Establishment of metrological parameters of the method for measuring the protein mass fraction in fish food products by the Kjeldahl method

In laboratory practice, there are many protein quantification methods, and all of them have their own advantages and disadvantages. The most common and widely used method for the protein analysis in food products, including fish, is the Kjeldahl method. However, the current standards for measurement methods for the determination of the protein content in fish food products do not provide for the use of devices that meet the modern level of technical development, and also do not contain metrological indicators that guarantee the reliability of the results obtained. The aim of the study was to substantiate the method for measuring the protein mass fraction in fish food products by the Kjeldahl method on an automatic analyzer and to establish metrological parameters. The assessment of the quality indicators of the Kjeldahl measuring method was carried out using a Kjeltec System 2300 Nitrogen Analyzer (Foss Analytical AB, Sweden) in the form of a characteristic of the measurement error and its components, which will provide results with the required accuracy.

Pulsed Nanoelectrospray Ionization Boosts Ion Signal in Whole Protein Mass Spectrometry

Electrospray ionisation (ESI) is renowned for its ability to ionise intact proteins for sensitive detection by mass spectrometry (MS). However, the use of a conventional direct current ESI voltage can result in the formation of relatively large initial droplet sizes, which can limit efficient ion desolvation and sensitivity. Here, pulsed nanoESI (nESI) MS using nanoscale emitters with inner diameters of ~250 nm is reported. In this approach, the nESI voltage is rapidly pulsed from 0 to ~1.5 kV with sub-nanosecond rise times, duty cycles from 10 to 90%, and repetition rates of 10 to 350 kHz. Using pulsed nESI, the performance of MS for the detection of intact proteins can be improved in terms of increased ion abundances and decreased noise. The absolute ion abundances and signal-to-noise levels of protonated ubiquitin, cytochrome C, myoglobin, and carbonic anhydrase II formed from standard denaturing solutions can be increased by up to 82% and 154% using an optimal repetition rate of ~200 kHz compared to conventional nESI-MS. Applying pulsed nESI-MS to a mixture of four proteins resulted in the signal for each protein increasing by up to 184% compared to the more conventional nESI-MS. For smaller ions (≤1032 m/z), the signal can also be increased by the use of high repetition rates (200–250 kHz), which is consistent with the enhanced performance depending more on general factors associated with the ESI process (e.g., smaller initial droplet sizes and reduced Coulombic repulsion in the spray plume) rather than analyte-specific effects (e.g., electrophoretic mobility). The enhanced sensitivity of pulsed nESI is anticipated to be beneficial for many different types of tandem mass spectrometry measurements.

Transcriptome-Based Identification of a Functional Fasciola hepatica Carboxylesterase B

Bioinformatics analysis of the complete transcriptome of Fasciola hepatica, identified a total of ten putative carboxylesterase transcripts, including a 3146 bp mRNA transcript coding a 2205 bp open reading frame that translates into a protein of 735 amino acids, resulting in a predicted protein mass of 83.5 kDa and a putative carboxylesterase B enzyme. The gene coding for this enzyme was found in two reported F. hepatica complete genomes stretching 23,230 bp, containing two exons of 1282 and 1864 bp, respectively, as well as a 20,084 bp intron between the exons. The enzymatic activity was experimentally assayed on F. hepatica protein extracts by SDS-PAGE zymograms using synthetic chromogenic substrates, confirming both the theoretical molecular weight and carboxylesterase enzymatic activity. Further bioinformatics predicted that this enzyme is an integral component of the cellular membrane that should be active as a 167 kDa homodimer complex and polyacrylamide gel electrophoresis (PAGE) zymograms experiments confirmed the analysis. Additional bioinformatics analysis showed that DNA sequences that code for this particular enzyme are highly conserved in other parasitic trematodes, although they are labeled hypothetical proteins.

Genetic variability of productivity traits and evaluation of exterior of Holstein cows depending on body type

