Study of proteolysis in river buffalo mozzarella cheese using a proteomics approach.

The guarantee of the origin and quality of raw material is essential for the protection and valorization of Campana buffalo mozzarella cheese. The risk of utilization of semifinished products and stored milk in substitution for fresh milk is increasing, due to the continuous desire to reduce production costs. A proteomics approach and electrophoresis survey of retail mozzarella cheeses indicated different rates of proteolysis in the production of dairy industries. The use of fresh milk and correct cheesemaking protocol yielded only γ-caseins, which are derived from ß-casein by plasmin, and para-κ-casein, which is derived from κ-casein by chymosin. The detection of abnormal hydrolysis resulting in ß- and αS1-casein fragments, identified by mass spectrometry, indicates the use of stored milk or stored and pressed curd, or the reuse of unsold mozzarella cheese, to produce mozzarella. The formation of γ-caseins and other fragments during a long storage of raw materials at room or refrigeration temperature was ascribed to plasmin (endogenous milk enzyme), whereas formation of αS1-casein fragments, mainly αS1-I(6P)- and αS1-I(7P)-casein during the storage of curd was ascribed to the action of chymosin (exogenous enzyme) from rennet. Sodium dodecyl sulfate-PAGE and alkaline urea-PAGE permitted us to evaluate the freshness of the raw materials used in the manufacturing of buffalo mozzarella cheese and to reveal possible inappropriate preservation.

Polymeric protein formation during pasta-making with barley and semolina mixtures, and prediction of its effect on cooking behaviour and acceptability.

Spaghetti were produced in a pilot plant from semolina and semolina blended with increasing amounts of barley flour. According to size exclusion high-performance liquid chromatography (SE-HPLC), barley proteins interact with semolina proteins during pasta making, forming polymers of high molecular weight. Of these, the unextractable polymeric proteins (UPP) were at significantly higher concentrations than in spaghetti made from semolina. The decrease of both S-S bonds and -SH free groups in barley semolina spaghetti, with respect to that made of semolina, suggested that polymerisation among the different classes of proteins involves a new bonding arrangement. Due to ß-glucan hydrophilicity and competition with starch for water, the replacement of increasing amounts of semolina with barley flour was able to increase the optimal cooking time. The sensory properties of composite spaghetti were judged as better than the control because of the higher firmness and the lower bulkiness and stickiness of the former.

Interactions between Lactobacillus sakei and CNC (Staphylococcus xylosus and Kocuria varians) and their influence on proteolytic activity.

AIMS: To evaluate interactions between Lactobacillus sakei and coagulase negative cocci (CNC) (Staphylococcus xylosus and Kocuria varians) and to investigate the influence of these interactions on their own proteolytic activity. METHODS AND RESULTS: Interactions occurring between strains of Lact. sakei and CNC were assessed by spectrophotometric analysis. The growth of 35 strains of Lact. sakei, used as indicators, was compared to that obtained combining the same strains with growing cells or cell-free supernatants of 20 CNC (18 Staph. xylosus and 2 K. varians). The proteolytic activity expressed by single strains or by their combinations was assessed on sarcoplasmic protein extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results evidenced that interactions are able to affect not only the growth but also the in vitro proteolytic activity of Lact. sakei and CNC used in combination. CONCLUSIONS: A relationship between the presence of interactions among useful strains and the strength of technological characteristics, such as proteolysis, was defined. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlighted that CNC are able to stimulate the growth of some Lact. sakei strains. At the same time, this interaction positively influences the proteolytic activity of strains used in combination. Given the importance of proteolysis during the ripening of fermented meats, this phenomenon should be taken into account to select meat starter cultures.

Hot topic: Gene duplication at the α-lactalbumin locus: finding the evidence in water buffalo (Bubalus bubalus L.).

