Alfonso Clemente

Enzymatic protein hydrolysates in human nutrition

Abstract Protein hydrolysates constitute an alternative to intact proteins and elemental formulas in the development of special formulations designed to provide nutritional support to patients with different needs. The production of extensive protein hydrolysates by sequential action of endopeptidases and exoproteases coupled with the development of post-hydrolysis procedures is considered the most effective way to obtain protein hydrolysates with defined characteristics. This paper reviews the development and use of protein hydrolysates for dietary treatment of patients with phenylketonuria, food allergy and chronic liver failure.

The Tomato NBARC-LRR Protein Prf Interacts with Pto Kinase in Vivo to Regulate Specific Plant Immunity

Immunity in tomato (Solanum lycopersicum) to Pseudomonas syringae bacteria expressing the effector proteins AvrPto and AvrPtoB requires both Pto kinase and the NBARC-LRR (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4 fused to C-terminal leucine-rich repeats) protein Prf. Pto plays a direct role in effector recognition within the host cytoplasm, but the role of Prf is unknown. We show that Pto and Prf are coincident in the signal transduction pathway that controls ligand-independent signaling. Pto and Prf associate in a coregulatory interaction that requires Pto kinase activity and N-myristoylation for signaling. Pto interacts with a unique Prf N-terminal domain outside of the NBARC-LRR domain and resides in a high molecular weight recognition complex dependent on the presence of Prf. In this complex, both Pto and Prf contribute to specific recognition of AvrPtoB. The data suggest that the role of Pto is confined to the regulation of Prf and that the bacterial effectors have evolved to target this coregulatory molecular switch.

Protein isolates from chickpea (Cicer arietinum L.): chemical composition, functional properties and protein characterization

Two types of protein isolates were prepared from ground chickpea seeds by alkaline extraction, with (Isolate-B) and without (Isolate-A) sodium sulphite, and acid precipitation of the proteins at the isoelectric point (pI 4.3). The percentage of protein recovered from chickpea flour in the preparation of Isolates-A and B were 65.9 and 62.1%, respectively. Chemical composition, main functional properties and protein composition of chickpea flour and protein isolates were determined. Isolates-A and B contained 78 and 88.1% of protein, respectively, and had a balanced content of essential amino acids, with respect to the FAO pattern. The in vitro protein digestibility ranged between 95.6 and 96.1%. Isolate-A showed a partial dissociation of the 11S protein because of the high pH used for the protein extraction, and this probably explains the differences observed in the functional characteristics of both isolates.

2S Albumin Storage Proteins: What Makes them Food Allergens?

2S albumin storage proteins are becoming of increasing interest in nutritional and clinical studies as they have been reported as major food allergens in seeds of many mono- and di-cotyledonous plants. This review describes the main biochemical, structural and functional properties of these proteins thought to play a role in determining their potential allergenicity. 2S albumins are considered to sensitize directly via the gastrointestinal tract (GIT). The high stability of their intrinsic protein structure, dominated by a well-conserved skeleton of cysteine residues, to the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier to sensitize the mucosal immune system and/or elicit an allergic response. The flexible and solvent-exposed hypervariable region of these proteins is immunodominant and has the ability to bind IgE from allergic patients´ sera. Several linear IgE-binding epitopes of 2S albumins spanning this region have been described to play a major role in allergenicity; the role of conformational epitopes of these proteins in food allergy is far from being understood and need to be investigated. Finally, the interaction of these proteins with other components of the food matrix might influence the absorption rates of immunologically reactive 2S albumins but also in their immune response.

Protein quality of chickpea (Cicer arietinum L.) protein hydrolysates

Abstract Chickpea protein isolate (CPI) was used as the starting material in the production of chickpea protein hydrolysates (CPHs). To obtain a highly extensive hydrolysate with a degree of hydrolysis higher than 50%, a sequential utilisation of endoprotease (Alcalase) and exoprotease (Flavourzyme) was necessary. Molecular weight patterns of CPHs were determined by gel filtration chromatography. As a result of the enzymatic activity, differences in the chromatographic pattern of CPHs were observed. Although significant ( P ⩽0.05) decreases of Phe and Arg were observed after hydrolysis, adequate amounts of essential amino acids in relation to the reference pattern of FAO (FAO/WHO/ONU, 1985. Energy and requirements. Technical report series No. 724. Geneva) were found. In vitro protein digestibility of CPHs (95%) were similar to that of the starting material (CPI), and TIA was not detected in any case. A high increase of solubility in CPHs, with respect to CPI, was observed, one CPH being totally soluble over a wide pH range (2–10) when the enzymes were added sequentially. Due to their high protein quality and solubility, CPHs might be considered as potential ingredients in the food industry.