The aim of the research is to study the selection and genetic parameters of the characteristics of productivity and the exterior of Holstein first-calf cows, depending on their body type. Research methods. Based on the data from the SELEX database, we researched the performance indicators and measurements of the trunk of first calving cows in the amount of 4049 heads. Based on the RENUMF90 software shell of the BLUPF90 program, we obtained digital values of the genetic variability of productivity indicators and the external structure of animals in accordance with the equation of the mixed model. Results. The most broad-bodied animals classified as airysomal type had the highest milk yield in the first lactation, and exceeded animals of the leptosomal type by 563.3 kg (p ≤ 0.001). According to the "B" system, broad-bodied cows are superior to narrow-bodied animals: stature – by 1 point (p ≤ 0.001); body depth – by 0.8 points (p ≤ 0.01); rump width – by 1.9 points (p ≤ 0.001); rump angle – by 0.9 points (p ≤ 0.01); fore udder attachment – by 1 point (p ≤ 0.01). Animals of the narrow-bodied type reliably (p ≤ 0.001) had a high genetic relationship between milk yield in 305 days of first lactation and the mass fraction of protein in milk (0.59 ± 0.005). At the same time, the leptosomal type of cows also had the greatest correlation between milk yield and mass fraction of fat (0.51 ± 0.005) (p ≤ 0.001). According to body measurements, the highest correlation coefficients were found in animals of the airysomal type – 0.24 ... 0.50. All Holstein heifers of different body types have high heritability coefficients in terms of fat mass fraction (h2 = 0.37 ... 0.49) and protein mass fraction (h2 = 0.42 ... 0.51). Scientific novelty. For the first time, studies of the genetic variability of productivity indicators and the exterior of Holstein cows, depending on their body type, were conducted.

Technology of production and quality assessment of direct-squeezed juice and concentrated elderberry growing in the Republic of Belarus

Авторами статьи впервые разработана технология производства соков прямого отжима и высокой концентрации из плодов бузины садовой и дикорастущей, собранных на территории Беларуси. Сок прямого отжима получали при помощи ферментации с последующей фильтрацией. Концентрированный сок был получен из сока прямого отжима и экстрагирования выжимок. Для максимального извлечения сока после дробления при ферментативной обработке была использована температура 50 °С в течение 240 мин, количество ферментного препарата составляло 400 см/т. Были исследованы органолептические и физико-химические показатели сока прямого отжима и сока концентрированного: массовая доля сухих веществ, aктивная кислотность (рН), массовая доля пектиновых веществ, cодержание антоцианов, белка, массовая доля органических кислот. Исследованы показатели безопасности продукции, впервые определен состав аминокислот (качество и количество) в концентрированном соке бузины. Установлено, что совокупность физико-химических показателей позволяет отнести сок прямого отжима из бузины к профилактическим напиткам. В состав сока бузины высокой концентрации входят 18 аминокислот в количестве 4,84 г/100 мл. Незаменимых аминокислот выявлено 7 в количестве 1,51 г/100 мл. Также сок суммарно содержит фенолы в соотношении 42,95 мг-экв галловой кислоты/г сухого вещества и большое количество гидролизованных танинов. При этом большинство полифенольных соединений избежали разрушения в процессе концентрации. The authors of the article developed for the first time a technological process for the production of direct-squeezed juice and concentrated juice from the fruits of garden and wild elderberry growing in the Republic of Belarus. Direct-squeezed juice was obtained by fermentation followed by filtration. Concentrated juice was obtained by processing direct-squeezed juice and extracting the marc. For maximum juice extraction after crushing during enzymatic treatment, a temperature of 50 °C was used for 240 min, the dose of the enzyme preparation was 400 cm/t. Direct-squeezed juice and concentrated juice were studied by organoleptic and physicochemical parameters: mass fraction of solids, active acidity (pH), mass fraction of pectin substances, content of anthocyanins, protein, mass fraction of organic acids. The product safety indicators were studied, the qualitative and quantitative composition of amino acids in concentrated elderberry juice was first determined. It has been established that by the combination of physicochemical parameters, direct squeezed juice from elderberry can be considered a preventive drink. The composition of concentrated elderberry juice includes 18 amino acids, in the amount of 4.84 g/100 ml. 7 essential amino acids were detected in the amount of 1.51 g/100 ml. The concentrated juice contained phenols in the quantity of 42.95 mEq gallic acid / gram dry matter and a large number of hydrolyzed tannins. Most polyphenolic compounds were preserved after the juice concentration.

Technology of enzymatic-acid hydrolysis of bone raw material in production of gelatine

Bone gelatin is an important and irreplaceable item widely used in the food industry and pharmaceutical production; it is also widely used in tissue engineering and other spheres. Due to widespread use of gelatin it is necessary to search for new safe and effective technologies for bone gelatin production. This research represents the results of enzymatic-acid hydrolysis of raw material in the process of gelatin production. The article presents the results of hydrolysis analyzes, the results of the main quality parameters of the obtained gelatin samples; and the major technological scheme for gelatin production is proposed here. As result of developed technology of enzymatic-acid hydrolysis of bone raw material with the ratio of raw material mass to the volume of solvent (HCl 1M and pepsin with an enzymatic activity of 40 units) as 1:9, duration of exposure: 180 minutes (3 hours), at the stage of demineralization, liming and de-ashing, we obtained samples of gelatin at yield rate of 12.1% from the initial mass of raw materials, which is 6.9% higher in comparison with the lowest yield of gelatin according to the proposed schemes. It is shown that the samples have a high protein mass fraction 91.4%, and a low fat mass fraction 0.4%, the obtained results indicate the high technological qualities of the obtained gelatin sample, this is also confirmed by high strength of gel according to Bloom scale, which value varies within the range of 290 ± 0.7 units.

