Per Einar Granum

An absolute method for protein determination based on difference in absorbance at 235 and 280 nm.

Abstract Because of the importance of quantitative determination of protein in the research laboratory as well as in the food and feed industries (1), search for the ideal method continues unabated after many years. Methods available include nitrogen determination (Kjeldahl (2) and Dumas (3)), hydrolysis of the protein, derivatization of the amino acids with phthalaldehyde and fluorescence determination (4), determination of bound or free lysine (5) or glutamate (4), and the Lowry (6), biuret (7) dye-binding (8–11) turbidity (12) and spectral methods (13). With the exception of the spectral methods, the methods involve destruction of the sample. In this paper we report the use of difference in absorbance between 235 and 280 nm for determination of protein concentration.

Clostridium perfringens toxins involved in food poisoning

Clostridium perfringens is a Gram-posit ive spore-forming, anaerobic and nonmotile rod. It is a normal soil inhabitant, and plays an important part in the putrification process. C. perfringens is also readily isolated from dust, raw meat and the intestinal tract of man and animals. It is divided into five types based on the production of four major lethal toxins, as shown in Table I. Table I also shows the different types of diseases caused by C. perfringens. The enterotoxin has been shown to be produced by types A, C and D (Table I), but is not used for typing C. perfringens. C. perfringens is responsible for two very different types of food poisoning (Table I). Type A causes the mild classic form of food poisoning, while the severe type of food poisoning known as necrotic enteritis is caused by type C. The symptoms of the C. perfringens type A food poisoning are: acute abdominal pain and diarrhoea with nausea and fever, with vomiting being rare. The symptoms usually appear 8-12 h (6-24 h) after ingestion of contaminated food (106-107 cells/g) and can last between 12-24 h (Hobbs, 1969). The poisoning is seldom fatal, although a few deaths have been reported in elderly and infirm (Parry, 1963; Sutton and Hobbs, 1965). The true incidence of C. perfringens food poisoning is not known, because of the relative mildness of the syndrome. Nevertheless, in several countries C. perfringens has been reported as the most important cause of food poisoning, only exceeded in incidence by food infections caused by Salmonella (Cliver, 1987; Reynolds, 1987). Necrotic enteritis is a rare and often fatal illness. The symptoms are: acute sudden onset of severe abdominal pain and diarrhoea (often bloody), sometimes with vomiting, and necrotic inflammation of the small intestine.

Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin

A new method for preparing non-parenchymal rat liver cells (NPC) is described. The liver cell suspension, prepared by perfusing the liver with collagenase, was treated with enterotoxin from Clostridium perfringens for 15 min. The enterotoxin made the parenchymal cells leaky, and these cells could be separated from the NPC by centrifugation in a solution containing Nycodenz (20%, w/v). During the centrifugation, the NPC floated, while the parenchymal cells sedimented. The yield of NPC per liver (200 g rat) was about 250 X 10(6) cells. The NPC were further separated into endothelial cells, Kupffer cells and stellate cells by centrifugal elutriation. This method was particularly useful for preparing endothelial cells in high yield (100 X 10(6) cells per liver). Intravenously injected formaldehyde-treated albumin was selectively taken up by the endothelial cells. Isolated endothelial cells in suspension as well as in surface culture maintained their ability to endocytose this ligand.

Chymotrypsin treatment increases the activity of Clostridium perfringens enterotoxin.

Chymotrypsin treatment of the enterotoxin from Clostridium perfringens results in the loss of 36 amino acids from the N-terminus and 3 amino acids from the C-terminus. This processing results in a 3.2 fold increase in activity on Vero cells, which is close to what had been found for trypsin treatment following the loss of 25 amino acids from the N-terminus.

Enterotoxin formation by Clostridium perfringens during sporulation and vegetative growth

Abstract Growth, sporulation and enterotoxin production have been followed for three different strains of Clostridium perfringens on a vegetative (FTG) medium and a sporulation (DS) medium. Enterotoxin production was followed by use of enzyme-linked immunosorbent assay (ELISA). All strains produced enterotoxin under both vegetative growth and sporulation in spite of the fact that one strain is known to be enterotoxin negative. The presumed negative strain produced about the same amount of enterotoxin on both media (1 ng/ml). The two other strains produced about 1000–2000 times more enterotoxin during sporulation than during vegetative growth.

