SDS-PAGE Profiles Research Articles

The Application of SDS-PAGE Protein and HPTLC Amino Acid Profiling for Verification of Declared Variety and Geographical Origin of Honey

Proteins and amino acids are minor components of honey that are rarely used for its quality evaluation, although these components create its biological activity as well as can serve for overheating detection. The aim of the study was to use these indicators to confirm a declared on the label variety. Fifty-eight honey samples of 8 different varieties meeting the commercial quality requirements were used, including 28 local Polish and 30 commercial (mainly imported from EU and non-EU) honeys. For honey protein profiling previously used, polyacrylamide electrophoresis in denaturing conditions (SDS-PAGE) was applied whereas the free amino acid profile was analyzed by high-performance thin-layer chromatography (HPTLC) for the first time. As auxiliary indicators, the colorimetric determination of the protein content by the Bradford method, the activity of 5 glycolytic enzymes, including diastase, β-galactosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-glucosidase, and the content of proline were used. It has been shown that based on the determined model protein SDS-PAGE profiles for selected monofloral honeys, it is possible to detect honeys of questionable variety based on the lack of specific protein bands or their diverging intensity. The HPTLC amino acid analysis can serve as a supporting control tool, capturing differences in the amino acid profile. Due to a great variation of multifloral honey, such assays are effective for monofloral honey only. The colorimetric assays, especially for total protein and β-galactosidase, can be also useful. The applied tools can be proposed for the initial verification of honey variety for cost reduction of officially recognized melissopalynological analysis.

Antibacterial Activity From Goat’s Milk Whey Hydrolized by Protease Enzyme of Bacillus Lincheniformis

Food additives had been used to prevent and inhibit the growth of microorganisms and extend shelf life of food. Based on the safety of their used, it has an impact such as microorganism resistance to synthetic antimicrobials. This encourages the development of antimicrobials derived from natural ingredients, especially from milk. The bioactive components of milk protein have not always existed in their natural state, including precursors or peptides that will only be active if they have been hydrolyzed in vitro from their natural proteins. Hydrolysis of milk protein can be carried out using proteolytic enzymes from various sources, including protease extracted from Bacillus lincheniformis. This study used different concentrations of the protease extracted from Bacillus lincheniformis (0.5%, 1% and 1.5%) to produce hydrolyzate which has antibacterial activity in terms of protein content, SDS PAGE profile and the size of the inhibition zone (disc method). The results showed that the best activity of the whey hydrolyzed on Gram-negative Escherichia coli was 1% concentration of protease enzyme. In comparison, Gram positive Staphylococcus aureus can be inhibited with 0.5% concentration of protease enzyme.

Impacts of Electroextraction Using the Pulsed Electric Field on Properties of Rice Bran Protein.

The pulsed electric field (PEF) was applied to improve the extraction yield and properties of rice bran proteins from two rice varieties ("Kum Chao Mor Chor 107" and "Kum Doi Saket"). As compared to the conventional alkaline extraction, PEF treatment at 2.3 kV for 25 min increased the protein extraction efficiency by 20.71-22.8% (p < 0.05). The molecular weight distribution detected by SDS-PAGE and amino acid profiles of extracted rice bran proteins was likely unchanged. The PEF treatment influenced changes in the secondary structures of rice bran proteins, especially from the β-turn to the β-sheet structure. Functional properties of rice bran protein including oil holding capacity and emulsifying properties were significantly improved by PEF treatments by about 20.29-22.64% and 3.3-12.0% (p < 0.05), respectively. Foaming ability and foam stability increased by 1.8- to 2.9-fold. Moreover, the in vitro digestibility of protein was also enhanced, which was consistent with the increment of DPPH and ABTS radical-scavenging activities of peptides generated under in vitro gastrointestinal digestion (37.84-40.45% and 28.46-37.86%, respectively). In conclusion, the PEF process could be a novel technique for assisting the extraction and modification of the protein's digestibility and functional properties.

