Chemical and physical changes induced by cold plasma treatment of foods: A critical review.

Cold plasma treatment is an innovative technology in the food processing and preservation sectors. It is primarily employed to deactivate microorganisms and enzymes without heat and chemical additives; hence, it is often termed a "clean and green" technology. However, food quality and safety challenges may arise during cold plasma processing due to potential chemical interactions between the plasma reactive species and food components. This review aims to consolidate and discuss data on the impact of cold plasma on the chemical constituents and physical and functional properties of major food products, including dairy, meat, nuts, fruits, vegetables, and grains. We emphasize how cold plasma induces chemical modification of key food components, such as water, proteins, lipids, carbohydrates, vitamins, polyphenols, and volatile organic compounds. Additionally, we discuss changes in color, pH, and organoleptic properties induced by cold plasma treatment and their correlation with chemical modification. Current studies demonstrate that reactive oxygen and nitrogen species in cold plasma oxidize proteins, lipids, and bioactive compounds upon direct contact with the food matrix. Reductions in nutrients and bioactive compounds, including polyunsaturated fatty acids, sugars, polyphenols, and vitamins, have been observed in dairy products, vegetables, fruits, and beverages following cold plasma treatment. Furthermore, structural alterations and the generation of volatile and non-volatile oxidation products were observed, impacting the color, flavor, and texture of food products. However, the effects on dry foods, such as seeds and nuts, are comparatively less pronounced. Overall, this review highlights the drawbacks, challenges, and opportunities associated with cold plasma treatment in food processing.

Effect of solvent composition on the extraction of proteins from hemp oil processing stream.

BACKGROUND: Hempseed meal, a by-product of the hempseed oil processing stream, is a potential alternative source for food proteins. Efficient extraction of proteins from hempseed meal is challenging owing to differences in the structure and solubility of various protein fractions present in the seed. In the present study, protein was extracted from hempseed meal using four different solvents, including aqueous NaOH, KOH, NaHCO3 and NaCl, at four different concentrations with the aim of improving the recovery of protein fractions rich in essential amino acids. RESULTS: Extraction using alkaline solvents provided superior protein recovery (60-78%) compared with NaCl solution and control extractions (20-48% and 21%, respectively). The concentration of alkali or salt (0.25-1 mol L-1 ) had a minor but significant impact on the yield. Amino acid composition analysis revealed that hempseed meal contains 24% (54.5 ± 0.19 mg g-1 ) essential amino acids of total amino acids, and extraction with NaOH, KOH, NaHCO3 or NaCl did not improve the selective extraction of essential amino acids compared to control experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis allowed the identification of edestin and albumin in the extracts obtained with NaHCO3 and NaCl solvents, with results further showing that the type of extraction solvent influences protein extraction selectivity. CONCLUSION: Although alkali solvents provide superior extraction yields, extraction with water resulted in extracts containing the highest proportion of proteins bearing essential amino acids. According to the results of SDS-PAGE, extraction using alkali solvents induced protein crosslinking. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Ultrasound processing of coffee silver skin, brewer's spent grain and potato peel wastes for phenolic compounds and amino acids: a comparative study.

Awareness towards utilizing food-processing by-products are increasing in health as well as environmental purview. Coffee silver skin (CSS), potato peel (PP) and brewer's spent grain (BSG) are voluminous by-products in their respective processing industries. The present study compared these three by-products for their prospective utilization in producing polyphenols-rich aqueous extracts by using ultrasound-assisted extractions (UAE). A probe-type sonicator was used for ultrasound treatments. The total phenolic contents in the extracts were assessed by Folin-Ciocalteu assay, while the phenolic profiles of the extract was characterized by LC-Q-TOF mass spectrometry. The microstructure of the samples after UAE was evaluated by scanning electron microscopy (SEM). Ultrasound treatment enhanced the rate of extraction and recovered 2.79, 2.12 and 0.66 mg gallic acid equivalents/g of TPC from CSS, PP and BSG, respectively in 30 min, which correspond to recoveries of 97.6%, 84.5% and 84.6%, respectively, compared to conventional solid-liquid extractions carried out for 24 h. The extraction yield was dependent on the particle size of the raw materials and the highest yield was obtained from the materials with 100-250 µm particle size. The SEM imaging revealed that ultrasound treatment caused prominent tissue damage. Extracts contained mainly hydroxycinnamic acid derivatives of phenolic acids. PP and CSS had the highest amounts of umami free amino acids (0.13 mg/g in each), while BSG contained the highest amount of essential amino acids (92 mg/g). The present work shows that CSS, PP and BSG are good sources of polyphenols and UAE can be employed to enhance the extraction efficiency as means of a green approach.

