Molecular Weight Proteins Research Articles

Chemical Composition of Acid Soluble Collagen (ASC) Isolated from Indonesia Local “Kacang” Goat Skin (<i>Capra aegagrus hocus</i>)

Collagen from the local "Kacang” goat skin is a natural raw material in the halal food industry in Indonesia. This study aims to isolate acid-soluble collagen (ASC) from “Kacang” goat skin and characterize its chemical properties. The collagen was derived from one-year-old goat skin and cured in acid condition for 48 hours at 4°C to eliminate meats, fat, and hair. The cleaned skins were treated at 1:10 (w/v) of 0.1 M NaOH for 0, 24, and 48 h at 4°C. It was then neutralized in distilled water and extracted with 0.5 M acetic acid at a 1:10 (w/v) ratio for 24, 48, and 72 h at 4°C. The yield of ASC was 21%, characterized by chemical composition, soluble protein, differential scanning calorimeter (DSC), and protein molecular weight. The chemical composition of ASC was 11.15% (moisture), 9.04% (protein), 0.98% (fat), and 0.052% (ash). ASC has the highest collagen solubility in NaCl 5% at pH 2. ASC also has thermal stability with a low profile pattern of molecular weight. In conclusion, “Kacang” goat skin from Indonesia might be used to make a value-added product because it has a high moisture content and low fat level.

Extraction and Nutritional Value of Soybean Meal Protein Isolate

The increasing protein demand driven by global population growth necessitates the search for an alternative protein source. Soybean meal (SM), with approximately 47–49% proteins, is a viable option. Soymeal protein isolate (SMPI) is a cost-efficient protein source with a well-balanced amino acid profile, making it suitable for addressing this demand. This study attempts to address the lack of information regarding the extractability and nutritional potential of SMPI obtained utilizing a weak base and recoverable solvent. In this work, the structural and compositional studies of SMPI, as well as the aqueous extractability of ammonium hydroxide (NH4OH), are investigated. Furthermore, we examined the effects of heat treatment during oil extraction, where a comparison between protein isolates from soymeal and whole soybeans was carried out. The maximum extraction yield of 24.1% was achieved by increasing the concentration of NH4OH from 0.25–1%. In comparison to the soy protein isolate from whole soybeans (SPI), the compositional analysis report revealed that SMPI had higher levels of crude protein, moisture, and ash content, but lower levels of fat and carbohydrates. Furthermore, the examination of the amino acid composition confirms the existence of vital amino acids in SMPI. The amino acid score indicates that methionine, lysine, and threonine are the limiting amino acids. SMPI and SPI share structural and functional group similarities, as demonstrated by Fourier-transform infrared spectroscopy. Gel electrophoresis using sodium dodecyl sulfate–polyacrylamide shows that the protein molecular weight distributions of SPI and SMPI are similar. This in-depth evaluation emphasizes the advantages of SM by advocating its application in other sectors beyond conventional animal feed, such as nutritional supplements and bio-based products, and by improving the environmental sustainability and global food chains.

Elucidating the gas cell stabilization mechanism of buckwheat-wheat steamed bread induced by transglutaminase: A focus on the foaming and air-water interfacial properties of dough liquor

This work investigated the improvement mechanism of transglutaminase (TG) on the buckwheat-wheat steamed bread qualities from the view of protein structural, foaming and air-water interfacial properties of dough liquor (DL). TG reduced the hardness, ameliorated the crumb structure and enhanced the specific volume of buckwheat-wheat steamed bread by up to 9.0%. The DL yield, protein and water recovery were notably increased as the TG concentration increased to 0.2 U/g, then decreased with the higher TG concentrations. The low TG concentrations (<0.2 U/g) increased the protein molecular weight, and decreased the protein hydrophobicity, free amino and sulfhydryl groups content of DL. The high TG concentrations (>0.2 U/g) tended to induce both protein intermolecular and intramolecular cross-linking, which reduced the average particle size from 9.6 to 4.3 μm as the TG concentration increased from 0.2 to 1.0 U/g. TG markedly improved the foaming capacity and foam stability, decreased the surface tension and strengthened the interfacial films by enhancing the viscoelasticity modulus of buckwheat-wheat DL interfacial protein films. The topography of the air-water interfacial layer revealed that the TG-induced protein cross-linking led to a more homogeneous and denser topographical structure. This work will provide fresh insight into the effects of TG-induced protein cross-linking on gas cell stabilization and relate them to the buckwheat-wheat steamed bread quality properties.

