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.

Isolation and rapid sequence characterization of two novel bovine beta-lactoglobulins I and J.

Two novel bovine beta-lactoglobulins I and J have been isolated from bovine milk and characterized by isoelectric focusing. Their primary structure was determined by a very rapid method consisting of a combination of Edman sequencing, mass analysis, and ladder sequencing by mass spectrometry. We found that both new beta-lactoglobulins are of the bovine beta-lactoglobulin B-variant type. beta-lactoglobulin I shows Gly instead of Glu at position 108, whereas beta-lactoglobulin J shows a Pro-to-Leu exchange at position 126.

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.

Inactivation kinetics of gamma-glutamyltransferase during the heating of milk.

The inactivation of gamma-glutamyltransferase (GGT, E.C.2.3.2.2) during the heating of milk to between 65.0 degrees C and 76.0 degrees C for periods of 5 s to 1000 s follows a first-order reaction (energy of activation 457 kJ/mol) and can be used to monitor the process of milk pasteurisation. GGT activity in raw milks from individual cows showed only little variation (5.92 +/- 0.59 units). Residual GGT activity in 17 commercial milks ranged between 1% and 20%, indicating a heat treatment at the upper limit of the permitted pasteurisation conditions.

A method to mimic and to study the release of flavour compounds from chewed food.

A method for analysing the flavour release from chewed food has been developed. Flavour release is studied in an artificial mouth simulating the process of chewing and using fluid model systems, in our case aromatised oil in water emulsions. The fast transfer of volatile substances from the chewpulp into the gaseous phase is followed up by comparing six quickly taken gas samples. Volatile substances are analysed by means of a special technique which includes cryofocusing and capillary gas chromatography. As a wide spectrum of individual volatile substances is considered, systematic investigations into the flavour release from food under mouth-typical conditions are possible.

Fast and sensitive determination of furosine.

Sensitive determination of furosine in acid hydrolysates of foods was achieved by isocratic ion-pair reversed-phase HPLC and direct UV-detection within a run time of 5 minutes and levels lower than 1.5 mg per kg of protein. The formation of furosine during hydrolysis of food samples with hydrochloric acid of varying concentration was studied. Furosine formation increased linearly with acid concentration (4 to 8 mol/L).

Studies on the formation of lysinomethylalanine and histidinomethylalanine in milk products.

From reaction mixtures consisting of N-acetyldehydroaminobutyric acid methyl ester and N alpha-acetyl-L-lysine or N alpha-acetyl-L-histidine, respectively, distinct amounts of the cross-link amino acids N epsilon-(2-amino-2-carboxy-1-methyl-ethyl)-L-lysine (lysinomethylalanine, LMeAL) and N tau-(2'-amino-2'-carboxy-1'-methyl-ethyl)-L-histidine (histidinomethylalanine, HMeAL) were isolated via preparative ion-exchange chromatography and identified by 1H- and 13C-nuclear magnetic resonance. In the amino acid chromatogram, both compounds eluted clearly separated from other basic amino acids. However, neither LMeAL nor HMeAL could be detected in numerous acid hydrolysates of a range of milk products. In model studies, threonine showed a significantly lower tendency for an alkali-induced beta-elimination reaction compared to serine. The reactivity of the resulting dehydroaminobutyric acid towards nucleophiles was more than tenfold lower as compared to dehydroalanine. Thus, the formation of LMeAL as well as of HMeAL during food processing is negligible.

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.

A simple method for the preparation of furosine and pyridosine reference material.

After heating N alpha-acetyllysine and glucose for 4 h at 90 degrees C in the dry state and subsequent acid hydrolysis with 7.8 N HCl, preparative fractionation of the dihydrochlorides of furosine and pyridosine was achieved by cation-exchange chromatography. The lysine derivatives could be prepared with high yield and sufficient purity for the use as reference material.

Detection and identification of the cross-linking amino acids N tau- and N pi-(2'-amino-2'-carboxy-ethyl)-L-histidine ("histidinoalanine", HAL) in heated milk products.

An unknown ninhydrin positive compound, X, was detected in acid hydrolysates of heated skim milk samples by amino acid analysis, eluting between phenylalanine and pyridosine in the chromatogram. The formation of X correlated with heating time and temperature. preparative ion-exchange chromatography enabled the isolation of X and a second minor compound from a milk protein hydrolysate and from a model mixture consisting of N alpha-acetylhistidine and methyl-2-acetamidoacrylate (acetyldehydroalaninemethylester), in a relative abundance of 8 to 1. By 1H-NMR spectroscopy, the two compounds could be identified as the N tau- and N pi-isomers of N-(2'-amino-2'-carboxy-ethyl)-L-histidine (histidinoalanine), a cross-link amino acid that has not been described in food proteins up to now. In a number of foods containing milk protein, the N tau-histidinoalanine contents were between 50 and 1800 mg/kg protein, which is in a concentration range comparable to the potential nephrotoxic cross-link lysinoalanine, which was determined simultaneously.

Application of a microbial sensor for determination of short-chain fatty acids in raw milk samples.