Dissociation and reduction of covalent ß-lactoglobulin-quinone adducts by dithiothreitol, tris(2-carboxyethyl)phosphine, or sodium sulfite.

Covalent protein-phenol adducts, generated by reaction of protein nucleophiles with quinones, have recently attracted increased attention because the interactions change the functionality and physicochemical properties of proteins in biological and food systems. The formation of such covalent adducts between ß-lactoglobulin (ß-LG) and the quinone of 4-methylcatechol, 4-methylbenzoquinone (4MBQ), and subsequent reduction by dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or sodium sulfite was investigated by mass spectrometry. The results showed that 19.0 ± 8.8% of ß-LG reacted with 4MBQ when present in equimolar ratio at 20°C (pH 8.0) to yield the protein-phenol adduct (ß-LG-Q). Following treatment with sulfite, DTT, or TCEP, 75, 68, or 36%, respectively, of the formed ß-LG-Q adduct dissociated. Different reaction mechanisms were proposed for the reduction of ß-LG and ß-LG-Q by each of the reducing agents. These results show that on reductive sample preparation for analysis of protein samples, not only are protein polymers formed through oxidative disulfide bonds reduced into the individual protein constituents but also a large part of any protein-phenol adducts present will dissociate and, thus, give a false picture of the level of protein-protein interactions that have occurred in the sample.

Resolving fluorescence spectra of Maillard reaction products formed on bovine serum albumin using parallel factor analysis.

Formation of Maillard reaction products (MRPs) is increasingly studied by the use of fluorescence spectroscopy, and most often, by measuring single excitation/emission pairs or use of unresolved spectra. However, due to the matrix complexity and potential co-formation of fluorescent oxidation products on tryptophan and tyrosine residues, this practice will often introduce errors in both identification and quantification. The present study investigates the combination of fluorescence excitation emission matrix (EEM) spectroscopy and parallel factor analysis (PARAFAC) to resolve the EEMs into its underlying fluorescent signals, allowing for better identification and quantification of MRPs. EEMs were recorded on a sample system of bovine serum albumin incubated at 40 °C for up to one week with either glucose, methylglyoxal or glyoxal added. Ten unique PARAFAC components were resolved, and assignment was attempted based on similarity with fluorescence of pure standards of MRPs and oxidation products and reported data from literature. Of the ten fluorescent PARAFAC components, tyrosine and buried and exposed tryptophan were resolved and identified, as well as the formation of specific MRPs (argpyrimidine and Nα-acetyl-Nδ-(5-methyl-4-imidazolon-2-yl)ornithine) and tryptophan oxidation products (kynurenine and dioxindolylalanine). The formation of the PARAFAC resolved protein modifications were qualitatively validated by liquid chromatography-mass spectrometry.

Quantification of protein thiols using ThioGlo 1 fluorescent derivatives and HPLC separation.

A method for quantification of total soluble protein-derived thiols in beer was developed based on the formation of fluorescent adducts with the maleimide compound ThioGlo 1. The problem of interference from fluorescent adducts of sulfite and ThioGlo 1 was solved by HPLC separation of the adducts followed by fluorescence detection. Using standard addition of GSH, a detection limit of 0.028 µM thiols was achieved. The application and validation of the method was demonstrated for beers with different color intensities, and the application range is in principle for any biological system containing thiols. However, the quantification of cysteine was complicated by a lower fluorescence response of its ThioGlo 1 adducts. Based on the studies of the responses of a series of cysteine-derived thiols and (1)H NMR studies of the structures of ThioGlo 1 adducts with GSH and cysteine, it was concluded that thiols with a neighboring free amino group yield ThioGlo 1 adducts with a reduced fluorescence intensity.

Covalent bonding between polyphenols and proteins: Synthesis of caffeic acid-cysteine and chlorogenic acid-cysteine adducts and their quantification in dairy beverages.

