Aspects of high hydrostatic pressure food processing: Perspectives on technology and food safety.

The last two decades saw a steady increase of high hydrostatic pressure (HHP) used for treatment of foods. Although the science of biomaterials exposed to high pressure started more than a century ago, there still seem to be a number of unanswered questions regarding safety of foods processed using HHP. This review gives an overview on historical development and fundamental aspects of HHP, as well as on potential risks associated with HHP food applications based on available literature. Beside the combination of pressure and temperature, as major factors impacting inactivation of vegetative bacterial cells, bacterial endospores, viruses, and parasites, factors, such as food matrix, water content, presence of dissolved substances, and pH value, also have significant influence on their inactivation by pressure. As a result, pressure treatment of foods should be considered for specific food groups and in accordance with their specific chemical and physical properties. The pressure necessary for inactivation of viruses is in many instances slightly lower than that for vegetative bacterial cells; however, data for food relevant human virus types are missing due to the lack of methods for determining their infectivity. Parasites can be inactivated by comparatively lower pressure than vegetative bacterial cells. The degrees to which chemical reactions progress under pressure treatments are different to those of conventional thermal processes, for example, HHP leads to lower amounts of acrylamide and furan. Additionally, the formation of new unknown or unexpected substances has not yet been observed. To date, no safety-relevant chemical changes have been described for foods treated by HHP. Based on existing sensitization to non-HHP-treated food, the allergenic potential of HHP-treated food is more likely to be equivalent to untreated food. Initial findings on changes in packaging materials under HHP have not yet been adequately supported by scientific data.

Postprandial metabolic responses to ingestion of bovine glycomacropeptide compared to a whey protein isolate in prediabetic volunteers.

PURPOSE: Whey protein was shown to reduce blood glucose responses in humans and various other positive effects have been attributed to this protein. In contrast, studies using glycomacropeptide (GMP) as part of the whey fraction of bovine milk are rare. We, therefore, studied the postprandial responses to GMP administration in humans with impaired glucose tolerance compared to the effects of pure whey protein in a random design. METHODS: Fifteen prediabetic volunteers received on different occasions one of three test drinks containing 50 g of maltodextrin19 (MD19) alone or in combination with either 50 g GMP or 50 g whey protein isolate (WPI). Blood was collected over 4 h with analysis of blood glucose and hormones, gastric emptying rate as well as plasma amino- and fatty acids, ß-hydroxybutyrate and acylcarnitines. RESULTS: The WPI drink reduced the AUC of venous blood glucose compared to the MD19 drink in the prediabetic group by 11% (p = 0.0018) whereas GMP reduced the AUC by 18% (p < 0.0001), significantly different to the WPI drink (p = 0.0384). The reduction in blood glucose after the GMP drink was accompanied by a significantly lower AUC of insulin (- 34%) than for the WPI drink. Levels of C-peptide and of glucose insulinotropic polypeptide (GIP) were highly increased after the WPI drink over the MD19 control drink but remained in essence unaffected by the GMP. CONCLUSION: GMP reduced the glycemic response more potently than whey protein, whereas insulin output was less affected making GMP an interesting protein to control postprandial glucose responses.

Effect of Hay Steaming on the Estimated Precaecal Digestibility of Crude Protein and Selected Amino Acids in Horses.

