Generation Of Free Amino Acids Research Articles

Effect of ultrasound and enzymatic pre-treatments on the profile of bioactive peptides of beef liver hydrolysates

The use of meat by-products as a potential source of protein hydrolysates rich in bioactive peptides is of growing interest to valorise these products and improve their sustainable use. This study aimed to evaluate the effect of different pre-treatments (hydrolysis with pepsin, ultrasounds, and ultrasounds-assisted pepsin hydrolysis) on the peptide profile and bioactivity of beef liver hydrolysed with flavourzyme. The pre-treatments with pepsin increased the degree of hydrolysis and generation of free amino acids in the liver hydrolysates, whereas the subsequent hydrolysis with flavourzyme influenced the molecular weight distribution of the peptides. Samples pre-treated with pepsin and ultrasounds-assisted pepsin hydrolysates showed the highest values of antioxidant and inhibitory activity of ACE-I, ECE-I, DPP-IV, and neprilysin enzymes, and therefore these hydrolysates were characterised to obtain the bioactive peptide profiles. A total of 995 peptides were identified by tandem mass spectrometry in the most active fractions separated by reversed-phase liquid chromatography, and 20 of them were predicted as potentially responsible for the observed bioactivities and subsequently characterised by in silico tools. These results evidenced the potential of hydrolysis pre-treatments with pepsin and ultrasounds-assisted pepsin to obtain beef liver hydrolysates rich in bioactive peptides that could exert multifunctional bioactivities such as antioxidant, antihypertensive, and antidiabetic, while giving added value to this meat by-product.

Fermentation of Texturized Pea Protein in Combination with Proteases for Aroma Development in Meat Analogues.

The potential use of texturized pea protein in meat analogues was investigated by comparing the effects of fermentation on pea and myofibrillar pork proteins in a model system including additives, microbial starters, and proteases. Model fermentation was controlled for 15 days by a pH decrease and microbial count and free amino acid increase. Besides, volatile production and sensory properties were evaluated at the end of fermentation. Protein type affected free amino acid generation and volatile profile. Models supplemented with proteases showed an increase in amino-acid-derived compounds (branched aldehydes and alcohols) and fruity odor notes. During fermentation, protease addition significantly reduced the production of linear aldehydes (pentanal, hexanal, and octanal) in vegetal models, while pyrazine compounds were not affected. This changes in the volatile profile reduced the legume beany odor but increased the perception of toasted cereal-like notes generated by the texturization process.

Storage Stability of Plant-Based Drinks Related to Proteolysis and Generation of Free Amino Acids

The market for plant-based drinks (PBDs) is experiencing a surge in consumer demand, especially in Western societies. PBDs are a highly processed food product, and little is known about this relatively new food product category when compared to bovine milk. In the present study, the storage stability, proteolysis and generation of free amino acids were investigated in commercially available PBDs over the course of a one-year storage period. Generally, pH, color and protein solubility were found to be stable in the PBDs during storage, except for the pea-based product, which showed less protein solubility after storage. The pea-based drinks also had higher initial levels of free N-terminals prior to storage compared with levels for the other plant-based drinks, as well as significantly increasing levels of total free, and especially bitter free, amino acids. The development of free amino acids in the oat-based drink indicated that the released amino acids could be involved in various reactions such as the Maillard reaction during the storage period.

In vitro digestion of a mixed gel of pork muscle and resistant starch: Salt-soluble protein perspective

The in vitro digestion of a mixed gel (MS) of pork muscle and resistant starch (RS) was investigated and the role of the salt-soluble protein (SSP) in the function promotion of the mixed gel was clarified. The results showed that the mixed muscle gel (MS) and the addition of RS to muscle gel (M + S) presented an improved protein digestion, as indicated by a reduced particle size of the hydrolysates, more degradation of proteins with large molecular weight and more generation of free amino acids compared with the RS-free muscle gel (M). Meanwhile, the hydrolysates of the M + S and MS showed intensified DPPH radical scavenging activities. Specifically, the MS exerted preferable properties in protein digestion and antioxidant activity. Similar digestion characteristics were noticed in mixed SSP gels.The current study revealed that the reinforced functionality of the mixed muscle gel was associated with the binding relationships between SSP and RS during cooking.

