytic enzymes in the feedstuffs of ruminants even though it is one of the alternatives to improve protein intake of sheep in arid and semi-arid regions in general and, particularly during lean periods of feed availability.

Looking into the paucity of literature on the adversity of trypsin in ruminants, microbial protein production (MBP) was taken as an appropriate indicator to evaluate its overall effect on the rumen environment. MBP production in rumen has not only quantitative significance to provide quality protein to the animal down the gut but also, an ultimate marker for optimum conditions of nutrient fermentation in rumen. This investigation was conducted with the objectives; 1. To increase the solubility of bound protein in the tree leaves using trypsin as proteolytic enzyme and, 2.  To evaluate exogenous trypsin affect on the MBP production and nutrient utilization in sheep.

Materials and methods 

Substrate and trypsin hydrolysis

Sun dried Alianthus excelsa leaves were taken as substrate for trypsin hydrolysis. Trypsin from bovine pancreas (1:3000, bovine pancreas, MW 23, 281, M/s Merck India Ltd.) was used for hydrolysis. A stock solution of 50 g of trypsin prepared in 250 ml of 10^-4 N HCl (pH 4) and stored in refrigerator at 4^oC till further use.  Enzymatic hydrolysis of substrate was performed in 20 L capacity polypropylene buckets in an open shed at 40^oC of ambient air temperature. A 50 ml of trypsin stock solution was diluted to 2 L with tap water to make the final concentration of enzyme to 500 mg/100ml.    This was added to 2 Kg of Alianthus excelsa leaves  and kept for 12 h. Contents in the bucket were thoroughly mixed at every 2-3 h intervals.  After 12 h of soaking, 3 kg of concentrate supplement (CS; consisted groundnut cake 25, maize 30, wheat bran 40, mineral mixture 3 and common salt 2 parts) was mixed thoroughly with the tree leaves to adsorb any hydrolyzed protein that was leached into the solution from the leaves. This complete feed mix was again hydrated with another 1 L of water and kept overnight to complete the reaction of any residual enzyme in the substrate. Trypsin treated Feed mix (TFM) was exposed to air for 8 h to reduce moisture content as sheep prefers dry feeds over mash. Simultaneously, a blank diet was also prepared without adding trypsin to the solution.  TFM was prepared every day for 45 d using the trypsin stock solution.

Animals and feeding

Twenty Malpura breed of indigenous male sheep of average age 8.6 + 0.4 mo and body weight (BW) of 15.9 + 0.7 kg were randomly divided into two equal groups. Animals were dewormed prior to experimentation. Animals in both the groups were fed 200 g of Alianthus excelsa leaves at 08:00 h. Uneaten tree leaves were removed at 14:00 h, followed by feeding either 500 g of TFM (experimental group; EG) or a similar quantity of blank diet  (control group; CG) that is without trypsin treatment. Drinking water was provided to the sheep 4 times in a day in 4-6 h interval. Weight change in sheep was recorded at 5 d intervals for a period of 45 d.

Metabolism trial

After 25 d of preliminary feeding, a metabolism trial was conducted for 7d. Animals were accommodated in metabolic cages 2 d before the sampling for acclimatization. Urine collection bucket was acidified by adding 10% H₂SO₄ (v/v) solution. Faecal and urine output was measured at 08:30 h every day. Faecal samples were sub-sampled to 1/50^th part in 2 quantities. One part was oven dried at 100 + 5 ⁰C for 24 h. The other part was acidified with 25% H₂SO₄ (v/v) in a glass bottle and kept for nitrogen (N) estimation. Urine quantity was measured and diluted uniformly to 2 L with tap water. Diluted urine was mixed, filtered through glass wool and 20 ml was stored in 50 ml capacity polypropylene bottles on each day. Urine samples stored at –20 ⁰C to estimate purine derivatives. Another sub-sample of 20 ml of urine was pooled separately for 7 d to estimate Nitrogen (N). Feed offered and orts samples were collected every day, oven dried and pooled for 7 d. At the end of metabolism trial sheep were shifted to individual stalls and, feeding continued for another fortnight.

