Developing Soybean Protein Gel-Based Foods from Okara Using the Wet-Type Grinder Method

Okara, a by-product of tofu or soymilk, is rich in dietary fibers (DFs) that are mostly insoluble. A wet-type grinder (WG) system was used to produce nanocellulose (NC). We hypothesized that the WG system would increase the dispersion performance and viscosity of okara. These properties of WG-treated okara improve the gel-forming ability of soybean proteins. Here, the suspensions of 2 wt% okara were treated with WG for different passages (1, 3, and 5 times). The particle size distribution (PSD) and viscosity of WG-treated okara decreased and increased, respectively, with different passages. The five-time WG-treated okara homogeneously dispersed in water after 24 h, whereas untreated okara did not. The breaking stress, strain, and water holding capacity of soybean protein isolate (SPI) gels increased upon the addition of WG-treated okara. This effect increased as the number of WG treatments increased. The breaking stress and strain of SPI gels to which different concentrations of the five-time WG-treated okara were added also increased with increasing concentrations of WG-treated okara. These results suggest that NC technology can improve the physicochemical properties of okara and are useful in the development of protein gel-based foods.

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis

Background Soybean (Glycine max) is a major protein crop, because soybean protein has an amino acid score comparable to that of beef and egg white. However, many allergens have been identified among soybean proteins. A decrease in allergenic protein levels would be useful for expanding the market for soybean proteins and processed foods. Recently, the CRISPR/Cas9 system has been adopted as a powerful tool for the site-directed mutagenesis in higher plants. This system is expected to generate hypoallergenic soybean varieties. Results We used two guide RNAs (gRNAs) and Agrobacterium-mediated transformation for simultaneous site-directed mutagenesis of two genes encoding the major allergens Gly m Bd 28 K and Gly m Bd 30 K in two Japanese soybean varieties, Enrei and Kariyutaka. We obtained two independent T0 Enrei plants and nine T0 Kariyutaka plants. Cleaved amplified polymorphic sequence (CAPS) analysis revealed that mutations were induced in both targeted loci of both soybean varieties. Sequencing analysis showed that deletions were the predominant mutation type in the targeted loci. The Cas9-free plants carrying the mutant alleles of the targeted loci with the transgenes excluded by genetic segregation were obtained in the T2 and T3 generations. Variable mutational spectra were observed in the targeted loci even in T2 and T3 progenies of the same T0 plant. Induction of multiple mutant alleles resulted in six haplotypes in the Cas9-free mutants derived from one T0 plant. Immunoblot analysis revealed that no Gly m Bd 28 K or Gly m Bd 30 K protein accumulated in the seeds of the Cas9-free plants. Whole-genome sequencing confirmed that a Cas9-free mutant had also no the other foreign DNA from the binary vector. Our results demonstrate the applicability of the CRISPR/Cas9 system for the production of hypoallergenic soybean plants. Conclusions Simultaneous site-directed mutagenesis by the CRISPR/Cas9 system removed two major allergenic proteins from mature soybean seeds. This system enables rapid and efficient modification of seed components in soybean varieties.

Features of сalibration еequations for IR scanners in determining the amino acid composition of soy proteins

Из 20 аминокислот, входящих в состав растительных белков, 17 лучше всего определяются с помощью высокоэффективной жидкостной хроматографии. Но эта технология затратна по времени, в том числе из-за подготовки проб, что делает ее малопригодной при проведении массовых анализов, например при оценке селекционного материала. В этом случае наиболее приемлемы технологии, основанные на сканировании в ближнем инфракрасном диапазоне излучения. Несмотря на то что ИК-сканеры способны по одному калибровочному уравнению выявлять большое количество компонентов, необходима постоянная коррекция при определении состава аминокислот и приведении его в процентное соотношение. В статье рассматриваются варианты создания калибровочных уравнений для расчета аминокислотного состава белков сои с помощью компьютерных программ (Nir 42, ISI), обеспечивающих работу ИК-сканеров типа NIR-4250 или FOSS NIRSystem 5000. Установлено, что при создании калибровочных уравнений содержание каждой аминокислоты наиболее корректно выражать в абсолютных единицах (г на 100 г белка), а не относительных (%). 17 of the 20 amino acids, included in the composition of plant proteins, are most effectively determined using liquid chromatography. The technology of high-performance liquid chromatography is to a certain extent costly in time, among other things because of sample preparation that makes it unsuitable for mass analysis, for example, when evaluating a breeding material. In this case, the technology based on scanning in the near infrared radiation band are the most acceptable. Despite the fact that IR scanners are able to determine a sufficiently large number of components on the basis of one calibration equation, a constant correction is required when determining the composition of amino acids and reducing it to a percentage ratio. The options for creating calibration equations for determining the amino acid composition of soybean proteins for computer programs (Nir 42, ISI), which provide the operation of IR scanners, such as NIR-4250 or FOSS NIRSystem 5000 are considered in the article. It was found that when creating calibration equations, it is most correct to set for each amino acid its mass content (g per 100 g of protein), and not the relative portion (in %).