Protein-polyphenol interactions affect the structure, stability, and functional properties of proteins and polyphenols. Oxidized polyphenols (o-quinones) react rapidly with the sulfhydryl group of cysteine (Cys) residues, inducing covalent bonding between proteins and polyphenols. However, quantitative data on such reactions remain elusive, despite the importance of depicting the significance of such interactions on food structure and function. This work reports the synthesis, purification, and characterization of caffeic acid-cysteine (CA-Cys) and chlorogenic acid-cysteine (CGA-Cys) adducts and their stable isotope analogs, CA-[13C3,15N]Cys and CGA-[13C3,15N]Cys. A sensitive LC-MS/MS isotope dilution method was developed to simultaneously quantify these adducts in foods and beverages. Protein-bound CA-Cys and CGA-Cys were detected in the micro-molar range in milk samples with added CA and CGA, confirming covalent bonding between milk proteins and CA/CGA. These adducts were detected in commercial coffee-containing beverages but not in cocoa-containing drinks. Furthermore, the adducts were found to be partially stable during enzymatic protein hydrolysis.

Temperature affects the kinetics but not the products of the reaction between 4-methylbenzoquinone and lysine.

Quinones are electrophilic compounds that can undergo Michael addition or Schiff base reaction with nucleophilic amines, but the effect of temperature has not been systematically studied. The aim of this study was to characterize how temperature affects the reaction mechanism and kinetics of 4-methylbenzoquinone (4MBQ) with lysine (Lys), Nα-acetyl Lys or Nε-acetyl Lys. The products were identified and characterized by LC-MS/MS, which revealed formation of Michael addition products, Schiff base, and a di-adduct in Lys and Nα-acetyl Lys-containing reaction mixtures. The product profiles were not affected by temperature in the range of 15-100 °C. NMR analysis proved that Michael addition of Nα-acetyl Lys occurred on the C5 position of 4MBQ. Rate constants for the reactions studied by stopped-flow UV-vis spectrophotometry under pseudo-first-order conditions where the amines were present in excess in the range 15 °C to 45 °C showed the α-amino groups of Lys are more reactive than the ε-groups. The kinetics results revealed that the temperature dependence of reaction rates followed the Arrhenius law, with activation energies in the order: Lys < Nε-acetyl Lys < Nα-acetyl Lys. Our results provide detailed knowledge about the temperature dependence of the reaction between Lys residues and quinones under conditions relevant for storage of foods.

Phenolic Acid-Amino Acid Adducts Exert Distinct Immunomodulatory Effects in Macrophages Compared to Parent Phenolic Acids.

Caffeic acid (CA) and chlorogenic acid (CGA) are commonly found phenolic acids in plant-derived foods and beverages. Their corresponding adducts with cysteine (Cys) have been detected in coffee-containing beverages. However, despite the well-documented antioxidant and anti-inflammatory activity of CA and CGA, the immunomodulatory activities of the Cys adducts (CA-Cys and CGA-Cys) are unknown. The adducts were therefore synthesized, and their immunomodulatory effects were studied in lipopolysaccharide (LPS)-treated RAW 264.7 cells and compared to the activity of the parent phenolic acids. CA and CGA generally down-regulated the inflammatory responses. However, RNA-sequencing showed that the LPS-induced pathways related to Toll-like receptor signaling, chemokine signaling, and NOD-like receptor signaling, and JAK-STAT/MAPK signaling pathways were upregulated in adduct-treated cells relative to parent phenolic acids, while neurodegenerative disorder-related pathways and metabolic pathways were downregulated. Production of prostaglandin E2 (PGE2), interleukin-6, tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS) was all inhibited by CA and CGA (P < 0.05). PGE2 and TNF-α were further suppressed in adduct-stimulated cells (P < 0.05), but ROS production was increased. For example, TNF-α produced by 100 µM CGA-stimulated cells and 100 µM CGA-Cys adduct-stimulated cells were 4.46 ± 0.23 and 1.61 ± 0.18 ng/mL, respectively. Thus, the addition of the Cys moiety drastically alters the anti-inflammatory activity of phenolic compounds.

Site-Specific Characterization of Heat-Induced Disulfide Rearrangement in Beta-Lactoglobulin by Liquid Chromatography-Mass Spectrometry.