Laboratory analysis

DM content was determined by drying samples in a forced draught oven at 100 ^oC for 24 h (AOAC 1995). Oven dried samples were weighed and ground in a lab mill (M/s Jaico laboratory, India) using 1 mm sieve. Organic matter (OM) content was obtained by difference after ashing the dried sample in a muffle furnace at 600 ^oC (AOAC 1995).  N in feces and urine was determined by Kjeldahl procedure (CP = 6.25 X N) and ether extract (EE) by petroleum ether (AOAC 1995). Solubility of protein was analysed as suggested by Licitra et al (1996). Non Protein Nitrogen (NPN) was determined as difference between total N and true Protein N (TP; estimated by precipitating protein by trichloroacetic acid, AOAC 1995).  Buffer insoluble protein (PBIP) was analyzed by soaking 5 g of sample in 500 ml of borate-phosphate buffer (pH 6.7) for 4 h at room temperature, filtered and N content in the residue was estimated by standard Kjeldahl method. PBIP values were subtracted from TP to derive true soluble protein (Licitra et al 1996). Cell wall bound N fractions such as NDF insoluble (NDIP) and ADF insoluble (ADIP) protein were analyzed from NDF and ADF residues respectively, by Kjeldahl method (AOAC 1995). NDIP was subtracted from PBIP to derive NDS soluble protein (fraction B₂). Difference between NDIP and ADIP was taken as protein insoluble in NDS but soluble in ADS. ADIP was considered as indigestible (Licitra et al 1996, Chalupa and Sniffen 1996). Neutral detergent fiber (NDF) analysis did not use amylase and expressed inclusive of residual ash (Van Soest et al 1991). Acid detergent fiber without adjustment for residual ash (ADF), cellulose and, lignin were analyzed according to methods of AOAC (1995). Hemicelluloses were expressed as difference between NDF and ADF (Van Soest et al 1991). Gross (GE) and digestible energy (DE) was estimated by adiabatic bomb calorimeter (M/s Gallenkamp Bomb Calorimeter, UK). Metabolizable energy (ME), heat increment (HI) and energy efficiency were determined based on the empirical models recommended by ARC (1990).

Microbial protein analysis

MBP was estimated from the purine derivatives excreted in the urine (Fujihara et al 1987, Chen et al 1995).  Urine samples were stored in refrigerator at 4⁰ C. These samples were then thawed to 25⁰ C and diluted further 10 times to maintain the concentration of purine derivatives within the range of 5-50 mg l^-1.  Diluted urine samples were pooled into triplicate for each animal. Allantoin standards were prepared for 10 to 60 mg l^-1 with 10 mg increment (nr. A-7878, Sigma, USA). Allantoin concentration was determined by colorimetric method at 522 nm using UV-visible spectrometer (Cintra 10e Double beam, M/s GBC Scientific Equipments, Australia). Xanthine and hypoxanthine were estimated by degrading to uric acid using Xanthine Oxidase (nr. X-1875, Sigma, USA). Standards of uric acid (nr.U-0881, Sigma, USA) were prepared in the concentration 20 to 100 mg/l with 20 mg increment and absorbance read at 293 nm. Uric acid was determined by uricase (M/s Sigma Chemicals, USA) method and standard working concentrations were prepared 5, 10, 20, 30 and 40 mg/l. Allantoin concentration in urine was calculated using linear regression analysis. Uric acid estimations derived from the natural logarithmic function of Ln (X) and Ln (Y). Purines absorption (X) was calculated using following equation on the basis of total purine excreted (Y) in urine. 
 

Y = 0.84X + (0.150 W^0.75 e^-0.25X)

Calculation of X from Y was performed by means of the Newton-Raphson iteration process as below:

where,

ƒ (x) = 0.84X + 0.150 W^0.75 e^-0.25X  - Y  and,

the derivative of ƒ’(X_n) = 0.84-0.038 W^0.75 e^-0.25X .

The supply of microbial nitrogen (MN) to the duodenum was calculated as:

MN (g/day) = X(70 x 0.83 x 0.116x1000) = 0.727 X

Where,

the digestibility of microbial purine was 0.83,

N concentration in purine (mg/mmol) was 70 and,

0.116 was the ratio of purine-N : total-N in mixed rumen microbes.

Therefore, efficiency of microbial N synthesis used here was expressed as grams of microbial N per kilogram of digestible organic matter (DOM) apparently digested in the rumen (DOMR). DOMR derived by multiplying DOM with a factor of 0.65.