Effect of cooking on protein quality of chickpea (Cicer arietinum) seeds

Amino acid composition and in vitro protein digestibility of cooked chickpea were determined and compared to raw chickpea seeds. Heat treatment produced a decrease of methionine, cysteine, lysine, arginine, tyrosine and leucine, the highest reductions being in cysteine (15%) and lysine (13.2%). Protein content declined by 3.4% and in vitro protein digestibility improved significantly from 71.8 to 83.5% after cooking. The decrease of lysine was higher in the cooked chickpea seeds than in the heated protein fractions, globulins and albumins. The structural modification in globulins during heat treatment seems to be the reason for the increase in protein digestibility, although the activity of proteolytic inhibitors in the albumin fraction was not reduced. Results suggest that appropriate heat treatment may improve the bioavailability of chickpea proteins.

The cytotoxic effect of Bowman–Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases

Bowman-Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non-malignant colonic fibroblast CCD-18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9+/-2.3 and 48.3+/-3.5 microM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose-dependent manner, with cells becoming blocked in the G0-G1 phase. Chemically inactive soybean BBI had a weak but non-significant effect on the proliferation of HT29 cells. The anti-proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins.

Monomer and Linkage Type of Galacto-Oligosaccharides Affect Their Resistance to Ileal Digestion and Prebiotic Properties in Rats

A detailed study was performed to compare the in vivo ileal digestibility and modulatory effects in fecal microbiota of novel galacto-oligosaccharides (GOS) derived from lactulose [GOS-Lu; degree of polymerization (DP) ≥2, 14.0% trisaccharides] and commercial GOS derived from lactose (GOS-La; DP ≥3, 35.1% trisaccharides) in growing rats (5 wk old). Rats were fed either a control diet or diets containing 1% (wt:wt) of GOS-Lu or GOS-La for 14 d. Quantitative analysis of carbohydrates from dietary and ileal samples demonstrated that the trisaccharide fraction of GOS-Lu was significantly more resistant to gut digestion than that from GOS-La, as indicated by their ileal digestibility rates of 12.5 ± 2.6% and 52.9 ± 2.7%, respectively, whereas the disaccharide fraction of GOS-Lu was fully resistant to the extreme environment of the upper digestive tract. The low ileal digestibility of GOS-Lu was due to the great resistance of galactosyl-fructoses to mammalian digestive enzymes, highlighting the key role played by the monomer type and linkage involved in the oligosaccharide chain. The partial digestion of GOS-La trisaccharides showed that glycosidic linkages (1→6) and (1→2) between galactose and glucose monomers were significantly more resistant to in vivo gastrointestinal digestion than the linkage (1→4) between galactose units. The absence of GOS-La and GOS-Lu digestion-resistant oligosaccharides in fecal samples indicated that they were readily fermented within the large intestine, enabling both types of GOS to have a potential prebiotic function. Indeed, compared with controls, the GOS-Lu group had significantly more bifidobacteria in fecal samples after 14 d of treatment. The number of Eubacterium rectale also was greater in the GOS-Lu and GOS-La groups than in controls. These novel data support a direct relationship between patterns of resistance to digestion and prebiotic properties of GOS.

Factors affecting the in vitro protein digestibility of chickpea albumins

In vitro protein digestibility (IVPD) of chickpea albumins and its possible relationship to their structure and the presence of trypsin inhibitor activity (TIA) have been studied. Trypsin digestion of the albumin fraction under non-reducing conditions was incomplete, while the reduction of inter- and intramolecular disulphide bonds caused an improvement in the accessibility of sites susceptible to trypsin digestion. Trypsin inhibitor activity in the chickpea albumin fraction was dependent upon both temperature and heating time. Although heating the albumin fraction at 100 °C for 30 min reduced the TIA by more than 50% with respect to the initial activity, an important TIA rate was attributable to heat-resistant trypsin inhibitor. The TIA decrease was not related to an increase in the rate of IVPD. However, we observed a significant (P ≤ 0.05) increment in IVPD in the presence of β-ME, confirming the essential role of disulphide bonds in stabilising the protein structure of the albumin fraction. © 2000 Society of Chemical Industry

Characterization of galactooligosaccharides derived from lactulose.

Galactooligosaccharides are non-digestible carbohydrates with potential ability to modulate selectively the intestinal microbiota. In this work, a detailed characterization of oligosaccharides obtained by transgalactosylation reactions of the prebiotic lactulose, by using β-galactosidases of different fungal origin (Aspergillus oryzae, Aspergillus aculeatus and Kluveromyces lactis), is reported. Oligosaccharides of degree of polymerization (DP) up to 6 were detected and quantified by HPLC-ESI MS from a complex mixture produced by transgalactosylation reaction with A. oryzae (GOSLuAo), whereas only carbohydrates up to DP4 and DP5 were found for those obtained from the reaction with β-galactosidases from K. lactis (GOSLuKl) and A. aculeatus (GOSLuAa), respectively. Disaccharides (galactosyl-galactoses and galactosyl-fructoses) and trisaccharides were characterised in the three mixtures by GC-MS as their trimethylsilyl oximes. Galactosyl- and digalactosyl-glycerols were produced during the transgalactosylation reaction of lactulose with β-galactosidases from A. aculeatus and K. lactis, due to the presence of glycerol as enzyme stabiliser.