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.

Changes in physicochemical properties of proteins in Kayserian Pastirma made from the M.semimembranosus muscle of cows during traditional processing

In the current study,we examined the effects of beef processing to produce pastirma on the physicochemical properties of proteins in M.semimembranosus(SEM)muscle.Protein concentrations significantly increased in pastirma muscles(P<0.01),as a result of the salting and curing process.The surface hydrophobicity values of processed samples were higher than those without processing at all guanidine-HCl concentrations,suggesting hydrophobicity increased,which may attribute to the new generated peptides during the traditional pastirma-making process.The metmyoglobin content greatly increased(by as much as 89%)in pastirma samples compared with the unprocessed samples.The images of histology also demonstrate that the pastirma processing had no negative impact on the structure of the muscle.The results from this study suggest that the traditional pastirma-making process catalyzed the enzymatic digestion of muscle proteins,and the differences in some physicochemical parameters between the control and pastirma samples were thus likely to be contributable to protein digestion.Thus,the traditional pastirma-making process results in the degradation of many proteins into peptides,which might then be obtainable as functional components to treat human dietand lifestyle-related diseases such as hypertension,hyperglycemia syndromes or to be used as nutraceuticals.©2013 Beijing Academy of Food Sciences.Production and hosting by Elsevier B.V.All rights reserved.

Effect of Bacillus subtilis and Bacillus licheniformis supplementation in diets with low-and high-protein content on ileal crude protein and amino acid digestibility and intestinal microbiota composition of growing pigs

Background： Bacillus spp. seem to be an alternative to antimicrobial growth promoters for improving animals＇ health and performance. However, there is little information on the effect of Bacillus spp. in combination with different dietary crude protein（CP） levels on the ileal digestibility and microbiota composition. Therefore, the objective of this study was to determine the effect of Bacillus spp. supplementation to low-（LP） and high-protein diets（HP） on ileal CP and amino acid（AA） digestibility and intestinal microbiota composition.Methods： Eight ileally cannulated pigs with an initial body weight of 28.5 kg were randomly allocated to a rowcolumn design with 8 pigs and 3 periods of 16 d each. The assay diets were based on wheat-barley-soybean meal with two protein levels： LP（14% CP, as-fed） and HP diet（18% CP, as-fed）. The LP and HP diets were supplemented with or without Bacil us spp. at a level of 0.04%（as-fed）. The apparent ileal digestibility（AID） and standardized ileal digestibility（SID） of CP and AA was determined. Bacterial community composition from ileal digesta was analyzed by Illumina amplicon sequencing and quantitative real-time PCR. Data were analyzed as a 2 × 2 factorial design using the GLIMMIX procedures of SAS.Results： The supplementation with Bacillus spp. did not affect both AID and SID of CP and AA in growing pigs.Moreover, there was no difference in AID of CP and AA between HP and LP diets, but SID of cystine, glutamic acid,glycine, and proline was lower（P 〈 0.05） in pigs fed the HP diets. The HP diets increased abundance of Bifidobacterium spp. and Lactobacillus spp.,（P 〈 0.05） and by amplicon sequencing the latter was identified as predominant genus in microbiota from HP with Bacillus spp., whereas dietary supplementation of Bacillus spp. increased（P 〈 0.05） abundance of Roseburia spp.Conclusions： The HP diet increased abundance of Lactobacillus spp. and Bifidobacterium spp.. The supplementation of Bacil us spp. resulted in a higher abundance of healthy gut associated bacteria without affecting ileal CP and AA digestibility, whereas LP diet may reduce the flow of undigested protein to the large intestine of pigs.

Phytochemical, anti nutrient, invitro-protein digestibility and functional properties of (Balanites aegyptiaca (L.) Delile) Aduwa protein meals, Protein Concentrate and Isolate

Background:Protein meals,Concentrate and Isolates are widely use as bioactive ingredients in functional food.In this study,the effect of toasting of Aduwa(Balanites aegyptiaca)seed on the phytochemicals,Anti nutrient,in vitro protein digestibility and functional properties of Aduwa Protein Meals,Protein Concentrate and Isolate were investigated.Methods:Aduwa seeds toasted at 70°C to make Aduwa protein meal(APM)yield extensive resolved biomaterials.The meal were resolved into DAPM,APC and API.Results:Phytochemicals analysis revealed reduction as APM sample is being resolved to protein concentrate and isolate.Similar trend was also observed in Anti-nutrients content significant reduction as material meal are resolved into protein concentrate and isolate.The in-vitro protein digestibility showed that APM(59.81%)and API(76.41%)had high percentage protein digestibility.Functional properties declined as meals materials were been resolved into protein concentrate and isolate.Swelling capacity revealed that resolved and unresolved samples leached biomolecules.Conclusion:The results obtained showed nutritional potential,and safety of biomaterials from Aduwa meals,an alternative food ingredient for protein supplementation.