Deep‐eutectic‐solvent‐assisted hydrothermal carbonization of sewage sludge for green wood bioadhesive production

AbstractThis study reports the use of deep‐eutectic‐solvent‐ (DES‐) assisted hydrothermal carbonization (HTC) to disrupt the floc structure of sewage sludge (SS) for deep carbonization, with the resulting hydrochars employed in the preparation of formaldehyde‐free plywood bioadhesives. Sewage‐sludge‐based bioadhesive exhibits an excellent wet shear strength, complying with the requirements of Chinese national standard GB/T 9846–2015 (≥0.7 MPa). The molecular weight of proteins and the formation of covalent bonds via dehydration have a notable role in improving adhesive performance (wet shear strength). The Maillard reaction is a key reaction during HTC to destroy the secondary structure of proteins, resulting in the release of more OH and NH2. The main reaction during hot‐press treatment is dehydration. High ash content in bioadhesives improves flame resistance potential, particularly on addition of DES. A plausible mechanism is proposed for this. This work provides a new method for the valorization of SS‐derived hydrochars and contributes to the development of greener formaldehyde‐free wood bioadhesives.

Partitioning of proteins and small molecular weight compounds from alfalfa juice during ultrafiltration

Ribulose-1,5-biphosphate-carboxylase/oxygenase (RuBisCO) from alfalfa is a promising source of alternative food protein, but it is challenging to purify. Ultrafiltration shows potential for concentrating and fractionating RuBisCO from low molecular weight compounds present in the extracted juice, but there is limited information on their partitioning depending on the membrane pore size. Therefore, the objective of this study was to evaluate the partitioning of various compounds from the juice depending on membrane pore size. It was hypothesized that both membrane pore sizes will retain RuBisCO but might partition low molecular weight compounds within the matrix differently. RuBisCO was fully retained by both the 300 and 100 kDa polyethersulfone (PES) membranes, while ash (29.7%–35.4% dry matter (DM)), non-protein nitrogen (2.38%–2.53% DM), and free N-terminals (10.13–10.34 mg glutamic equivalent/100 mg DM) were transmitted through both membranes. The retentates showed a distinct yellow color when filtering with 100 kDa membranes, but not when using 300 kDa membranes. Although no significant differences in total polyphenol permeation were observed through a spectrophotometric assay, a metabolomics screening analysis showed that the 100 kDa membranes had less transmission of the flavonoids apigenin, chrysoeriol, tricin, and tricin glycosides, which might contribute to the yellow color. These results reiterate the importance of measuring individual polyphenols using hyphenated techniques as opposed to spectrophotometric total reducing capacity. Sixteen saponins were identified and their permeation was lower in the 100 than the 300 kDa membranes, indicating a better potential for higher degree of protein refinement for the latter. This study demonstrates for the first time the difference in partitioning of the small molecular weight components, using PES membranes of similar chemistry but different size, during ultrafiltration of alfalfa juice.

Ultrasonication assisted enzymatic hydrolysis for generation of pulses protein hydrolysate having antioxidant and ACE-inhibitory activity