Effect of food matrix and fermentation on angiotensin-converting enzyme inhibitory activity and β-glucan release after in vitro digestion in oat-based products

The aim of this study was to determine the effects of fermentation and food matrix on the ACE inhibitory activities of the peptides obtained after in vitro gastrointestinal digestion, protein profiles (SDS-PAGE) and β-glucan amounts of oat products. Furthermore, the physicochemical and microbiological properties of fermented oat drinks and oat yogurt-like product obtained from oat fermentation were evaluated. Oat grains were mixed with a certain ratio of water 1:3 w/v (oat:water, yogurt consistency) and 1:5 w/v (oat:water, drink consistency), and this mixture was fermented with yogurt culture and probiotic Lactobacillus plantarum and fermented drinks and yogurt were produced. The results indicated that the fermented oat drink and the oat yogurt-like product had L. plantarum viability over 107 cfu/g. After the in vitro gastrointestinal digestion of the samples, the hydrolysis levels ranged from 57.70 % to 82.06 %.The hydrolysis level of the samples with fermented-drink consistency was significantly higher than the samples with yogurt consistency (p < 0.05).The SDS-PAGE profiles of the non-digested samples showed that the bands had molecular weights of 12–15 kDa and around 35 kDa. Bands whose molecular weights were around 35 kDA disappeared after gastric digestion. ACE inhibitory activities of the fractions composed of molecular weights of 2 kDa and 2–5 kDa obtained after in vitro gastrointestinal digestion of the oat samples were in the range of 46.93–65.91 %. The effect of fermentation on the ACE inhibitory activities of the peptide mixture with molecular weights between 2 and 5 kDa was not statistically significant, however, fermentation caused an increase in the ACE inhibitory activities of the peptide mixture with a molecular weight<2 kDa (p < 0.05). The β-glucan amounts of fermented and non-fermented oat products were in the range of 0.57–1.28 %. The β-glucan amounts detected after gastric digestion decreased considerably and β-glucan could not be detected in the supernatant after gastrointestinal digestion. This indicated that β-glucan did not solubilize in the supernatant (bioaccessible) and remained in the pellet. In conclusion, fermentation is a valuable process for releasing peptides with moderately high ACE inhibitory effects from the parent oat proteins.

Characterization of the recombinant GH10 xylanase from Trichoderma orientalis EU7-22 and its synergistic hydrolysis of bamboo hemicellulose with α-glucuronidase and α-L-arabinofuranosidase

Here the recombinant GH10 xylanase (HoXyn10) was investigated for biochemical characteristics and synergistic effect with α-glucuronidase (AnGus67) and α-L-arabinofuranosidase (AnAxh62A) on extracted Maso bamboo hemicellulose (BCH) to produce xylooligosaccharides (XOS). The GH10 endo-β-1, 4-xylanase from Trichoderma orientalis EU7–22 is heterologously expressed in Pichia pastoris GS115. The crude HoXyn10 showed three bands on the SDS-PAGE profile and one of the proteins was N-glycosylated. The optimum pH and temperature of HoXyn10 were pH= 6.0 and 65 °C, respectively. It showed a wide range of pH stability and remained more than 80% of optimal activity at pH4.5–8.0 after holding at 40 °C for 60 min. HoXyn10 was highly stable at 50 °C without substrate and the residue xylanase activities were retained 97% and 83% after incubating for 2 h and 18 h, respectively. HoXyn10 displayed high activities and almost the same catalytic efficiency to oat-spelt xylan (OSX), Beechwood xylan (BWX) and wheat arabinoxylan (WAX). Notably, the HoXyn10 exhibited apparent activity on Avicel, but showed no activity on CMC-Na. The hydrolytic products from oligosaccharides (X3-X6) by HoXyn10 were all xylose (X1) and xylobiose (X2). The results from FT-IR and NMR showed the backbone of extracted BCH was (1→4)-linked β-D-xylopyranose and the main side chains were α-L-arabinofuranose residues and 4-O-methyl-D-glucuronic acid residues. Based on the characterized structure, when BCH was synergistically hydrolyzed with HoXyn10, AnGus67 and AnAxh62A using appropriate strategy, the yield of xylooligosaccharide (XOS) and xylose were 50.3% and 14.7%. The results indicated HoXyn10 might be a promising tool for various hemicellulose hydrolysis and Maso bamboo hemicellulose is a promising source for XOS production.