The amino acid sequence of the enterotoxin from Clostridium perfringens type A

The amino acid sequence of the enterotoxin from Clostridium perfringens type A was determined by analysis of peptides derived from the protein by digestion with trypsin chymotrypsin, thermolysin, pepsin, a lysinespecific protease, S. aureus V8 protease and a proline‐specific protease, and fragments generated by cleavage with cyanogen bromide or by dilute acetic acid in 7 M guanidine HCl. The sequence which is complete except for the definite order of 3 small peptides between residues 88 and 103 consists of 309 amino acids and contains a correction to our preliminary announcement [(1984) FEMS Symp. 24, 329‐330].

Studies on α-amylase inhibitors in foods

Abstract Different foods have been tested for α-amylase inhibitor activity on human salivary α-amylase. High activity was found in wheat flour (590 units/g), whole wheat flour (351 units/g) and whole rye flour (186 units/g) but oat flour and barley flour had no activity. Bread baking reduced the α-amylase inhibitor activity in white bread and rye bread about 80–90% and no activity was left in whole wheat bread. Spaghetti had high activity (248 units/g) before boiling but less than 2% of the activity remained after 15 min of boiling. Red beans had some activity before boiling (41 units/g) but no activity was left after boiling for 1 1 2 h . Split peas, brown rice, potato, carrot and swede did not contain any α-amylase inhibitor activity.

Purification and physicochemical properties of an extra-cellular cycloamylose (cyclodextrin) glucanotransferase from Bacillus macerans

An extracellular cycloamylose (cyclodextrin) glucanotransferase (EC 2.4.1.19) from Bacillus macerans was purified to homogeneity by adsorption on starch, ammonium sulfate fractionation, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of 67,000 and consisted of one polypeptide chain. The isoelectric point was pH 5.4. Temperature and pH optima were 60 degrees and 5.4--5.8, respectively. The purified enzyme was quite stable at 50 degrees (pH 6.0), but lost approximately 80% of its activity at 60 degrees for 30 min (pH 6.0). Prolonged digestion by trypsin did not affect the catalytic properties of the enzyme. The Km for starch was 5.7 mg/ml.

Inhibition of protein synthesis by a tryptic polypeptide of Clostridium perfringens type a enterotoxin

The biological activity of Clostridium perfringens enterotoxin can be tested more precisely and with a much higher sensitivity by using the inhibition of protein synthesis by Vero cells, rather than the guinea pig skin test. Tryptic peptides of the enterotoxin produced in the presence of different concentrations of sodium dodecyl sulfate (0-1%) have been tested for biological activity (Vero cells) and inhibitory effect on cell-free protein synthesis (rabbit reticulocyte lysate). A fraction of tryptic peptides, about 16,000 daltons, was able to inhibit the cell-free protein synthesis, while the native enterotoxin had no such effect. The 16 kDa fraction had, however, lost the ability to disrupt the Vero cells (normal biological activity). It is probable that the enterotoxin has the double function (A and B chain), known from several other toxins, confined in its single polypeptide chain.

The amylase-producing microflora of semi-preserved canned sausages: identification of the bacteria and characterization of their amylases.

ZusammenfassungDreizehn Stämme von Amylase produzierenden Bakterien wurden von halbkonservierten Dosenwürstchen und ihren Bestandteilen isoliert. Alle gehörten zum GenusBacillus und konnten in 4 verschiedenen Gruppen geteilt werden. Zwei der Gruppen waren verschiedene Stämme vonB. subtilis, eine warB. amyloliquefaciens und die letzte Gruppe warB. macerans. Die Identifizierung der verschiedenen Bakterienarten wurden nach Wachstum durch Diskgelelektrophorese der überstehenden Kulturflüssigkeit unterstützt. Die Amylasen wurden durch auf Temperatur-, pH-Optimum und Thermostabilität charakterisiert. Obwohl einige der Amylasen sehr thermostabil sind, scheint die einzige Erklärung des Stärkeabbaues in halbkonservierten Nahrungsmittel das Überleben der Sporen mit nachfolgender Auskeimung, Wachstum und Amylaseproduktion zu sein.SummaryThirteen strains of amylase-producing bacteria were isolated from semi-preserved canned sausages and their ingredients. All belonged to the genusBacillus, and could be separated into 4 diffärent groups. Two groups were diffärent strain ofB. subtilis, one wasB. amyloliquefaciens and the last wasB. macerans. The identification of the different bacteria species was supported by disc gel electrophoresis of the supernatant culture fluid, after growth. The amylases were chracterizied with regard to temperature optimum, pH optimum and thermostability. Although some of the amylases appear to be quite thermostable, the only explanation for starch degradation in semi-preserved foods seems to be the amylase production from outgrowing spores which survived the heat treatment.