I. De Noni

Role of polysaccharides in food, digestion, and health

ABSTRACT Polysaccharides derived from plant foods are major components of the human diet, with limited contributions of related components from fungal and algal sources. In particular, starch and other storage carbohydrates are the major sources of energy in all diets, while cell wall polysaccharides are the major components of dietary fiber. We review the role of these components in the human diet, including their structure and distribution, their modification during food processing and effects on functional properties, their behavior in the gastrointestinal tract, and their contribution to healthy diets.

The evolution of chemical and microbiological properties of fresh goat milk cheese during its shelf life

This study investigated the changes in chemical and microbiological properties of fresh goat milk cheese stored in an open deck refrigerated display cabinet (6 ± 2°C) or in a dark cold room (4 ± 1°C). The effects of partial-vacuum packaging and fluorescent lighting were studied during the cheese shelf life (45 d) and 15 d after. Storage conditions did not affect the pH values (4.3), whereas a slight decrease in moisture (ca. 1%) and in water activity (<0.01 units) was recorded. Proteolysis monitored by Kjeldahl determination increased significantly during storage of all samples. The highest increase from 8.5 to 13.0% of soluble nitrogen (expressed as percentage of total nitrogen) was measured in cheese packaged in the presence of air and stored in a lighted cabinet. The proteolytic trend was also studied through capillary zone electrophoresis by monitoring the degradation of the main casein fractions and the formation of new peptides. In particular, 2 indices, based on peak area ratio of new-formed peptides and casein fractions were related to cheese age. Lipolysis, measured by solid-phase microextraction gas chromatography coupled to mass spectrometry of volatile fatty acids, was unaffected by air or light and did not proceed through storage. As expected, hexanal formed mainly in cheeses stored under light and packaged in air. Evaluation of sensorial quality, performed using a hedonic scale, showed significantly lower scores of cheeses kept under light compared with those kept in the dark, both at 45 and 60 d storage. Overall, the microbiological and chemical results suggested that the shelf life of soft goat milk cheese would be extended from 45 to 60 d. Such conclusion was supported also by the sensory quality evaluation.

Nutritional Quality of Milk Proteins

The first part of this chapter outlines general aspects concerning dietary proteins, i.e. protein requirements in human diet, role and nutritional quality of proteins and methods for its evaluation (CS, Protein Digestibility Corrected AA Score [PDCAAS]), protein digestibility and efficiency of protein utilisation.

Rapid determination of sodium in milk and milk products by capillary zone electrophoresis

A capillary zone electrophoresis method for the determination of Na in milk and milk products was developed and compared with an International Organization for Standardization/International Dairy Federation standard method that is based on flame atomic absorption spectrometry. The adoption of a background electrolyte consisting of 10 mM imidazole adjusted to pH 3.75 by the addition of oxalic acid allowed baseline separation of Na from other milk cations and from Li ion, which was adopted as an internal standard. Method validation was performed and the results for linearity, precision, limit of detection, limit of quantification, and recovery are presented. The procedure was tested on commercial milk samples differing in fat content (whole, semiskimmed, and skimmed) and processing conditions (pasteurization, UHT sterilization, and microfiltration). The reliability of the method was confirmed for different varieties of cheese and other milk products. The method enables the routine measurement of Na content by a rapid and accurate capillary zone electrophoresis procedure.

Antibacterial activity and immunomodulatory effects on a bovine mammary epithelial cell line exerted by nisin A-producing Lactococcus lactis strains