Isolation, Characterization, and Effect on Biofilm Formation of Bacteriocin Produced by Lactococcus lactis F01 Isolated from Cyprinus carpio and Application for Biopreservation of Fish Sausage.

The aim of this work was the screening of bacteriocin-producing LABs isolated from fish, the selection of promising/prominent strain(s), the characterization of the bacteriocin produced, and the evaluation of its potential to be used as biopreservative(s). Amplification and sequencing of the 16S rRNA gene of the bacteriocin-producing strain was performed. Then a partial purification of the produced bacteriocin, using a combination of ammonium sulfate and chloroform-methanol precipitation, was done. Its molecular weight was determined by SDS-PAGE. In addition, the action spectrum, the hemolysis test, and its ability to inhibit biofilm formation were analyzed. A total of 88 isolates of lactic acid bacteria (LAB) including one bacteriocin producer, which was identified as Lactococcus lactis F01, were collected. The bacteriocin was partially purified with an estimated yield of 40%. Regarding the SDS-PAGE profile, the secreted bacteriocin has molecular weight of about 3.5 kDa and was identified as class I bacteriocin. The antimicrobial test showed that the bacteriocin inhibits pathogenic and/or spoilage bacteria, 10 Gram-positive and 16 Gram-negative bacterial species. Moreover, it can inhibit biofilm formation from 1.3% (Escherichia coli) to 63.92% (Pseudomonas aeruginosa ATCC15692) depending on the strain. The hemolytic activity of novel bacteriocin was observed at the concentration of 10 μg/ml of bacteriocin crude extract, which was 0.7 ± 0.0029%. In addition, it exhibited good thermal and pH stability with retained antibacterial activity of 85.25% after treatment at 121°C for 20 min, as well as at a pH range between 2.0 and 10.0. Moreover, this bacteriocin showed the ability to inhibit the growth of bacterial culture load in fish sausage stored at 8°C for 28 days. Considering the results obtained, bacteriocin could be potentially exploited as an alternative to chemical preservatives or as a substitute for antibiotics.

Sterilising effect of high power pulse microwave on Listeria monocytogenes

In the present work, Listeria monocytogenes was used as the target strain to investigate the sterilising potential and mechanism of high power pulse microwave (HPPM). Results showed that the inactivation was positively correlated with the pulse frequencies and operating times. The count of Listeria monocytogenes was decreased by 5.09 log CFU/mL under 200 Hz for 9 min, which was used as the optimised condition to further explore the sterilisation mechanism. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images showed that the L. monocytogenes cells of untreated group presented intact surfaces, clear boundary, and its intracellular contents distributed uniformly in the cytoplasm. Following treatment, the cell wall surfaces began to deform in small areas, and cell membranes were severely ruptured, thus resulting in the appearance of electron transmission areas. Extracellular protein and nucleic acid contents, represented by OD260 nm and OD280 nm, increased with the increase in operating time significantly. After treatment, SDS-PAGE profiles of whole-cell proteins displayed that the protein bands became lighter or even disappeared. Na+ K+-ATPase activities and intracellular ATP content decreased by 72.97 and 79.09%, respectively. This was consistent with the cell viability of L. monocytogenes observed by confocal laser scanning microscopy. Overall, the sterilisation mechanism of HPPM on L. monocytogenes may be caused by membrane damage, intracellular component leakage, and energy metabolism hindrance.

