G. Humbert

Method for the measurement of lipase activity in milk

Lipolysis has many effects in dairy technology. Undesirable consequences include deleterious effects on the organoleptic properties of milk and some cheeses; however, it is desirable in some other cheeses. The usual methods for detecting lipolysis in milk measure the amounts of free fatty acids liberated from triacylglycerols (International Dairy Federation, 1991). Lipase activity is detected by incubation of samples with substrates such as tributyrin (Castberg et al . 1975) or triolein (Nilsson-Ehle & Schotz, 1976; Shirai et al . 1982). A few reports describe methods using synthetic chromogenic substrates, e.g. p -nitrophenyl butyrate (Shirai & Jackson, 1982), β-naphthyl caprylate (McKellar, 1986; McKellar & Cholette, 1986) or 4-methylumbelliferyl oleate (Stead, 1983). Tsakalidou et al . (1992) have developed the detection of esterase activities on electrophoresis gels.

Role of the O-phosphoserine clusters in the interaction of the bovine milk αs1-, β-, κ-caseins and the PP3 component with immobilized iron (III) ions

Abstract α s1 - and β-Caseins have a sequence cluster -Ser( P )-Ser( P )-Ser( P )-Glu-Glu- which is not present in κ-casein and the whey PP3 component. The affinity of these phosphoproteins for the iron(III)-iminodiacetic acid (IDA) complex immobilized on Sepharose was studied a a function of pH, urea concetnration, calcium ion concentration, enzymatic dephosphorylation and temperature. The affinity of the three polyphosphorylated proteins ( α s1 - and β-caseins, PP3) was similar. The sequence cluster was not a specific recognition pattern for the iron(III) ion. These three proteins presented a site of high affinity and a site of weak affinity. κ-Casein, which had only one Ser( P ) residue, presented only the site of weak affinity. Their primary site which was absent after dephosphorylation or calcium ion addition required the presence of at least two Ser( P ) residues close in space. Their secondary site was sensitive to the presence of urea. It was sensitive to pH variation for PP3 and κ-casein. The study of the affinity of a few free amino acids towards iron(III)-IDA showed that the secondary site involved tryptophan and tyrosine residues for α s1 - and β-caseins, histidine residues for PP3 and cysteine residues for κ-casein.

Enzymatic assays for native plasmin, plasminogen and plasminogen activators in bovine milk.

Rapid and sensitive assays for plasmin, plasminogen and plasminogen activators (PA) were developed and applied to bovine milk. The reaction medium was clarified by addition of a dissolving agent after hydrolysis of a fluorescent substrate specific for plasmin. This final step enabled the use of larger sample amount with higher substrate concentration than other methods, and avoided previous sample preparation. The use of 4 g gelatin/l in buffers preserved plasmin activity, thus avoiding risks of overestimation of the assays results. Sensitivity, detection level, repeatability and analysis run time of plasmin and plasminogen assay were improved over previous enzymatic methods with synthetic substrates. The PA assay was assessed by measuring conversion of exogenous plasminogen into plasmin. A new kinetic approach was used to enable the direct determination of global PA activities on raw milk samples without interference from indigenous plasmin.

Measurement of proteolysis in milk and cheese using trinitrobenzene sulphonic acid and a new dissolving reagent.

Milk contains indigenous plasmin-like and thrombin-like proteinases and also exogenous proteinases, mainly extracellular enzymes of psychrotrophic bacteria. Their activities lead to protein hydrolysis which may have important effects on milk quality and cheese yield. It is also useful to estimate proteolysis and its development during cheese ripening. Milk opacity or turbidity due to calcium phosphocaseinate micelles and fat globules necessitates preliminary sample treatment, i.e. precipitation and filtration before spectrophotometric measurement. It is possible to eliminate these firs steps of sample treatment by dispersing the colloidal constituents. Some authors have already used different dissolving mixtures for sample analysis. Nakai & Anh Chi Le (1970) were the first to determine protein contents of milk and dairy products by absorbance measurement at 280 nm after clarification. Bosset & Blanc (1977) used the Biuret method. Owen & Andrews (1984) measured free amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS). Humbert et al. (1982) and Stead (1987) used synthetic or specific substrates for proteinase determination. In this work, we report the application of a new dissolving reagent to measure free amino groups in milk and cheese.

Development of a microparticle‐enhanced nephelometric immunoassay for the quantification of beta‐casein in milk

Beta‐casein determination has been performed in cows milk by a microparticleenhanced nephelometric immunoassay. Hydrophilic monodisperse beta‐casein coated microspheres, specifically conceived as an antigen‐antibody reaction support, are agglutinated by anti‐beta‐casein antiserum. Free beta‐casein inhibits this immunoagglutination, allowing its fast (30 min) and sensitive (1 μg l‐1) quantification with accuracy and precision, by nephelometric measurement of the light scattered by the beta‐casein coated microsphere agglutinates. This immunoassay of beta‐casein is easy to perform in milk (no washing, no phase separation, nor sample pretreatment). Microparticle‐enhanced nephelometric immunoassay appears as an alternative method to measure a large variety of molecules in dairy analysis.