A microbial sensor system, based on the use of immobilized Arthrobacter nicotiana and an oxygen electrode, was applied to determine free short-chain fatty acids in raw milk samples and the result was compared with gas chromatography (GC) and a titrimetric method. The sensor response was linearly related to the concentration of short-chain fatty acids obtained by GC (n = 10, r = 0.92) and to the total concentration of free fatty acids obtained by titrimetric measurement (n = 10, r = 0.78). This result suggests that the present microbial sensor can selectively determine free short-chain fatty acids in raw milk samples and may be useful as a very fast detection method of rancidity in milk.

Evaluation of the extent of the early Maillard-reaction in milk products by direct measurement of the Amadori-product lactuloselysine.

A new method for the evaluation of the extent of lysine modification caused by the Maillard reaction is presented, based on the direct determination of the Amadori product lactuloselysine plus unmodified lysine after complete enzymatic hydrolysis by ion-exchange chromatography. Using a simple mathematical relation, the amount of modified lysine in milk products can be estimated without knowledge of the initial lysine value before heating or storage.

Genetic variants of bovine beta-lactoglobulin. A novel wild-type beta-lactoglobulin W and its primary sequence.

A novel bovine beta-lactoglobulin W has been isolated and its complete primary structure is presented. It was isolated by chromatofocusing of a beta-lactoglobulin AW heterozygote and purified by recrystallization. During sequencing of the oxidized protein, it became evident that the new beta-lactoglobulin W is a subtype of variant B with a single difference at position 56. This Ile----Leu substitution, which means a shift of a methyl group from C-beta to C-gamma of the amino-acid side chain causes a change of pI of 0.007 units, which can be detected by high resolution electrophoresis. This Ile56 amino-acid residue is among the most conserved residues with the exception of kangaroo beta-LG. The structures of other bovine beta-lactoglobulins and their relationships are discussed.

Isoelectric focusing in immobilized pH gradients with carrier ampholytes added for high-resolution phenotyping of bovine beta-lactoglobulins: characterization of a new genetic variant.

The genetic variants of bovine beta-lactoglobulin (beta-lg) from the "Murnau-Werdenfelser" breed were analyzed in three different isoelectric focusing (IEF) systems. While carrier ampholyte IEF with a pH gradient of 0.2 pH/cm did not resolve the new variant W from the B variant and IEF in immobobilized pH gradients (IPG) with 0.1 pH/cm only partially resolved it, adequate separation was achieved with IPG-IEF in a pH 5.25-pH 5.7 gradient, in presence of 0.8 % w/v carrier ampholytes, both over a 10 and 17 cm separation distance. Apparent isoelectric points (pI's) and genetic frequencies (f) were as follows: beta-lg A, pI = 5.370, f = 0.364; beta-lg B, pI = 5.485, f = 0.480; beta-lg W, pI = 5.492, f = 0.076; and beta-lg D, pI = 5.610, f = 0.080. The small difference of delta pI = 0.007 between beta-lg B and beta-lg W respectively, seems to originate from a "silent" substitution of neutral amino acid residues as compared to the larger delta pI's of the other genetic variants of beta-lg, which result from substitution of charged amino acids.

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.

Indoxylsulfate in milk.

Indoxylsulfate in 27 individual milk samples ranged from 25.4 to 111 micrograms/l (average 52.3 micrograms/l); pooled milk samples from 12 farms contained 81.1 micrograms/l (46.4-146 micrograms/l); the variation in indoxylsulfate concentration of dried skimmed milk over a period of one year amounted to 23%. This variability is likely attributable to regional and seasonal, and hence to feeding effects. The indoxylsulfate content of milk seems also to be dependent upon the degree of fermentation during processing of milk; yoghurt contained very low amounts of this component (6.4 micrograms/kg). On the other hand, heat treatment of the milk (HTST, UHT, sterilization) apparently does not affect its indoxylsulfate content. Indoxylsulfate concentrations in milk correlated positively with blood-serum indoxylsulfate content (r = 0.752, n = 20) and with the urea content of milk (r = 0.61, n = 12 pooled milks). Further research is suggested on the use of indoxylsulfate determinations as an aid to determine sweet whey added to dried skimmed milk, also as an analytical tool to differentiate bovine and sheep milks.

[Demonstration and identification of a new Maillard compound in heated milk]. / Nachweis und Identifizierung einer neuen Maillard-Verbindung in erhitzter Milch.

By means of polarography a new Maillard product (5a) was detected in heated milk. The compound was isolated from a glycine/lactose mixture, which had been heated in buffer at pH 6,8, and then freeze dried by extraction with ethylacetate/methanol. According to its spectroscopic properties, 5a is 4-0-beta-D-galactopyranosyl-2-hydroxy-2-methyl-2H-pyran-3-(6H)-one. Though the amount of 5a formed during heat treatment of milk corresponds to the severity of heat stress, the usefulness of 5a as an indicator with respect to intensity of heat treatment is limited. 5a is rather rapidly converted to galactosyl isomaltol; also unknown peaks develop in the polarogram.

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.