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Summary The heat stability of the trypsin inhibitors commonly present in meat products has been investigated. This includes the naturally occurring inhibitors in meat of serum, and some of the soybean proteins usually added to minced meat and sausages. The stability of the inhibitors are investigated both alone, and when mixed with meat and other compounds.

Yersinia ruckeriproduces an antibacterial factor which inhibits the growth of a wide spectrum of Gram-negative and Gram-positive bacteria, though not other strains of Y. ruckeri.The antibacterial factor was produced at low temperatures (4–20°C), but not at 37°C. The activity was lost after treatment of the supernatant with chloroform, UV-light and after boiling of the supernatant. One did not succeed in obtaining the antibacterial factor in a sterile solution.

Thirty-two Escherichia coli colonies were taken from the primary step of cultivation of the jejunal contents of each of 10 dead piglets which had suffered from diarrhea. The organisms of each colony were examined for the presence of adhesion fimbria (F4 (K88) and F5 (K99)), production of heat-stable and heat-labile enterotoxin and of colicins.

A simple semiquantitative method for detecting amylase or amylase inhibitors in biological materials is described. The substrate consists of a 0.25% starch-1.5% agar gel slab buffered at pH 6.5 with 0.1 M phosphate-0.01 M sodium chloride. Three millimeters wide cellulose strips saturated with a solution to be examined for inhibitor are placed parallel on the gel slab for 2 hours at 37°. The strips are removed and other 3-mm wide cellulose strips saturated with amylase solutions are placed at right angles across the first strips. The system is incubated for 6 or 18 hours at 37°. After flooding the slab with Lugol (an iodine containing) solution, amylase activity is shown by clear lysis zones on a deep-purple background. Presence of inhibitors is indicated by interruption or narrowing of the lysis zone where the inhibitor-containing and amylase-containing strips crossed. A variation of this method using amylase or amylase-inhibitor mixtures placed into 7-mm wells cut into the starch-agar gel slab is also described. The starch-agar gel slab methods were compared with the Bernfeld method of determining amylase activity.

Altogether 235 strains of Escherichia coli isolated from jejunal content of piglets with neonatal diarrhea were examined for serological, enterotoxin-producing and certain biochemical properties. Of 198 strains examined, 84 (42 %) belonged to O-group 149, while 14 (7 %)strains belonged to each of the O-group s 8 and 64. Seventy strains (30%) could not be grouped with the sera used. The remaining strains were distributed among the following O-groups with only a few strains in each group: 2,6,9,32,45,98 and 141. Eighty out of 84 E. coli strains of O-group 149 possessed the K88 antigen and produced heat labile enterotoxin (LT). Besides, LT production was demonstrated in 3 out of 14 strains of E. coli 08. K88 antigen was demonstrated in only 1 strain not belonging to O-group 149. Among strains of E. coli 064 12 out of 14 were K99 positive. This antigen was not demonstrated in E. coli strains of other O-groups. A close relationship was demonstrated between strains of E. coli 0149 possessing the K88 antigen and the ability to ferment both raffinose and adonitol. This ability was only detected in 2 other strains of E. coli not belonging to O-group 149.

Enterotoxigenic reference strains of Staphylococcus aureus were cultivated in sterile whole and skim milk for 18 h at 37°G. Staphylococcal enterotoxin A, B, and C were detected directly in the milk by an enzyme linked immunosorbent assay (ELISA), sensitive down to 1 ng/ml. Enterotoxins in the range of 1 ng–20 µg/ml milk were detected without any concentration or extraction. Skim and whole milk were almost identical as medium for enterotoxin production.

The serological relationship between bovine and swine trypsins, and bovine α-chymotrypsin has been studied with rabbit antisera at different stages in the immunization period. By using paper electrophoresis to distinguish between the naturally occurring inhibitors and the antienzymes in the γ-globulin fractions, combined with the casein precipitating inhibition test (electrophoretic CPI-test) it was found that at 18 days after immunization the antienzymes inhibited only the homologous enzymes. After an additional 12 and 24 days the anti- bovine trypsin also inhibited swine trypsin and α-chymotrypsin, and anti-swine trypsin inhibited bovine trypsin, while antia-chymotrypsin inhibited only the homologous enzyme. The enzyme inhibition in the heterologous systems was about 1/10 of that in the homologous systems. Similar results were obtained by applying the Kunitz test to isolated γ-globulins. The total trypsin inhibitory activity of the whole anti- bovine trypsin serum increased 50 % from the beginning to the end of the immunization period (tested on bovine trypsin).

By the use of the electrophoretic casein precipitating inhibition test (CPI-test) the serological relationship between proteolytic enzymes produced by different species within the genera Clostridium and Bacillus has been tested. The proteases produced by Clostridium botulinum types A, B, C, D and F cross-reacted with each other. Clostridium botulinum strain 84 was inhibited by antiproteases produced against Clostridium sporogenes, Clostridium botulinum types C and F (protease F I and F II), but not by antiproteases against Clostridium botulinum types B and F (protease II), Clostridium bifermentans and Clostridium perfringens. The protease of the newly described Clostridium botulinum strain 89 (type G) was inhibited by Clostridium sporogenes antiprotease, but not by any of the other antiproteases. It is not possible to differentiate between Clostridium botulinum, Clostridium sporogenes and Clostridium perfringens by use of serological differentiation of their proteolytic enzymes. The protease of Clostridium bifermentans is not serologically related to any of the species tested in this investigation. Proteases produced by different Bacilli were not inhibited by antiproteases from Clostridium botulinum types B, C and F, Clostridium sporogenes, Clostridium bifermentans, and the two strains of Clostridium perfringens tested. This investigation indicates a serological relationship between proteases from different Clostridium species, but not a serological relationship between proteases produced by the Clostridium species and Bacillus species tested.

Commercial ovomucoid trypsin inhibitors (OMTI) and egg white were exposed to various doses of gamma irradiation, using a cobalt-60 source. The inhibitory activity of non-irradiated and irradiated samples on bovine trypsin was tested by the Kunitz method (Kunitz1947). The activity of the inhibitors was shown to decrease exponentially with increasing radiation dose. The D10for OMTI was 0.6 Mrad and for the trypsin inhibitors in the crude egg white diluted 1: 5 in saline 3.0 Mrad under the conditions used. The activity of the non-irradiated and irradiated inhibitors upon various animal, microbial and plant proteinases was also determined by using the crosswise casein precipitating inhibition test (crosswise CPI-test) (Fossum1970a). By this method, which is more sensitive than the Kunitz method, trypsin inhibitory activity in eggwhite could be detected after exposure to an irradiation dose of 9 Mrad. No inhibitory activity could be found against any enzyme after an irradiation dose of 15 Mrad.

It has been shown that lignosulphonic acids have an inhibitory effect on various animal and microbial proteinases. The activity of 0.005 mg swine trypsin, 0.007 mg bovine a-chymotrypsin and the proteinase activity in 0.004 ml of jejunum content from pig is inhibited 70–75 % by 0.25–0.30 ml of a solution containing 250 diffusion units of peptide-precipitating lignosulphonic acids per 50 µl.