Bovine seminal plasma aSFP: localization of disulfide bridges and detection of three different isoelectric forms.

Acidic seminal fluid protein (aSFP) is a major 13 kDa protein isolated from bull seminal plasma and characterized as a new growth factor which stimulates in vitro cell division and progesterone secretion by ovarian cells. Here, we establish that the four cysteines of oxidized aSFP form two disulfide bridges between nearest-neighbour residues. This pattern is conserved in boar spermadhesins, with which aSFP shares up to 50% amino acid sequence identity, and other proteins of the recently identified CUB domain family. Using isoelectric focusing in combination with sulfhydryl group-specific blotting, the three forms of aSFP were identified as completely oxidized (pI 4.7), partly reduced (pI 4.8) and fully reduced at pI 5.1. These results indicate that native aSFP possesses two pairs of cysteine residues of different reactivity. The observation that aSFP can protect sperm from oxidative damage might be explained by its reduction/oxidation behaviour.

Heat induced crosslinks in milk proteins and consequences for the milk system.

The protein system of milk is rather unusual, there are nearly no interchain crosslinks found. Even intrachain crosslinks, especially disulfide bridges, are present only in about every fourth protein molecule. Heating causes dramatic changes in the structure of milk proteins, resulting in the formation of polymeric networks. The contribution of individual milk proteins, namely the beta-lactoglobulins, alpha-lactalbumin and chi-casein, to the formation of crosslinks is studied with respect to heating temperature and time, pH and atmosphere. Measured are changes in molecular weights and in the SH/SS-levels as well as the formation of dehydroalanine, lysinoalanine, lanthionine and isopeptide bonds. Some practical aspects of crosslinking in milk proteins are discussed.

Production of Lipase by Byssochlamys fulva.

Byssochlamys fulva exhibited maximum growth and lipase production at 30°C in 6 d at pH 7.0. Aeration enhanced lipase yield by 22%. Growth medium containing maltose showed maximum production of lipase followed by glucose, mannitol, fructose, xylose, sucrose and galactose in decreasing order. Among the nitrogen sources tested. lipase yield was maximum with 2% casamino acids. Tributyrin induced lipase synthesis, whereas other lipids inhibited lipase production.

[Proteolytic activities of Ps. fluorescens in milk: determination with azocasein in comparison with HPA]. / Proteolytische Aktivitäten von Ps. fluorescens in Milch: Bestimmung mit Azocasein und vergleichsweise mit HPA.

Proteolytic activity of psychotrophie bacteria in milk can be measured in a practical and comparatively sensitive manner using Azocasein as a substrate. The necessary parameters were exemplarily developed and demonstrated with a typical, strongly proteolytic strain of Pseudomonas fluorescens (No. 112). Comparison of the improved Azocasein method with a modified version of the HPA method of Cliffe and Law--they had proposed it for predicting the stability of UHT milk--showed nearly the same sensitivity with six test strains of Pseudomonas fluorescens. The first significant detection of proteolytic activity could be made approximately at the same time except with one strain of the biotype II (miscellaneous strains). It is discussed, whether the demonstrated methods can be judged (by the bacterial counts of detection) as sufficiently sensitive for to prediction of term spoilage during storage of UHT products.

[Studies on the determination of gelatine in milk products especially in yoghurt (author's transl)]. / Zur Bestimmung von Gelatine in Milcherzeugnissen, speziell in Joghurt

Gelatine may be determined in yoghurt and similar milk products after total hydrolysis with HCl in the presence of SnCl2 via the content of hydroxyprolin. The high proportion of carbohydrates requires the use of high amounts of SnCl2, since the stannic ions cannot be removed without great lossess of hydroxyprolin. The lossess, however, are well reproducible so that the determination can be performed with an accuracy of +/- 5% by means of calibration curves. Since the gelatine qualities used in the dairy industry are varying in their content of hydroxyprolin by +/- 10%, the determination of the gelatine portion in yoghurt may be performed within a range of 0.05 -- 1.00% with an accuracy of aroung +/- 15%.

[Formation of dehydroalanine, lanthionine and lysinoalanine during heat treatment of beta-lactoglobuline A (author's transl)]. / Bildung von Dehydroalanin, Lanthionin und Lysinoalanin beim Erhitzen von beta-Lactoglobulin A.