Microbiota and volatilome of dry-cured pork loins manufactured with paprika and reduced concentration of nitrite and nitrate

Dry-cured pork loin is a very popular meat product in Mediterranean countries. Pork-loin is manufactured rubbing curing salts, nitrite and nitrate, and spices on the surface of the loin which is then dry-cured or smoked for several months. Although nitrite-derived compounds are crucial for the microbiological safety and development of a distinct flavour, there have been recent concerns about the adverse health effects of nitrite-derived compounds driving to the reduction of curing agents in meat products. In this study, we have evaluated the differences in microbiota and aroma of dry-cured pork loins manufactured with or without paprika and reduced ingoing amounts of nitrate and nitrite. Staphylococcus dominated the microbiota of pork loins without paprika, regardless of the nitrite and nitrate reduction. On the contrary, the reduction of nitrite and nitrate in loins with paprika had an important effect on the microbiota. In these loins a codominance of Staphylococcus and Bacillus together by Enterobacteriaceae occurred. Moreover, paprika addition and reduction of nitrite and nitrate seemed to promote proliferation of lactic acid bacteria. Occurrence of these genera was correlated with the generation of free amino acids and their derived volatile compounds setting clear differences in the aroma profile of dry-cured loins.

Impacts of deep-sea aging on quality of greater amberjack (Seriola dumerili) and bluefin tuna (Thunnus orientalis) meats

This is the first report on the effective utilization of the deep sea for improving food quality. Long-term aged fish has recently received substantial attention because of improved taste quality and the sustainable utilization of fishery resources. However, problems such as the refrigeration energy and space limitations for aging remain. Therefore, effects of deep-sea aging at a 2034 m depth for 34 days on meat quality of greater amberjack and bluefin tuna were investigated with a focus on changes in taste components and texture. Our results showed that free amino acid (FAA) contents in the deep-sea-aged greater amberjack were higher than those in the laboratory-aged sample. The deep-sea method promoted the proteolysis involved in FAA generation and mitigated the degradation of inosine-5′-monophosphate in greater amberjack. Conversely, deep-sea aging did not improve the quality of bluefin tuna. The present study also showed that the increase in the total viable bacteria count could be suppressed in greater amberjack during deep-sea aging. Thus, the deep sea can be utilized for sustainable aging of fish with superior quality, even though its suitability might depend on the fish species and depths related to the pressure level.

Plasma membrane V-ATPase controls oncogenic RAS-induced macropinocytosis.

Oncogenic activation of Ras is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumor cell fitness. Mutant Ras cells are endowed with the capability to internalize and degrade extracellular protein via a fluid–phase uptake mechanism termed macropinocytosis1. There is a growing appreciation for the role of this Ras-dependent process in the generation of free amino acids that can be used to support tumor cell growth under nutrient limiting conditions2. However, little is known about the molecular steps that mediate the induction of macropinocytosis by oncogenic Ras. Here we identify vacuolar ATPase (v-ATPase) as an essential regulator of Ras-induced macropinocytosis. Oncogenic Ras promotes the translocation of v-ATPase from intracellular membranes to the plasma membrane (PM) via a pathway that requires protein kinase A (PKA) activation by a bicarbonate-dependent soluble adenylate cyclase (sAC). PM accumulation of v-ATPase is necessary for the cholesterol-dependent association of Rac1 with the PM, a prerequisite for the stimulation of membrane ruffling and macropinocytosis. These observations identify a link between v-ATPase trafficking and nutrient supply by macropinocytosis that could be exploited to curtail the metabolic adaptation capacity of mutant Ras tumor cells.

Relationship among pH, generation of free amino acids, and Maillard browning of dried Japanese common squid Todarodes pacificus meat

Relationship among pH, generation of free amino acids (FAAs), and Maillard browning was investigated in Japanese common squid during air drying at 40 °C to prevent its discoloration. In the surface color measurement, increase in the b* value which is an indicator for Maillard browning of the dried squid was mitigated at pH 4.0–6.0 and accelerated at pH 7.5–9.0. Adjusting the pH to 5.5 effectively suppressed increasing of the b* value (p < 0.05). Arginine (Arg) generation involved in the Maillard reaction, was inhibited in the dried squid at pH 5.5 (p < 0.05). Maillard reactivities between ribose and amino acids were lower at pH 4.0 than at pH 5.5 (p < 0.05). These results indicate that browning of the dried squid is significantly suppressed at pH 5.5. Moreover, suppression of Arg generation is more effective than weakening of Maillard reactivity between ribose and Arg, in mitigating Maillard browning of the dried squid at an acidic pH.