Steaming hay is increasingly used to treat low-quality forage because it was proven to reduce inhalable allergens such as mould spores, bacteria, and airborne dust particles. Preliminary results have shown a substantial loss of precaecal (pc) digestibility (D) of crude protein (CP) and amino acids (AA). For this purpose, six different batches of hay from central Germany were divided into four subsamples, and each one was individually steamed. Native hay and four replicates of each steamed subsample were analysed for CP, AA, neutral detergent insoluble crude protein (NDICP), neutral detergent soluble crude protein (NDSCP) as well as pepsin insoluble CP (piCP). Based on the analytical parameters, pcD of CP, protein solubility (PS), piCP (% CP) and precaecal digestible (pcd) CP and pcdAA contents were calculated. Selected Maillard reaction products (MRP), namely furosine and carboxymethyllysine (CML), were also analysed. Steaming did not affect CP content (native = 69, steamed = 67 g/kg dry matter, DM; p > 0.05), but it had an impact on the insoluble part of CP. Thus, NDICP increased by 57% (native = 27, steamed = 42 g/kg DM; p < 0.05) and piCP by 15% overall (native = 40, steamed = 46% of CP; p < 0.05). This could be a consequence of the heat damage and the associated increase in MRP. The content of furosine rose by 67% (native = 17.6, steamed = 29.4 mg/100 g DM; p < 0.05). The content of CML increased by 120% (native = 5.1, steamed = 11.3 mg/100 g DM; p < 0.05). We chose to analyse these two MRPs because they represent the reaction products with the limiting AA lysine. In contrast, the soluble fractions of CP declined, while PS as a percentage of CP decreased by 38% as a result of the treatment, and NDSCP was reduced by as much as 41% (p < 0.05). In line with this, the steaming process decreased the pcD of CP (native = 56%, steamed = 35%; p < 0.05) and pcdCP (native = 37.9, steamed = 22.5 g/kg DM; p < 0.05), respectively. The same effects were shown for selected AA; e.g., sulphuric AA pcd methionine plus pcd cysteine decreased by 45%, pcd threonine decreased by 41%, and the limited AA pcd lysine decreased by more than 50% (p < 0.05). In conclusion, the high temperatures generated during steaming lead to protein damage and consequently to a reduction in the pcD of CP and essential AA. Nevertheless, steaming successfully reduces viable microorganisms and binds dust particles. Therefore, steamed hay is still a proper and sometimes the only possible roughage for horses suffering from respiratory diseases such as equine asthma. Essentially, horse diets based on steamed hay should be balanced accordingly.

Insights at the molecular level into the formation of oxo-bridged trinuclear uranyl complexes.

Reaction of 1,3,4,6-tetra-O-acetyl-N-(2-hydroxy)-naphthylidene glucosamine (HL(Ac)) with uranyl acetate in ethanol leads to formation of dinuclear [(UO2)2(L)2] (1). In a second step 1 is quantitatively transferred into the trinuclear oxo-bridged complex [(UO2)3(µ3-O)(L)3]2- (22-) via deprotonation and coordination of a water molecule. This transformation was followed by NMR and UV/Vis spectroscopy and it proved possible to selectively introduce 18O into the µ3-bridge.

High-Pressure Processing of Kale: Effects on the Extractability, In Vitro Bioaccessibility of Carotenoids & Vitamin E and the Lipophilic Antioxidant Capacity.

High pressure processing (HPP) represents a non-thermal preservation technique for the gentle treatment of food products. Information about the impact of HPP on lipophilic food ingredients (e.g., carotenoids, vitamin E) is still limited in more complex matrices such as kale. Both the variation of pressure levels (200-600 MPa) and different holding times (5-40 min) served as HPP parameters. Whereas a slightly decreasing solvent extractability mostly correlated with increasing pressure regimes; the extension of holding times resulted in elevated extract concentrations, particularly at high-pressures up to 600 MPa. Surprisingly, slightly increasing bioaccessibility correlated with both elevated pressures and extended holding times, indicating matrix-dependent processes during in vitro digestion, compared to results of extractability. Moreover, the verification of syringe filters for digest filtration resulted in the highest relative recoveries using cellulose acetate and polyvinylidene difluoride membranes. The α-tocopherol equivalent antioxidant capacity (αTEAC) and oxygen radical antioxidant capacity (ORAC) assays of treated kale samples, chopped larger in size, showed increased antioxidant capacities, regarding elevated pressures and extended holding times. Consequently, one may conclude that HPP was confirmed as a gentle treatment technique for lipophilic micronutrients in kale. Nevertheless, it was indicated that sample pre-treatments could affect HP-related processes in food matrices prior to and possibly after HPP.