Solutions of beta-lactoglobulin A were heated at 97.5 degrees C at pH 6.9 and 9.8 80 min. The formation of dehydroalanine, lanthionine and lysinoalanine as well as changes in the levels of cysteine, cystine and the basic amino acids were recorded. Lanthionine (equal amounts of the 1- and the mesoform) was formed only in small amounts, while the lysinoalanine levels were rather high. Beside this, lysine was converted to unknown compounds. The main sources for the formation of dehydroalanine are the cystine moieties of the protein. Cysteine residues are first oxidized to cystine before beta-elimination occurs.

Heat inactivation of exogenous proteinases from Pseudomonas fluorescens. I. Possibility of inactivation in milk.

The inactivation reaction of the proteinase of a P. fluorescens strain of biotype I in milk was investigated at 130-150 degrees C, also in milk and in buffer with and without added CaCl2 at temperatures below 100 degrees C. The decline in activity corresponded to first order kinetics in the UHT region; Ea = 115 kJ/mol. D values were 290 (130 degrees C), 124 (140 degrees C) and 54 s (150 degrees C); therefore, the usual temperature time combinations of UHT treatment are not sufficient to achieve the required rates of inactivation. At temperatures below 80 degrees C, inactivation corresponded increasingly to second order kinetics with considerably higher reaction rates; at 55 degrees C, an inactivation reaction corresponding to that induced by UHT treatment could be achieved at a thermal stress lower by a factor of 500. This "low temperature inactivation" was observed in a further 20 strains representing the spectrum of P. fluorescens. The average rates of inactivation following heat treatment in milk for 20 min are 47% at 55 degrees C and 44% at 60 degrees C. This can be regarded as the most effective temperature range for the inactivation of the proteinases in milk. Clear connections can be seen between the biotype groups and the optimum temperature for inactivation: biotype group I ca. 55 degrees C, group II (with a few exceptions) less than or equal to 50 degrees C and group III greater than or equal to 60 degrees C. The inactivation reaction is systematically influenced by the proteins and Ca++ ions present in milk.

A new metalloproteinase from Pseudomonas fluorescens biotype I.

Isolation and purification of a metalloproteinase from Pseudomonas fluorescens Biotype I are described. The molecular mass of the enzyme is 46 kDa, its isoelectric point is 8.1, its activity is trypsin-like. The amino-acid composition of the single chain protein is given. One molecule of the enzyme contains 1 atom of zinc and 9 atoms of calcium.

[Detection and determination of protein-bound formaldehyde. II. Improved recovery of formaldehyde by reduction with sodium cyanoborohydride (NaCNBH3)]. / Nachweis und Bestimmung von Protein-gebundenem Formaldehyd. II. Verbesserte Wiederfindung von Formaldehyd durch Reduktion mit Natriumcyanoborhydrid (NaCNBH3).

When using NaCNBH3 as a reductant, it is possible to recover 70% of added formaldehyde in foods--in this skim milk powder. Formaldehyde is attached to the epsilon-Aminogroups of lysine and is detected after reduction in form of N epsilon-methyllysine in an automated amino acid analyzer system. After addition of N alpha-acetyllysine the reduction is carried out by incubating the skim milk powder at 70 degrees C for 1 h. The best combination of pH, concentration of reductant, time and temperature of incubation is described.

On the influence of non-enzymatic crosslinking of caseins on the gel strength of yoghurt.

After storage of UHT milk at 37 degrees C resp. 50 degrees C, yoghurt was prepared. For a storage temperature of 37 degrees C, breaking strength of the yoghurt samples increased from 2.7 to 5.8 N with increasing storage duration of the UHT milk. A plateau is reached after 17 days of storage. This increase in breaking strength correlates with a significant increase in non-reducible casein oligomerization from 14% for fresh UHT milk to 25% measured using size exclusion chromatography under reducing and denaturing conditions and calculated as sum of predominantly formed dimers and trimers at the total casein fraction. At a storage temperature of 50 degrees C, a less increase in breaking strength from 2.7 to 4.6 N with a plateau after 17 days was observed while casein oligomerization increased to 63%. After acid hydrolysis, only lysinoalanine and histidinoalanine were detected in the caseinate samples via amino acid analysis. The quantified concentration of lysinoalanine and histidinoalanine could not explain the observed casein oligomerization. Thus, unknown crosslinked amino acids must have been formed during storage, inducing significant changes in the functional properties of milk proteins.

Transpeptidation in sequence analysis. Investigations concerning a native and a synthetic hexapeptide.

