Content and biological value of protein in grain of spring barley accessions

Цель и задачи исследования . Целью исследования было выделение генотипов с высоким содержанием белка в зерне как материала для селекционных исследований по созданию сортов ячменя ярового пищевого направления использования. Для этого было исследовано изменчивость содержания белка у различных образцов ячменя – пленчатых и голозерных с обычным и waxy крахмалом. С целью определения ценности белка была определена его переваримость. Материалы и методы. Исследования проводили в Институте растениеводства им. В.Я. Юрьева НААН в 2015–2017 гг. Исходным материалом были 70 линий и сортов ячменя ярового различного эколого-географического происхождения. Сорта и линии выращивали в опытах сортоиспытания, площадь делянки 10 м 2 . Существенность различий между вариантами определяли с помощью дисперсионного анализа, апостериорное сравнение – по Homogenous groups (Fisher LSD) по программе STATISTICA 10. Содержание белка в зерне определяли на ИнфраЛЮМ ФТ-10М 09495. Переваримость белка определяли в лаборатории медико-биологических проблем технологии пищевых продуктов на кафедре химии, микробиологии и питания Государственного университета питания и торговли на основе методики О. Покровского и И. Эртанова. Обсуждение результатов. Установлено, что в условиях 2015–2017 гг. содержание белка в зерне образцов ячменя ярового в зависимости от условий выращивания существенно не изменяется и определяется, в основном, генотипом. У образцов голозерного ячменя этот показатель существенно выше (12,05–15,46 %), чем у пленчатых (10,91–13,82 %). Среди пленчатых сортов содержание белка существенно выше среднего по опыту было только у сорта Резерв (13,82 %), а у голозерных – у 11 сортов из 16 изученных. У шестирядных ячменей, как пленчатых, так и голозерных, содержание белка ниже, чем у двурядных. По переваримости белка как показателю биологической ценности выделены сорта Парнас и Беркут (69,80 и 74,20 мг тирозина на 1 г белка соответственно). Сорт Беркут создан отбором из гибридной популяции Парнас / Омский голозерный 1, поэтому Парнас является возможным источником белка с высокой переваримостью, представляющим интерес для селекции сортов ячменя для производства продукции детского и диетического питания. Выводы. Таким образом, в результате трехлетних исследований выделены образцы со стабильно высоким содержанием белка. Это голозерные сорта отечественной селекции Гатунок (15,46 %), Козацький (15,16 %), Ахіллес (14,61 %) и зарубежной Merlin (15,23 %), Alamo (14,93 %), Mebere (14,54 %), Millhouse (14,29 %), Голозерный 1 (14,56 %), Майский (14,29 %). Установлено, что характерным есть более низкое содержание белка у шестирядных образцов в сравнении с двурядными во всех выделенных группах. По показателю биологической ценности белка выделены сорта Парнас и Беркут (69,80 и 74,20 мг тирозина на 1 г белка соответственно). Сорт Парнас является возможным источником белка с высокой переваримостью. Это имеет значение для селекции сортов ячменя для производства продукции детского и диетического питания.

sportsmen, military servicemen and men of reproductive age, the requirement for proteins is even higher -2.0-3.0 g per kg of body weight [3,4].
In addition to protein content in foods, one should know the level of protein digestibility by the human body. This parameter determines the biological value of protein, which is due to the presence of irreplaceable amino acids, their ratio to replaceable ones and digestibility in the gastrointestinal tract. Digestibility of protein can be measured biochemically by enzymatic hydrolysis in conditions simulating those in a living organism. In this case, in vitro digestibility is determined, which comprehensively characterizes the protein under study related to the reference protein (casein) [3].
Given the paramount importance of protein to the human life, creation of high-protein cultivars of agricultural crops, including barley, one of the most valuable cereals for functional nutrition, is among the objectives of breeding. However, while breeders place a high priority on protein content, its quality is mainly studied in the context of forage for livestock husbandry. The same applies to definition of requirements for the barley grain quality as a foodstuff [5].
The protein content in grain is strongly influenced both by the growing conditions and by the genotype, and the growing condition effect is stronger. Concentration of available for plants nitrogen in soil is a factor increasing the protein content in grain. However, high protein content achieved owing to nitrogen fertilizers, is associated with an increase in replaceable amino acid (glutamic acid, proline, glutamine) amounts and with a relative decrease in amounts of irreplaceable ones (lysine, tryptophan) [1]. Negative correlation between the protein content and lysine amount in protein is typical for most barley cultivars, i.e., the higher the protein content in barley grain is, the lower its biological value is [6].