Effects of Different Acetic Acid Immersion Time on the Properties of Collagen from Pangasius Skin

Highlight Research Experimental use of pre-treated pangasius skin as collagen raw material using different acetic acid immersion time Effects of immersion time on the yield of collagen from pangasius skin Effects of immersion time on the proximate content of collagen from pangasius skin Presenting SDS-Page profile of collagen from pangasius skin Abstract Pangasius sp. production in Indonesia has reached 384,310 tons in 2019. One of the main waste in pangasius fillet industry is the skin, which reached to 6% of body mass. Fish skins are alternative for making collagen because it has no restrictions for certain religions and ethnicities. Collagen is a protein biomaterial which acts as the main component of connective tissue. Extracting collagen using organic acids such as acetic acid is the most common extraction method. Herein, we report the effects of different immersion time of acetic acid to collagen properties from pangasius skin. In this study, pangasius skin was treated using 10% alcohol with the ratio 1:10 (w/v) for defatting and 0.1M NaOH with the ratio 1:10 (w/v) for eliminating non-collagenous protein. The immersion of 0.5M acetic acid was carried out on pangasius skin with the ratio of 1:20 (w/v), for 24, 48, and 72h at temperature less than 15oC. The sample was salted-out for 24 hours with NaCl until the concentration reached 2.5M, then the sample was freeze-dried at -40oC. This study investigated that longer immersion time affected the properties of pangasius skin collagen. Different immersion time significantly affected the yield of collagen from pangasius skin (p&lt;0.05). Pangasius skin has a potential to be used as collagen raw material, and 72h of immersion time (P3) is suggested to produce pangasius skin collagen with the highest yield and protein content (6.15 % and 9.26 %). Production of collagen from the fish skin will contribute to increase pangasius waste valorization in pharmaceutical industry.

Optimization of ultrasound‐assisted protein extraction from defatted mustard meal and determination of its physical, structural, and functional properties

Protein isolates were prepared from the defatted mustard meal (DMM) by ultrasound-assisted alkaline extraction and precipitation at the isoelectric point (pH 4.5). Response surface methodology was used to determine optimum conditions for the extraction of protein from the DMM. Box–Behnken design includes independent variables such as pH (8, 9.5, and 11), particle size (150, 375, and 600 μm), ultrasonic amplitude (50%, 70%, and 90%), and ultrasound treatment time (8, 10, and 12 min), which were used to get the optimal conditions for highest protein yield (dependent variable). Maximum yield (44.87%) was obtained when pH, particle size, amplitude, and time were kept at 11, 375 μm, 90%, and 10 min, respectively. Functional properties were studied for the protein isolate obtained at optimal conditions with the highest yield. Results showed that ultrasound pretreatment increased the protein yield as compared to conventional methods. Protein isolate obtained by ultrasound assessment had comparable functional properties to those produced by traditional methods. SDS-PAGE profile of proteins does not show any change in the molecular weight. XRD analysis showed that the difference between the particle distance of protein obtained with and without ultrasound-assisted extraction was very small. The protein content (dry basis), water holding capacity, oil holding capacity, emulsifying activity, foaming capacity, and foam stability were 93.33 ± 0.57%, 0.68 ± 0.01 g/g, 1.86 ± 0.05 g/g, 63.16 ± 0.25%, 58 ± 0.47%, and 63 ± 0.84%, respectively. Practical applications Mustard seed is used for edible oil production and the meal is its by-product. The meal contains a good amount of protein, which can be utilized in two ways: for nutrition and as a functional ingredient in various food products. The use of ultrasound-assisted extraction increases the yield as well as modifies the functional properties. This process can be used practically to add the value of the mustard meal.