Studies on milk proteins revealed that a qualitative and quantitative polymorphism may often be found regarding alpha-lactalbumin (alpha-LA). In mammals, a similar phenomenon was widely documented in the alpha-globin system as the result of a gene duplication. The presence of several differently expressed alpha-lactalbumin gene (LALBA) products suggests that the mechanism underlying this phenomenon may involve nonallelic genes. To check this hypothesis, an experiment was set up to investigate the LALBA gene arrangement of a water buffalo exhibiting an alpha-LA phenotype characterized by a double-band pattern on PAGE isoelectric, focusing analysis of milk protein. In particular, the relative amount of protein inferred from the different intensity of the bands was consistent with a gene duplication. Thus, leukocyte DNA was extracted from a blood sample of the buffalo and amplified with 4 primers (2 RV-IVFW for PCR and 4 FW-IRV for nested PCR). The intergenic segments of the assumed duplicated gene were then amplified with 2 different PCR protocols. First, the segment limited by the third exon in the upstream gene and the second exon in the downstream gene was amplified by simple PCR, which gave aspecific results. Second, this PCR product was subjected to nested PCR, amplifying the segment limited by the fourth exon in the upstream gene and the first exon in the downstream gene, yielding an amplified nucleotide fragment of about 6,200 bp. Blood samples from an additional 15 buffalos were then analyzed in the same manner. The results obtained from the new samples confirmed the presence of an amplified nucleotide fragment of about 6,200 bp in most of them, though they all were characterized by an alpha-LA monomorphic phenotype. A couple of 6,200-bp fragments obtained were purified, cloned in pGEM-T easy vector system (Promega, Madison, WI) and sequenced. The sequence of the large DNA segments, containing the intergenic portion, was aligned with the LALBA gene (accession number AF194373; http://www.ncbi.nlm.nih.gov/Database/index.html). They both were found to coincide with the portion containing exon 4 and the untranslated region at the 3' end of the upstream gene and with the portion containing exon 1 and the untranslated region at the 5' end of the downstream gene. These results confirm the hypothesis that a tandemly repeated copy of the LALBA gene is present in water buffalo.

Characterization and genetic study of the ovine alphaS2-casein (CSN1S2) allele B.

An interesting and quite complex protein pattern has been described at ovine milk proteins but the genetic control of the variation observed was assessed only in few cases. The aim of this work was to characterize the ovine alpha ( s2 )-casein (CSN1S2) B variant, first observed in the Italian Gentile di Puglia, a fine-wooled ovine breed, and to investigate its occurrence in two further breeds, the Sarda and Camosciata, which are the most widespread dairy breeds in Italy. The B variant differs from the most common form A with two amino acid exchanges: Asp(75) --> Tyr(75) and Ile(105) --> Val(105). The first substitution, resulting in a loss of a negative charge, is responsible for the higher isoelectric point of the B protein variant, which allows its detection by isoelectric focusing electrophoresis (IEF). The occurrence of CSN1S2*B in Sarda and Comisana was demonstrated. Since the Asp(75) --> Tyr(75) substitution modifies the protein electric charge, milk properties may result affected to some extent.

Short communication: Relationships between milk quality and acidification in the production of table Mozzarella without starters.

The effect of some quality parameters of the milk (refrigeration time, pH, protein, and fat/protein ratio) on the extent of acidification in the production technology of table Mozzarella without starters was investigated. A screening phase carried out at the laboratory level demonstrated that variations of the milk characteristics require different levels of acidification to keep constant the quality of the cheese. The elaboration of the data collected throughout the successive experimentation on industrial scale allowed us to find a mathematical model to describe the relationships between the pH of the curd at stretching time and the milk characteristics, of which the protein concentration and the refrigeration time play the main roles.

Application of capillary electrophoresis to determine the technological properties of wheat flours by a glutenin index.

Capillary electrophoresis was used to characterize glutenin proteins from ancient varieties of Southern Italy common wheat and to determine the technological properties of wheat flours based on a glutenin index. Three zones were identified in the electropherograms, indicated as A, B, and C according to electroelution order. The three zones corresponded to the low molecular weight glutenin subunits and to the y- and x-type high molecular weight subunits, respectively. The ratio B/C was correlated to the alveographic parameter P/L. These results indicated that flours resulting in a B/C ratio lower than 2 produced elastic doughs whereas flours resulting in a B/C ratio higher than 2 produced doughs more resistant to extension. This study showed that capillary electrophoresis is useful for determining the types and quantities of gluten proteins in the evaluation of wheat-flour technological properties of a limited number of noncommercial varieties as evidenced by the x-type content which seems to strongly influence the flour technological parameters.

Occurrence of beta-casein fragments in cold-stored and curdled river buffalo (Bubalus bubalis L.) milk.

