The cleavage site preference of the porcine pepsin on the N-terminal α1 chain of bovine type I collagen: a focal analysis with mass spectrometry.

Bovine type I collagen consists of two α1 and one α2 chains, containing the internal triple helical regions and the N- and C-terminal telopeptides. In industries, it is frequently digested with porcine pepsin to produce a triple helical collagen without the telopeptides. However, the digestion mechanism is not precisely understood. Here, we performed a mass spectrometric analysis of the pepsin digest of the N-terminal telopeptide pQLSYGYDEKSTGISVP (1-16) in the α1 chain. When purified collagen was digested, pQLSYGY (1-6) and pQLSYGYDEKSTG (1-12) were identified, while DEKSTG (7-12) was not. When the N-terminal telopeptide mimetic synthetic peptide pQLSK(MOCAc)GYDEKSTGISK(Dnp)P-NH2 was digested, pQLSK(MOCAc)GYDEKSTG (1-12) and ISK(Dnp)P-NH2 (13-16) were readily identified, pQLSK(MOCAc)GY (1-6) and DEKSTGISK(Dnp)P-NH2 (7-16) were weakly detected, and DEKSTG (7-12) was hardly identified. These results suggest that pepsin preferentially cleaves Tyr6-Asp7 and less preferentially Gly12-Ile13. They also suggest that the former cleavage requires native collagen structure, while the latter cleavage does not.

Alfalfa Silage Diet Improves Meat Quality by Remodeling the Intestinal Microbes of Fattening Pigs.

Because the demand for pork is increasing, it is crucial to devise efficient and green methods to improve the quality and quantity of meat. This study investigated the improvement in pork quality after the inclusion of alfalfa meal or alfalfa silage in pig diet. Our results indicated that alfalfa silage improved meat quality more effectively in terms of water-holding capacity, drip loss, and marbling score. Besides, an alfalfa silage diet can affect the level of fatty acids and amino acids in pork. Further, alfalfa silage was found to improve meat quality by remodeling intestinal microbiota and altering the level of SCFAs, providing a viable option for improving meat quality through forage.

Effects of alfalfa meal on quality and function of pork meatballs.

Alfalfa (Medicago sativa L.) is abundant in dietary fiber, alfalfa saponins, and other active ingredients. However, the application of alfalfa is scarce in food. Meatball is one of the most popular meat products in daily life, but eating too many meatballs could result in obesity, hyperlipidemia, and other diseases. With increasing attention to healthy diet, how to keep the original color, aroma, taste, and shape of food with low fat and nutrition has become an urgent problem to be solved. In this study, different amounts of alfalfa meal or extruded alfalfa meal were added to pork meatballs to explore the optimal adding ratio of two kinds of alfalfa meal in pork meatballs. Further animal experiments were conducted for two weeks to prove the efficacy of two kinds of alfalfa balls in lowering blood lipid and body weight. The results showed that 0.5% alfalfa meal and 1% extruded alfalfa meal could improve the quality of prepared pork meatballs. Animal experiments demonstrated that two kinds of alfalfa meal pork meatballs had a good effect of reducing blood lipid, and the alfalfa meal pork meatballs had a better effect on reducing serum cholesterol and average daily weight gain of mice. This study provided a theoretical basis for making healthy and nutritious pork meatballs, which could provide more delicious food for people, especially people who are obese and the elderly.

Comparative studies on the activities of collagenases from Grimontia hollisae and Clostridium hystoliticum in the hydrolysis of synthetic substrates.

The collagenase produced by a gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently than that produced by a gram-positive bacterium Clostridium histolyticum (Chcol), which is currently the most widely used collagenase in industry [Teramura et al. (Cloning of a novel collagenase gene from the gram-negative bacterium Grimotia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis. J Bacteriol 2011;193:3049-3056)]. Here, we compared the Ghcol and Chcol activities using two synthetic substrates. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2, 4-dinitrophenyl)-L-2, 3-diaminopropioyl]-L-Ala-L-Arg-NH2, Ghcol exhibited 350-fold higher activity than Chcol in the absence of CaCl2 and NaCl. The Ghcol activity markedly decreased with increasing concentrations of buffer, CaCl2 or NaCl, while the Chcol activity did not, suggesting that the Ghcol activity was sensitive to solvent components. In the hydrolysis of N-[3-(2-furyl)acryloyl]-L-Leu-Gly-L-Pro-Ala, Ghcol exhibited 16-fold higher activity than Chcol in the absence of CaCl2 and NaCl, and both enzyme activities did not decrease with increasing concentrations of buffer, CaCl2 or NaCl. pH dependences of activity revealed that the ionizable group responsible for acidic pKe may be Glu for Ghcol and Chcol, while that for alkaline pKe may be His for Ghcol and Tyr for Chcol. These striking differences suggest that the catalytic mechanism of Ghcol might be considerably different from that of clostridial collagenases.