Development of sorghum mutants with improved in vitro protein digestibility by CRISPR/Cas9 editing of kafirin genes

Sorghum(Sorghum bicolor(L.) Moench) is a major world crop that is a reliable source of fodder and food grain in arid regions. However, unlike other cereals, sorghum grain has low nutritional value, owing mainly to the resistance of its storage proteins(kafirins) to protease digestion. Changing the composition of kafirins or their primary structure may address this problem. To induce mutations in kafirin-encoding genes that were expected to disturb their accumulation in endosperm cells, we used a genome-editing approach. By Agrobacterium-mediated genetic transformation of immature embryos of cv. Avans, we obtained 14 transgenic plants with genetic constructs for site-directed mutagenesis of the k1C5 and g KAF1 genes encoding 22 k Da a-and 28 kDa γ-kafirins, respectively. Sequencing of 5 regenerants obtained by using k1C5-addressing vector revealed two plants with mutations. T_1 progeny of these mutants had higher in vitro digestibility of endosperm proteins(86%–92%), in comparison with the donor Avans(63%–67%). The kernels of these plants had a thick vitreous endosperm. A mutant with increased in vitro protein digestibility and vitreous endosperm, carrying a mutation in the target sequence, was also obtained by use of the gKAF1-addressing vector. Thus, using genome editing technology, we have obtained mutants with improved kafirin digestibility that can be used in sorghum breeding.

Binary Vector Construction for Site-Directed Mutagenesis of <i>Kafirin</i>Genes in Sorghum

Sorghum (Sorghum bicolor (L.) Moench) is one of the world’s leading cereal crops in agricultural production, which has a special importance in the arid regions. However, unlike other cereals, sorghum grain has a lower nutritional value, which is caused, inter alia, by the resistance of its seed storage proteins (kafirins) to protease digestion. One of the effective approaches to improve the nutritional value of sorghum grain is to obtain mutants with partially or completely suppressed synthesis or altered amino acid composition of kafirins. The employment of genome editing may allow to solve this problem by introducing mutations into the nucleotide sequences of the α- and γ-kafirin genes. In this study, genomic target motifs (23 bp sequences) were selected for the introduction of mutations into the α- and γ-KAFIRIN genes of sorghum. The design of the gRNAs was conducted using the online tools CRISPROR and CHOPCHOP. Two most suitable targets were chosen for α-KAFIRIN (k1C5) and two for γ-KAFIRIN (gKAF1) genes. The insertion of respective sequences in the generic vector pSH121 was performed at the BsaI (Eco31I) sites. Validation of the cloning procedure was performed by DNA sequencing. Subcloning of the resulting constructs was performed using the SfiI restriction sites into the compatible binary vector B479p7oUZm-LH. The correct assembly of binary vectors was confirmed by restriction analysis using the MluI and SfiI cleavage sites. The four vectors created (1C - 4C) were transferred by electroporation into the Agrobacterium tumefaciens strain AGL0. Currently, this vector series is used for stable transformation of sorghum using immature embryo explants.

Performance of broiler chickens offered nutritionally-equivalent diets based on two red grain sorghums with quantified kafirin concentrations as intact pellets or re-ground mash following steam-pelleting at65 or 97℃ conditioning temperatures

The Liverpool Plains is a fertile agricultural region in New South Wales, Australia. Two sorghums from the2009 Liverpool Plains harvest, sorghums #3 and #5, were extensively characterised which included concentrations of kafirin and phenolic compounds plus rapid visco-analysis(RVA) starch pasting profiles. Diets based on these two sorghums were formulated to be iso-nitrogenous and iso-energetic and were offered to male Ross 308 broiler chicks from 7 to 28 days post–hatch as either intact pellets or reground mash following steam-pelleting at conditioning temperatures of either 65 or 97°C. Thus the feeding study consisted of a 2×2×2 factorial array of dietary treatments: two sorghum varieties, two feed forms and two conditioning temperatures. Each of the eight treatments was replicated six times with six birds per replicate cage.Assessed parameters included growth performance, nutrient utilisation, apparent starch and protein(N) digestibility coefficients and disappearance rates from the distal jejunum and distal ileum. Intact pellets supported higher(P <0.001) feed intakes and weight gains by 9.83 and 9.08%, respectively, than reground mash diets. Feed conversion ratios of broilers offered diets steam-conditioned at 97°C were 2.46% inferior(P <0.001) in comparison to 65°C diets and both apparent metabolizable energy(AME) and N-corrected AME(AMEn) were compromised. Broilers offered sorghum #3-based diets significantly(P <0.001) outperformed their sorghum #5 counterparts in terms of weight gain by 3.75%(1,334 versus 1,223 g/bird), FCR by 4.81%(1.524 versus 1.601), AME by 1.06 MJ(13.61 versus 12.55 MJ/kg), ME:GE ratio(ME:GE) by 4.81%(0.806 versus0.769) and AMEn by 1.03 MJ(12.38 versus 11.35 MJ/kg). The inferiority of sorghum #5 appeared to be associated with higher concentrations of kafirin(61.5 versus 50.7 g/kg) and conjugated phenolic acids,including ferulic acid(31.1 versus 25.6 mg/g). There were no significant differences in jejunal and ileal starch and protein(N) digestibility coefficients between the two sorghums. However, starch to protein(N) disappearance rate ratios from the distal jejunum were significantly(P <0.001) correlated with ME:GE and AME. The multiple linear regression equations indicated that energy utilisation was enhanced by coupling rapidly digestible protein with slowly digestible starch, which suggests that bilateral bioavailability of starch and protein is pivotal to efficient energy utilisation.

