Development of PCR-based markers for identification of wheat HMW glutenin Glu-1Bx and Glu-1By alleles.

BACKGROUND: In common wheat (Triticum aestivum L.), allelic variations in the high-molecular-weight glutenin subunits Glu-B1 locus have important effects on grain end-use quality. The Glu-B1 locus consists of two tightly linked genes encoding x- and y-type subunits that exhibit highly variable frequencies. However, studies on the discriminating markers of the alleles that have been reported are limited. Here, we developed 11 agarose gel-based PCR markers for detecting Glu-1Bx and Glu-1By alleles. RESULTS: By integrating the newly developed markers with previously published PCR markers, nine Glu-1Bx locus alleles (Glu-1Bx6, Glu-1Bx7, Glu-1Bx7*, Glu-1Bx7 OE, Glu-1Bx13, Glu-1Bx14 (-) , Glu-1Bx14 (+)/Bx20, and Glu-1Bx17) and seven Glu-1By locus alleles (Glu-1By8, Glu-1By8*, Glu-1By9, Glu-1By15/By20, Glu-1By16, and Glu-1By18) were distinguished in 25 wheat cultivars. Glu-1Bx6, Glu-1Bx13, Glu-1Bx14 (+)/Bx20, Glu-1By16, and Glu-1By18 were distinguished using the newly developed PCR markers. Additionally, the Glu-1Bx13 and Glu-1Bx14 (+)/Bx20 were distinguished by insertions and deletions in their promoter regions. The Glu-1Bx6, Glu-1Bx7, Glu-1By9, Glu-1Bx14 (-), and Glu-1By15/By20 alleles were distinguished by using insertions and deletions in the gene-coding region. Glu-1By13, Glu-1By16, and Glu-1By18 were dominantly identified in the gene-coding region. We also developed a marker to distinguish between the two Glu-1Bx14 alleles. However, the Glu-1Bx14 (+) + Glu-1By15 and Glu-1Bx20 + Glu-1By20 allele combinations could not be distinguished using PCR markers. The high-molecular-weight glutenin subunits of wheat varieties were analyzed by ultra-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the findings were compared with the results of PCR analysis. CONCLUSIONS: Seven Glu-1Bx and four Glu-1By allele detection markers were developed to detect nine Glu-1Bx and seven Glu-1By locus alleles, respectively. Integrating previously reported markers and 11 newly developed PCR markers improves allelic identification of the Glu-B1 locus and facilitates more effective analysis of Glu-B1 alleles molecular variations, which may improve the end-use quality of wheat.

In vitro protein digestibility of RuBisCO-enriched wheat dough: a comparative study with pea and gluten proteins.

Growing demand for sustainable, plant-based protein sources has stimulated interest in new ingredients for food enrichment. This study investigates the nutritional and digestive implications of enriching wheat dough with RuBisCO, in comparison to pea protein-enriched and gluten-enriched doughs. The protein quality and digestibility of these enriched doughs were analysed through dough characterization, in vitro digestion experiments and biochemical analysis of digesta. Our findings indicate that an enrichment at 10% of RuBisCO or pea proteins improves the chemical score and the in vitro PDCAAS (IV-PDCAAS) score of wheat dough as compared to the control dough. Digestibility assays suggest that RuBisCO introduction modifies the protein hydrolysis kinetics: the nitrogen release is lower during gastric digestion but larger during intestinal digestion than other samples. The analysis of the protein composition of the soluble and insoluble parts of digesta, using size-exclusion chromatography, reveals that the protein network in RuBisCO-enriched dough is more resistant to gastric hydrolysis than the ones of other doughs. Indeed, non-covalently bound peptides and disulfide-bound protein aggregates partly composed of RuBisCO subunits remain insoluble at the end of the gastric phase. The digestion of these protein structures is then mostly performed during the intestinal phase. These results are also discussed in relation to the digestive enzymatic cleavage sites, the presence of potential enzyme inhibitors, the protein aggregation state and the secondary structures of the protein network in each dough type.