The safeguard of river buffalo Mozzarella cheese, a Protected Designation of Origin dairy product, has prompted an analytical study to trace the milk and curd used as raw material in cheesemaking. This is to prevent the illegal use of milk or curd from different geographical areas outside of those indicated in the official production protocol. For this purpose, we studied primary proteolysis occurring in fresh and frozen milk and curd to identify a molecular marker that could indicate the raw material used. Whole casein from frozen river buffalo milk was separated using cation-exchange chromatography and sodium dodecyl sulfate-PAGE, and a protein component with an estimated molecular weight of 15.3 kDa was detected. This protein component was revealed in fresh river buffalo milk as a faint electrophoresis band, which drastically increased in intensity in refrigerated and frozen milk as well as in curd and was found to be associated with beta-CN through hydrophobic interaction. By using matrix-assisted laser desorption/ionization-time of flight peptide mass mapping, this component was identified as the C-terminal fragment f(69-209) of beta-CN (expected molecular weight of 15,748.8 Da). beta-Casein f(69-209), originating from the early hydrolysis of Lys(68)-Ser(69) by plasmin, has no counterpart in bovine milk. The increased rate of hydrolysis by plasmin toward the cleavage site Lys(68)-Ser(69) has to be ascribed to the elevated proline content of the peptide 61-73. The favored production of beta-CN f(69-209) has also drawn attention to the complementary proteose peptone beta-CN f(1-68) that is presumed to play a physiological role in inducing milk secretion similar to that of beta-CN f(1-29). The higher in vivo and in vitro production rate, compared with gamma(1)-CN formation, indicates that beta-CN f(69-209) and its complementary fragment are candidate molecular markers to evaluate milk and curd freshness. We suggested [corrected] indirect ELISA analysis based on the determination of remaining nonhydrolyzed beta-CN to perform a quantitative evaluation of proteolysis.

Effect of dietary energy content on the intramuscular fat depots and triglyceride composition of river buffalo meat.

The production of river buffalo meat in Italy has long been under discussion due to poor acceptance by consumers. In order to understand whether dietary energy content may affect the organoleptic characteristics of buffalo meat, two groups of river buffalo calves were fed on two diets, with high (H) and low (L) energy contents. The animals were slaughtered at 4-monthly intervals starting from 6 months old (10, 14 and 18 months) and five muscles were dissected on the half-carcass: Caput longum tricipitis brachii (CloTB), Gluteobiceps (Gb), Semitendinosus (St), Semimembranosus (Sm) and Longissimus dorsi (LD). The results showed that from 6 to 10 months of age the meat lipid content decreases and protein content increases for both diets. The lipid content increases slowly with both diets from 10 to 14 months. In the last experimental period (from 14 to 18 months) an increase in the percentage of lipids with diet H and a decrease with diet L was observed. At all slaughtering ages the meat from the animals fed on diet H had a higher energy content. The different energy content of the two diets did not significantly influence the composition of triglycerides only formed by SFA and triglycerides with a higher degree of unsaturation. The triglycerides with an unsaturated fatty acid in position sn 2 did not show the same behaviour in relation to age and diet. The energy content of the feed did influence the unsaturated fatty acid composition: MUFA increased with an increased energy level of the diet, while PUFA increased with a reduction in the energy level of the diet. The muscle LD showed a significantly higher (P<0.05) content of SFA and lower (P<0.05) of MUFA and PUFA than the other muscles. On the basis of our results, the better TAG's composition is found in the meat of animals fed on diet H and slaughtered at 4 months of age.

Primary structure of alpha-globin chains from river buffalo (Bubalus bubalis L.) hemoglobins.

Primary structure analysis of the four river buffalo alpha-globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two alpha-globin chains, Ialpha1 and IIalpha3, which differ at positions 129 and 131: Ialpha1 has 64 Ala, 129 Phe, 131 Asn; IIalpha3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two alpha-globin chains, Ialpha2 and IIalpha4, which differ at positions 10 and 11: Ialpha2 has 10 I1e, 11 Gln, 64 Asn; IIalpha4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic alpha-globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo alpha-globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.

Milk identification of different species: 13C-NMR spectroscopy of triacylglycerols from cows and buffaloes' milks.

Triacylglycerols from cows and buffaloes' milk fat were investigated by 13C nuclear magnetic resonance (NMR) spectroscopy. By the addition of pure triacylglycerols standards, we identified the resonances of both milk fats, and the peaks were used for qualitative and quantitative analysis of acyl groups. Multivariate analysis treatment of triacylglycerols distribution and composition parameters enabled us to identify milk. This study shows that NMR can safely be used to quantitate milk fatty acid content, providing unique information for milk identification of different animal species.

Identical marker alleles in Podolic cattle (Bos taurus) and Indian zebu (Bos indicus).