Is the kafirin profile capable of modulating the ileal digestibility of amino acids in a soybean meal-sorghum diet fed to pigs?

The effects of kafirins on protein and amino acid ileal digestibility have not been evaluated in vivo in pigs.The aim of this study was to determine the effects of protein profile on apparent ileal digestibility(AID)of amino acids. We used a sorghum hybrid with low tannin content(<0.5%). The same hybrid was harvested from 2 different plots with different kafirin profile. Sorghum with greater content of total kafirins had less content of y-and al-kafirins and higher content of β-and a2-kafirins than that with lower content of total kafirins. Two sorghum-soybean meal(SBM) diets were formulated: 1) low kafirin(LK) content(32.2 g/kg) and 2) high kafirin(HK) content(48.1 g/kg). A control diet(maize-SBM) and a reference SBM-diet were also prepared. The reference diet was fed to all pigs following the experimental period and was used to estimate the AID of cereals by the difference method. 'T' cannulas were fixed in the distal ileum of 18 barrows(6 by treatment), divided into 2 groups of 9 pigs. The pigs were fed 2.5 times their maintenance requirement of digestible energy(110 kcal/kg BW00.75). The AID of dry matter,protein, amino acids, and energy of the experimental diets was measured;the AID of cereals(maize, LK sorghum and HK sorghum) was estimated by the difference method. The maize-SBM diet was more digestible than the sorghum-SBM diets, only with respect to valine(P < 0.05). The AID of valine in the maize-SBM diet was higher than that in sorghum-SBM diets. The changes in kafirin profile between the diets only affected the AID of threonine(P < 0.01), which decreased by 9.5 percentage units in LK diet compared with HK diet. Regarding the AID of cereals, maize exhibited greater AID than sorghum, with respect to valine(P < 0.01) and serine(P < 0.10). A comparison of sorghum with LK and HK content showed that the AID of threonine and serine increased by 50.5(P < 0.001) and 19.2 percentage units(P < 0.05) in the latter, respec:tively. The higher content of γ-kafirins in LK sorghum negatively affected threonine and serine digestibility, implying that the AID of amino acids is affected more by the profile than the content of kafirins.

影响高粱饲用价值主要内在因素及其对策

大量研究结果显示,饲用高粱含有3种物质不利于高粱在动物体内的消化,一是酚类化合物,二是醇溶蛋白,三是植酸。本文从3类化合物的特征、对消化酶的抑制作用、醇溶蛋白与淀粉以及氨基酸的消化率、植酸和植酸磷以及酶制剂在高粱型畜禽饲粮中的应用等方面,就高粱在动物饲料应用的研究动态和进展进行了简要综述。

丁香酚对高粱醇溶蛋白可食性膜结构及性能的影响

为开发天然的可降解、可食性包装材料,以高粱醇溶蛋白为原料,采用溶液共混的方法制备可食性丁香酚/高粱醇溶蛋白复合膜,分析不同浓度丁香酚对可食性高粱醇溶蛋白膜物理性能及微观结构的影响并探讨其变化机理。结果表明,添加4%丁香酚可优化蛋白膜的机械性能,提升膜的拉伸强度(TS)和断裂伸长率(EAB);添加丁香酚不影响蛋白膜的水蒸气透过系数(WVP),但略微提高了蛋白膜的溶解度;添加4%丁香酚可增加蛋白膜对紫外光和可见光的吸光度值,即增强膜的光阻隔性能。DSC测量显示,添加丁香酚后降低了高粱醇溶蛋白的玻璃态转变温度(Tg),表明丁香酚提高了丁香酚/高粱醇溶蛋白复合膜的延展性;FTIR分析结果表明,添加丁香酚后使得高粱醇溶蛋白二级结构中的α-螺旋、无规则卷曲转变为β-折叠、β-转角,表明丁香酚有助于提高丁香酚/高粱醇溶蛋白复合膜的机械性能;SEM结果显示,4%丁香酚与高粱醇溶蛋白的相容性良好,制备的复合膜截面光滑紧致。本研究结果为可降解、可食性膜新材料的研究及应用推广提供了理论参考。