Degradation of ribosome and chaperone proteins is attenuated during the Differentiation of Replicatively Aged C2C12 Myoblasts

Age-related impairments in myoblast differentiation may contribute to reductions in muscle function in older adults, however, the underlying proteostasis processes are not well understood. Young (P6-10) and replicatively aged (P48-50) C2C12 myoblast cultures were investigated during early (0h-24h) and late (72h-96h) stages of differentiation using deuterium oxide (D2O) labelling and mass spectrometry. The absolute dynamic profiling technique for proteomics (Proteo-ADPT) was applied to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins. Proteo-ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P<0.05, FDR <5 %) different changes in abundance between young and aged cells at early and later periods of differentiation. Young cells exhibited a steady pattern of growth, protein accretion and fusion, whereas aged cells failed to gain protein mass or undergo fusion during later differentiation. Maturation of the proteome was retarded in aged myoblasts at the onset of differentiation, but their proteome appeared to "catch up" with the young cells during the early phase of the differentiation period. However, this "catch up" process in aged cells was not accomplished by higher levels of protein synthesis. Instead, a lower level of protein degradation in aged cells was responsible for the elevated gains in protein abundance. Our novel data point to a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts and highlights dysregulation of protein degradation, particularly of ribosomal and chaperone proteins, as a key mechanism that may contribute to age-related declines in the capacity of myoblasts to undergo differentiation.

Integration of proteomic and genetic approaches to assess developmental muscle atrophy

Muscle atrophy, or a decline in muscle protein mass, is a significant problem in the aging population and in numerous disease states. Unraveling molecular signals that trigger and promote atrophy may lead to a better understanding of treatment options; however, there is no single cause of atrophy identified to date. To gain insight into this problem, we chose to investigate changes in protein profiles during muscle atrophy in Manduca sexta and Drosophila melanogaster. The use of insect models provides an interesting parallel to probe atrophic mechanisms since these organisms undergo a normal developmental atrophy process during the pupal transition stage. Leveraging the inherent advantages of each model organism, we first defined protein signature changes during Manduca intersegmental muscle (ISM) atrophy and then used genetic approaches to confirm their functional importance in the Drosophila dorsal internal oblique muscles (DIOMs). Our data reveal an upregulation of proteasome and peptidase components and a general downregulation of proteins that regulate actin filament formation. Surprisingly, thick filament proteins that comprise the A band are increased in abundance, providing support for the ordered destruction of myofibrillar components during developmental atrophy. We also uncover the actin filament regulator Ciboulot (Cib) as a novel regulator of muscle atrophy. These insights provide a framework towards a better understanding of global changes that occur during atrophy and may lead to eventual therapeutic targets.

EXPRESS: On the Limit of Detection in Infrared Spectroscopic Imaging

Infrared (IR) spectroscopic imaging instruments’ performance can be characterized and optimized by an analysis of their limit of detection (LoD). Here we report a systematic analysis of the LoD for Fourier transform IR (FT-IR) and discrete frequency IR (DFIR) imaging spectrometers. In addition to traditional measurements of sample and blank data, we propose a decision theory perspective to pose the determination of LoD as a binary classification problem under different assumptions of noise uniformity and correlation. We also examine three spectral analysis approaches, namely absorbance at a single frequency, sum of absorbance over selected frequencies and total spectral distance – to suit instruments that acquire discrete or contiguous spectral bandwidths. The analysis is validated by refining the fabrication of a bovine serum albumin protein microarray to provide eight uniform spots from 2.8 nL of solution for each concentration over a wide range (0.05 -10 mg/mL). Using scanning parameters that are typical for each instrument, we estimate a LoD of 0.16 mg/mL and 0.12 mg/mL for widefield and line scanning FT-IR imaging systems, respectively, usingthespectraldistanceapproach,and0.22mg/mLand0.15mg/mL using an optimal set of discrete frequencies. As expected, averaging and the use of post-processing techniques such as minimum noise fraction (MNF) transformation results in LoDs as low as 0.075 mg/mL that correspond to a spotted protein mass of 112 fg/pixel. We emphasize that these measurements were conducted at typical imaging parameters for each instrument and can be improved using the usual trading rules of IR spectroscopy. This systematic analysis and methodology for determining the LoD can allow for quantitative measures of confidence in imaging an analyte’s concentration and a basis for further improving IR imaging technology.