The main objective of this work was to investigate the impact of ultrasonication assisted enzymatic treatment on the physicochemical and bioactive properties of broad bean (BBP), lentil bean (LBP), and mung bean (MBP) protein isolates. The protein was extracted using alkaline acid precipitation method, ultrasonicated at a frequency of 20 kHz, temperature 20–30 °C and then hydrolysed using alcalase enzyme (1 % w/w, pH 8.5, 30 min, 55 οC). The generated hydrolysates were characterized by degree of hydrolysis (DH), SDS, FTIR, surface hydrophobicity, amino acid composition, antioxidant and antihypertensive properties. Results showed that the degree of hydrolysis was found to increase in ultrasonicated protein hydrolysate (18.9 to 40.71 %) in comparison to non- ultrasonicated protein hydrolysate (11 to 16.3 %). SDS-PAGE results showed significant changes in protein molecular weight profiles (100–11kDa) in comparison to their natives. However, no substantial change was found in ultrasonicated and non-ultrasonicated protein hydrolysates. The FTIR spectrum showed structural alterations in ultrasonicated and non-ultrasonicated protein hydrolysates, suggesting modifications in secondary structure such as amide A, amide I and amide II regions. The essential amino acid content varied in the range of 60.09 mg/g to 73.77 mg/g and 28.73 to 50.26 mg/g in case of ultrasonicated and non-ultrasonicated protein hydrolysates, and non-essential content varied in the range of 49.42 to 65.93 mg/g and 43.12 to 47.12 mg/g. Both antioxidant and antihypertensive activities were found to increase significantly in ultrasonicated and non-ultrasonicated protein hydrolysates in comparison to their native counterparts, highlighting their potential as functional ingredients for management of hypertension. It was concluded that ultrasonication assisted enzymatic hydrolysis is an efficient approach for production of bioactive pulse protein hydrolysates with enhanced nutracutical properties, thus offering promising avenues for their utilization in the food industry and beyond.

Contribution of yeast freezing to the quality of frozen dough: Rheological properties, protein depolymerization, gluten conformation and water state

The present study has comparatively investigated the variation in yeast leachates in frozen suspension and dough systems and assessed the effects of leachate released from frozen yeast cells on dough rheology, protein molecular weight distribution, gluten conformation, and water state. The results revealed that yeast in the dough was more susceptible to freezing than that in the suspension and released more low-molecular-weight metabolites (e.g., trehalose, glutathione, and amino acids) into the dough matrix. Dynamic rheological measurements showed that frozen yeasted dough had weaker viscoelastic properties than non-yeasted dough. Fourier transform infrared spectroscopy revealed a high fraction of β-turns and random coils at the expense of α-helices and β-sheets, demonstrating a flexible protein secondary structure and increase in weakened hydrogen bonds in the gluten network. The presence of yeast in the frozen dough facilitated dough depolymerization via the cleavage of interchain disulfide bonds of gluten protein and softened the gluten network structure. Yeast leachates released from frozen yeast cells caused water redistribution in the dough and increased water release from the weakened gluten network, increasing freezable water content. This study has revealed the essential role of yeast freezing in gluten depolymerization and the deterioration of dough quality.

Physicochemical, structural and functional properties of pomelo peel pectin extracted by combination of pulsed electric field and cellulase hydrolysis

In this study, pectin extracted from pomelo peel was investigated using three different combination methods of pulsed electric field (PEF) and cellulase. Three action sequences were performed, including PEF treatment followed by enzymatic hydrolysis, enzymatic hydrolysis followed by PEF treatment, and enzymatic hydrolysis simultaneously treated by PEF. The three corresponding pectins were namely PEP, EPP and SP. The physiochemical, molecular structural and functional properties of the three pectins were determined. The results showed that PEP had excellent physiochemical properties, with the highest yield (12.08 %), total sugar (80.17 %) and total phenol content (38.20 %). The monosaccharide composition and FT-IR analysis indicated that the three pectins were similar. The molecular weights of PEP, EPP and SP were 51.13, 88.51 and 40.00 kDa, respectively. PEP showed the best gel properties, emulsification stability and antioxidant capacity among the three products, due to its high galacturonic acid and total phenol content, appropriate protein and low molecular weight. The mechanism of PEF-assisted cellulase hydrolysis of pomelo peel was also revealed by SEM analysis. These results suggested that PEF pretreatment was the best method, which not only improved the efficiency of enzymatic extraction, but also reduced resource waste and increased financial benefits.

Effect of dry- and moist-heat treatment processes on the structure, solubility, and in vitro digestion of macadamia protein isolate.