Physical and chemical changes of rapeseed meal proteins during toasting and their effects on in vitro digestibility

Background： Toasting during the production of rapeseed meal（RSM） decreases ileal crude protein（CP） and amino acid（AA） digestibility. The mechanisms that determine the decrease in digestibility have not been fully elucidated. A high protein quality, low-denatured, RSM was produced and toasted up to 120 min, with samples taken every 20 min. The aim of this study was to characterize secondary structure and chemical changes of proteins and glucosinolates occurring during toasting of RSM and the effects on its in vitro CP digestibility.Results： The decrease in protein solubility and the increase of intermolecular β-sheets with increasing toasting time were indications of protein aggregation. The contents of NDF and ADIN increased with increasing toasting time.Contents of arginine, lysine and O-methylisourea reactive lysine（OMIU-RL） linearly decreased with increasing toasting time, with a larger decrease of OMIU-RL than lysine. First-order reactions calculated from the measured parameters show that glucosinolates were degraded faster than lysine, OMIU-RL and arginine and that physical changes to proteins seem to occur before chemical changes during toasting. Despite the drastic physical and chemical changes noticed on the proteins, the coefficient of in vitro CP digestibility ranged from 0.776 to 0.750 and there were no effects on the extent of protein hydrolysis after 120 min. In contrast, the rate of protein hydrolysis linearly decreased with increasing toasting time, which was largely correlated to the decrease in protein solubility, lysine and OMIU-RL observed. Rate of protein hydrolysis was more than 2-fold higher for the untoasted RSM compared to the 120 min toasted material.Conclusions： Increasing the toasting time for the production of RSM causes physical and chemical changes to the proteins that decrease the rate of protein hydrolysis. The observed decrease in the rate of protein hydrolysis could impact protein digestion and utilization.

Overload type and optimization of meadow carrying capacity in Maqin County in the Three-River Source Region,China

Alpine meadows,comprising the main ecosystem of the Three-River Source Region,play an important role in the economic development of western pastoral areas.To determine whether the grazing level was too intense or the nutrient-carrying capacity of the meadows was overloaded,and to offer solutions in Maqin County,Qinghai Province,China,the meadows carrying capacity was determined by combining grass-yield and nutrients data for different seasons across different meadows.The results showed that the levels of crude protein(CP),ether extract(EE),ash,soluble sugars(SS),gas production,and energy value of forage in the summer–autumn grazing meadows were higher than those of the winter–spring meadows.Neutral detergent fiber(NDF)and acid detergent fiber(ADF)of forage in the summer–autumn grazing meadows were lower than those of the winter–spring meadows.We found that no pastures were overloaded with metabolizable energy(ME)for the whole year but digestible crude protein(DCP)was overloaded in winter–spring.And there were differences in the amount carrying capacity(forage yield)of seasonal grazing meadows for the different herds,the overload types were annual overloading(herd A),overloaded in summer–autumn(herd B),overloaded in winter–spring(herd C),and not overloaded(herd D).Compared with the previous grazing mode,theoretical carrying capacity,DCP carrying capacity,and ME carrying capacity of herd A increased by 20,19,and 27 sheep units,respectively,after optimization;herd B:36,31,and 45 sheep units,respectively;herd C:28,23,and 44 sheep units,respectively;and herd D:43,40,and 61 sheep units,respectively.In the Three-River Source Region and similar alpine pastoral areas,the grassland-livestock structure should be optimized to improve grassland vegetation status and increase the theoretical carrying capacity.