Possible interaction between pectin and gluten alters the starch digestibility and texture of wheat bread.

This study incorporated citrus pectin in wheat bread, aiming to develop breads with both desirable texture and slow starch digestibility. Results showed that starch digestibility in wheat bread decreased over the addition of pectin, and the maximum starch digested amount decreased by 6.6 % after the addition of 12 % pectin (wheat flour weight basis). The addition of pectin transferred part of the rapidly digestible starch into slowly digestible starch, and reduced the binding rate constant between slowly digestible starch and digestive enzymes, resulting in overall reduced starch digestibility. Furthermore, the addition of 4 % pectin contributed to the development of wheat bread with softer texture and increased specific volume. Mechanistically, the lowered starch digestibility of wheat bread after the pectin addition was due to (1) residual outermost swollen layer of starch granules, (2) protein and pectin interactions, and (3) increased short-range ordering of starch. This study, therefore, suggests that the addition of an appropriate amount of citrus pectin has the potential to develop bread with both a low glycemic index and desirable texture.

Generation of major glutelin-deficient (GluA, GluB, and GluC) semi-dwarf Koshihikari rice line.

KEY MESSAGE: We generated a new Koshihikari rice line with a drastically reduced content of glutelin proteins and higher lodging resistance by using new and conventional plant breeding techniques. Using CRISPR/Cas9-mediated genome editing, we generated mutant rice with drastically decreased contents of major glutelins. A Koshihikari rice mutant line, a123, lacking four glutelins (GluA1, GluA2, GluB4, and GluB5) was used as a host, and another five major glutelin genes (GluA3, GluB1a, GluB1b, GluB2, and GluC) were knocked out through two iterations of Agrobacterium-mediated transformation. Mutant seeds were deficient in the GluA family, GluB family, and GluC, and the line obtained was named GluABC KO. Glutelin content was much lower in GluABC KO than in the existing low-glutelin rice mutant LGC-1. A null segregant of GluABC KO was selected using new-generation sequencing and backcrossing, and the sd-1 allele for the semi-dwarf trait was introduced to increase lodging resistance.

Intestinal mRNA expression analysis of polarity-related genes identified the discriminatory ability of CRB3 as a diagnostic marker for celiac disease.

BACKGROUND: Celiac disease (CD) is a chronic autoimmune disorder characterized by an abnormal immune response to gluten, a protein found in wheat, barley, and rye. It is well established that the integrity of epithelial tight junctions (TJs) and adherens junctions (AJs) plays a crucial role in the pathogenesis of CD. These junctional complexes contribute to the apical-basal polarity of the intestinal epithelial cells, which is crucial for their proper functioning. METHODS: Sixty CD subjects, and 50 controls were enrolled in the current study. Mucosal samples were obtained from the distal duodenum, total RNA was extracted and complementary DNA was synthesized. The relative expression levels of the desired genes were evaluated by quantitative real-time polymerase chain reaction based on ΔΔCt method. The gene-gene interaction network was also constructed using GeneMANIA. RESULTS: CRB3 (p = .0005), LKB1 (p < .0001), and SCRIB (p = .0005) had lower expression in CD patients compared to controls, while PRKCZ expression did not differ between groups (p > .05). CRB3 represented a significant diagnostic value for differentiating CD patients from the control group (p = .02). CONCLUSION: The aim of the current study was to evaluate the changes in the mRNA expression levels of SCRIB, PRKCZ, LKB1, and CRB3 genes in the small intestinal biopsy samples of CD patients in comparison to the healthy control subjects. Our data uncover the importance of polarity-related genes (especially CRB3) in CD pahtomechanism, that may facilitate the planning of the future studies looking for finding innovative diagnostic and therapeutic strategies for CD.