High Pressure Induced Effects on Free Amino Acid Generation in Mung Bean Sprouts during Preservation after Trigger Pressurization

High Pressure Induced Effects on Free Amino Acid Generation in Mung Bean Sprouts during Preservation after Trigger Pressurization

The effect of organic salts on the browning of dried squid products processed by air-drying

To control discoloration of dried Japanese common squid (Todarodes pacificus) product, influence of 0–1% organic salts, including sodium gluconate (Na-gluconate), sodium citrate (Na-citrate), sodium phytate (Na-phytate), sodium benzoate (Na-benzoate) and sodium tartrate (Na-tartrate), on Maillard browning in the squid meat during air-drying was investigated. Changes in surface color of the dried squid were mitigated by the addition of organic salts. Organic salts also showed inhibitory effects on autolysis and generation of free amino acids (FAAs), especially arginine, in both the dried squid meat and the model solution. Moreover, Maillard reaction degree of the dried squid was decreased by using organic salts. The addition of 1% Na-citrate or 1% Na-phytate well suppressed the browning of the dried squid (p < 0.05). The results indicated that organic salts having strong chelating ability, including Na-citrate and Na-phytate, can prevent the browning of the dried squid product by inhibiting the generation of FAAs.

A peptidomic approach of meat protein degradation in a low-sodium fermented sausage model using autochthonous starter cultures

Fermented sausage technology is currently compromised in decreasing the addition of NaCl. Use of starter cultures with peptidogenic potential could be a valuable strategy that can mask or hide off flavors produced by the use of NaCl substituents. In the present work, the peptidogenic potential of four lactic acid bacteria species was evaluated in a low-sodium beaker sausage (BS) model. Using a peptidomic approach, a total of 86 low molecular weight (LMW) peptides were accurately identified, mostly derived from myofibrillar proteins, especially actin, which generated 53 peptides. The BS inoculated with L. curvatus CRL705 generated 56 LMW peptides, followed by Enterococcus (E.) mundtii CRL35 with 43 peptides. In addition, BS inoculated with Lactobacillus (L.) plantarum and with L. sakei produced higher amino acid amounts over time as compared to the rest of BS models, highlighting the importance of both, time and sample effect on the overall free amino acid generation. The presence of each LAB strain in BS models generated a unique profile of small peptides and amino acids that could serve as a distinctive biochemical trait to differentiate specific fermented products. According to these results, E. mundtii and L. sakei, which are compatible between them, are proposed as the most efficiently adapted to low-sodium conditions. The use of selected strains during the processing of low-sodium fermented sausages could have a positive effect on the production of small peptides and free amino acids.

The influence of proteases on the browning of dried squid products processed by air-drying

To clarify why mantle meat from Japanese common squid (Todarodes pacificus) dried products discolor dramatically when processed by air-drying, the role of endogenous protease(s) on browning during the processing of dried squid products was studied. The involvement of endogenous protease(s) in the generation of Free Amino Acids (FAAs) participating in the Maillard reaction was characterized. The browning and myosin heavy chain degradation during air-drying were obviously mitigated by the addition of metalloprotease inhibitors, especially EGTA and 1,10-phenanthroline, followed by serine proteinase inhibitor, especially PMSF. The amount of total FAAs in dried products increased by only 0.17% with the addition of 1,10-phenanthroline and 5.0% with added EGTA. In autolysis models, protein autolysis was almost completely inhibited by 1,10-phenanthroline. The addition of, 1,10-phenanthroline inhibited the increase in total FAAs including arginine (Arg), followed by EGTA and PMSF. The results indicated that endogenous metalloproteases and serine proteases might influence the generation of FAAs including Arg, and contribute to product discoloration during air-drying.

Dietary effect of green tea extract on hydration improvement and metabolism of free amino acid generation in epidermis of UV-irradiated hairless mice

Purpose: Ultraviolet (UV) irradiation decreases epidermal hydration, which is maintained by reduction of natural moisturizing factors (NMFs). Among various NMFs, free amino acids (AA) are major constituents generated by filaggrin degradation. This experiment was conducted to determine whether or not dietary supplementation of green tea extract (GTE) in UV-irradiated mice can improve epidermal levels of hydration, filaggrin, free AAs, and peptidylarginine deiminase-3 (PAD3) expression (an enzyme involved in filaggrin degradation). Methods: Hairless mice were fed a diet of 1% GTE for 10 weeks in parallel with UV irradiation (group UV+1%GTE). As controls, hairless mice were fed a control diet in parallel with (group UV+) or without (group UV-) UV irradiation. Results: In group UV+, epidermal levels of hydration and filaggrin were lower than those in group UV-; these levels increased in group UV+1% GTE to levels similar to group UV-. Epidermal levels of PAD3 and major AAs of NMF, alanine, glycine and serine were similar in groups UV- and UV+, whereas these levels highly increased in group UV+1% GTE. Conclusion: Dietary GTE improves epidermal hydration by filaggrin generation and degradation into AAs.