Modulating molecular interactions in pea protein to improve its functional properties

Proteins exist in numerous spatial arrangements and are stabilized by various inter- and intra-molecular forces. The objectives of this study were to investigate the functional properties of pea protein isolate in terms of water/oil holding capacity, emulsifying and foaming properties, solubility, and gelation by modulating protein covalent and non-covalent interactions with specific denaturants/modifiers and to understand the physicochemical characteristics (e.g., free amino group content, free sulfhydryl group content, surface hydrophobicity, SDS-PAGE profile, secondary structures) of the unfolded pea proteins that are responsible for the functional changes. All the denatured proteins possessed significantly increased solubility. Both urea and SDS unfolded proteins had significantly higher water holding capacity and oil holding capacity of up to 5.01 and 5.09 g H 2 O/g, and 3.06 and 2.84 g oil/g compared with the control pea protein (4.12 g H 2 O/g and 1.29 g oil/g), respectively. The proteins unfolded with urea or SDS also showed improved emulsification properties. The trypsin hydrolyzed protein exhibited the highest foaming capacity and better gelation properties among all the treatments. Principal component analysis indicated strong associations between protein functional and physicochemical properties and molecular interactions. This study provides useful fundamental knowledge in tailoring protein structures in order to enhance its functionalities and broaden the food applications. • Both urea and SDS unfolded pea proteins had improved water/oil holding capacity. • Trypsin hydrolyzed protein had improved foaming and gelation properties. • All the denatured proteins possessed increased solubility. • Proteins unfolded with urea or SDS showed improved emulsifying properties. • The study provides knowledge in tailoring protein structure for improved functionality.

Effects of Different Heat Processing on Molecular Weight and Allergenicity Profile of White Shrimp (Litopenaeus vannamei) and Mud Crab (Scylla serrata) from Indonesian Waters

Shrimp and crab are fishery products with high nutritional value, especially as protein sources. However, they belong to the crustacean group known to cause many allergies in Southeast Asian countries. Not only affecting the nutritional composition, processing also has the potential in reducing the allergenicity of a food ingredient. This study aims to analyze the effects of various heat processing on nutrient composition, soluble protein content, protein molecular weight profile, and allergenicity of white shrimp and mud crab. The processing was carried out by boiling, frying, and autoclaving. Changes in macronutrient content were determined using proximate analysis. Soluble protein content was analyzed using the Bradford method, while molecular weight and allergenicity profiles were analyzed using SDS-PAGE electrophoresis and ELISA techniques. Results showed that processing treatment could significantly reduce protein solubility. SDS-PAGE profile showed that heating shrimp and crab using autoclave at 121.1 oC and pressure of 0.2 MPa for 10 min was able to remove protein bands of 35-38 kDa, which were suspected as the allergen bands. However, boiling (100 oC for 10 min) and deep frying in palm oil (160 oC for 10 min) treatments of the samples gave stronger 35-38 kDa bands intensity than the unprocessed sample. Autoclaving was able to significantly reduce IgE reactivity to shrimp extract better than the boiling and frying process. In contrast, IgE reactivity of crab extract was increased by heat processing especially frying. Thus, autoclaving can be used in the white shrimp processing to produce hypoallergenic food.

Biochemical and Microstructural Properties of Lizardfish (Saurida tumbil) Scale Collagen Extracted with Various Organic Acids.

The purpose of this research was to extract collagen from the scales of lizardfish (Saurida tumbil) using various acids. Acetic acid-extracted collagen (AScC) produced a higher yield (1.8 mg/g) than lactic acid-extracted collagen (LScC) and citric acid-extracted collagen (CScC) although not significantly different (p > 0.05). All extracted collagens were categorized as type I collagens with the presence of alpha chains (α1 and α2) based on the SDS-PAGE profiles. The triple-helical structure of the collagen was maintained in the AScC, LScC, and CScC as confirmed by the FTIR spectra. The UV-vis and X-ray diffraction spectra observed in all collagens were in agreement with previous work on fish scale and calfskin (commercial) collagens. The thermal stability of AScC (Tmax = 31.61 °C) was greater than LScC (Tmax = 30.86 °C) and CScC (Tmax = 30.88 °C). The microstructure of acid-extracted collagens was characterized as complex, fibrous, and multilayered, with irregular sheet-like structures. All samples were highly soluble in acidic pH (1.0–4.0) and in low concentrations of NaCl (0–20 g/L). In conclusion, the lizardfish scale collagen, particularly AScC, may be used as an alternative to terrestrial animal collagen.