Utilization of Small Size Bolti (<i>Talibia nilotica</i>) and Sardine (<i>Sardina pilchardus</i>) Fish in Preparing Canned Weaning Food Purees

Utilize small size of bolti and sardine fish in preparing fish based weaning foods rich in protein, essential amino acids, fat high in omega 3 fatty acids, minerals and vitamins was investigated. The proximate composition, nutritional value, physical and sensorial properties were evaluated, The results of the chemical composition showed that the moisture content of fish based weaning food purees ranged from 67.73% to 72.05%. On dry weight basis, the five prepared formulations considered rich in protein (37.39% - 44.86%), fat (26.55% - 30.43%), ash (6.53% - 12.23%) and low in carbohydrates (20.70% - 22.18%). Using fish bones with muscles in preparing weaning food puree caused a marked rise in its content of Ca, P, Fe, and Zn levels especially in purees formulated from bolti. Weaning food purees containing sardine fish rich, in fat, were higher in vitamin A and D than those formulated from bolti one. Palmitic acid (C16:0), stearic acid (C18:0), and myristic acid (C14:0) represented the major saturated fatty acids in the prepared formulations. Weaning food purees containing sardine fish had relatively higher levels of sulfur-containing amino acids and slight lower values of lysine, alanine and glycine than these including bolti fish. The In vitro protein digestibility index (IVPDI) of the different formulations was found to be 100%. The visual colour of such products can be described as yellowish. Except hardness, the other texture characteristics;cohesiveness, springiness, gumminess, and chewiness of the weaning food purees did not significantly change. The children’s mothers accepted the appearance, flavor, and mouth feel of all weaning food purees prepared in this study.

Effect of Additional Feed Supplement Fermentation Shrimp Waste Extract on Digestibility in Sentul Chicken Growth Phase

This study aims to determine the effect of adding feed supplements of fermented shrimp waste extract in the ration on the digestibility of local chicken rations in the growth phase.The research was carried out in Jatinangor District as well as the Laboratory of Ruminant Animal Nutrition and Animal Feed Chemistry,Faculty of Animal Husbandry,Padjadjaran University,Sumedang.The method used in this study was experimental with a Complete Randomized Design(RAL).The data were analyzed by fingerprint test(ANOVA)with further tests using the Dunnet test.The object of this study consisted of 20 Sentul chickens raised from the age of 1 day to 12 weeks,divided into 5 treatments and 4 tests.The treatment consists of P0=Basal ration without the addition of feed supplement fermented shrimp waste extract,P1=Basal ration+0.5%feed supplement fermented shrimp waste extract,P2=Basal ration+1.0%feed supplement fermented shrimp waste extract,P3=Basal ration+1.5%feed supplement fermented shrimp waste extract,and P4=Basal ration+2.0%feed supplement fermented shrimp waste extract.The changes observed are the digestibility of dry matter,the digestibility of organic matter,and the digestibility of proteins.The results showed that the addition of feed supplements for fermented shrimp waste extract had a significantly different influence on the digestibility of dry matter,the digestibility of organic matter,and the digestibility of protein.This study concludes that the addition of a feed supplement of 2%fermented shrimp waste extract in the ration can produce a high ration digestibility value for Sentul chicken in the growth phase.

Protein digestive dynamics of meat and bone meals in broiler chickens

This study determined the variations in protein digestibilities and digestion rates in broiler chickens offered diets containing 7 different meat and bone meals(MBM).A total of 252 male Ross 308 broiler chickens were offered 7 atypical diets largely based on maize and MBM from 24 to 28 d post-hatch.Each experimental diet was offered to 6 replicates with 6 birds per replicate cage.Excreta were collected in their entirety from 25 to 27 d post-hatch and on 28 d post-hatch.Digesta samples were collected from the proximal jejunum,distal jejunum,proximal ileum and distal ileum.Apparent digestibilities of protein were determined in each segment and apparent digestibilities of amino acids were measured in the distal ileum.There were significant differences in apparent protein digestibility coefficients in the proximal jejunum(P=0.006),where broiler chickens offered the high ash beef meal(diet 7)generated the lowest protein digestibility in the proximal jejunum(0.318).Similarly,there were significant differences in apparent digestibility coefficients in the distal jejunum(P<0.001)and distal ileum(P<0.001)but not in the proximal ileum.More pronounced differences were found in the disappearance rate of protein and there were significant differences in all 4 segments of the small intestine(P<0.001).Broiler chickens offered the high ash beef meal had the lowest protein disappearance rate(P<0.001).No difference was observed in the predicted protein digestion rate(P=0.486)but chickens offered the high ash beef meal had the lowest potential digestible protein(0.662,P=0.034)whereas the highest potential digestible protein(0.739)was detected in diet 5(containing a beef meal).This study contributed to the establishment of a preliminary database to include digestion rates of starch and protein into practical diet formulation.