The harmonized INFOGEST in vitro digestion method: From knowledge to action

Within the active field of in vitro digestion in food research, the COST Action INFOGEST aimed to harmonize in vitro protocols simulating human digestion on the basis of physiologically inferred conditions. A harmonized static in vitro digestion (IVD) method was recently published as a primary output from this network. To validate this protocol, inter-laboratory trials were conducted within the INFOGEST network. A first study was performed using skim milk powder (SMP) as a model food and served to compare the different in-house digestion protocols used among the INFOGEST members. In a second inter-laboratory study applying the harmonized protocol, the degree of consistency in protein hydrolysis was investigated. Analysis of the hydrolyzed proteins, after the gastric and intestinal phases, showed that caseins were mainly hydrolyzed during the gastric phase, whereas β-lactoglobulin was, as previously shown, resistant to pepsin. Moreover, generation of free amino acids occurred mainly during the intestinal phase.The study also showed that a few critical steps were responsible for the remaining inter-laboratory variability. The largest deviations arose from the determination of pepsin activity. Therefore, this step was further clarified, harmonized, and implemented in a third inter-laboratory study.The present work gives an overview of all three inter-laboratory studies, showing that the IVD INFOGEST method has led to an increased consistency that enables a better comparability of in vitro digestion studies in the future.

Reduction of biogenic amine accumulation in silver carp sausage by an amine-negative Lactobacillus plantarum

The ability of an amine-negative Lactobacillus plantarum ZY-40 to reduce biogenic amine accumulation during the fermentation of silver carp sausage was studied. And the relative parameters such as microbiological growth, pH and free amino acid generation were also evaluated. Inoculation with L. plantarum ZY-40 caused a rapid pH decrease and growth inhibition of undesirable bacteria such as Pseudomonas and Enterobacteriaceae. Putrescine, cadaverine and tyramine were the main amines formed during sausage fermentation. L. plantarum ZY-40 was able to reduce the contents of putrescine and cadaverine by more than 70%, but did not affect tyramine accumulation. On the other hand, the sausages with and without L. plantarum ZY-40 demonstrated a marked increase in the content of free amino acids. No correlation between free amino acid generation and biogenic amine accumulation was observed. The use of an amine-negative starter culture is advisable to produce safer fish sausages with low biogenic amine contents.

Neonatal Skin Maturation—Vernix Caseosa and Free Amino Acids

Neonatal skin hydration decreases rapidly postnatally and then increases, indicating adaptive changes in stratum corneum water handling properties. Transition from high to low humidity at birth may initiate filaggrin proteolysis to free amino acids. Neonatal skin with vernix caseosa retained is more hydrated than skin with vernix removed. This study examines the potential roles of free amino acids and vernix in postnatal adaptation of infant stratum corneum in vivo. Specifically, the ontogeny of free amino acid generation in neonatal stratum corneum and the role of vernix caseosa in postnatal adaptation were examined using high performance liquid chromatography. Free amino acids were quantified for infant skin samples collected at (i) birth and 1 month and (ii) birth and 24 hours after vernix caseosa retention or removal and compared to neonatal foreskin, vernix caseosa, and adult stratum corneum using t-tests, analysis of variance, or univariate procedures. Free amino acids were extremely low at birth, significantly higher 1 month later but lower than in adults. Vernix caseosa retention led to significantly higher free amino acids 24 hours after birth compared to infants with vernix caseosa removed, and it paralleled the higher stratum corneum hydration of vernix caseosa-retained skin. Vernix caseosa contained free amino acids, with glutamic acid and histidine levels higher than in infants. Free amino acids in vernix caseosa-retained skin appear to originate from vernix caseosa. Free amino acids were lower in neonatal foreskin than adult forearm stratum corneum. Arginine was higher than citrulline at birth, but levels were comparable in older infants. The free amino acid increase at 1 month may be initiated by the humidity transition at birth and supports results in animals. The findings have implications for infant skin care practices.

Effect of sucrose on the generation of free amino acids and biogenic amines in Chinese traditional dry-cured fish during processing and storage.