Improvement of a method for the measurement of lactoperoxidase activity in milk

Abstract Lactoperoxidase (LPO) is one of the two most used indigenous enzymes to evaluate milk heat treatment, alkaline phosphatase being the other. In the present work, LPO activity was measured using Clarifying Reagent ® to facilitate spectrophotometric measurement in milk. Satisfying repeatability was obtained with a CV of 5.2% ( n =33). Results obtained by this method correlated well with the classic Rothenfusser method ( R 2 =0.97; n =20) and also with another method using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate diammonium salt) ( R 2 =0.98; n =21). This very easy rapid colorimetric method avoided preliminary casein precipitation and filtration steps. It has been shown to be suitable for routine characterisation of heat treatment of milk.

Camel (camelus dromedarius) milk PP3: evidence for an insertion in the amino-terminal sequence of the camel milk whey protein.

The camel (camelus dromedarius) milk proteose peptone 3 (PP3) was purified successively by size exclusion fast protein liquid chromatography and reversed phase high performance liquid chromatography and then characterized by amino acid residue composition determination and chemical microsequencing after CNBr or trypsin cleavages. In comparison with the previously reported structure of camel milk whey protein, the camel PP3 contains an insertion in the N-terminal region which has approximately 24 residues, whereas the remaining C-terminal regions of these two homologous proteins are essentially identical. The camel PP3 seems to contain a potential O-glycosylation site localized in this insertion and 2 or 3 phosphorylated serine residues. PP3 belongs to the glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) family and could therefore play an immunological role in the camel or its suckling young.

SPECIFIC INTERACTION BETWEEN ANIONIC PHOSPHOLIPIDS AND MILK BOVINE COMPONENT PP3 AND ITS 119-135 C-TERMINAL FRAGMENT

Abstract The behaviour of component PP3, a bovine milk protein with emulsifying properties, was investigated at the air–water interface and in a lipidic environment using the monolayer technique. The amphipathic 119–135 C-terminal fragment of PP3 was also tested since we proposed, on the basis of structural analysis, that this region is probably responsible for the surface-active properties of the protein. This hypothesis was confirmed by the tensiometric measurements at the air–water interface in which the addition of the C-terminal peptide increased the surface pressure with a similar amplitude as the whole protein. Penetration measurements into lipidic monolayers indicated that the insertion of component PP3 and its C-terminal peptide is the highest with anionic phospholipids in a gel state. Moreover, the electrostatic attractions provided by anionic phospholipids are essential for the peptide interaction. We also showed by Fourier transform infrared spectra study, that the peptide displays a β-type conformational state in aqueous solution and in the presence of solvant or anionic phospholipid (DPPG). In contrast, the protein adopts in aqueous solution an α helical conformation which remains the dominant conformational state in the presence of DPPG although the apparition of β-structure is detected.

Microparticle-enhanced nephelometric immunoassay for caseinomacropeptide in milk

A microparticle-enhanced nephelometric immunoassay has been developed for the determination of caseinomacropeptide (CMP) in bovine milk. It is based on the nephelometric quantification of the competitive immunoagglutination of a microparticle-CMP conjugate with an anti-κ-casein (κ-CN) antiserum. This one step immunoassay was sensitive (detection limit in reaction mixture, 16 μg/l), accurate (linear recovery of CMP in dilution overloading) and reproducible (CV 7-14% for within and between run precision). Because of the specificity of the polyclonal antiserum used, it was necessary to separate CMP from κ-CN by ultrafiltration before the quantification of bovine milk CMP. Under the conditions of milk ultrafiltration used, κ-CX was entirely retained (>99.5%) but the concentration of CMP measured in milk ultrafiltrates was underestimated (by ∼25%) compared with its concentration in whole milk. Microparticle-enhanced nephelometric immunoassay of CMP, with a calibration range from 0.32 to 20 mg/l for 20fold diluted milk ultrafiltrate, allowed contamination of bovine milk by rennet whey as low as 5 ml/l to be detected. Applied to ultrafiltrates from milk stored at 4 °C, this immunoassay also detected proteolysis of κ-CN not revealed by measurement of κ-CN concentration in milk. A statistical lower limit of 3.21 mg/l was determined as the increase in CMP concentration in milk ultrafiltrates that indicated probable κ-CN proteolysis in the milk sample. Previously demonstrated to be an easy to perform method for assaying the main proteins of bovine milk, microparticle-enhanced nephelometric immunoassay thus also appeared to be appropriate to quantify CMP so as to detect slight contamination of milk by whey and to indicate the proteolysis of κ-CN during milk storage at low temperature.

Specificity of chymosin on immobilized bovine B-chain insulin

1. The specificity of chymosin on immobilized bovine B-chain insulin is studied. 2. Eight sites of hydrolysis are determined.