Guy Linden

Characterization of α -casozepine, a tryptic peptide from bovine α s1-casein with benzodiazepine-like activity

Caseins are a known source of biologically active peptides. In this study, we have shown evidence of a novel anxiolytic activity in a tryptic hydrolysate of bovine αs1‐casein. Injection of 3 mg/kg of this hydrolysate significantly reduced the epileptic symptoms caused by pentylenetetrazole in rats. Anxiety reduction was also observed when the hydrolysate was tested in the elevated plus‐maze and in the conditioned defensive burying rat models. Peptides isolated from the hydrolysate were examined for their affinity for the γ‐amino‐butyric acid (GABA) type A receptor. Only one peptide, named α‐casozepine, corresponding to the 91–100 fragment from bovine αs1‐casein, expressed affinity for GABAA receptor. In vitro, the peptide had 10,000 less affinity for the benzodiazepine site of the GABAA than did diazepam. However, in the conditioned defensive burying paradigm it was 10‐fold more efficient than diazepam. The difference observed between the in vitro and in vivo activity of α‐casozepine could not been explained by an action via the peripheral‐type benzodiazepine receptor; α‐casozepine had no affinity for this receptor. The α‐casozepine amino acid sequence could be related to the carboxy‐terminal sequence of the polypeptide diazepam binding inhibitor, an endogenous ligand of the central GABAA and peripheral‐type benzodiazepine receptors.

A 16 kDa protein family overexpressed by Streptococcus thermophilus PB18 in acid environments

The one- and two-dimensional protein patterns of Streptococcus thermophilus PB18 in the exponential and stationary phases of growth were analysed. One-dimensional SDS-PAGE showed that a 16 kDa protein was overexpressed in stationary phase as well as 2 h after an acid shock, and that it was not expressed when the bacteria reached the stationary phase in medium with limiting lactose concentrations (5 or 10 g l-1), in which the pH (5.5) was not as acid as in control cultures (pH 4.7, lactose 20 g l-1). The results support the idea that this protein is expressed in response to the acidic environment and not in response to the growth phase. Two-dimensional PAGE showed that nine proteins were expressed only during the exponential phase and ten others only during the stationary phase. The 16 kDa band seen in one-dimensional SDS-PAGE corresponded to a 16 kDa protein family observed on two-dimensional SDS-PAGE/IEF gels, whose expression was increased 8.5-fold when the extracellular pH reached a critical value below 5.0. The N-terminal sequences of proteins from two spots on the two-dimensional gels (members of the 16 kDa family) were determined and found to be identical. The physiological role of this protein family has not yet been elucidated.

New ingredients in food processing: biochemistry and agriculture.

Growth and functional properties of intermediate foodstuffs Manufacturing processes Vegetable-based intermediate foodstuffs Milk and egg products Meat products Marine products Products from sugar Starches Lipids Colourings and flavourings.

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.

Biologically active factors in bovine milk and dairy byproducts: Influence on cell culture

Substantial progress has been made in our knowledge of the biological properties of mammal milks. Many nutritional, biochemical, immunological, or other biological properties have been studied in mature or industrially processed bovine milk as well as in human milk and colostrum. This article is a critical review of selected publications covering (1) the use of bovine milk or dairy byproducts (processed acid and enzymatic whey fractions) as a serum substitute for cell cultures, (2) specific factors in bovine milk and industrially processed milk the affect cell proliferation, and (3) the known functional and biological roles of two whey proteins: beta-lactoglobulin and the PP3 component.

Effect of temperature on the salt balance of milk studied by capillary ion electrophoresis

Many inorganic species, such as calcium, phosphate and magnesium, are in equilibrium between the liquid and colloidal phases of milk and hence are of importance with respect to the coagulation properties of milk. Capillary ion electrophoresis makes possible the determination of anions and cations in less than 6 min. The soluble phase of milk was obtained by ultrafiltration and samples had to be diluted 250-fold before analysis. Cold storage increased soluble calcium and phosphate concentrations, and warm-up of the milk restored the initial ionic equilibria. More drastic heat treatments (80-90 degrees C) caused precipitation of tricalcium phosphate and calcium citrate.

Surface active and emulsifying properties of casein micelles compared to those of sodium caseinate

Emulsions prepared using casein micelles and sodium caseinate as the emulsifying agent are compared. It is found that the ultrastructure of the casein proteins influences the properties of the emulsion.

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.

Restriction/modification in Streptococcus thermophilus: isolation and characterization of a type II restriction endonuclease Sth455I

Streptococcus thermophilus strain CNRZ 455 produces a type II restriction endonuclease designated Sth455I. This enzyme was isolated from cell extracts by anionic and cationic exchange chromatography. This yielded an enzyme preparation free of non-specific nucleases. The optimal reaction conditions for Sth455I are: MgCL2, 30 mm; pH range, 8–9; incubation temperature, 37–42°C; and a high NaCl concentration, 100–200 mm. The results of single- and double-digestion experiments indicates that Sth455I is an isoschizomer of BstNI and EcoRII showing different sensitivity to methylation. The enzyme exhibits restriction activity on the DNA of three bacteriophages of S. thermophilus and no activity on the phage lytic for strain CNRZ 455. The restriction/modification system associated with this strain is discussed.

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.