响应面优化超声波-微波协同提取高粱醇溶蛋白工艺

以高粱种子为原料,采用超声波-微波协同辅助提取高粱醇溶蛋白。在单因素试验结果的基础上利用响应面法优化高粱醇溶蛋白提取工艺,建立提取工艺回归数学模型。结果表明:最佳提取工艺条件为超声时间26 min、液料比10∶1(mL/g)、微波时间260 s,在该条件下高粱醇溶蛋白的得率为6.79%,与理论得率接近,可为实际生产提供理论依据。

高粱种子醇溶蛋白的提取条件

以高粱种子为材料,研究了不同提取剂、提取剂用量、浓度和还原剂浓度对高粱种子醇溶蛋白提取的影响.蛋白浓度测定和SDS-PAGE电泳表明,DDT浓度为4%的60%叔丁醇溶液,样品质量比提取剂用量为1∶8(g/mL)是提取高粱种子醇溶蛋白的最佳条件.该条件下,提取高粱属不同种的种子醇溶蛋白,得到的高粱属种子醇溶蛋白的"指纹"图谱,可以用来区分高粱属内的种间差异.

高粱醇溶蛋白对浓香型白酒窖泥味的消减机制

窖泥味在浓香型白酒中被视为一种异臭味,添加高粱醇溶蛋白对浓香型白酒中的窖泥味有消减作用,对高粱醇溶蛋白消减窖泥味的作用机制进行了研究。浓香型白酒中的3-甲基吲哚和4-甲基苯酚是使其产生窖泥味的关键气味物质。香气轮廓实验表明,高粱醇溶蛋白的加入可显著降低浓香型白酒中的窖泥味,而对其他风味无显著影响。采用顶空固相微萃取法分析发现,高粱醇溶蛋白可使模拟酒样中的3-甲基吲哚和4-甲基苯酚的挥发度分别降低38.99%和46.39%。且当添加200 mg/L高粱醇溶蛋白后,3-甲基吲哚和4-甲基苯酚的气味阈值分别提高到1.50μg/L和0.31μg/L,气味阈值分别为添加高粱醇溶蛋白前的4.59和1.73倍。为进一步解释高粱醇溶蛋白对窖泥味物质消减作用机制,分别在25、45、65℃下进行紫外-可见分光光度法分析,发现高粱醇溶蛋白与3-甲基吲哚、4-甲基苯酚可自发络合形成复合物,高粱醇溶蛋白与3-甲基吲哚络合物的生成反应为焓变驱动,高粱醇溶蛋白与4-甲基苯酚络合物的生成反应为吉布斯自由能驱动。研究应用分子对接模型实验表明:高粱醇溶蛋白分子表面的疏水口袋通过空间匹配和分子间作用力将3-甲基吲哚和4-甲基苯酚包裹或连接在内,主要的分子间相互作用力为氢键和疏水作用。希望研究结果可为降低浓香型白酒中的异臭味提供解决方案,为食品中异臭味成分的消减研究提供新思路。

蛋白类抗营养因子的抗营养效应及其解决方案

饲料中的蛋白类抗营养因子主要包括蛋白酶抑制因子、致敏蛋白、凝集素和醇溶蛋白等。蛋白酶抑制因子通过降低胰蛋白酶活性、引起胰腺增生和肿大、降低蛋白质消化率等发挥抗营养效应;致敏蛋白通过致敏作用导致肠道形态结构损伤,进而影响消化吸收功能和肠道健康;凝集素与肠道上皮细胞特异性结合,影响肠道结构和功能;醇溶蛋白能包裹谷物淀粉,影响谷物淀粉的消化率。蛋白类抗营养因子通过不同方式直接或间接影响营养物质的消化、吸收和利用,甚至影响畜禽健康和生产性能。深入研究蛋白类抗营养因子的特性和作用机理,探索消除或减轻其抗营养效应的解决方案,对于优化饲料配方、提升饲料品质、促进肠道健康、提升综合效益具有重要意义。