Twenty-nine strains of mastitis pathogens were used to study the antibacterial activity of the cell-free supernatants (CFS) of 25 strains of Lactococcus lactis ssp. lactis. Out of the tested strains, only the CFS of L. lactis LL11 and SL153 were active, inhibiting and killing most of the pathogens. By means of ultra-performance liquid chromatography/high resolution mass spectrometry, they were shown to produce nisin A, a class I bacteriocin. A variable sensitivity to nisin A-containing CFS was observed among Streptococcus uberis and Enterococcus faecalis strains. Nonetheless, Streptococcus agalactiae, Strep. uberis, and E. faecalis displayed high minimum inhibitory concentration values, reaching 384 arbitrary units/mL. Interestingly, the minimum inhibitory values and the bactericidal concentrations were almost identical among them for each of the 2 stains, LL11 and SL153. Staphylococci were, on average, less sensitive than streptococci, but the 2 CFS inhibited and killed, at different dilutions, strains of methicillin-resistant Staphylococcus aureus. The immune response to nisin A-containing CFS was tested using the bovine mammary epithelial cell line BME-UV1. Application of CFS did not damage epithelial integrity, as demonstrated by the higher activity of N-acetyl-β-d-glucosaminidase (NAGase) and lysozyme inside the cells, in both treated and control samples. On the other hand, the increase of released NAGase after 15 to 24h of treatment with LL11 or SL153 live cultures demonstrated an inflammatory response of epithelial cells. Similarly, a significantly higher lysozyme activity was detected in the cells treated with LL11 live culture confirming the stimulation of lysosomal activity. The treatment of epithelial cells with SL153 live culture induced a significant tumor necrosis factor-α downregulation in the cells, but did not influence IL-8 expression. The control of tumor necrosis factor-α release could be an interesting approach to reduce the symptoms linked to clinical intramammary infections. Due to their antibacterial activity and to the stimulation of lysosomal activity of mammary epithelial cells, the L. lactis strains SL153 and LL11 could be of interest for the development of alternative intramammary treatments to control cow mastitis.

Release of angiotensin converting enzyme-inhibitor peptides during in vitro gastrointestinal digestion of Parmigiano Reggiano PDO cheese and their absorption through an in vitro model of intestinal epithelium

The occurrence of 8 bovine casein-derived peptides (VPP, IPP, RYLGY, RYLG, AYFYPEL, AYFYPE, LHLPLP, and HLPLP) reported as angiotensin converting enzyme-inhibitors (ACE-I) was investigated in the 3-kDa ultrafiltered water-soluble extract (WSE) of Parmigiano Reggiano (PR) cheese samples by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry via an electrospray ionization source. Only VPP, IPP, LHLPLP, and HLPLP were revealed in the WSE, and their total amount was in the range of 8.46 to 21.55 mg/kg of cheese. Following in vitro static gastrointestinal digestion, the same ACE-I peptides along with the newly formed AYFYPEL and AYFYPE were found in the 3 kDa WSE of PR digestates. Digestates presented high amounts (1,880-3,053 mg/kg) of LHLPLP, whereas the remaining peptides accounted for 69.24 to 82.82 mg/kg. The half-maximal inhibitory concentration (IC50) values decreased from 7.92 ± 2.08 in undigested cheese to 3.20 ± 1.69 after in vitro gastrointestinal digestion. The 3-kDa WSE of digested cheeses were used to study the transport of the 8 ACE-I peptides across the monolayers of the Caco-2 cell culture grown on a semipermeable membrane of the transwells. After 1h of incubation, 649.20 ± 148.85 mg/kg of LHLPLP remained in the apical compartment, whereas VPP, IPP, AYFYPEL, AYFYPE, and HLPLP accounted in total for less than 36.78 mg/kg. On average, 0.6% of LHLPLP initially present in the digestates added to the apical compartment were transported intact to the basolateral chamber after the same incubation time. Higher transport rate (2.9%) was ascertained for the peptide HLPLP. No other intact ACE-I peptides were revealed in the basolateral compartment. For the first time, these results demonstrated that the ACE-I peptides HLPLP and LHLPLP present in the in vitro digestates of PR cheese are partially absorbed through an in vitro model of human intestinal epithelium.

Detection of commondashwheat (Triticum aestivum) flour in Durum-wheat (Triticum durum) semolina by reverse-phase high-performance liquid chromatography (RP-HPLC) of specific albumins

A method for detecting commondashwheat flour in Durum-wheat semolina and low-heat dried pasta by means of RP-HPLC of water-soluble protein is described. This method provides separation of the same specific protein fractions as those determined by IEF according to the Italian Official Method (Official Italian Journal, No. 4, 5 January 1980). A faster sample preparation on solid-phase extraction (SPE) of protein can be adopted. This RP-HPLC method proves to be repeatable (coefficient of variation less than 4%), to have high sensitivity (less than 1% of commondashwheat flour detectable), and an accuracy comparable to that of the IEF method. The RP-HPLC method cannot be applied to protein extracted from high-heat dried pasta. The HPLC patterns obtained are poorly resolved and a correct quantification of commondashwheat flour cannot be achieved.

Composition, proteolysis, and volatile profile of Strachitunt cheese.

