Stefano Cattaneo

Spaghetti from durum wheat: Effect of drying conditions on heat damage, ultrastructure and in vitro digestibility

The effects of low (LT) or high (HT) temperature drying on ultrastructural, molecular and in vitro digestibility properties of cooked spaghetti were studied. Starch swelling and denaturation/aggregation of proteins occurring at diverse stages, LT or HT drying and cooking, resulted in different in vitro digestibility of spaghetti. For the first time, these differences were assessed in terms of the release of free AA and simple sugars. Indeed, at the end of in vitro digestion, the total amount of released maltotriose, maltose and glucose significantly differentiated digestates of LT and HT spaghetti (12.6 and 15.9 g 100g⁻¹). In the same samples, diverse amounts (16.3 and 12.5 g 100g⁻¹ protein) of free amino acids were found. Chemical artifacts occurring at protein level impaired release of lysine in cooked HT spaghetti after in vitro digestion. These results increase the knowledge on digestibility of LT and HT cooked spaghetti.

Liquid infant formulas: technological tools for limiting heat damage.

In a study considering 15 commercial samples of liquid milk-based infant formulas (MBF) from different manufacturers, the levels of selected molecules, that is, furosine (FUR), galactosyl-beta-pyranone (GAP), lactulose (LCT), and lysinoalanine (LAL), have been measured to provide estimation of the heat damage in these products. The ranges of the studied markers were as follows: FUR=153-600 mg 100 g(-1) of protein, GAP=0.5-4.3 mg L(-1), LCT=226-1511 mg L(-1), and LAL=1.0-16.1 mg 100 g(-1) of protein. The highest levels were found in MBF intended for the youngest babies. Experimental samples were produced in an industrial plant to evaluate the relative contribution of individual technological aspects to the final heat damage. About 90% of both GAP and LCT contents was due to the ultrahigh-temperature sterilization process itself. This effect was more than halved when the pH of the ingredient mixture was adjusted from 7.2 to 6.9 before sterilization or when the product recirculated in the plant was discarded. Up to 60 and 20%, respectively, of the FUR and LAL levels in the finished product were already present in protein ingredients (whey powder, whey protein concentrate). Accurate optimization of processing conditions and scrupulous selection of raw materials proved to be effective means to minimize heat damage in such special food products.

Occurrence and fate of ACE-inhibitor peptides in cheeses and in their digestates following in vitro static gastrointestinal digestion

The occurrence of the casein-derived angiotensin converting enzyme-inhibitor (ACE-I) peptides VPP, IPP, RYLGY, RYLG, AYFYPEL, AYFYPE, LHLPLP and HLPLP were investigated in 12 different cheese samples by Ultra Performance Liquid Chromatography/High-Resolution Mass Spectrometry. The total amount of ACE-I peptides was in the range 0.87-331mgkg(-1). VPP and IPP largely prevailed in almost all cheeses. Following in vitro static gastrointestinal digestion of Cheddar, Gorgonzola, Maasdam and Grana Padano cheeses, type and amount of ACE-I peptides changed, and only VPP, IPP, HLPLP and LHLPLP were detected in the intestinal digestates. The results evidenced that the degree of proteolysis itself cannot be regarded as a promoting or hindering factor for ACE-I peptide release during cheese digestion. Moreover, the data indicated that the ACE-I potential of cheeses cannot be inferred based on the type and amount of ACE-I peptides present in undigested samples.

Occurrence of galactosyl isomaltol and galactosyl β‐pyranone in commercial drinking milk

Occurrence of galactosyl isomaltol (GAI) and galactosyl beta-pyranone (GAP), two advanced glycosylation end products arising from the Maillard reaction of lactose via 1-deoxyosone pathway, was studied in commercial drinking milk. Galactosyl isomaltol was extracted from milk spiked with this isomaltol glycoside avoiding usage of any deproteinizing agent and was determined by a sensitive and interference-free HPLC method. No quantifiable amount of GAI proved to be present in any type of drinking milk, suggesting that some data reported in literature arise from uncontrolled conversion of GAP into GAI. The standard molecule of GAP was produced by heating a model system containing lysine and [U-14C]lactose, purified by solid phase extraction (SPE) on a C18 cartridge eluting with water, separated by the inverse distance function (IDF) standard HPLC method specified for lactulose determination, and characterized by both spectroscopic data and tandem mass spectrometry. The behaviour of formation of GAP and GAI in model systems containing lysine and lactose, heated under conditions of in bottle sterilization of milk, was studied in a wide range of values of the molar ratio lysine to lactose. While GAP easily forms as soon as lysine is present in the system, GAI does not form below a value of 0.1 of this molar ratio, so explaining why this compound is not present in commercial drinking milk. Amounts of GAP varying from 0.04 to 43.1 mumol/l were found in the different types of drinking milk ranging from high temperature pasteurized to in bottle sterilized, proving that this compound is a stable and sensible marker for evaluating the extent of the advanced Maillard reaction, hence the heating severity of commercial drinking milk. Moreover, GAP can be determined after conversion into GAI under acid warm conditions with a yield of 0.5 mol GAI from 1 mol GAP. Values of GAP obtained on commercial milk samples either by the direct HPLC method or after conversion into GAI were rather comparable, but the latter method needs further study in view of routine application.