Antioxidant potential and amino acid profile of ultrafiltration derived peptide fractions of spent hen meat protein hydrolysate.

Spent hen meat is considered as a category of waste generated by the poultry sector which can lead to serious environmental concerns if not disposed and utilized properly. In this work, spent hen meat was hydrolysed by 2% Flavourzyme (6.5 pH, 55°C) followed by ultrafiltration to produce three peptide fractions with molecular weights > 10kDa, 5-10kDa and < 5kDa. These fractions were evaluated for antioxidant potential, SDS PAGE and amino acid profile. The SDS PAGE profile demonstrated bands in the low molecular weight (< 10kDa) region. Peptide fractions of < 5kDa exhibited highest antioxidant activity and, essential as well as hydrophobic amino acid composition than whole hydrolysate and other peptide fractions. Incorporation of the identified hydrolysate fraction in food could improve its shelf stability while serving as a preventive component against human degenerative diseases.

Effect of gastrointestinal alterations mimicking elderly conditions on in vitro digestion of meat and soy proteins

Among the physiological functions declining with aging, decreased secretion of gastric fluid, achlorhydria, is commonly observed. We evaluated the digestion of meat (chicken, beef, and pork) and soy proteins using in vitro conditions mimicking gastrointestinal (GI) digestion in adults (control, C) and elderly individuals with achlorhydria (EA). Changes in degrees of hydrolysis (DH), SDS-PAGE profiles, peptide concentrations, and proteomic profiles during digestion were investigated. Digestion under the EA conditions markedly decreased DH, especially for soy proteins. SDS-PAGE profiling and proteomics showed that myofibrillar/sarcoplasmic proteins from meat and glycinin/beta-conglycinin from soy were most affected by digestion conditions. Our results indicated that differences in the digestibility of meat protein between EA and control conditions gradually narrowed from the gastric to the intestinal phase for meat protein, while a pronounced difference persisted in the intestinal phase for soy protein. Our work provides new insight of value for future dietary recommendations for elderly individuals.

Molecular characterization of a 42 kDa subunit pili protein of Salmonella typhi causes typhoid fever

Abstract. Darmawati S, Ethica SN, Prastiyanto ME, Depamede SN, Putri EO, Kamaruddin M. 2022. Molecular characterization of a 42 kDa subunit pili protein of Salmonella typhi causes typhoid fever. Biodiversitas 23: 962-968. Blood culture is the gold standard for diagnosing typhoid fever, but it has limitations such as media and laboratory equipment, specimen volume, and examination time. However, the Academy of Pediatrics does not recommend serology due to its low sensitivity.The purpose of this study was to determine the molecular properties of the protein pilli of Salmonella typhi(S. typhi) that the findings can be used to develop a typhoid fever diagnostic reagent. The SDS-PAGE method was used, as well sequence analysis with ProtParam, ProtScale, and PSIPRED. The SDS-PAGE profile reveals one major protein (42 kDa) and fourteen minor proteins. The pili protein subunit 42 kDa had an amino acid (AA) sequence with a length of 390 AA, according to bioinformatics analysis. According to the ProtParam results, the pili protein subunit 42 kDa has good stability with a value of 40 and is a hydrophilic protein with an average GRAVY value of -0.950. PSIPRED results show that among the secondary structural elements, coil strand predominates, followed by -helix and -strand. It is concluded that this protein is immunogenic and that it can be used to develop a more specific and sensitive diagnostic reagent for typhoid fever.

