Protease Inhibitors in Porcine Colostrum: Potency Assessment and Initial Characterization

Q Zhou,S R Liao,X Li,R G He

doi:10.5713/ajas.2003.1822

Abstract

Porcine colostrum and milk were separated into the acid-soluble and casein fractions by acidification followed by centrifuge. The acid-soluble fraction of porcine colostrum was further separated by liquid chromatography and anisotropic membrane filtration. Trypsin and chymotrypsin inhibitory capacity in porcine colostrum, milk and their components was determined by incubating bovine trypsin or chymotrypsin in a medium containing their corresponding substrates with or without addition of various amounts of porcine colostrum, porcine milk or their components. The inhibition of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) degradation in pig small intestinal contents by porcine colostrum was measured by incubating iodinated IGF-I or EGF with the intestinal contents with or without addition of porcine colostrum. Degradation of labeled IGF-I or EGF was determined by monitoring the generation of radioactivity soluble in 30% trichloroacetic acid (TCA). The results showed that porcine colostrum had high levels of trypsin and chymotrypsin inhibitory activity and increased the stability of IGF-I and EGF in pig intestinal contents. The inhibitory activity declined rapidly during lactation. It was also found that trypsin and chymotrypsin inhibitory activity and the inhibition on IGF-I and EGF degradation in the acid-soluble fraction were higher than that in the casein fraction. Heat-resistance study indicated that trypsin inhibitors in porcine colostrum survived heat treatments of 100°C water bath for up to 10 min, but exposure to boiling water bath for 30 min significantly decreased the inhibitory activity. Compared with the trypsin inhibitors, the chymotrypsin inhibitors were more heat- sensitive. Separation of the acid-soluble fraction of porcine colostrum by liquid chromatography and anisotropic membrane filtration revealed that the trypsin and chymotrypsin inhibitory capacity was mainly due to a group of small proteins with molecular weight of 10,000-50,000. In conclusion, the present study confirmed the existence of high levels of protease inhibitors in porcine colostrum, and the inhibition of porcine colostrum on degradation of milk-borne growth factors in the pig small intestinal tract was demonstrated for the first time. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 12 : 1822-1829)

Highlights

The trypsin and chymotrypsin inhibitory activity in the casein fraction (CF) of Statistical analysis was carried out using a computer porcine milk was too low to be detected in the present assay package: Minitab (Minitab Inc., USA, 1994)
The present study demonstrated that porcine colostrum contained high levels of trypsin-chymotrypsin inhibitory activity and one ml of porcine colostrum could inactivate 1 mg of bovine trypsin
The trypsin inhibitory activity has been previously reported in porcine colostrum (Laskowski et al, 1957; Kress et al, 1971; Westrom et al, 1982; Xu et al, 1996), and the inhibitory activity is highest at the time of parturition and declines rapidly with the progress of lactation (Laskowski et al, 1957; Westrom et al, 1982)

Summary

Introduction

Protease inhibitors have been found in the mammary secretions of human (Laskowski and Laskowski, 1951; Lindberg, 1979; Lindberg et al, 1982), rat (Carlsson et al., 1975; Westrom et al, 1976; Telemo et al, 1982; Rao et al., 1990; Rao et al, 1993), cow (Laskowski and Laskowski, 1951; Weber and Nielsen, 1991) and pig (Laskowski et al., 1957; Kress et al, 1971; Carlsson et al, 1980; Westrom et al, 1982; Xu et al, 1996). The amount of trypsin inhibitors in sow colostrum is to a group of colostrum-specific protease inhibitors (Westrom et al, 1982). It has been shown that colostrum-specific protease inhibitors enhance absorption of intact colostrum proteins in neonatal pigs, probably by inhibiting the gastrointestinal proteolysis (Westrom et al, 1985). Little is known about the physical and chemical characteristics of protease inhibitors in porcine colostrum. Whether porcine colostrum enhances the survival rates of milk-borne growth factors, which have been speculated to play a role in the development of the gastrointestinal (GI) tract in suckling young (Koldovsky, 1996; Xu, 1996), is largely unknown.

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