This study aimed to enhance the solubility and digestibility of macadamia protein isolate (MPI) for potential utilization in the food industry. The impact of dry- and moist-heat treatments at various temperatures (80, 90, and 100°C) and durations (15 and 30min) on macadamia protein's microstructure, solubility, molecular weight, secondary and tertiary structure, thermal stability, and digestibility were investigated and evaluated. The heating degree was found to cause roughening of the MPI surface. The solubility of MPI after dry-heat treatment for 15 min at 100°C reached 290.96±2.80% relative to that of untreated protein. Following heat treatment, the bands of protein macromolecules disappeared, while MPI was stretched by vibrations of free and hydrogen-bonded hydroxyl groups. Additionally, an increase in thermal stability was observed. After heat treatment, hydrophobic groups inside the protein are exposed. Heat treatment significantly improved the in vitro digestibility of MPI, reaching twice that of untreated protein. The results also demonstrated that dry- and moist-heat treatments have distinct impacts on MPI, while heating temperature and duration affect the degree of modification. With a decreased ordered structure and increased random coil content, the dry-heat treatment significantly enhanced the in vitro digestibility of MPI. The digestibility of MPI after dry-heat treatment for 30 min at 90°C increased by 77.82±2.80% compared to untreated protein. Consequently, compared to moist-heat treatment, dry-heat treatment was more effective in modifying macadamia protein. Dry-heat treatment of 30 min at 90°C was determined as the optimal condition. PRACTICAL APPLICATION: Heat treatment enhances MPI characteristics, potentially advancing macadamia-derived food production, including plant-based beverages and protein supplements.

Develop targeted protein drug carriers through a high-throughput screening platform and rational design.

Protein-based drugs offer advantages such as high specificity, low toxicity, and minimal side effects compared to small molecule drugs. However, delivery of proteins to target tissues or cells remains challenging due to the instability, diverse structures, charges, and molecular weights of proteins. Polymers have emerged as a leading choice for designing effective protein delivery systems, but identifying a suitable polymer for a given protein is complicated by the complexity of both proteins and polymers. To address this challenge, we developed a fluorescence-based high-throughput screening platform called ProMatch to efficiently collect data on protein-polymer interactions, followed by in vivo and in vitro experiments with rational design. Using this approach to streamline polymer selection for targeted protein delivery, we identified candidate polymers from commercially available options and developed a polyhexamethylene biguanide (PHMB)-based system for delivering proteins to white adipose tissue as a treatment for obesity. We also developed a branched polyethylenimine (bPEI)-based system for neuron-specific protein delivery to stimulate optic nerve regeneration. Our high-throughput screening methodology expedites identification of promising polymer candidates for tissue-specific protein delivery systems, thereby providing a platform to develop innovative protein-based therapeutics. This article is protected by copyright. All rights reserved.

Acid-soluble collagen (ASC) hydrolysate from Javanese goat (Capra aegagrus hocus) skin: effect of pepsin hydrolysis and antioxidant activity

Abstract Javanese goat (Capra aegagrus hocus) is an animal species which have many populations in Indonesia and it is used because more selective in feed, especially Leguminosae and seeds which of rich in collagen. The study aims to determine the impact of hydrolysis time and antioxidant activities on the extraction of skin Javanese goats using the acid-soluble collagen (ASC) method. The ASC used in this investigation was taken from another study and hydrolyzed with pepsin throughout the course of 0, 30, 60, 90, and 120 minutes of incubation. The success of the hydrolysis process can be shown by the percent degree of hydrolysis. Methods are used to determine the percent degree of hydrolysis of the TCA method. Results showed that hydrolyzing for 60 min yields the highest total soluble collagen of ASC, with a value of 0.77 mg/ml. Finally, the ASC which is hydrolysis use pepsin for 60 min has a degree of hydrolysis, and total soluble collagen is the highest. The protein molecular weight of ASC which is the hydrolysis of 60 min is smaller than before hydrolysis and it has radical scavenging activity of 56.42% ± 1.45. So far, ASC from Javanese goat (Capra aegagrus hocus) has been hydrolyzed in this study has high antioxidant activity and can be recommended as a substitute for functional food.

Physicochemical Properties of Different Sulfated Polysaccharide Components from Laetiporus sulphureus and Their Anti-Proliferative Effects on MDA-MB-231 Breast Cancer Cells.