Alterações das propriedades da gelatina da pele do bagre tra (Pangasianodon hypophthalmus) pela substituição alcalina por enzima no processo pré-tratado

ABSTRACT: The effect of pretreated method to remove the non-collagenous protein by using alkaline and enzyme Alcalase, as well as the temperature and time for extracting on the properties of gelatin from tra catfish skin were investigated. Yields of gelatin extracted at 70 °C for 1h from pretreated skin by enzyme method (16.2%) was significantly higher than that of the sample by alkaline method (12.14%). However, the gel strength of gelatin from skin treated via enzyme Alcalase was lower than gelatin sample pretreated by alkaline while the turbidity values was higher than gelatin from skin pretreated via alkaline. From SDS-PAGE profile, gelatin from skin pretreated by alkaline consisted of two different α- chains in protein pattern while enzymatic gelatin had low molecular weight peptides. The FT-IR spectra showed the lower wavenumber in amide I and III of enzymatic gelatin in compare to alkaline gelatin by the loss of triple helical structure during enzyme treatment. From the results, the using enzyme for pretreated material has potential to replace the alkaline method for gelatin production with purpose to reduce chemical waste caused serious ecological issues.

Evaluation of Proprietary MDZenPro Formulation by Zenherb Labs in Mediating Protein Digestion under INFOGEST in-vitro Simulated Gastrointestinal Conditions

Protein breakdown by endogenous enzymes in the gastrointestinal (GI) tract results in generation of peptides and amino acids that act as building blocks in essential biological functions. Exogenous proteases possess immense potential as digestive enzyme supplements that can assist protein digestion in the GI system. Plant proteases, in addition to their promising activity, are considered to be safe in nature. The present study evaluated the potential application of a plant protease based proprietary formulation - MDZenPro in digesting raw whey protein, whey protein isolate and plant protein under INFOGEST simulated GI conditions. The gastric and GI digested protein products were analyzed for determining the degree of hydrolysis. The protein profiles were evaluated using SDS-PAGE. The results of the degree of hydrolysis study revealed that MDZenPro facilitated gastric and GI digestion of proteins. This increase in degree of hydrolysis was noted to be higher than that observed in proteins that were not treated with MDZenPro. The SDS-PAGE profile further supported these findings wherein, the MDZenPro treated protein samples displayed low molecular weight fragmented peptides in contrast to the profile of undigested proteins. The present study thus highlights the promising application of ‘MDZenPro’ as an effective supplement for protein digestion.

Association of bull semen protein estimates and SDS-PAGE profiles on semen freezability

It is vital to identify the ejaculate with good freezability by determining the biochemical makeup of the ejaculate at the pre-freeze stage. The present study targeted to assess the use of the protein estimates and profiles at the pre-freeze stage as markers of freezability in Frieswal populations. Storing the proteins for proteomic studies is always tricky in the case of animal studies, where accessibility to liquid nitrogen is limited. Hence alternative storing approaches need to be optimized. The second part of this study examined the protein concentration and protein profiles of RNALater and frozen stored sperm cells to assess the use of RNALater preservation in sperm proteomic studies. Sperm and seminal plasma protein concentrations were quantified using Bradford assay, and total protein quantities were derived. The seminal plasma and sperm protein profiles were generated with SDS-PAGE. The protein estimates and SDS-PAGE profiles of good and poor freeze-groups were similar. Also, sperm and seminal plasma protein concentration were not correlated with the semen volume and sperm count. Even though the yield was comparatively less, the protein profiles of sperm preserved by RNALater were similar to that of frozen sperms. The present study results indicate that the protein estimates and qualitative profiles of sperm and seminal plasma proteins may not be sufficient to reveal the differences in the proteome of good and poor freezable bulls at the macro level. Hence, the protein estimates and profiles of neat semen may not be helpful for the prediction of freezability at the pre-freeze stage. Secondly, this study indicates that RNALater preservation helps store sperms for proteome analysis studies.