高粱醇溶蛋白消化性研究

该文介绍高粱醇溶蛋白的提取方法,特性和消化性测定方法,并简单分析高粱醇溶蛋白发展前景。

反溶剂法制备姜黄素-高粱醇溶蛋白复合颗粒及其特性分析

以姜黄素为芯材,高粱醇溶蛋白为壁材,采用反溶剂法制备包埋姜黄素的高粱醇溶蛋白复合颗粒(简称复合颗粒),并对其理化性质及稳定性进行研究。结果表明:姜黄素与高粱醇溶蛋白制备复合颗粒的最佳芯壁比(质量比)为1∶10,产品平均粒径为13.17μm,电位为19.38 m V,得率为87.51%,包封率为62.61%,负载率为6.51%。相比于高粱醇溶蛋白颗粒,扫描电子显微镜显示:复合颗粒呈球形,但表面有多孔结构;红外光谱结果显示:复合颗粒位于1 534、1 655 cm^(-1)等处的波峰均发生红移,α-螺旋含量减少,β-折叠含量增加。24 h紫外光照射后,高粱醇溶蛋白包埋的姜黄素光稳定性提高33%。复合颗粒的粒径和多分散性系数在30 d室内环境贮藏过程中均无明显变化,但在环境p H 5~6,颗粒易聚集。结果表明:高粱醇溶蛋白的包埋作用有益于提高姜黄素的稳定性,可为姜黄素和高粱醇溶蛋白的高值化利用提供理论依据。

高粱醇溶蛋白结构与功能活性的研究进展

高粱醇溶蛋白是高粱中的主要贮藏蛋白,可应用于食品加工和医疗保健领域。从高粱醇溶蛋白组成、结构和功能及活性方面进行了阐述,综述了高粱醇溶蛋白在运输生物活性成分、制备包装薄膜、改善高粱蛋白消化率、生产无麸质面团、制备生物活性肽等领域的研究现状,并对其发展前景进行了展望。

高粱作为饲料原料的营养与应用特性

作为一种新型的饲料资源,高粱的应用正受到饲料企业的关注和重视。文章回顾了高粱的应用趋势,分别阐述了高粱的营养性能、抗营养因子、物理与加工特性,并概述了酶制剂在高粱日粮领域的应用潜力与前景。

以高粱醇溶蛋白和酪蛋白酸钠为壁材制备粉末油脂的工艺优化

以高粱醇溶蛋白和酪蛋白酸钠作为壁材,采用喷雾干燥法制备大豆油粉末油脂。以包埋率为指标,进行单因素和Box-Behnken响应面试验优化粉末油脂的制备工艺条件。结果表明:大豆油粉末油脂的最佳制备工艺为高粱醇溶蛋白与大豆油质量比2.3∶1,酪蛋白酸钠添加量5 g/L,进风温度110℃。在此条件下,包埋率为40.15%。与市售产品相比,自制粉末产品感官品质较好,水分含量低,具有良好的溶解度和分散性,休止角<40°,可满足食品工业的应用。

RNAi靶向沉默opaque2基因对高粱醇溶蛋白含量的影响

为研究opaque2基因沉默对高粱醇溶蛋白含量的影响,采用RNAi技术构建opaque2基因抑制表达载体,利用农杆菌介导转化高粱幼胚,测定转基因植株籽粒中醇溶蛋白含量,SDS-PAGE电泳检测醇溶蛋白带型,并使用透射电镜观察胚乳细胞中蛋白质体变化。结果表明,与野生型相比,5株转基因植株中4株籽粒的醇溶蛋白含量显著降低(P<0.05);野生型和各转基因植株的醇溶蛋白SDS-PAGE电泳带型相同;野生型胚乳细胞中淀粉粒排列紧密,部分大淀粉粒融合成大块状,蛋白质体数量较多且有一些体积较大;而转基因植株淀粉粒排列疏松,细胞中存在多处孔隙,蛋白质体数量较少,体积较小。opaque2基因沉默引起胚乳细胞蛋白质体数量变少,体积变小,进而影响胚乳细胞内部结构和籽粒醇溶蛋白含量。