O.I. Rybalka with colleagues extensively studies the breeding of barley cultivars for functional nutrition. For example, they found that the protein content in naked accessions was always higher (17.14-21.06%) than in chaffy ones (12.5-14.05%), which was observed both for winter and for spring accessions. They also found that there was no significant correlation between the grain size and protein content in grain of naked cultivars, whereas a clear negative relationship was established for chaffy ones. When the digestibility of protein in barley groats from naked cultivars and in commercial groats was evaluated, solubility of protein in 50% 1-propanol was found to be significantly higher for all the samples of barley groats than for samples of groats from wheat cultivar Selianka (21.7%). The fraction of soluble proteins in groats from naked cultivars was also higher (33.4-45.9%) than that in commercial groats (29.3%). As to the growing conditions, the soluble protein content increases under dry conditions [1,7].
In arid 2012, Russian researchers recorded the highest protein content in barley accessions, and in 2013 with sufficient water availability -the lowest. Cultivars Hetman, Bezynchukskyi 3, Povolzhskiy 65, Vitiaz were defined as sources of high protein content [8]. In other studies, it was found that naked two-row barley Omskiy Golozyornyy 1 exceeded naked six-row Omskiy Golozyornyy 2 and chaffy Omskiy 90 in terms of protein content (18.82%, 16.19% and 16.00%, respectively). In addition, the grain evenness in the multi-row cultivar was significantly lower (49.6% vs. 74.0-76.3% in other cultivars) [9].
Researchers from the Republic of Tatarstan found that the protein content in grain of 22 barley cultivars of different eco-geographical origin was determined rather by air temperature than by varietal characteristics. The protein content was 12.21-16.69% in dry years, and in years with sufficient water availability it was reduced to 9.9-11.8% [10].
VV Glukhovtsev drew similar conclusions on the influence of weather conditions on the protein content in barley grain [11], namely -the protein content was more influenced by air temperature in June than during the grain filling phase. In addition, he found that the protein content significantly positively correlated with the contents of lysine, threonine and valine (r = 0.85-0.99). As for the content of irreplaceable amino acids, other researchers revealed that their amounts in all protein fractions of flour were 6-12% lower than those in barley grain [12].
Research on medical and preventive properties of barley grain is huge in the world. The amino acid composition of protein, reference intake [13], potential preventive effects of barley grain [14,15], grain quality and composition, etc. are studied. [16].
Chinese scientists studied 10 two-row barley cultivars and found that the protein content varied greatly, depending on the sowing date and cultivar, but there was no relationship between the sowing date and cultivar. A significant difference between cultivars was noticed in the contents of hordeins B, C and D [17], which confirmed the findings of J.L. Molina-Cano et al. [18].
In a study of 30 barley accessions with various protein content, correlation were found between the protein content, molecular structure and starch granule size: the protein content significantly negatively correlated with the amylose content, and positively -with the amilopectin content. The starch granule size positively correlated with the starch content [19].
The Plant Production Institute named after V.Ya. Yuriev of NAAS consistently creates cultivars with increased protein content. In studies of 60 barley cultivars of different ecogeographical origin, the protein content in grain was found to depend on the growing conditions (including predecessor) and genotype. In waterlogged 2005, the protein content dropped to 9.44-9.48% in cv. Scarlett, Aspekt and Efekt. In arid years, this parameter rose to 15.13-15.82% (Parnas, Merkurii) [20]. In 2013, which was very unfavorable for barley, the experiments were conducted on 17 cultivars in three locations with various soil and climatic conditions. It was found that cv. Zdobutok, Dokaz and Dyvohliad had the highest protein contents (15.38-15.59%), while in cv. Parnas and Dokaz Proof this parameter was the most stable [21].
The aim and tasks of the study. Thus, studies on dependence of protein content on the growing conditions and genotype are widely conducted in breeding. However, results of these studies differ both by location and by cultivars. Therefore, such experiments do not lose relevance. As for the quality of barley protein, the number of such studies in breeding is very limited. Proceeding from the unmet need for food cultivars, it is necessary to deepen investigations of quality features of barley grain and their variability, depending on the growing conditions and genotype.
Materials and methods. The study was conducted at the Plant Production Institute named after V.Ya. Yuriev of NAAS in 2015-2017. Seventy spring barley lines and cultivars of different eco-geographical origin were taken as the test material. The cultivars and lines were grown in variety trial plots with an area of 10 m 2 . Significance of differences between variants was tested by analysis of variance; post hoc comparisons to generate Homogeneous groups (Fisher LSD) were performed in the STATISTICA software package. The protein content in grain was measured on an Infralume FT-10M 09495.
The protein digestibility was evaluated in the Laboratory of Medical and Biological Problems of Food Technology at the Chair of Chemistry, Microbiology and Nutrition of Kharkiv State University of Nutrition and Trade. The in vitro protein digestibility by proteolytic enzymes was assessed by O. Pokrovsky and I. Yertanov's method [22], which is based on a gradual action of a proteinase system consisting of crystalline pepsin and trypsin on proteins.