Proteomic analysis of oocytes retrieved from in vitro cultured ovine small antral follicles and large antral follicles cultured with amphiregulin, neuregulin-1 and tumour necrosis factor-a

Protein components change markedly during follicular development in the ovary. In this study, a proteomics approach was used to evaluate changes in proteins during different size follicles viz small antral follicles (SAFs) and large antral follicles (LAFs) culture exposed with different growth factors. The optimum and elevated doses of amphiregulin (AREG) or neuregulin (NRG-I) or tumour necrosis factor-α (TNF-α) were used for culture of SAFs and LAFs and then oocytes were retrieved for analysis of secretory protein by SDS-PAGE. SAFs were cultured in media containing 150 ng of AREG, NRG1 and TNF-α and LAFs in media containing 50 ng of AREG, NRG1 and TNF-α. The average yield of protein from oocytes retrieved from SAFs when cultured with 150 ng of AREG, NRG-1 and TNF-α was 0.0012, 0.0016 and 0.0010 mg/ml respectively. When cultured with oocytes retrieved from LAFs in 50 ng AREG, 50 ng NRG-I and 50 ng TNF-α, the corresponding values were 0.0026, 0.0021 and 0.0014 mg/ml respectively. The SDS-PAGE profile of the oocytes retrieved from culturing of SAFs and LAFs in different doses of AREG, NRG-I and TNF-α revealed no peptide bands.

Selected Quality Attributes of Freshwater Mussel Powder as a Promising Ingredient for Pet Food.

Simple SummaryBivalves such as clams, mussels, and oysters provide a good source of protein, glycogen, and minerals. The high-value compounds and bioactive properties of these organisms are well known and confirmed by numerous scientific studies. During the last two decades, the freshwater mussel Sinanodonta woodiana as one of the most invasive species, has penetrated into Europe along with fish shipments from Asia. Numerous studies indicate that both the soft tissues and mussel shells of this low-grade species may constitute promising biomaterial with potential processing ability. Due to growing interest in new raw materials having been observed recently in the pet food market, this study aimed to obtain and evaluate selected quality attributes of freshwater pond mussel powder. After initial ecotoxicity testing in vitro, powdered mussel tissue was evaluated with respect to its basic physicochemical and functional properties, such as solubility, emulsifying capacity, and gel-forming ability. The study also provides novel information about electrophoretic protein profile and amino acid composition, including taurine content. Based on the results obtained in this study, it can be concluded that tissues of mussels available locally from lakes and ponds can be proposed as an interesting source of new ingredients for developing formulated pet food products.The aim of this study was to describe the quality attributes of a freeze-dried preparation obtained from freshwater mussel Sinanodonta woodiana (SW) soft tissue in respect to its potential as a novel pet food ingredient. After ecotoxicological testing of the raw material with MARA (Microbial Assay for Risk Assessment), the basic physico-chemical properties of the powder, such as approximate composition, bulk density, color parameters, water activity, electrophoretic analysis (SDS-PAGE), solubility, gelling and emulsifying capacity, were analyzed. The powder with a water activity of 0.43 offers a toxically safe preparation that contains over 34% protein/100 g of dry matter (DM). The SDS-PAGE profile showed twelve protein bands with a molecular weight (MW) ranging from >250 to 10 kDa. Taurine content has been estimated at an essential amount above 150 mg/100 g of DM. The powder possessed desirable emulsifying properties with 230 mL per 1 g and demonstrated the ability to form a firmer gel with a strength of 152.9 g at a temperature above 80 °C with at least 10% protein content. The L*, a*, and b* values characterizing powder color were found to be 69.49, 16.33, and 3.86, respectively. The SW mussel powder seems to be a promising ingredient that can be added with other binding or gelling agents in order to improve both the taste and acceptance of the final pet food products.