高粱醇溶蛋白-卡拉胶复合纳米颗粒负载姜黄色素的特性研究

本研究以姜黄色素为芯材,高粱醇溶蛋白为壁材,卡拉胶自组装,采用反溶剂法制备负载姜黄色素的中空和实心复合纳米颗粒,并对其理化性质及体外消化特性进行研究。结果表明,制备中空(C-HNP)和实心纳米颗粒(C-SNP)的最佳芯壁比为1∶10,其粒径约为71 nm和135 nm,电位为-37.7 mV和-38.2 mV,包埋率为89.6%和84.1%,负载率为8.01%和7.49%。透射电镜显示两种纳米粒子呈规则的球形形态,C-HNP具有内部中空结构。红外光谱结果显示复合颗粒中酰胺峰Ⅰ和Ⅱ均发生蓝移,卡拉胶和姜黄色素一些吸收峰发生相应偏移,说明它们与蛋白质分子发生相互作用。体外消化模拟实验显示复合纳米颗粒显著地增加了姜黄色素的体外溶解释放率,C-HNP具有更高的控释能力。结果表明,高粱醇溶蛋白是一种适合疏水性分子包埋与控释的生物原材料。

高粱醇溶蛋白的研究进展

高粱醇溶蛋白是种子主要贮藏的蛋白,可广泛应用于食品和医药行业。研究从高粱醇溶蛋白组成、消化特性、表达调控、提取及应用等方面进行阐述,为高粱醇溶蛋白深入研究提供参考。

Outlook:Sorghum as a feed grain for Australian chicken-meat production

This review is an outlook for sorghum as a feed grain for broiler chickens based on a survey of relevant stake-holders and recent research outcomes.Australian grain sorghum production will probably continue to generate a harvest in the order of 2.5×10~6t of which some 7.9×10~5t will be used as a feed grain for poultry and pigs.Feed grains are included primarily to provide energy from starch,but energy utilisation by broiler chickens offered sorghum-based diets is relatively inferior,because of incomplete starch digestion.Kafirin,the dominant protein fraction,‘non-tannin’phenolic compounds and phytate are 3‘starch extrinsic’factors in sorghum that compromise starch digestibility and energy utilisation in broiler chickens offered sorghum-based diets.Kafirin concentrations in 6 sorghum varieties were negatively correlated with metabolizable energy to gross energy(ME:GE)ratios(r=0.891;P<0.02)or the efficiency of energy utilisation in broiler chickens.Importantly,kafirin proportions of sorghum protein may be increasing with time in Australia.If so,this represents a fundamental challenge to sorghum breeders which presumably could be met by the development of sorghum varieties with different characteristics,especially in relation to the g-and b-kafirin fractions.White sorghum varieties contain lower polyphenol concentrations which should be advantageous as concentrations of total phenolic compounds were negatively correlated to ME:GE ratios(r=0.838;P<0.04)in 6 sorghum varieties.It would be desirable if more white varieties were to become available.It is suggested that responses to exogenous phytase in birds offered sorghum-based diets would be more robust if sorghum were to contain lower concentrations of kafirin and phenolic compounds.Paradoxically,while better sorghum varieties almost certainly could be developed,it may not necessarily follow that they will command a price premium from poultry and pig producers.

The potential of rapid visco-analysis starch pasting profiles to gauge the quality of sorghum as a feed grain for chicken-meat production

Thirteen extensively characterised grain sorghum varieties were evaluated in a series of 7 broiler bioassays. The efficiency of energy utilisation of broiler chickens offered sorghum-based diets is problematic and the bulk of dietary energy is derived from sorghum starch. For this reason, rapid visco-analysis(RVA)starch pasting profiles were determined as they may have the potential to assess the quality of sorghum as a feed grain for chicken-meat production. In review, it was found that concentrations of kafirin and total phenolic compounds were negatively correlated with peak and holding RVA viscosities to significant extents across 13 sorghums. In a meta-analysis of 5 broiler bioassays it was found that peak, holding,breakdown and final RVA viscosities were positively correlated with ME:GE ratios and peak and breakdown RVA viscosities with apparent metabolizable energy corrected for nitrogen(AMEn) to significant extents. In a sixth study involving 10 sorghum-based diets peak, holding and breakdown RVA viscosities were positively correlated with ME:GE ratios and AMEn. Therefore, it emerged that RVA starch pasting profiles do hold promise as a relatively rapid means to assess sorghum quality as a feed grain for chicken-meat production. This potential appears to be linked to quantities of kafirin and total phenolic compounds present in sorghum and it would seem that both factors depress RVA starch viscosities in vitro and, in turn, also depress energy utilisation in birds offered sorghum-based diets. Given that other feed grains do not contain kafirin and possess considerably lower concentrations of phenolic compounds, their RVA starch pasting profiles may not be equally indicative.