Production of the prolyl endoprotease (PEP) from Aspergillus sp. FSDE 16 by solid-state fermentation (SSF) and use for producing a gluten-free beer.

Beer is a beverage that contains gluten and cannot be consumed by people with celiac disease. In this context, the enzyme prolyl endoprotease (PEP) can be used to reduce the gluten content in beer. The present study aimed to produce the PEP from Aspergillus sp. FSDE 16 using solid-state fermentation with 5 conditions and comparing with a similar commercial enzyme produced from Aspergillus niger in the production of a gluten-free beer. The results of the performed cultures showed that during the culture, the most increased protease activity (54.46 U/mL) occurred on the 4th day. In contrast, for PEP, the highest activity (0.0356 U/mL) was obtained on the 3rd day of culture in condition. Regarding beer production, cell growth, pH, and total soluble solids showed similar behavior over the 7 days for beers produced without enzyme addition or with the addition of commercial enzyme and with the addition of the enzyme extract produced. The addition of the enzyme and the enzyme extract did not promote changes, and all the beers produced showed similar and satisfactory results, with acid pH between 4 and 5, total soluble solids ranging from 4.80 to 5.05, alcohol content ranging from 2.83% to 3.08%, and all beers having a dark character with deep amber and light copper color. Gluten removal was effectively using the commercial enzyme and the enzyme produced according to condition (v) reaching gluten concentrations equal to 17 ± 5.31 and 21.19 ± 11.28 ppm, respectively. In this way, the production of the enzyme by SSF and its application in the removal of gluten in beer was efficient.

Synergistic effects of ternary mixture formulation and process parameters optimization in a sequential approach for enhanced L-asparaginase production using agro-industrial wastes.

A novel ternary mixture of inexpensive and nutrient-rich agro-substrates comprising groundnut de-oiled cake, corn gluten meal, and soybean meal has been explored to enhance the L-asparaginase production in solid-state fermentation. To achieve the aim, a hybrid strategy was implemented by utilizing a combination of a mixture design and artificial neural networks. The study initiated with the judicious selection of the agro-substrates based on their low C/N content in comparison to the control using the CHNS elemental analysis. The mixture composition of soybean meal (49.0%), groundnut de-oiled cake (31.5%), and corn gluten meal (19.5%) were found optimum using the simplex lattice mixture design. The agro-industrial substrates mix revealed synergistic effects on the L-asparaginase production than either of the substrates alone. The maximum L-asparaginase activity of 141.45 ± 5.24 IU/gds was observed under the physical process conditions of 70% moisture content, autoclaving period of 30 min and 6.0 pH by adopting the machine learning-derived artificial neural network (ANN) methodology. The ANN modeling showed excellent prediction ability with a low mean squared error of 0.7, a low root mean squared error of 0.84, and a high value of 0.99 for regression coefficient. Moisture content (%) was assessed to be the most sensitive process parameter in the global sensitivity analysis. The net outcome from the two sequential optimization designs is the selection of the ideal mixture composition followed by the optimum physical process parameters. The application of the enzyme demonstrated significant cytotoxicity against leukemia cell line and therefore exhibited an anti-cancer effect. The present study reports a novel mixture combination and methodology that can be used to lower the cost and enhance the production of L-asparaginase using an agro-industrial substrate mixture.

Sulfated polysaccharides accelerate gliadin digestion and reduce its toxicity.

Celiac disease and other types of gluten intolerance significantly affect the life quality of patients making them restrict the diet removing all food produced from wheat, rye, oat, and barley flour, and some other products. These disorders arise from protease resistance of poorly soluble proteins prolamins, contained in gluten. Enhanced proteolytic digestion of gliadins might be considered as a prospective approach for the treatment of celiac disease and other types of gluten intolerance. Herein, we tested a range of sulfated polymers (kappa-carrageenan, dextran sulfate and different polysaccharides from brown seaweeds, and a synthetic polystyrene sulfonate) for the ability to activate gliadin digestion by human digestive proteases, pepsin and trypsin. Sulfated polysaccharide from Fucus evanescens enhanced proteolytic digestion of gliadins from wheat flour and reduced its cytotoxicity on intestinal epithelial Caco-2 cell culture. Regarding the non-toxic nature of fucoidans, the results provide a basis for polymer-based drugs or additives for the symptomatic treatment of gluten intolerance.