Cross-linking of type I collagen with microbial transglutaminase: identification of cross-linking sites.

Collagen is a popular biomaterial. To deal with its lack of thermal stability and its weak resistance to proteolytic degradation, collagen-based materials are stabilized via different cross-linking procedures. Regarding the potential toxicity of residual cross-linking agents, enzyme-mediated cross-linking would provide an alternative and nontoxic method for collagen stabilization. The results of this study show that type I collagen is a substrate for mTG. However, epsilon-(gamma-glutamyl)lysine cross-links are only incorporated at elevated temperatures when the protein is partially or completely denatured. A maximum number of 5.4 cross-links per collagen monomer were found for heat-denatured collagen. Labeling with the primary amine monodansylcadaverine revealed that at least half of the cross-links are located within the triple helical region of the collagen molecule. Because the triple helix is highly ordered in its native state, this finding might explain why the glutamine residues are inaccessible for mTG under nondenaturing conditions.

Modification of collagen in vitro with respect to formation of Nepsilon-carboxymethyllysine.

Developing new biopolymer-based materials with bio-identical properties is a significant challenge in modern science. One interesting route to this goal involves the biomineralization of collagen, a pre-structured and widely available protein, into a material with interesting properties. A prerequisite for biomineralization is the ability of cations (e.g., calcium) to bind to the protein and to result in concert with appropriate anions (e.g., phosphate) in composite material with e.g., bone-like properties. In order to increase the number of binding sites it is necessary to modify the protein prior to mineralization. For this glucuronic acid (GA) was used due to its carbonyl and carboxyl groups to derivatize proteinogenic amino groups transferring them into negatively charged carboxyl groups. Our experiments showed for the first time, that Nepsilon-carboxymethyllysine is the major product of in vitro non-enzymatic glycosylation of collagen by glucuronic acid. For an unequivocal determination of the reaction products, the lysine residues of collagen and of the model peptide were carboxymethylated through a reductive alkylation with glyoxalic acid and compared to the glucuronic acid derivatives. Beside their identical mass spectra the common structure elements could be confirmed with FTIR. Thus, in the context of matrix engineering, by producing Nepsilon-carboxymethyllysine, glucuronic acid offers a convenient way of introducing additional stable acidic groups into protein matrices.

No evidence for the involvement of serotonin or the 5-HTTLPR genotype in intertemporal choice in a larger community sample.

BACKGROUND: Serotonin has been implicated in impulsive behaviours such as temporal discounting. While animal studies and theoretical approaches suggest that reduced tonic serotonin levels increase temporal discounting rates and vice versa, evidence from human studies is scarce and inconclusive. Furthermore, an important modulator of serotonin signalling, a genetic variation in the promoter region of the serotonin transporter gene (5-HTTLPR), has not been investigated for temporal discounting so far. OBJECTIVE: First, the purpose of this study was to test for a significant association between 5-HTTLPR and temporal discounting. Second, we wished to investigate the effect of high/low tonic serotonin levels on intertemporal choice and blood oxygen-level-dependent response, controlling for 5-HTTLPR. METHODS: We tested the association of 5-HTTLPR with temporal discounting rates using an intertemporal choice task in 611 individuals. We then manipulated tonic serotonin levels with acute tryptophan interventions (depletion, loading, balanced) in a subsample of 45 short (S)-allele and 45 long (L)/L-allele carriers in a randomised double-blind crossover design using functional magnetic resonance imaging and an intertemporal choice task. RESULTS: Overall, we did not find any effect of serotonin and 5-HTTLPR on temporal discounting rates or the brain networks associated with valuation and cognitive control. CONCLUSION: Our findings indicate that serotonin may not be directly involved in choices including delays on longer timescales such as days, weeks or months. We speculate that serotonin plays a stronger role in dynamic intertemporal choice tasks where the delays are on a timescale of seconds and hence are therefore directly experienced during the experiment.