Laetiporus sulphureus is an edible and medicinal mushroom widely used in folk medicine for treating cancer and gastric diseases. This study aimed to investigate the physicochemical properties of different sulfated polysaccharide (SPS) components (F1, F2, and F3) isolated from L. sulphureus and evaluate their activity against MDA-MB-231 breast cancer cell proliferation. Compared with F1 and F3, the results showed that F2 exhibited the most potent anti-proliferative activity on MDA-MB-231 cells, which could be attributed to the sulfate and protein contents, molecular weight, and monosaccharide composition. F2 inhibited breast cancer cell proliferation by blocking the cell cycle at the G0/G1 phase but not triggering cell apoptosis. In addition, F2 also showed selective cytotoxicity on breast cancer cells. It modulated the expression of proteins involved in G0/G1 phase progression, cell cycle checkpoints, DNA replication, and the TGFβ signaling pathway in MDA-MB-231 cells. This study demonstrated that F2, the medium-molecular-weight SPS component of L. sulphureus, possessed the most potent inhibitory effect on MDA-MB-231 cell proliferation by arresting the cell cycle at the G0/G1 phase. The main factors contributing to the differences in the potency of anti-breast cancer activity between F1, F2, and F3 could be the sulfate and protein contents, molecular weight, and monosaccharide composition of SPS.

Moderate regulation of wheat B-starch ratio: Improvement of molecular structure, spatial conformation, aggregation behavior of reconstituted fermented doughs and its processing suitability

Aiming to investigate the changes and effects of different particle sizes of wheat A/B starch during dough fermentation, the present study reconstituted A/B starch fractions in ratios of 100:0, 75:25, 50:50, 25:75, and 0:100, further blended with gluten and subjected to slight (20 min), medium (30 min), and high (60 min) fermentation processes by yeasts. Results showed that fermentation gas production promoted gluten network extension, inducing starch granule exposure and dough surface roughness. Also, fermentation fractured protein intermolecular disulfide bonds and decreased α-helix and β-folded structure content, contributing to GMP, LPP, and SPP content decreases. Moreover, moderately increasing the B-starch ratio in the dough can improve gluten network stability, continuity, and air-holding capacity. The 25A-75B steam bread exhibited optimal processing suitability (better morphology, texture, and quality) due to its higher GMP and polymer protein content with lower free sulfhydryl and monomeric protein content. Further, conformational relationships indicated the key indicators influencing dough products' properties were free sulfhydryl content, GMP content, protein molecular weight distribution, and secondary structure. The obtained findings contributed to understanding the effect of wheat starch granule size distribution on dough processing behavior, and future targeted breeding for wheat cultivars with high B-starch content for improved fermentation pasta product qualities.

Physicochemical characteristics and protein profile of oviductal and uterine fluids from domestic sheep

Oviductal (OF) and uterine (UF) fluids are a complex mixture of ions and macromolecules dissolved in water, derived from the secretions of secretory cells and transudates of the circulatory system. Through proteomics, OF and UF have been analyzed in different domestic species throughout the estrous cycle or during the first days of pregnancy. Therefore, the aim of this study was to evaluate the volume, osmolarity, concentration and distribution pattern of proteins, as well as the identification of OVGP1, HSP70 and ezrin proteins for their importance in reproductive physiology, in OF and UF from adult criollo type domestic sheep during the early luteal phase of the estrous cycle. An average of 3.2±1.5 uL OF and 17±0.5 uL UF per reproductive system were obtained; osmolarity was 343±20.8 mOsm kg-1 and 280±96.2 mOsm kg-1 and protein concentration was 71.9±23.8 g L-1 and 21.8±1.1 g L-1, respectively. In the protein distribution pattern, 20 bands were observed in the OF and 14 bands in the UF. Of these, 14 and 8 were specific for OF and UF, respectively, and 6 were common for both. The spectra of the protein molecular weights were 24–324 and 29–353 kDa for OF and UF, respectively. The presence of OVPG1, HSP70 and ezrin proteins in both fluids was identified, being in greater quantity in the OF (P &lt; 0.0005). The volume recovered from the UF was five times greater than that of the OF. Both osmolarity and protein concentration were higher in OF than in UF (1.2 and 3 times higher). The pattern of protein distribution between the OF and UF was different, being more complex in the OF. OVGP1, HSP70 and ezrin were identified in the OF and UF, and were found in greater quantities in the OF.