Statistical analysis

Body weight changes were subjected to repeated measure analysis by taking 9 levels (weeks) for within group. Intake, digestibility, N and energy and ruminal microbial protein synthesis data were analysed by paired sample student t- test that included the standard error between two means. Variations among means with P < 0.05 to 0.001 were accepted as representing tendencies to differences among the groups. These analyses used the standard procedures of statistical package for social sciences (SPSS), V 14.0 for Windows.

Results 

Chemical composition of CS, Alianthus excelsa leaves and feed mix is presented in table 1.

No significant variation was observed in the chemical composition of feed mix (P>0.05) between CG and EG except CP (P<0.01) content. NPN was high (P<0.05) in treated mix than in blank diet (table 2). 

Other than NPN, no change in any other soluble and indigestible protein (P> 0.05) was observed. However, both NPN and true soluble protein together were increased while cell wall bound and insoluble protein fractions were decreased after trypsin treatment of feed with probability of significance was 0.13 (P < 0.13) and 0.20 (P < 0.20), respectively.

BW changes were not significantly different between both the groups till 30d of feeding (Figure 1).

Thereafter, weight gain in EG was significantly (P< 0.05) lower than CG. Sheep in CG gained 1.54 kg higher BW during 45 d compared to EG (table 3).

Contrary to 2-fold lower growth rate, dry matter intake (DMI) of sheep in EG was significantly higher (P< 0.01) than CG. Digestibility of CP (P< 0.05) and hemicelluloses (P< 0.01) were significantly different between two groups. Digestible CP intake was higher in CG than EG (P< 0.05) but contrary was true for GE intake (P< 0.05).

Allantoin, xanthine and hypoxanthine and, uric acid in urine were 41 (P<0.05), 15 (P> 0.05) and 36 per cent (P< 0.01) lesser in EG compared to CG (table 4).

Total purine derivatives excreted in the urine of EG were 39 per cent lesser than CG. Contrary to expectation, TFM had negative affect on the MBP absorbed (P< 0.05) or microbial N flow to intestine (P<0.05). Efficiency of microbial N synthesis in terms of DOMI or DOMR (P<v0.05) also decreased due to trypsin treatment of feed mix.

N absorbed from the gut was 2.51+0.91 g/d higher in CG than EG (P<0.05). On contrary, urinary N excretion was 0.65+0.29 g/d (P<0.05) lesser in EG compared to CG (Table 5).

However, the overall metabolizable N was lower in EG than CG (P<0.05). There was no difference in DE or ME between both the groups. The q-value of the diet (ME/GE) was 0.82 and 0.83 in CG and EG respectively. ME available for each gram of metabolizable N (MN) was 0.75 and 1.48 MJ in CG and EG, respectively. Although the ratio between MN and ME was narrow on EG (P< 0.05), heat increment was 9 per cent more (P< 0.05) in EG than CG.

Discussion

Alianthus excelsa tree leaves are one of the widely used and good protein sources among top feeds in semi -arid regions (Kumawat and Chaudhary 2004). These tree leaves are important component of the diet of small ruminants during November to July in the environmentally challenged, underdeveloped semi-arid regions. Apart from shortage of grazing resource in these regions, decrease in the availability of protein from top feeds as a result of binding to cell wall polysaccharides with maturity may further affect the productivity of sheep. Like many other tree leaves, CP content in Alianthus excelsa leaves were comparable with CS. However, even with higher CP content in leaves, earlier studies reported negative N balance (Aganga and Tshwenyane 2003, Salem et al 2005). This was mainly due to cell wall bound fraction that was around ¹/₃ of the total protein in Alianthus excelsa. Normally cell wall bound fraction may range between 9 to 43% of total  CP content of tree leaves used for livestock feeding in India (Bhadauria et al 2002). 