Disulfides are important for maintaining the protein native structure, but they may undergo rearrangement in the presence of free Cys residues, especially under elevated temperatures. Disulfide rearrangement may result in protein aggregation, which is associated with in vivo pathologies in organisms and in vitro protein functionality in food systems. In a food context, it is therefore important to understand the process of disulfide rearrangement on a site-specific level in order to control aggregation. In the present study, a liquid chromatography-mass spectrometry (LC-MS)-based bottom-up site-specific proteomic approach was optimized to study disulfide rearrangements in beta-lactoglobulin (ß-LG) under different heat treatments (60-90 °C). Artifactual disulfide rearrangement observed during sample preparation using a conventional protocol was detected and minimized by blocking the remaining free Cys residues with iodoacetamide in the presence of urea after heat treatment. Use of endoproteinase Glu-C for enzymatic hydrolysis allowed, for the first time, identification and comparison of the relative intensity of all theoretically possible ß-LG disulfide cross-links formed by the heat treatments. Non-native disulfides were formed from heat treatment at approx. 70 °C where ß-LG started to unfold, while higher levels of inter-molecular disulfide links were formed at ≥80 °C, in agreement with ß-LG aggregation detected by size exclusion chromatography analysis. Collectively, the Cys residues of the surface-located native disulfide Cys66-Cys160 were proposed to be more reactive, participating in heat-induced disulfide rearrangement, compared to other Cys residues. The abundant signal of non-native disulfide bonds containing Cys66, especially Cys66-Cys66, observed after heating suggested that Cys66 is a key disulfide-linked Cys residue in ß-LG participating in heat-induced inter-molecular disulfide bonds and the corresponding protein aggregation.

Chemical Stability of Proteins in Foods: Oxidation and the Maillard Reaction.

Protein is a major nutrient present in foods along with carbohydrates and lipids. Food proteins undergo a wide range of modifications during food production, processing, and storage. In this review, we discuss two major reactions, oxidation and the Maillard reaction, involved in chemical modifications of food proteins. Protein oxidation in foods is initiated by metal-, enzyme-, or light-induced processes. Food protein oxidation results in the loss of thiol groups and the formation of protein carbonyls and specific oxidation products of cysteine, tyrosine, tryptophan, phenylalanine, and methionine residues, such as disulfides, dityrosine, kynurenine, m-tyrosine, and methionine sulfoxide. The Maillard reaction involves the reaction of nucleophilic amino acid residues with reducing sugars, which yields numerous heterogeneous compounds such as α-dicarbonyls, furans, Strecker aldehydes, advanced glycation end-products, and melanoidins. Both protein oxidation and the Maillard reaction result in the loss of essential amino acids but may positively or negatively impact food structure and flavor.

Quantification of advanced glycation end products and amino acid cross-links in foods by high-resolution mass spectrometry: Applicability of acid hydrolysis.

An analytical method was developed and validated for simultaneous identification and quantification of advanced glycation end products (AGEs), amino acid cross-links, lysine and arginine in foodstuffs based on acid hydrolysis, hydrophilic interaction chromatography and high-resolution mass spectrometry. The method proved to be sensitive, reproducible and accurate for furosine, N-Ɛ-(carboxymethyl)lysine, N-Ɛ-(carboxyethyl)lysine, methylglyoxal and glyoxal-derived hydroimidazolones (MG-H and GO-H isomers, respectively), glyoxal lysine dimer, lysinoalanine, lanthionine, lysine and arginine. LOD and LOQ values in water were found to be 0.9-15.5 ng/mL and 2.8-47 ng/mL, respectively, and increased to 1.4-60 ng/mL and 4.4-182 ng/mL in liquid infant formula. Recovery values ranged from 76 to 118% in four different food matrices. Microwave-assisted hydrolysis for 11 min had similar efficiency as conventional hydrolysis, which requires overnight incubation. Acid stability of each compound was determined during microwave and conventional hydrolysis, and showed that the MG-H1 isomer is partially converted to the MG-H3 isomer during acid hydrolysis.

Maillard reaction products and amino acid cross-links in liquid infant formula: Effects of UHT treatment and storage.

The formation of Maillard reaction products, including Amadori compounds (determined as furosine), advanced glycation end products (AGEs), α-dicarbonyl and furfural compounds, as well as amino acid cross-links (lysinoalanine and lanthionine) was investigated in direct (DI) and indirect (IN) UHT-treated experimental liquid infant formula (IF) during storage at 40 °C. IN-IF had higher concentrations of all investigated compounds compared to DI-IF and low pasteurized IF. IN UHT treatment induced significantly higher concentrations of α-dicarbonyl compounds (glyoxal, methylglyoxal, 3-deoxyglucosone and 3-deoxygalactosone) compared to DI, which facilitated increased formation of AGEs (N-Ɛ-(carboxymethyl)lysine, methylglyoxal- and glyoxal-derived hydroimidazolones) in unstored IFs. During storage for 6 months, concentrations of furosine and AGEs increased while α-dicarbonyl compounds decreased. Principal component analysis indicated that differences between IN-IF and DI-IF disappeared after 2 months of storage. IN-IF had higher concentrations of lysinoalanine and lanthionine and lower concentrations of available lysine and arginine than DI-IF indicating higher loss of protein quality in IN-IF.