Antibody fragments targeting the extracellular domain of follicular stimulating hormone receptor for contraception in male dogs and cats

The increased LH levels resulting from the absence of negative feedback after castration has been linked to long-term health issues. A need exists for an alternative contraceptive agent that functions without interfering the LH pathways. This study aimed to develop antibody fragments against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and evaluate its effects on Sertoli cell functions. Phage clones against the extracellular domain of dog and cat FSHr selected from an antibody fragment phagemid library were analyzed for binding kinetics by surface plasmon resonance. Sertoli cells were isolated from testes of adult animals (five dogs and five cats). Efficacy test was performed by treating Sertoli cell cultures (SCCs) with anti-FSHr antibody fragments compared with untreated in triplicates. Expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB) and vascular endothelial growth factor A (VEGFA) mRNA in SCCs were quantified by RT-qPCR. The results demonstrated that the molecular weight of the purified dog and cat anti-FSHr antibody fragment was 25 kDa and 15 kDa, respectively. Based on protein molecular weight, the antibody fragment of dogs and cats was therefore, so-called single-chain variable fragments (scFv) and nanobody (nb), respectively. The binding affinity with dissociation constant (KD) was 2.32 × 10−7 M and 2.83 × 10−9 M for dog and cat anti-FSHr antibody fragments, respectively. The cross-binding kinetic interactions between the dog anti-FSHr scFv and the cat ECD of FSHr could not be fitted to the curves to determine the binding kinetics. However, the cross-binding affinity KD between the cat anti-FSHr nb and the dog ECD FSHr was 1.75 × 10−4 M. The mRNA expression of ABP, IHBB and VEGFA in SCCs was less (P < 0.05) in both dogs (12.26, 4.07 and 5.11 folds, respectively) and cats (39.53, 14.07 and 20.29 folds, respectively) treated with anti-FSHr antibody fragments, indicating the Sertoli cell functions were suppressed. In conclusion, this study demonstrated the establishment of species-specific antibody fragments against FSHr in SCCs for dogs and cats. The fragment proteins illustrate potential to be developed as non-surgical contraceptive agent targeting FSHr in companion animals.

Genome-wide investigation and analysis of NAC transcription factor family in Populus tomentosa and expression analysis under salt stress.

The NAC transcription factor family is one of the largest families of TFs in plants, and members of NAC gene family play important roles in plant growth and stress response. Recent release of the haplotype-resolved genome assembly of P. tomentosa provide a platform for NAC protein genome-wide analysis. A total of 270 NAC genes were identified and a comprehensive overview of the PtoNAC gene family is presented, including gene promoter, structure and conserved motif analyses, chromosome localization and collinearity analysis, protein phylogeny, expression pattern, and interaction analysis. The results indicate that protein length, molecular weight, and theoretical isoelectric points of the NAC TF family vary, while gene structure and motif are relatively conserved. Chromosome mapping analysis showed that the P. tomentosa NAC genes are unevenly distributed on 19 chromosomes. The interchromosomal evolutionary results indicate 12 pairs of tandem and 280 segmental duplications. Segmental duplication is possibly related to amplification of P. tomentosa NAC gene family. Expression patterns of 35 PtoNAC genes from P. tomentosa subgroup were analysed under high salinity, and seven NAC genes were induced by this treatment. Promoter and protein interaction network analyses showed that PtoNAC genes are closely associated with growth, development, and abiotic and biotic stress, especially salt stress. These results provide a meaningful reference for follow-up studies of the functional characteristics of NAC genes in the mechanism of stress response and their potential roles in development of P. tomentosa.