Strachitunt, a blue-veined Italian cheese, received the Protected Designation of Origin (PDO) label in 2014. Its unique technological feature is represented by the dual-curd method of production. Strachitunt is produced from raw bovine milk with or without the inoculation of natural starter cultures of lactic acid bacteria, and the addition of secondary cultures of mold spores is not permitted by the product specification. Physico-chemical properties, proteolysis, and volatile profile of Strachitunt were investigated in 10 cheese samples (ripened for 75 d) made throughout spring 2015 and provided by the main cheese maker. Overall, composition parameters showed a large variability among samples. Cheese was characterized by an acid paste (pH 5.46) and a lower extent of proteolysis compared with other blue-veined varieties. The main chemical groups of volatile organic compounds were alcohols and esters, whereas ketones represented only a minor component. The erratic adventitious contamination by mold spores of the cheese milk, the unique dual-curd method of cheese-making, and the large time variability between the piercing time and the end of ripening could be highlighted as the main causes of both the distinctive analytical fingerprint and the scarce standardization of this blue-veined cheese.

Formation of protein bound lysine-derived galactosyl and glucosyl pyrroles in heated model systems.

Residues of lysine-substituted AGEs (advanced glycosylation end-products) arising from the Maillard reaction and containing the carbon backbone of lactose or maltose, thus deriving from 1-desoxy-ketose degradation, were produced when casein was heated in the presence of the corresponding U14C labelled disaccharides. The enzymatic hydrolysates of the washed casein were purified by SPE and submitted to HPLC on a C18 column flushed with diluted acetic acid. A specific chromatographic peak (lambda max, 288.1 nm; MW, 416.2 Da) with a different retention time was obtained for each disaccharide reacted. On the basis of the value of the specific radioactivity, the two compounds appeared to contain the whole carbon backbone of the parent sugar. Analyses by MS/MS and NMR performed on the same two compounds extracted at preparative scale from lysine-lactose and lysine-maltose model systems allowed the structure assignment of 6-[2-acetyl-3-(beta-D-galactopyranosyloxy)-1-pyrrolyl] 2-amino hexanoic acid and 6-[2-acetyl-3-(alpha-D-glucopyranosyloxy)-1-pyrrolyl] 2-amino hexanoic acid, respectively. Both compounds submitted to enzymatic deglycosylation by specific alpha- or beta-glucosidases produced the lysine-derived acetyl pyrrole 6-(2-acetyl-3-hydroxy-1-pyrrolyl) 2-amino hexanoic acid (lambda max, 288.1 nm; MW, 254.1 Da). Galactosyl- and glucosyl-isomaltoles, extracted from the lysine-containing systems, identified with the reference molecules and heated in the presence of lysine under slightly alkaline conditions, gave the expected lysine-derived glycosyl pyrroles as identified above. The HPLC conditions were optimized by adjusting the composition of the eluting solvent and temperature of the column to achieve the best separation and identification of the AGEs in mixtures such as foods with possible interfering molecules like Trp and lysyl pyrrole aldehyde. Because of the reported presence of the two precursors isomaltol glycosydes in some foods, the corresponding lysine-derived glycosyl pyrroles can occur as both protein bound and in free form.

Extracellular thermostable proteolytic activity of the milk spoilage bacterium Pseudomonas fluorescens PS19 on bovine caseins.

We studied the thermostable proteolytic activity of Pseudomonas fluorescens PS19 isolated from raw bovine milk. The heat-treated cell-free supernatant (HT-CFS) contained a thermostable protease of approximately 45 kDa, as revealed by casein zymography. We assigned this enzyme to P. fluorescens AprX metalloprotease (UniProtKB Acc. No. C9WKP6). After concentration by ultrafiltration at 10 kDa, the HT-CFS showed 2 other thermostable proteolytic bands on zymogram, with molecular masses of approximately 15 and 25 kDa. The former resulted a fragment of the AprX protease, whereas the 25-kDa protease was not homologous to any known protein of Pseudomonas spp. Subsequently, we assessed the proteolytic activity of the HT-CFS on bovine αS-, β-, and κ-casein during in vitro incubation at 7 or 22°C. By means of ultra-performance liquid chromatography-tandem mass spectrometry we identified the released peptides (n=591). Some of them resisted proteolysis during the whole incubation period at both incubation temperatures and, therefore, they could be assumed as indicators of the proteolytic action of P. fluorescens PS19 on bovine caseins.