The Chinese traditional dry-cured grass carp fish (Layú) was processed with (A) and without (B) sucrose. Higher levels of free amino acids (FAA) and biogenic amines were detected in the final products when compared to the fresh fish. In the presence of sucrose, Layú A had higher total free amino acids (39.9g/kg DW) but lower total biogenic amines (112.5mg/kg DW) than those in Layú B (35.4g/kg DW and 143.7mg/kg DW, respectively) after ageing. After 60days, the products storaged at 4°C had lower amount biogenic amines (Layú A: 112.2~579.5; Layú B: 144~593.8mg/kg DW) than those at 20°C (Layú A: 112.2~974.8; Layú B: 144~773.4mg/kg DW), indicating that low temperature storage was a safer procedure.

Distinct distribution of GLUT4 and insulin regulated aminopeptidase in the mouse kidney

The physiological importance of the insulin responsive glucose transporter GLUT4 in adipocytes and muscle in maintaining glucose homeostasis is well established. A key protein associated with this process is the aminopeptidase IRAP which co-localizes with GLUT4 in specialized vesicles, where it plays a tethering role. In this study, we investigated the distribution of both GLUT4 and IRAP in the kidney to gain insights into the potential roles of these proteins in this organ. Both IRAP and GLUT4 immunostaining was observed in the epithelial cells of the proximal and distal tubules and thick ascending limbs in the cortex, but very little overlap between GLUT4 and IRAP immunoreactivity was observed. GLUT4 staining was consistent with a vesicular localization, whereas IRAP staining was predominantly on the luminal surface. In the principal cells of the inner medulla collecting duct (IMCD), IRAP immunoreactivity was detected throughout the cell, with limited overlap with the vasopressin responsive water channel aquaporin-2 (AQP-2). AQP-2 levels were observed to be two-fold higher in IRAP knockout mice. Based on our results, we propose that GLUT4 plays a role in shunting glucose across epithelial cells. In the kidney cortex, IRAP, in concert with other peptidases, may be important in the generation of free amino acids for uptake, whereas in the principal cells of the inner medulla IRAP may play a localized role in the regulation of vasopressin bioactivity.

Generation of Free Amino Acids and γ-Aminobutyric Acid in Water-Soaked Soybean by High-Hydrostatic Pressure Processing

The effects of high-hydrostatic pressure processing (HPP) on soybean cotyledon as a cellular biological material were investigated from the viewpoints of the cell structure and enzyme reaction system. Damage to cell structure was evaluated by measuring dielectric properties using the Cole-Cole arc, the radius of which decreased as pressure level increased. Results suggested that cell structure was damaged by HPP. The distribution of free amino acids was measured after HPP (200 MPa) of soybean soaked in water or sodium glutamate (Glu) solution. HPP resulted in high accumulation of free amino acids in water-soaked soybean, due to proteolysis. HPP of soybean in Glu solution caused higher accumulation of gamma-aminobutyric acid, suggesting that both proteolysis and specific Glu metabolism were accelerated by HPP. We concluded that HPP partially degraded cell structure and accelerated biochemical reactions by allowing enzyme activities to remain. These events can be considered "high-pressure induced transformation" of soybean.

Proteomic Identification of Actin-Derived Oligopeptides in Dry-Cured Ham

An intense proteolysis of muscle proteins, mainly due to the action of endogenous proteolytic enzymes, has been reported to occur during the processing of dry-cured ham. This gives rise to an important generation of free amino acids and peptides of small size that contribute directly or indirectly to flavor characteristics of the final product. The nature and properties of the free amino acids generated during postmortem proteolysis have been well established. However, little is known about the identity of peptides generated during the processing of dry-cured ham. In the present paper, we describe the isolation (by ethanol precipitation followed by size exclusion and reverse phase chromatographies) and identification (by matrix-assisted laser desorption/ionization time-of-flight MS and collision-induced dissociation MS/MS) in a Spanish dry-cured ham of the following four oligopeptides: (A) TKQEYDEAGPSIVHR, (B) ITKQEYDEAGPSIVHRK, (C) DSGDGVTHNVPIYE, and (D) DSGDGVTHNVPIYEG. This is the first time that these peptide fragments have been reported in dry-cured ham at the end of processing. Sequence homology analysis revealed that the four peptides originated from muscle actin, indicating an extensive hydrolysis of this protein during dry-curing. Some of the cleavage sites corresponding to these fragments in actin were reproduced by other authors through the incubation of this myofibrillar protein in the presence of cathepsin D (EC 3.4.23.5), thus supporting a relevant action of this enzyme during the processing of dry-cured ham.