On sequencing a hemoglobin a peptic hexapeptide with the amino acid composition Asp2, Gly, Val, Lys2 was isolated. Cleavage of this peptide with Tos-Phe-CH2Cl-trypsin resulted in the fragments Asp-Lys, Gly-Asp, Val-Lys, Asp--Lys-Gly-Asp, Asp-Lys-Val-Lys and Val-Lys-Gly--Asp. From these data two possible but inconsistent sequences for the hexapeptide can be derived: Asp-Lys-Val-Lys-Gly-Asp and Val-Lys-Asp-Lys-Gly-Asp. Fragments obtained by other cleavage procedures, direct sequencing of the globin peptide-chain with a sequenator as well as X-ray data of this hemoglobin show, that only the sequence starting with Asp occurs in the intact protein. Therefore the peptide Asp-Lys-Gly-Asp must have been formed by transpeptidation during sequence work. In order to verify this, Asp-Lys-Val-Lys-Gly-Asp was synthesized by an unequivocal, conventional procedure. Tryptic digestion of this hexapeptide also resulted in ASP-Lys-Gly-Asp in addition to the expected fragments. Thus it has been shown for the first time, that sequencing conditions may alter the constitution of a peptide.

[Polysaccharide additives in foods rich in proteins. i Sample preparation for estimation, especially in fresh cheese (author's transl)]. / Polysaccharidzusätze in eiweissreichen Lebensmitteln. I. Probenaufbereitung für die Bestimmung, speziell in Frischksen

Published procedures for the isolation of polysaccharides from foods rich in proteins, especially fresh cheese, proved not very useful for the concentration in question (0,01--0.2%). We, instead, propose the following sample preparation: 1. Removal of lipids by an ethanol--ethyl ether--petrol ether mixture; 2. complete hydrolysis of proteins with pepsin, pronase E and a mixture of aminopeptidase M and prolidase; 3. separation of the polysaccharides from the low molecular substances and from enzyme residues by gel filtration on Biogel P-2; 4. concentration of the polysaccharides containing fraction by freeze drying, ultrafiltration or evaporation. --The method was tested with quarg and double cream fresh cheese, it can, however, be adopted to use in other products.

Mechanisms of heat inactivation of a proteinase from Pseudomonas fluorescens biotype I.

Heat inactivation of a metalloproteinase, isolated from Pseudomonas fluorescens biotype I strain 112, was investigated in the temperature ranges 50-60 degrees C and 90-140 degrees C. At 90 degrees C the denaturation of the enzyme followed first-order kinetics with a decimal reduction time of 110 min and a velocity constant K of 3.5 X 10(-4) S-1. Activation energy Ea was 100 kJ/mol for this temperature range. In the 50-60 degrees C region the proteinase was inactivated by autolysis, as shown by electrophoresis and gel filtration. At 55 degrees C the decimal reduction time was approximately 22 s, at 57 degrees C it was 8 s. Rapid inactivation at 55 degrees C was only possible if the enzyme was heated from lower temperatures, but not if cooled down from 90 degrees C. This is due to a conformational change of the protein at this temperature. A model for the description of heat inactivation in the two temperature ranges is proposed.

Influence of EDTA and metal ions on a metalloproteinase from Pseudomonas fluorescens biotype I.

The inactivation of a metalloproteinase from Pseudomonas fluorescens Biotype I with EDTA was investigated at 22 degrees C and 37 degrees C. At 22 degrees C proteolytic activity decreases linearly with time and an inactive apoenzyme is obtained by dialysis. Proteolytic activity can be restored with several metal-ions, Ca2+, Zn2+, Mg2+, Sr2+ and co2+ give the best results. Activity and substrate specificity are influenced by the metal-ions. Reactivation depends on the concentration of the metal-ions, optimum concentration is 1 mM for Ca2+ and 50 microM for Zn2+. The isoelectric point of the apoenzyme is around 8.0, this is about 0.3 pH-units lower than the isoelectric point of the native proteinase. At 37 degrees C inactivation follows first order kinetics and is irreversible because of autolysis as shown by a gel filtration-experiment.

Determination of biogenic amines by RP-HPLC of the dabsyl derivates.

An automated derivatization with dabsyl chloride in combination with a high-performance liquid chromatographic analysis is described for the determination of biogenic amines in complex matrices. The sample clean-up procedure consists of an ultrafiltration step, resulting in average recoveries for cheese in the range of 88% to 100%. A linear relation between the area of the peak and amine concentration was observed between 0.5 and 500 pmol for all amines under investigation and the detection limits ranged between 0.34 and 0.76 pmol. The average repeatability of both the performance of the chromatographic determination and the whole method, including sample preparation, examined using cheese samples containing different amounts of biogenic amines was found to be between 2.0% and 3.7%. The method was applied to the analysis of several types of food and feed, selected examples are given by the separation of dabsyl derivates from cheese, wine, and salami-type sausage.