Fermentation was carried out in two 60-minute steps.
Step I was pepsin fermentation at pH = 2.0; step II -trypsin fermentation at pH 8.0.
Samples containing 150 mg of protein were transferred in 2 test tubes, and 15 mL of 0.02 N HCl was added. 15 mg of pepsin was placed in one test tube. The other (control) test tube had no enzyme. Samples were incubated at 38 С for 120 minutes with constant stirring. To determine soluble pepsinolysis products, 0.2 mL of digest was taken from the test tubes, and 2 mL of 10% trichloroacetic acid was added to the digest aliquot. The test tubes were incubated for at room temperature 20 minutes, and then they were centrifuged at 6,000 rpm for 10 minutes. The amount of soluble pepsinolysis products was measured in supernatants by the Lowry method, and the pepsin digestibility was calculated.
For trypsinolysis, the contents of the experimental and control test tubes were neutralized with 5M sodium hydroxide, and 15 mL of 0.08 N NaHCO3 was added. 30 mg of trypsin was placed in the experimental test tube. Trypsinolysis was carried out at 38 С for 120 minutes with constant stirring. To determine the total digestibility (the amount of soluble products of protein hydrolysis by successive action of pepsin and trypsin), 0.2 mL of digest was taken from the test tubes, and 2 mL of 10% trichloroacetic acid was added. The test tubes were incubated at room temperature for 20 minutes and then centrifuged at 6,000 rpm for 10 minutes. The amount of sol-uble hydrolysis products was evaluated by the Lowry method, and the pepsin and total digestibilities of protein were calculated.
Results and discussion. The weather conditions during the study years little differed; therefore, the protein content in grain of the accessions was similar across the years. The HTC over the study years exceeded 1.5. There was a severe drought associated with high temperatures during the grain filling phase in 2017, nevertheless, the negative effect of such weather conditions was compensated by waterlogging during the tillering and earing phases, which contributed to the high productive tillering capacity.
To determine the genotype effect on the protein content, all the accessions were grouped according to their features, namely: chaffy with wild-type starch (about 20% amylose, 80% amylopectin), chaffy with waxy starch (100% amylopectin), naked with wild-type starch and naked with waxy starch.
There were significant differences in the protein content depending on the genotype. For example, the protein content of chaffy cv. Rezerv was significantly higher (13.82%) than the average across the chaffy accessions (12.81%). This cultivar is characterized by high drought tolerance, therefore the increased protein content is inherent to it, like to all cultivars belonging to the steppe ecotype. This parameter was also high in cv. Zvershennia, Dzherelo (13.24%), Donetskyi 14 (13.22%), and Novosadsky 294 (13.18%) (see Table 1).
Six-row accessions generally had significantly lower protein contents compared to tworow ones in all the groups. Thus, with the average across the chaffy accessions of 12.11-12.37%, the protein content in six-row accessions was 10.91-11.74%. The exception was cultivar Amil with a protein content of 12.70% (see Table 1), but even this level was low in comparison with other chaffy accessions. Similarly, six-row naked cultivar Buck had a significantly lower protein content (12.05%) than the average across the naked accessions.
High protein content is a very important feature for food barley breeding and food production. Nevertheless, in addition to protein content, its biological value should be taken into account. Determination of the digestibility of plant protein by the human digestive system is a method for evaluating the biological value of protein.
Analysis of the digestibility of protein by proteolytic enzymes showed that the protein of cv. Parnas and Berkut (naked) was highly digestible (69.80-7420 mg/g) ( Table 3). The trypsinolysis data are especially conspicuous (54.60-48.70 mg/g). Cultivar Berkut was created by selecting from hybrid population Parnas / Omskiy Golozyornyy 1. Thus, Parnas can be a source of highly digestible protein. This is important for the breeding of cultivars for dietic and baby food production.

Conclusions.
Thus, it was established that under similar weather conditions the protein content in grain of spring barley accessions was mainly determined by the genotype. In naked barley accessions, this parameter was significantly higher (12.05-15.46%) than in chaffy ones (10.91-13.82%). Of the chaffy accessions, Rezerv was the only cultivar with the protein content (12.81%) significantly exceeding the average throughout the experiment (13.82%). Eleven of 16 naked accessions had the protein contents significantly exceeding the average. Cultivars Parnas and Berkut were noticeable for to the biological value of protein (69.80 and 74.20 mg of tyrosine per g of protein, respectively). Since cv. Berkut was created by selecting from a hybrid population Parnas / Omskiy Golozyornyy 1, Parnas is can be a source of highly digestible protein. This is important for the breeding of barley cultivars for the production of baby and dietic foods.