Physicochemical properties, structural characteristics and protein digestibility of pea protein-wheat gluten composited meat analogues prepared via high-moisture extrusion

Recently, plant-based meat analogues (MAs) have received increased attention as a sustainable diet. In this study, MAs were prepared from pea protein isolate (PPI) and wheat gluten (WG) via high-moisture extrusion. The effects of different WG contents (0%, 10%, 30%, 50% and 70%) on the product characteristics of MAs were investigated. Results revealed that the addition of WG significantly reduced the hardness and chewiness of MAs, but significantly improved their springiness, brightness and whiteness. MAs containing 30% WG and 50% WG both exhibited a distinct fibrous structure in terms of microstructure, with MAs containing 30% WG having the highest fibrous degree in terms of texture properties. After the addition of WG, there was a tendency for water molecules in MAs to shift from bound water and free water to immobilized water. In addition, the results of protein solubility analysis indicated that hydrogen bonds and disulfide bonds played a major role in forming and maintaining structures within MAs. With the increase of WG content, the ordered structure content, apparent viscosity, storage modulus, loss modulus and protein molecular weight of MAs increased significantly. Finally, the addition of 30% and 50% content of WG resulted in decreased in vitro protein digestibility and essential amino acid content of MAs, but significantly improved their flavor and sensory scores. This study provides useful information for improving the quality of PPI-based MAs to meet consumer needs.

Enhancing Machine-Learning Prediction of Enzyme Catalytic Temperature Optima through Amino Acid Conservation Analysis.

Enzymes play a crucial role in various industrial production and pharmaceutical developments, serving as catalysts for numerous biochemical reactions. Determining the optimal catalytic temperature (Topt) of enzymes is crucial for optimizing reaction conditions, enhancing catalytic efficiency, and accelerating the industrial processes. However, due to the limited availability of experimentally determined Topt data and the insufficient accuracy of existing computational methods in predicting Topt, there is an urgent need for a computational approach to predict the Topt values of enzymes accurately. In this study, using phosphatase (EC 3.1.3.X) as an example, we constructed a machine learning model utilizing amino acid frequency and protein molecular weight information as features and employing the K-nearest neighbors regression algorithm to predict the Topt of enzymes. Usually, when conducting engineering for enzyme thermostability, researchers tend not to modify conserved amino acids. Therefore, we utilized this machine learning model to predict the Topt of phosphatase sequences after removing conserved amino acids. We found that the predictive model's mean coefficient of determination (R2) value increased from 0.599 to 0.755 compared to the model based on the complete sequences. Subsequently, experimental validation on 10 phosphatase enzymes with undetermined optimal catalytic temperatures shows that the predicted values of most phosphatase enzymes based on the sequence without conservative amino acids are closer to the experimental optimal catalytic temperature values. This study lays the foundation for the rapid selection of enzymes suitable for industrial conditions.

Optimal and sustainable production of tailored fish protein hydrolysates from tuna canning wastes and discarded blue whiting: Effect of protein molecular weight on chemical and bioactive properties

Thousands tons of discards of blue whiting (BW) and tuna heads (YT) by-products are generated each year in Europe. BW is the species most discarded by European fishing fleet and, in some canning factories, YT are processed for the retrieval of oil rich in omega-3, but producing a huge amount of solid remains and effluents disposal as wastes. The development of optimal and sustainable processes for both substrates is mandatory in order to reach clean solutions under the circular economy precepts. This work focused on the mathematical optimization of the production of tailored fish protein hydrolysates (FPH), from blue whiting and tuna residues, in terms of controlling average molecular weights (Mw) of proteins. For the modeling of the protein depolymerization time-course, a pseudo-mechanistic model was used, which combined a reaction mechanistic equation affected, in the kinetic parameters, by two non-lineal equations (a first-order kinetic and like-Weibull formulae). In all situations, experimental data were accurately simulated by that model achieving R2 values higher than 0.96. The validity of the experimental conditions obtained from modeling were confirmed performing productions of FPH at scale of 5 L-reactor, without pH-control in most of cases, at the different ranges of Mw selected (1–2 kDa, 2–5 kDa and 5–10 kDa). The results showed that FPH from BW with lower Mw led to a remarkable yield of production (12 % w/w of substrate), largest protein contents (77 % w/w of BW hydrolysate), greatest in vitro digestibility (>95 %), highest essential amino acid presence (43 %) and the best antioxidant (DPPH = 62 %) and antihypertensive (IC50-ACE = 80 mg/L) properties. Our results prove that the proposed procedure to produce sustainable FPH, with specific Mw characterisitics, could be extended to other fish waste substrates. Tailored FPH may have the potential to serve as valuable ingredients for functional foods and high-quality aquaculture feed.