In the context of biochemical marker research and in order to add new information on native breeds, the present work focuses on a local Southern Italy cattle, namely Italian Podolic. We provide the complete structural characterisation of alpha-lactalbumins and beta-globin chains isolated from Podolic cattle (Bos taurus). Given the unavailability of the complete sequence for alpha-lactalbumin A of taurine cattle in the literature, we intended to check its structure in order to ascertain the absence of any possible silent mutation. Screening the Podolic cattle, we found a new beta-globin variant not detectable by conventional methods. The presence of such a new variant might be helpful in the study of the Podolic population genetic structure and for a better knowledge of the gene pool per se, and in comparison with the other breeds. Structural analyses showed that the new beta-globin Podolic variant exhibited the same sequence as beta-globin Azebu. The alpha-lactalbumin A was the same as that isolated from zebu cattle (Bos indicus). The results are discussed in relation to the possible involvement of the two markers in the debate on the origin of the Podolic breed.

Interaction of putrescine with nuclear oligopeptides in the enterocyte-like Caco-2 cells.

Intestinal cells are able both to synthesize and take up putrescine, the main compound of the metabolic polyamine pathway. Polyamine binding to nuclear macromolecules is thougth to modulate DNA synthesis and transcription. Our aim was to study the fate of putrescine when taken up from the medium in the enterocyte-like Caco-2 cells and to analyze its binding to nuclear proteins. After having incubated the cells with 14C-putrescine (0.8 microM), during cell replication and differentiation, the nuclei were separated by sequential centrifugations in a sucrose gradient. About 20% of the putrescine taken up by Caco-2 cells resulted in the nuclei in both proliferating and differentiated cells. The binding of polyamines to nuclear proteins was studied on nuclear extracts, separated by both alkaline polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and gel permeation chromatography (GPC). No radioactivity was found in nuclear protein extracts when using the SDS-PAGE method. Conversely, in replicating cells, GPC showed that the greatest amount of radioactivity was present in the nuclear peaks corresponding to oligopeptides with a molecular weight of 4,800-8,000 daltons. High-performance liquid chromatography analysis showed the presence of putrescine and spermidine in the 8, 000-dalton protein peak, whereas in the 4,800-dalton peak spermine was found in addition to putrescine. The radioactive count in the HPLC separated polyamines showed that a small percentage of the radioactivity present in the 8,000 and 4,800-dalton GPC peaks was linked to spermine and spermidine, suggesting an interconversion of the supplemented putrescine. Conversely, in differentiated cells, the nuclear oligopeptides did not reveal any radioactivity or any polyamines, suggesting that the binding of polyamines to nuclear oligopeptides is exclusively concerned with replicating cells.

Bovine hemoglobin alpha-globin chain polymorphism: primary structure determination of two new genetic variants by mass spectrometry and amino acid sequencing.

The present work describes the biochemical procedures used to identify the cause of a quantitative and qualitative hemoglobin polymorphism found in Podolian cattle. First, to analyze the different phenotypes, isoelectric focusing (IEF) of hemoglobins and RP-HPLC of globin chains was carried out; secondly, to determine accurately the globin molecular masses, electrospray mass spectrometry was performed and finally to check the entire amino acid sequences of the proteins, several enzymatic digests were analyzed by fast atom bombardment mass spectrometry (FAB-MS) and Edman degradation procedure. As to the qualitative polymorphism, the results of RP-HPLC show the presence of two alpha-globin variants to which the extensive mass spectrometric analysis attributed a molecular mass of 15,026.47 +/- 0.44 Da and 15,079.86 +/- 0.66 Da and whose respective primary structure differed from that of the common alpha-globin chain in the amino acid substitution Asn-->Ser at position 131 and the other in the replacement of the histidine residue at position 89 with tyrosine. As to the quantitative polymorphism, on the basis of the expression gradient found out in the duplicated alpha genes of several mammals, we conceive that the alpha 89 His-->Tyr is an allelic form of the I alpha gene while the alpha 131Asn-->Ser is an allelic form of the II alpha gene.

Identification of C-terminally truncated forms of beta-lactoglobulin in whey from Romagnola cows' milk by two dimensional electrophoresis coupled to mass spectrometry.

Four minor protein components were detected in whey from Romagnola cows' milk by polyacrylamide gel isoelectric focusing and two dimensional gel electrophoresis. Individual protein spots were transferred by electroblotting on to a polyvinylidene difluoride membrane and isolated by cutting out the relevant area. After in situ trypsinolysis, a portion of the digest was analysed directly by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass profile allowed us to establish a correlation between beta-lactoglobulins A and B and the four minor whey protein components. They were identified as C-terminally truncated beta-lactoglobulin A and B variants with missing N-terminal peptides, beyond residues in the range 100-123 and 136-147 respectively. Two of the minor components were related to beta-lactoglobulin A and two to beta-lactoglobulin B.

Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma 'anandamide amidohydrolase'.

The endogenous cannabimimetic substance, anandamide (N-arachidonoyl-ethanolamine) and the recently isolated sleep-inducing factor, oleoyl-amide (cis-9,10-octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of 'anandamide amidohydrolase' from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [14C]anandamide was found in both microsomal and 10,000 x g pellet fractions. The latter fractions, which displayed the highest Vmax for anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl-amide, with an apparent Km of 9.0 +/- 2.2 microM. [14C]anandamide- and [14C]oleoyl-amide-hydrolysing activities: (i) exhibited identical pH- and temperature-dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A2 inhibitor arachidonyl-trifluoromethyl-ketone with the same IC50 (3 microM); (iv) were competitively inhibited by both anandamide (or other polyunsaturated fatty acid-ethanolamides) and oleoyl-amide. Proteins solubilised from 10,000 x g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]anandamide and [14C]oleoyl-amide. These data suggest that 'anandamide amidohydrolase' enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep-inducing factor oleoyl-amide.

Electrospray mass spectrometric analysis of river buffalo (Bubalus bubalis) hemoglobins. Re-examination of alpha 1 and alpha 3 globin chain sequences.

1. The accurate molecular weight of the globin chains from river buffalo hemoglobin components has been determined by means of electrospray mass spectrometry. 2. The ES/MS analysis demonstrated that all the buffalo Hb components share a common beta chain whose mass value coincides with that expected on the basis of the reported sequence. 3. The AA phenotype alpha 1 and alpha 3 globin chains exhibited a 30 Da mass difference as compared to their predicted molecular weights. Careful re-examination of the two alpha globin sequences by FAB/MS revealed the occurrence of sequence errors and hence the correct primary structure of both alpha chains was established.

Haemoglobin I: a new beta-globin chain variant found in sheep of Italian breeds.

A rather common haemoglobin variant was detected in the Sardinian and Altamurana sheep breeds. The mutated globin chain appears to be produced under the control of an allele at the HBB locus and due to a neutral amino acid substitution. The variant will be provisionally referred to as the Hb I.

Evolution of ruminant hemoglobins. Thermodynamic divergence of ox and buffalo hemoglobins.

The ligand-binding properties of hemoglobins from two homozygote phenotypes (AA and BB) of water buffalo (Bubalus bubalis) have been characterized by equilibrium and kinetic techniques. In the case of the BB phenotype, the two constituent hemoglobins have been purified and separately analysed. Buffalo hemoglobins display the reduced sensitivity to organic phosphates characteristic of ruminant hemoglobins, their physiological effector probably being the chloride ion. In contrast to the other known hemoglobins from ruminants, all the hemoglobins from the water buffalo display a significant temperature sensitivity, the delta H for oxygen binding in the presence of physiological effectors approaching that of human hemoglobin (delta H = -30.5 kJ/mol O2). This discrepancy with the other ruminant hemoglobins (e.g. ox, delta H = -10.4 kJ/mol O2), whose primary structure is very similar to that of buffalo, hemoglobins might be correlated to the different habitat and phylogenetic history of the two subfamilies (Bos and Bubalus) of Bovidae.

River buffalo (Bubalus bubalis L.) AA phenotype haemoglobins: characterization by immobiline polyacrylamide gel electrophoresis and high performance liquid chromatography and determination of the primary structure of the constitutive chains by mass spectrometry.

1. Polyacrylamide gel electrophoresis in ultra-narrow immobilized pH gradient shifted the "Hb fast" band of AA buffalo phenotype haemoglobin into two components which were named Hb1 and Hb3. 2. Urea/Triton electrophoresis and reversed-phase HPLC demonstrated that Hb1 and Hb3 differ in the presence of two structurally distinct alpha chains (alpha 1 and alpha 3), also suggesting that the alpha chains must differ for neutral amino acid substitution. 3. Extensive mass spectrometric analysis on several digests (FAB overlapping) meant to determine the complete sequence of the constituent chains. 4. Two amino acid replacements (Lys 18----His and Asn 116----His) were present in the beta chain with respect to the bovine (A phenotype) chain, whereas the alpha 1 and alpha 3 globins were found to contain four amino acid replacements compared to the bovine alpha, three of which were identical (Glu 23----Asp, Glu 71----Gly and Phe 117----Cys) and, notably, an insertion of Ala at position 123-124. 5. Furthermore, alpha 1 contains Phe at position 130 whereas alpha 3 contains Ser at position 132 (following the modified numbering as a consequence of the Ala insertion).