ABSTRACT
Objective:To study the material basis of the drug effect of the raw material of lamb′s tripe and vitamin B12 capsule.Methods:Rennet and pepsin were extracted with 0.9% sodium chloride and were purified by saturated ammonium sulfate precipitation, diethylaminoethyl-cellulose 52 and high pressure chromatography (HPLC) chromatography. Relative molecular weight was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and liquid chromatography-triple quadrupole mass spectrometer. The amino acid composition and antioxidant activity of raw materials were analyzed with HPLC. The raw materials were completely extracted with water, phosphate buffer and sodium bicarbonate in turn, and in vitro 1, 1-diphenyl-2-picrylhydrazyl radil 2, 2-diphenyl-1-(2, 4, 6-trinitrophenyl)hydrazyl (DPPH) and 2, 2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) radical activity were measured. Glycoprotein from raw material was extracted with hot water and determined its growth-promoting activity gaginst Bifidobacterium adolescentis , Lactobacillus delbrueckii subsp. Bulgaricus and Enterococcus faecalis was measured. The composition of amino acid and monosaccharide were analyzed by HPLC and gas chromatography-mass spectrometry, respectively. Results:The enzyme activity of two purified rennet F6-2 and F7-2 which had different ionic strengths and pepsin F7-1 were 27 557.10, 17 532.60 and 17 728.15 U/g, respectively. The results of SDS-PAGE indicated that the molecular weights of three enzymes were similar, ranging from 35 000 to 40 000. The raw material contained 16 kinds of amino acids, of which hydrophobic amino acids accounted for 33.03% of the total amino acid content. When the sample concentration was 5 mg/mL, ABTS free radical scavenging activity of the three extracts was (37.80±0.45)%, (23.20±0.78)% and (62.80±0.74)%; DPPH free radical scavenging activity was (57.87±0.55)%, (5.03±0.25)% and (26.67±3.10)%, respectively. Glycoprotein extracts had promoted the growth of Bifidobacterium adolescentis, Lactobacillus delbrueckii subsp. Bulgaricus and Enterococcus faecalis, and there was statistically significant difference in the promotion of Lactobacillus delbrueckii subsp. Bulgaricus and Enterococcus faecalis ( P<0.05). The protein chain of glycoprotein was composed of 15 amino acids and the polysaccharide chain was composed of two monosaccharides, glucose and lactose. Conclusions:Two rennet and one pepsin are isolated and analyzed from raw material of lamb abomasum by various chromatographic methods. The raw material is rich in antioxidant active ingredients. The glycoprotein components of lamb abomasum has the activity of promoting the growth of probiotics.
ABSTRACT
Objective To investigate the influencing factors and improve potency methods of chymosin, to verify the stability and applicability of the national standard of chymosin.Methods The effects of different formula milk powder substrate and enzyme concentration on the determination of the activity of chymosin were studied.3 ×3 dose-response parallel line method was established.The results were compared with the different methods of absolute and relative methods.Results The different formula milk powder had a significant effect on the determination of the absolute potency of the activity of chymosin.The concentration of the enzyme was a power function relationship with the milk clotting time.Compared with the absolute potency, reproducibility of the relative potency of the results was better in different laboratories.The suitable doses in 3 ×3 dose-response parallel line method were 0.35,0.44,0.55U/mL.The confidence limit rate was less than 5%.The potency of the national standard of chymosin (140712-201302) was not significantly different between 2013 and 2015.In a certain dose range, the dose-response of the national standard of chimosin and gastropylor complex or lamb'tripe extract was linear, and the two lines were parallel.Conclusion A lot of factors can affect on the potency of chymosin.Relative potency is determinate by reference standard which can eliminate the influence of different substrates, different operators and endpoint judgment on the determination in order to make results have comparability between laboratories.The test design of 3 ×3 dose-response parallel line can control the test deviation better than the single point determination.The stability of the national standard of chymosin(140712-201302) is good, and is suitable for the potency of chymosin of the products of gastropylor complex and the extract of the lamb.
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The aim of this work was to study proteolytic activity of actinidin in comparison with chymosin and ficin on bovine milk substrate. The specific activities of purified ficin and actinidin were 7.9 and 8.3 unit/mg protein, respectively. The optimum clotting activity of both actinidin and ficin was at 45°C, although chymosin was relatively less sensitive to temperature. Increasing CaCl2 concentration resulted in an enhancement of the clotting activities of all coagulating enzymes, this effect noticeable for ficin. In ficin treated sample significant decrease of bands intensity in the range 25-30 KD and appearance of some of κ- casein in 20 KD regions was observed by using SDS-PAGE. In conclusion, the chymosin and actinidin gave similar relative activity at different temperatures, pH values and CaCl2 concentrations for bovine milk substrate. Comparable electrophoresis profile of actinidin, ficin and chymosin by analysis of the whey with SDS-PAGE indicates that actinidin could be a potential alternative for chymosin.
ABSTRACT
High quality DNA is essential for many molecular biology techniques. However, the reagents used for that purpose usually are expensive and/or cause a high environmental impact. Here, we describe two alternative protocols that use inexpensive reagents and are not hazardous to the environment. The first protocol utilizes the enzyme chymosin, normally used as rennet in cheese production and which is easily obtained on the commercial market. The second protocol uses rennet DNA extraction protocol combined with the DNA binding capacity of glass powder (glass milk), which can easily be home made. The first protocol is used when a high yield of DNA is needed, whereas the second protocol is used for production of a higher quality DNA, being able to work with sparse samples.