Effect of Processing of Whey Protein Ingredient on Maillard Reactions and Protein Structural Changes in Powdered Infant Formula.

The most widely used whey protein ingredient in an infant formula (IF) is the whey protein concentrate (WPC). The processing steps used in the manufacturing of both a powdered IF and a WPC introduce protein modifications that may decrease the nutritional quality. A gently processed whey protein ingredient (serum protein concentrate; SPC) was manufactured and used for the production of a powdered IF. The SPC and the SPC-based IF were compared to the WPC and the powdered WPC-based IF. Structural protein modifications were evaluated, and Maillard reaction products, covering furosine, α-dicarbonyls, furans, and advanced glycation end products, were quantified in the IFs and their protein ingredients. IF processing was responsible for higher levels of protein modifications compared to the levels observed in the SPC and WPC. Furosine levels and aggregation were most pronounced in the WPC, but the SPC contained a high level of methylglyoxal, revealing that other processing factors should be considered in addition to thermal processing.

Oxidation of Whey Proteins during Thermal Treatment Characterized by a Site-Specific LC-MS/MS-Based Proteomic Approach.

Thermal treatment is often employed in food processing to tailor product properties by manipulating the ingredient functionality, but these elevated temperatures may accelerate oxidation and nutrient loss. Here, oxidation of different whey protein systems [α-lactalbumin (α-LA), ß-lactoglobulin (ß-LG), a mix of α-LA and ß-LG (whey model), and a commercial whey protein isolate (WPI)] was investigated during heat treatment at 60-90 °C and a UHT-like treatment by LC-MS-based proteomic analysis. The relative modification levels of each oxidation site were calculated and compared among different heat treatments and sample systems. Oxidation increased significantly in protein systems after heating at ≥90 °C but decreased in systems with higher complexity [pure protein (α-LA > ß-LG) > whey model > WPI]. In α-LA, Cys, Met, and Trp residues were found to be most prone to oxidation. In ß-LG-containing protein systems, Cys residues were suggested to scavenge most of the reactive oxidants and undergo an oxidation-mediated disulfide rearrangement. The rearranged disulfide bonds contributed to protein aggregation, which was suggested to provide physical protection against oxidation. Overall, limited loss of amino acid residues was detected after acidic hydrolysis followed by UHPLC analysis, which showed only a minor effect of heat treatment on protein oxidation in these protein systems.

Detection of protein oxidation products by fluorescence spectroscopy and trilinear data decomposition: Proof of concept.

Current analytical methods studying protein oxidation modifications require laborious sample preparation and chromatographic methods. Fluorescence spectroscopy is an alternative, as many protein oxidation products are fluorescent. However, the complexity of the signal causes misinterpretation and quantification errors if single emission spectra are used. Here, we analyzed the entire fluorescence excitation-emission matrix using the trilinear decomposition method parallel factor analysis (PARAFAC). Two sample sets were used: a calibration set based on known mixtures of tryptophan, tyrosine, and four oxidation products, and a second sample set of oxidized protein solutions containing UV-illuminated ß-lactoglobulin. The PARAFAC model succeeded in resolving the signals of the model systems into the pure fluorophore components and estimating their concentrations. The estimated concentrations for the illuminated ß-lactoglobulin samples were validated by liquid chromatography-mass spectrometry. Our approach is a promising tool for reliable identification and quantification of fluorescent protein oxidation products, even in a complex protein system.

UHT treatment and storage of liquid infant formula affects protein digestion and release of bioactive peptides.