In order to improve the protein solubility in tree leaves, trypsin has been selected owing to two reasons; 1. Formation of peptides by trypsin hydrolysis has been reported to be more extensive than other proteolytic enzymes (Pintado et al 1999) which may effectively hydrolyze at the least 8% of all peptide bonds present (Pintado et al 1999, Kananen et al 2000) and, 2. Trypsin has been used effectively without any adverse affects in poultry feeds and in some dairy whey products consumed by human beings.  Increase in NPN fraction in TFM was due to improvement in the solubility of protein.  Such improvements have been observed in the cheese whey proteins also with the application of trypsin (Pouliot et al 1997, Kim et al 2004). However, as per the objective of the investigation, it is important that the increased solubility of protein should come from the bound fraction of CP rather than other cellular fractions whose availability has been reported to 92% (Nag and Matai 1991). Trypsin treatment decreased bound fractions of protein such as protein soluble in NDS and ADS and, indigestible fractions though the level of significance was 20%. Such variation within the leaves is also possible as maturity may vary within the leaves or even between plant parts. This however, was indicative of possibility in disassociation of bound protein in the tree leaves by trypsin.

Feeding tree leaves along with CS increased their acceptability by sheep and decreased the wastage.  Reducing the wastage of diet is also necessary in semi-arid regions for careful use of limited feed resources. Contrary to expectation, CP digestibility was reduced after trypsin treatment but hemicelluloses digestibility was enhanced. Partial hydrolysis of cell wall bound protein in TFM improved the availability of hemicelluloses which may also presumably have influence on disassociation of bound protein.  However, decrease in CP digestibility may probably due to HCl used in the stock solution of the enzyme that might have reduced the potential degradability of CP in the rumen besides the reduced MBP production. Higher DMI in EG with 50% lower average daily gains were also indicative of restriction in energy utilization as a result of low metabolizable N which is actually available for synthesis of organic body constituents and secretions. (Kebreab et al 2002).

Relative proportion of allantoin or uric acid excreted in both the groups was with in the normal range. TFM damaged the MBP production even after precautions were taken to dilute residual activity by adding water to feed mix (Beauchemin et al 1995, Feng et al 1996). Different schools of opinion prevail about the application of exogenous enzymes owing to strong proteolytic activity in the rumen. Many workers opined that the exogenous proteolytic enzymes might not be stable and any excess may be inactivated in the normal rumen pH range of 6.2 to 6.8 (Chesson 1994, Feng et al 1996, Kung 1998). Contrary to it, exogenous trypsin in the feed mix was found to be active in the rumen environment and damaged the production of  rumen MBP. MBP production in rumen would represent both protein and energy supply and utilization in rumen. Decrease in microbial N yield due to trypsin treatment of feed mix was indicative of a poor rumen fermentation and imbalance in the DOM and N in the diet.  Though the DOMI was apparently higher in EG, gram microbial-N synthesized per Kg of DOMI was 54 % lesser (P<.05) than CG. The functional inefficiency of MBP production per kg of DOMI in EG compared to CG was thus, indicative of inefficient utilization of CP in the diet (Tebot et al 2004). Trypsin treatment induced imbalance in the ratio between available N and DOM in rumen. There was no significant difference in the fermentative and fecal heat losses as well as ME between CG or EG.  Higher heat increment however, observed on EG was an indication of increased energy use during metabolism by the animal. Additional heat increment from diet may also be a disadvantage in semi-arid regions owing to high air temperature. Energy losses were therefore, because of more heat increment during post gut absorption than fecal or fermentative energy losses on TFM. This enumerated that the distortion in nutrient utilization occurred with application of trypsin mainly due to imbalance in the absorbed protein and energy across the gut (Blaxter 1989).

Conclusions

• Trypsin treatment of tree leaves could disassociate bound protein. It improved the bound protein solubility with decrease in cell wall bound fractious thus, indicating the possibility of improving the availability of this fraction from top feed resources.  Sheep feeding trial however, indicated exogenous trypsin was still active in the rumen environment and adversely affected the MBP production by 39 %. Trypsin altered the efficiency of synthesis of proactive rumen microbes adversely. It had cumulative affect on the post gut utilization of protein also and reduced the weight gains in sheep. Contrary to non-ruminants, exogenous trypsin effect was negative on the ruminants. An alternative proteolytic enzyme may have to be studied that is safe for the viability of rumen microbial mass apart from improving the solubility of bound protein.

Acknowledgement 

Authors are thankful to the Director, Central Sheep and Wool Research Institute for financial support and extending the facilities.

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Received 7 May 2008; Accepted 2 September 2008; Published 5 December 2008

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