Model studies on protein glycation: influence of cysteine on the reactivity of arginine and lysine residues toward glyoxal.

Mixtures of N alpha-hippurylarginin, N alpha-hippuryllysine, and glyoxal were incubated in the absence and presence of N alpha-acetylcysteine in order to assess the individual reactivity of these nucleophilic amino acid residues. The incubations were performed under atmospheric and high hydrostatic pressure (400 MPa), and, at the same time, beta-casein was reacted with glyoxal. The results showed that arginine is the main partner for glyoxal in the absence of cysteine, whereas a lysine derivatization was not apparent. In the presence of cysteine, however, arginine was almost completely protected from the reaction, whereas a noticeable formation of lysine derivatives, mainly carboxymethyllysine, was observed. Based on these findings, a reaction mechanism is proposed to explain the influence of cysteine on the reaction.

N-terminal glycation of proteins and peptides in foods and in vivo: evaluation of N-(2-furoylmethyl)valine in acid hydrolyzates of human hemoglobin.

Specific determination of N-(2-furoylmethyl)valine (FM-Val) together with furosine in acid hydrolyzates of human hemoglobin of healthy volunteers (n = 6) and diabetic patients (n = 14) by means of reversed-phase HPLC with electrospray ionization-time-of-flight mass spectroscopy is reported. Whereas FM-Val is formed during acid hydrolysis of the N-terminal hemoglobin adduct N-fructosylvaline, furosine results from acid degradation of lysine residues glycated at the epsilon-amino group. Quantification was based on the use of synthesized isotopomers, namely N-[2-(13C6)furoylmethyl]valine and N-epsilon-[2-(13C6)furoylmethyl]lysine, thus enabling interference-free detection and calibration. Taking the conversion factors into account, the amount of N-terminally bound N-fructosylvaline in human hemoglobin was between 518 and 774 pmol/mg protein for healthy volunteers and between 586 and 1426 pmol/mg protein for diabetic patients. Derivatization at the side chain of peptide-bound lysine residues to N-epsilon-fructosyllysine was from 1156 to 1753 pmol/mg protein for healthy controls and from 1191 to 2409 pmol/mg protein for diabetics. For these patients, the amount of N-fructosylvaline showed good correlation with the values for HbA(1c). The significantly higher relative extent of glycation at the N terminus compared to side-chain glycation points to a specific and intraindividual capacity for enzymatic deglycation in human erythrocytes, which can be assessed using the proposed method.

Risk-seeking for losses is associated with 5-HTTLPR, but not with transient changes in 5-HT levels.

RATIONALE: Serotonin (5-HT) plays a key role in different aspects of value-based decision-making. A recent framework proposed that tonic 5-HT (together with dopamine, DA) codes future average reward expectations, providing a baseline against which possible choice outcomes are compared to guide decision-making. OBJECTIVES: To test whether high 5-HT levels decrease loss aversion, risk-seeking for gains, and risk-seeking for losses. METHODS: In a first session, 611 participants were genotyped for 5-HTTLPR and performed a mixed gambles (MGA) task and two probability discounting tasks for gains and losses, respectively (PDG/PDL). Afterwards, a subsample of 105 participants (44 with S/S, 6 with S/L, 55 with L/L genotype) completed the pharmacological study using a crossover design with tryptophan depletion (ATD), loading (ATL), and balanced (BAL) conditions. The same decision constructs were assessed. RESULTS: We found increased risk-seeking for losses in S/S compared to L/L individuals at the first visit (p = 0.002). Neither tryptophan depletion nor loading affected decision-making, nor did we observe an interaction between intervention and 5-HTTLPR genotype. CONCLUSION: Our data do not support the idea that transient changes of tonic 5-HT affect value-based decision-making. We provide evidence for an association of 5-HTTLPR with risk-seeking for losses, independent of acute 5-HT levels. This indicates that the association of 5-HTTLPR and risk-seeking for losses is mediated via other mechanisms, possibly by differences in the structural development of neural circuits of the 5-HT system during early life phases.