Ready-to-feed liquid infant formulas (IF) were subjected to direct (D) or indirect (ID) ultra-high-temperature (UHT) treatment and then stored at 40 °C under aseptic conditions for 60-120 days simulating global transportation which accelerates the Maillard reaction. Low pasteurized and unstored IF (LP) was included as a control for the UHT treatments. Simulated infant in vitro digestion was conducted. SDS-PAGE indicated that protein aggregate formation correlated with thermal treatment, being greatest after 60 days of storage. Limited protein digestion was observed after pepsin treatment for 2 h. Beta-lactoglobulin (ß-Lg), alpha-lactalbumin (α-La) and protein aggregates remained undigested after 2 h of pepsin digestion in LP and D, but less ß-Lg and α-La remained in ID. The digestion of ß-Lg and α-La was enhanced in D and ID stored for 60 days, but aggregates remained undigested. After pepsin and pancreatin digestion, large amounts of ß-Lg remained undigested in the LP, but digestion increased after UHT treatment (ID > D) and increased further after storage for 60 and 120 days, indicating that heat treatment and storage facilitate the digestion of unaggregated proteins. No aggregates remained after pancreatin digestion of LP, D, ID and D stored for 60 days, but were present in ID stored for 60 days. Aggregates were mainly disulphide-linked, but dityrosine linkages were detected in D and ID stored for 120 days. LC-MS/MS indicated limited proteolysis arising from endogenous milk proteases prior to in vitro digestion, being highest in D. Peptide numbers increased following pepsin and further during pancreatin digestion (ß-casein > ß-Lg > ß-La), and released ß-Lg peptides, typically 5-8 amino acids in length, contained several bioactivities, e.g., dipeptidyl-peptidase IV (DPP-IV) and angiotensin converting enzyme (ACE) inhibition.

Covalent bonding of 4-methylcatechol to ß-lactoglobulin results in the release of cysteine-4-methylcatechol adducts after in vitro digestion.

Protein-polyphenol adducts are formed upon covalent bonding between oxidized polyphenols and proteins. 4-Methylcatechol (4MC) is a polyphenol with origin in coffee and is oxidized to 4-methylbenzoquinone (4MBQ) under conditions used during food processing. The present study characterizes 4MBQ-induced covalent modifications on ß-lactoglobulin (ß-LG) from bovine milk, (henceforth ß-LQ) and the effect on protein digestibility. Significant thiol and amine loss was found in ß-LQ compared to ß-LG. Site-specific 4MBQ-induced modifications were identified on Cys, Lys, Arg, His and Trp in ß-LQ. No significant differences between ß-LG and ß-LQ on in vitro digestibility were observed by assessment with SDS-PAGE, degree of hydrolysis and LC-MS/MS unmodified peptide intensities. Cys-4MC adduct (1.7 ± 0.1 µmol/g) was released from ß-LQ after in vitro digestion. Thus, it is relevant to investigate how released Cys-4MC adducts are absorbed in vivo in future studies.

Cysteine residues are responsible for the sulfurous off-flavor formed in heated whey protein solutions.

Odor-active volatile sulfur compounds are formed in heated food protein systems. In the present study, hydrogen sulfide (H2S) was found to be the most abundant sulfur volatile in whey protein solutions (whey protein isolate [WPI], a whey model system and single whey proteins) by gas chromatography-flame photometric detector (GC-FPD) analysis after heat treatments (60-90 °C for 10 min, 90 °C for 120 min and UHT-like treatment). H2S was detected in WPI after heating at 90 °C for 10 min, and was significantly increased at higher heat load (90 °C for 120 min and the UHT-like treatment). Site-specific LC-MS/MS-based proteomic analysis was conducted, monitoring desulfurization reactions in these protein systems to investigate the mechanism of H2S formation in heated WPI. Cysteine residues from beta-lactoglobulin were found to be responsible for the formation of H2S in heated WPI, presumably via beta-elimination.

Ultra-High Temperature Treatment and Storage of Infant Formula Induces Dietary Protein Modifications, Gut Dysfunction, and Inflammation in Preterm Pigs.

SCOPE: Ready-to-feed liquid infant formula is increasingly used for preterm infants when human milk is unavailable. These formulas are sterilized by ultra-high temperature treatment, but heating and storage may reduce bioactivity and increase formation of Maillard reaction products with potential negative consequences for immature newborns. METHODS AND RESULTS: Using preterm pigs as a model for sensitive newborn infants, the study tests the intestinal responses of feeding experimental liquid formula within 5 days. A pasteurized formula (PAST) with the same nutrient composition but less protein modifications serves as control to ultra-high temperature-treated formula without (UHT) and with prolonged storage (SUHT). Relative to PAST, UHT contains lower levels of lactoferrin and IgG. Additional storage (40 °C, 60 days, SUHT) reduces antimicrobial capacity and increases non-reducible protein aggregates and Maillard reaction products (up to 13-fold). Pigs fed SUHT have more diarrhea and show signs of intestinal inflammation (necrotizing enterocolitis) compared with pigs fed PAST and UHT. These clinical effects are accompanied by accumulation of Maillard reaction products, protein cross-links, and inflammatory responses in the gut. CONCLUSION: The results demonstrate that feeding UHT infant formulas, particularly after prolonged storage, adversely affects gut maturation and function in preterm pigs used as a model of preterm infants.