Different Analytical Approaches in Assessing Antibacterial Activity and the Purity of Commercial Lysozyme Preparations for Dairy Application

Hen egg-white lysozyme (LSZ) is currently used in the food industry to limit the proliferation of lactic acid bacteria spoilage in the production of wine and beer, and to inhibit butyric acid fermentation in hard and extra hard cheeses (late blowing) caused by the outgrowth of clostridial spores. The aim of this work was to evaluate how the enzyme activity in commercial preparations correlates to the enzyme concentration and can be affected by the presence of process-related impurities. Different analytical approaches, including turbidimetric assay, SDS-PAGE and HPLC were used to analyse 17 commercial preparations of LSZ marketed in different countries. The HPLC method adopted by ISO allowed the true LSZ concentration to be determined with accuracy. The turbidimetric assay was the most suitable method to evaluate LSZ activity, whereas SDS-PAGE allowed the presence of other egg proteins, which are potential allergens, to be detected. The analytical results showed that the purity of commercially available enzyme preparations can vary significantly, and evidenced the effectiveness of combining different analytical approaches in this type of control.

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.

Heat damage and in vitro starch digestibility of puffed wheat kernels

The effect of processing conditions on heat damage, starch digestibility, release of advanced glycation end products (AGEs) and antioxidant capacity of puffed cereals was studied. The determination of several markers arising from Maillard reaction proved pyrraline (PYR) and hydroxymethylfurfural (HMF) as the most reliable indices of heat load applied during puffing. The considerable heat load was evidenced by the high levels of both PYR (57.6-153.4 mg kg(-1) dry matter) and HMF (13-51.2 mg kg(-1) dry matter). For cost and simplicity, HMF looked like the most appropriate index in puffed cereals. Puffing influenced starch in vitro digestibility, being most of the starch (81-93%) hydrolyzed to maltotriose, maltose and glucose whereas only limited amounts of AGEs were released. The relevant antioxidant capacity revealed by digested puffed kernels can be ascribed to both the new formed Maillard reaction products and the conditions adopted during in vitro digestion.

Targeted peptides for the quantitative evaluation of casein plasminolysis in drinking milk

In addition to proteose peptones (PP), the extent of plasminolysis in different classes of drinking milk during storage has been evaluated by the quantification of the peptides αs2-CN (f1-25) 4P and αs2-CN (f1-21) 4P by UPLC/HR-MS. The rate of increase in the levels of all the studied peptides during storage depended on the heating process. The samples of drinking milk showed different levels of plasminolysis at their expiration dates, as revealed by αs2-CN (f1-25) 4P and αs2-CN (f1-21) 4P amounts. The different treatments applied during the manufacturing of extended shelf life (ESL) milk samples resulted in different levels of plasminolysis, confirming the heterogeneity of this class of drinking milk. The peptides from αs2-CN accumulated faster than PP in all the samples with the exception of UHT milk. Therefore, these peptides can be considered as sensitive indices of early plasminolysis in pasteurised and ESL milk.

Protein breakdown and release of β-casomorphins during in vitro gastro-intestinal digestion of sterilised model systems of liquid infant formula.

Protein modifications occurring during sterilisation of infant formulas can affect protein digestibility and release of bioactive peptides. The effect of glycation and cross-linking on protein breakdown and release of β-casomorphins was evaluated during in vitro gastro-intestinal digestion (GID) of six sterilised model systems of infant formula. Protein degradation during in vitro GID was evaluated by SDS-PAGE and by measuring the nitrogen content of ultrafiltration (3kDa) permeates before and after in vitro GID of model IFs. Glycation strongly hindered protein breakdown, whereas cross-linking resulting from β-elimination reactions had a negligible effect. Only β-casomorphin 7 (β-CM7) was detected (0.187-0.858mgL(-1)) at the end of the intestinal digestion in all untreated IF model systems. The level of β-CM7 in the sterilised model systems prepared without addition of sugars ranged from 0.256 to 0.655mgL(-1). The release of this peptide during GID was hindered by protein glycation.

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.