The why and how of amino acid analytics in cancer diagnostics and therapy.

Pathological alterations in cell functions are frequently accompanied by metabolic reprogramming including modifications in amino acid metabolism. Amino acid detection is thus integral to the diagnosis of many hereditary metabolic diseases. The development of malignant diseases as metabolic disorders comes along with a complex dysregulation of genetic and epigenetic factors affecting metabolic enzymes. Cancer cells might transiently or permanently become auxotrophic for non-essential or semi-essential amino acids such as asparagine or arginine. Also, transformed cells are often more susceptible to local shortage of essential amino acids such as methionine than normal tissues. This offers new points of attacking unique metabolic features in cancer cells. To better understand these processes, highly sensitive methods for amino acid detection and quantification are required. Our review summarizes the main methodologies for amino acid detection with a particular focus on applications in biomedicine and cancer, provides a historical overview of the methodological pre-requisites in amino acid analytics. We compare classical and modern approaches such as the combination of gas chromatography and liquid chromatography with mass spectrometry (GC-MS/LC-MS). The latter is increasingly applied in clinical routine. We therefore illustrate an LC-MS workflow for analyzing arginine and methionine as well as their precursors and analogs in biological material. Pitfalls during protocol development are discussed, but LC-MS emerges as a reliable and sensitive tool for the detection of amino acids in biological matrices. Quantification is challenging, but of particular interest in cancer research as targeting arginine and methionine turnover in cancer cells represent novel treatment strategies.

Structural changes of microbial transglutaminase during thermal and high-pressure treatment.

The activity of microbial transglutaminase (MTG) and the corresponding secondary structure, measured by circular dichroism (CD), was analyzed before and after treatment at different temperatures (40 and 80 degrees C) and pressures (0.1, 200, 400, 600 MPa). Irreversible enzyme inactivation was achieved after 2 min at 80 degrees C and 0.1 MPa. Enzyme inactivation at 0.1, 200, 400, and 600 MPa and 40 degrees C followed first-order kinetics. The enzyme showed residual activity of 50% after 12 min at 600 MPa and 40 degrees C. Mobility of aromatic side chains of the enzyme molecule was observed in all temperature- and/or pressure-treated samples; however, high-pressure treatment at 600 MPa induced a loss of tertiary structure and a significant decrease in the alpha-helix content. The relative content of beta-strand substructures was significantly increased after 30 min at 600 MPa and 40 degrees C or 2 min at 0.1 MPa and 80 degrees C. We conclude that the active center of MTG, which is located in an expanded beta-strand domain, is resistant to high hydrostatic pressure and pressure-induced inactivation is caused by destruction of alpha-helix elements with a corresponding influence on the enzyme stability in solution.

Free Maillard Reaction Products in Milk Reflect Nutritional Intake of Glycated Proteins and Can Be Used to Distinguish "Organic" and "Conventionally" Produced Milk.

Using LC-MS/MS and isotopically labeled standard substances, quantitation of free Maillard reaction products (MRPs), namely, N(ε)-(carboxymethyl)lysine (CML), 5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (pyrraline, PYR), N(δ)-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H), and N(ε)-fructosyllysine (FL), in bovine milk was achieved. Considerable variations in the amounts of the individual MRPs were found, most likely as a consequence of the nutritional uptake of glycated proteins. When comparing commercial milk samples labeled as originating from "organic" or "conventional" farming, respectively, significant differences in the content of free PYR (organic milk, 20-300 pmol/mL; conventional milk, 400-1000 pmol/mL) were observed. An analysis of feed samples indicated that rapeseed and sugar beet are the main sources for MRPs in conventional farming. Furthermore, milk of different dairy animals (cow, buffalo, donkey, goat, ewe, mare, camel) as well as for the first time human milk was analyzed for free MRPs. The distribution of their concentrations, with FL and PYR as the most abundant in human milk and with a high individual variability, also points to a nutritional influence. As the components of concentrated feed do not belong to the natural food sources of ruminants and equidae, free MRPs in milk might serve as indicators for an adequate animal feeding in near-natural farming and can be suitable parameters to distinguish between an "organic" and "conventional" production method of milk.

