APPENDIX 1 LABORATORY PRODUCTION OF LIVER ETRACT AND LIVER DIGEST FROM FRESH BOVINE PANCREASE OBTAINED FROM AN IN ZARIA.

Initial preparation of liver and pancrease for extraction using aseptic techniques. The

ABATTOIR

hands were washed with soap and disinfected with a hand disinfectant mixture (chlorhexidine and isopropyl) before proceeding. The following were then disinfected with boiled water either by pouring boiled water on the surface twice or by filling containers halfway with boiled water with the lids on and kept for 10minutes: 1. Mortar and pestle 2. Cutting boards and knives 3. All plastic and metal containers 4. Working bench surfaces The following were further disinfected with 10% hypochlorite solution (HOCL) which is 35,000ppm solution of chlorine. Ten percent (10%) HOCL solution was made by adding 100ml of concentrated jik bleach to 900ml of tap water: 1. Bench surfaces 2. Mortar and pestle 3. Cotton board and knives 4. All plastic and metal containers Ten percent (10%) HOCL was poured on bench surfaces and cleaned with cotton wool soaked in the solution. The containers were filled with 10% HOCL until they were half-full and closed with theirs lids. The fluid was swirled around so that it touched all the surfaces of the containers and they were then left on the bench for 10 minutes. The chlorine solution was discarded and the containers were rinsed thrice with boiled tap water. The liver and pancreas were washed with detergent using and cooled tap water and rinsed thoroughly to remove all traces of soap. The liver and pancreas were immersed in hypochlorite solution in separate containers previously disinfected and half-filled with

10%HOCL solution. This completed the surface disinfected. The surface disinfected liver and pancreas were placed on the previously disinfected cutting board and minced,(cut in very tiny bits),using a previously disinfected knife. Extraction of Liver and Pancreas Pastes. The minced liver and pancreas were each placed separately into a disinfected and rinsed mortar and pounded into smooth pastes. A disinfected small metal cup was used to fetch the minced liver and pancreas into a previously disinfected large plastic container in each case. The number of cups of minced liver and pancreas was noted. Boiled and cooled tap water was added to the liver and pancreatic pastes in covered containers in the following ratio: Liver: 1 part of liver pastes to four parts of boiled and cooled tap water. Pancreas: 1 part of pancreatic paste to 3 parts of boiled and cooled tap water. The suspensions were kept in covered containers and held in the laboratory for 3hours at 300+/_ 10C to effect extraction. After this period, decantation and filtration of extracts was done. The pH of the liver extract was adjusted to neutrality and then boiled in 200ml, 300ml and 400ml portions which were pasteurized, cooled and stored in the refrigerator. Preparation of Liver Digest Pancreatic extract was added to the extracted liver paste to effect digestion of the liver paste. One volume of pancreatic extract was added to one volume of liver paste. The suspension was incubated at 30-350C for 4 hours with occasional agitation to effect thorough mixing of the suspension. As digestion progresses, the suspension became acidic due to formation of acidic fermentation products. The pH of the suspension was monitored and the tendency of high acidity was counteracted by the addition of 40% KOH to the suspension, using Bromothymol blue color standard to

guide the pH adjustment. After 4hour of digestion with pancreatic enzymes, the process was terminated by boiling the suspension. The boiled suspension was filtered and final adjustment to neutrality was carried out. The liver digest was bottled in 200ml, 300ml and 500ml portions in plastic containers, pasteurized cooled and stored in the refrigerator.