Effect of Milk Sources on Whey Protein and Fractions of Casein

Aim: Milk from Cow, Buffalo and goat was used for the study of isolation and fractionation of casein and whey proteins. Buffalo and goat milk were subjected to physicochemical attribute studies and a detailed protein profile namely total protein, casein fractions α-, β- and κ casein and whey protein were separated by urea fractionation method. The aim of this study was to compare various casein fractions isolated from cows, Buffalo and Goat milk. The method of separating casein into its fractions is based on the solubility of the individual components in urea the solution. The separation of casein fractions was carried out in dissolving molar concentration of urea and the yield of fractions and total protein percentages were calculated. Buffalo and goat milk have comparatively higher total protein and whey protein content as compared to cow milk. Maximum whey proteins found in goat milk (20.58%) whereas cow milk had the lowest whey protein (19.29%). Among casein fractions, α fraction (16.64%) was found maximum in Buffalo milk followed by cow (14.92%) and goat milk (5.42%). The goat milk showed the highest level of β casein (17.81%) whereas, the lowest concentration of β casein was found in cow milk (9.38%). Study Design: A significant contribution to the total milk production of India comes from buffalo milk and goat milk. Despite that, Buffalo milk and goat milk are not being utilized for many products because of difference in composition of different species milk and their inherent problems associated in the production of products. Place and Duration of Study: Karnataka Veterinary Animal and Fisheries Sciences University (KVAFSU), Dairy Science College, Hebbal, Bangalore Karnataka, India Materials: The Goat milk samples were collected from Sinchana Goat and Sheep farm, Marenahalli village (Bengaluru Rural Dist) and Buffalo milk was obtained from Country Delight Pvt. Ltd., J. P. Nagar, Bengaluru, Karnataka. Cow milk used in this investigation was collected from SEDP, Dairy Science College, Hebbal, Bengalore.. All the glassware used was soaked in chromic acid solution, repeatedly washed with water, rinsed with distilled water and dried before use. For microbiological analysis dried test tubes, conical flasks, pipettes were cotton plugged and sterilized in a hot air oven. The chemicals and reagents used in this study were mainly of analytical grade procured from Prince Laboratory Company Pvt. Limited, Bangalore. The protein molecular weight markers used for the electrophoretic study were procured from Bangalore Genei Pvt. Ltd. Methodology: Standard urea fractionation method adopted by Hipp et al was used for isolation and fractionation of caseins. Skim milk was prepared by subjecting whole milk for centrifugation, the skim milk obtained was acidified using 1 % hydrochloric acid at 200 C to obtain casein precipitate. Obtained whole casein was dissolved in 3.3 M urea at pH 7.5 and adjusted to pH 4.6 which precipitates the bulk of α casein and leaving k-caseins into soluble filtrates. Again the supernatant was adjusted to pH 4.9 diluted to 1.0 M urea and warmed to 300 C precipitating the β-caeisn. Further, addition of ammonium sulphate was used to isolate k-casein from the precipitate. Results: The sources of protein had a significant influence on the total casein, casein fractions and whey protein content of different species. The highest quantity of total caseins (34.30g/l) and whey protein (8.87 g/l) were observed in buffalo milk followed by cow (28.52 g/l) and lowest was observed in goat milk (28.45 g/l). Total protein, casein and whey protein contents were greatly affected by the source of milk. Conclusion: The significant effect of source of milk on total protein, casein, its fractions and their yield was observed. highest yield of casein (54.31%) was observed in cow milk than buffalo milk (48.95 %) and lower yield was found in goat milk. Amongst the three species, highest per cent of β casein was noted in goat milk (54.05 %) followed by buffalo (36.03%) and cow milk (34.14%). Recommendation: Rasagulla will be consumed universally by all age groups globally. Thus, the consumption of Rasagulla helps in providing overall nutritional requirements. However, the study has been conducted on a pilot scale. Moreover, commercial production may necessitate huge cost on enzymatic modification of proteins and separation peptides for value addition.