Identification and Quantitation of 2-Acetyl-1-pyrroline in Manuka Honey (Leptospermum scoparium).

Manuka honey from New Zealand is known for its exceptional antibacterial activity, which is due to high amounts of the 1,2-dicarbonyl compound methylglyoxal (MGO). MGO in manuka honey is formed via non-enzymatic dehydration from dihydroxyacetone (DHA) during honey maturation. MGO and DHA are highly reactive substances, leading to a variety of unique chemical reactions. During Strecker reaction between proline and MGO, 2-acetyl-1-pyrroline (2-AP), an important aroma compound, is formed. Using liquid-liquid extraction and gas chromatography-mass spectrometry analysis, 2-AP was identified unambiguously in manuka honey for the first time. Quantitation was carried out via external matrix calibration, using a synthetic 2-AP standard and artificial honey. The 2-AP concentration in 11 commercial samples of manuka honey ranged from 0.08 to 0.45 mg/kg. For manuka honey samples containing MGO in concentrations above 250 mg/kg, significantly higher amounts of 2-AP were found when compared to non-manuka honeys. When high amounts of MGO were artificially added to non-manuka multifloral honey, an increase of the 2-AP concentration from 0.07 to 0.40 mg/kg after 12 weeks of storage at 37 °C was observed, concomitant with a significant increase in the concentration of 5-hydroxymethylfurfural (HMF). No increase of 2-AP was found during storage at ambient temperature. 2-AP together with MGO can be a suitable parameter for the quality control of manuka honey.

Cross-linking of hen egg white lysozyme by microbial transglutaminase under high hydrostatic pressure: localization of reactive amino acid side chains.

After incubation of hen egg white lysozyme (HEWL) with microbial transglutaminase (mTG) under high pressure (400-600 MPa for 30 min at 40 °C), the formation of HEWL oligomers was observed via SDS electrophoresis. At atmospheric pressure, HEWL represents no substrate for mTG. Likewise, enzymatic treatment following a pretreatment with high pressure did not lead to oligomerization. Reactive amino acid side chains were identified by peptide mapping after tryptic digestion using RP-HPLC with ESI-TOF-MS. Isopeptide-containing peptide fragments were found only in HEWL samples simultaneously treated with enzyme and pressure. It was found that mTG exclusively cross-links HEWL under high pressure by formation of an isopeptide between lysine at position 1 and glutamine at position 121 in the peptide chain. Therefore, a pressure-induced partial and reversible unfolding of the protein with exposure of lysine and glutamine side chains has to occur, resulting in a site-directed oligomerization of HEWL by mTG. The enzymatic modification of HEWL by mTG under high pressure offers interesting perspectives for further functionalization reactions.

Carboxymethylation of the fibrillar collagen with respect to formation of hydroxyapatite.

Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Highly acidic macromolecules are postulated intermediates in tissue mineralization, because they sequester many calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. A prerequisite for biomineralization is the ability of cations like calcium to bind to proteins and to result in concert with appropriate anions like phosphates or carbonates in composite materials with bone-like properties. For this mineralization process the proteins have to be modified with respect to acidification. In this study we modified the protein collagen by carboxymethylation using glucuronic acid. Our experiments showed unambigously, that N(epsilon)-carboxymethyllysine is the major product of the in vitro nonenzymatic glycation reaction between glucuronic acid and collagen. We hypothesized that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions so that a critical nucleus of ions can be formed, leading to the formation of the mineral. Thus, the self-organization of HAP nanocrystals on and within collagen fibrils was intensified by carboxymethylation.

Affinity of microbial transglutaminase to αs1-, ß-, and acid casein under atmospheric and high pressure conditions.

Kinetics for the reaction of microbial transglutaminase (MTG) with individual caseins in a TRIS-acetate buffer at pH 6.0 was evaluated under atmospheric pressure (0.1 MPa) and high pressure (400 MPa) at 40 °C. The reaction was monitored under the following limitations: The kinetics from the initial velocities was obtained from nonprogressive enzymatic reactions assuming that the individual catalytic constants of reactive glutamine residues are represented by the reaction between MTG and casein monomers. Enzyme reaction kinetics carried out at 0.1 MPa at 40 °C showed Henri-Michaelis-Menten behavior with maximal velocities of 2.7 ± 0.02 × 10(-3), 0.8 ± 0.01 × 10(-3), and 1.3 ± 0.30 × 10(-3) mmol/L · min and K(m) values of 59 ± 2 × 10(-3), 64 ± 3 × 10(-3), and 50 ± 2 × 10(-3) mmol/L for ß-, α(s1)-, and acid casein, respectively. Enzyme reaction kinetics of ß-casein carried out at 400 MPa and 40 °C also showed a Henri-Michaelis-Menten behavior with a similar maximal velocity of 2.5 ± 0.33 × 10(-3) mmol/L · min, but, comparable to a competitive inhibition, the K(m) value increased to 144 ± 34 × 10(-3) mmol/L. The reaction of MTG with α(s1)-casein under high pressure did not fit in to Henri-Michaelis-Menten kinetics, indicating the complex influence of pressure on protein-enzyme interactions.

Crosslinking of casein by microbial transglutaminase and its resulting influence on the stability of micelle structure.

The influence of enzymatic crosslinking by microbial transglutaminase (mTG) on the stability of casein micelles of ultrahigh temperature (UHT)-treated milk in the presence of EDTA (0-0.45 mM) or ethanol (0-74 vol%) as well as under high hydrostatic pressures up to 400 MPa was investigated. Disintegration of micelles and changes in micelle size were monitored by the measurement of turbidity as well as by dynamic light scattering. The results show that the incubation of UHTtreated milk with mTG resulted in an improved micelle stability toward disintegration on addition of EDTA, ethanol, or pressure treatment. Intramicellar formed isopetides significantly enhanced the stability of casein micelles. It is supposed that net-like crosslinks are formed within the external region of the micelles and they adopt the stabilizing role of colloidal calcium phosphate within the micelles, thus making the micelles less contestable for disrupting influences.

Modification of beta-lactoglobulin by microbial transglutaminase under high hydrostatic pressure: localization of reactive glutamine residues.

Microbial transglutaminase (mTG) mediated modification of bovine beta-lactoglobulin (bLG) at ambient and high hydrostatic pressure was investigated in order to characterize preferred sites of the crosslinking reaction by identifying reactive glutamine residues. bLG was labeled with triglycine (GGG) by incubation with mTG at ambient pressure or at 400 MPa, respectively, and was subjected to an enzymatic digestion with trypsin. The resulting peptides were separated and those containing glutamine residues modified with GGG were unambiguously identified using RP-HPLC with ESI-TOF-MS. For bLG treated with mTG at ambient pressure for 1 h at 40 degrees C, no labeling was observed, thus confirming that the native protein is no substrate for mTG. After incubation of the protein with mTG at 400 MPa for 1 h at 40 degrees C, four out of nine glutamine residues, namely at positions 5, 13, 35, and 59 were identified as accessible for the mTG catalyzed reaction, indicating partial unfolding of bLG under pressure and exposure of previously unaccesible glutamine residues. Thus, only a limited number of glutamine residues were substrates for mTG, which points to a pronounced substrate specificity of mTG toward individual glutamine residues within a protein.