Inhibition mechanism of rice glutelin on extruded starch digestion: From the structural properties of starch and enzyme activity.

To increase the anti-digestion ability of extruded rice starch (ERS), the influence of rice glutelin (RG) on digestive and structural characteristics of ERS were investigated. The resistant starch content increased from 4.49 % to 18.08 % as the RG content increased, while the digestion rate and digestion velocity constant were reduced by the incorporation of RG. Morphological observations showed that ERS was adhered and encapsulated by RG, and the specific area of starch granules were decreased after the addition of RG. The results of XRD and FTIR suggested that the long-range and short-range orders of ERS were improved due to the complexation with RG. The thickness of crystalline of ERS was increased while its amorphous region thickness was reduced by the supplementation with RG. The 1H NMR and 13C NMR data revealed that the branching degree and double helix content of ERS was increased by 46.24 % and 52.67 % when RG content reached to 12 %. Additionally, the addition of RG altered the molecular weight and chain length distribution of ERS. The α-amylase activity and glucoamylase activity was inhibited by RG. These results could provide a valuable basis for the application of RG in extruded rice starchy foods with lower glycemic index.

Down-Regulation of Rice Glutelin by CRISPR-Cas9 Gene Editing Decreases Carbohydrate Content and Grain Weight and Modulates Synthesis of Seed Storage Proteins during Seed Maturation.

The glutelins are a family of abundant plant proteins comprised of four glutelin subfamilies (GluA, GluB, GluC, and GluD) encoded by 15 genes. In this study, expression of subsets of rice glutelins were suppressed using CRISPR-Cas9 gene-editing technology to generate three transgenic rice variant lines, GluA1, GluB2, and GluC1. Suppression of the targeted glutelin genes was confirmed by SDS-PAGE, Western blot, and q-RT-PCR. Transgenic rice variants GluA1, GluB2, and GluC1 showed reduced amylose and starch content, increased prolamine content, reduced grain weight, and irregularly shaped protein aggregates/protein bodies in mature seeds. Targeted transcriptional profiling of immature seeds was performed with a focus on genes associated with grain quality, starch content, and grain weight, and the results were analyzed using the Pearson correlation test (requiring correlation coefficient absolute value ≥ 0.7 for significance). Significantly up- or down-regulated genes were associated with gene ontology (GO) and KEGG pathway functional annotations related to RNA processing (spliceosomal RNAs, group II catalytic introns, small nucleolar RNAs, microRNAs), as well as protein translation (transfer RNA, ribosomal RNA and other ribosome and translation factors). These results suggest that rice glutelin genes may interact during seed development with genes that regulate synthesis of starch and seed storage proteins and modulate their expression via post-transcriptional and translational mechanisms.

Effects of Aegilops longissima chromosome 1Sl on wheat bread-making quality in two types of translocation lines.

KEY MESSAGE: Two wheat-Ae. longissima translocation chromosomes (1BS·1SlL and 1SlS·1BL) were transferred into three commercial wheat varieties, and the new advanced lines showed improved bread-making quality compared to their recurrent parents. Aegilops longissima chromosome 1Sl encodes specific types of gluten subunits that may positively affect wheat bread-making quality. The most effective method of introducing 1Sl chromosomal fragments containing the target genes into wheat is chromosome translocation. Here, a wheat-Ae. longissima 1BS·1SlL translocation line was developed using molecular marker-assisted chromosome engineering. Two types of translocation chromosomes developed in a previous study, 1BS·1SlL and 1SlS·1BL, were introduced into three commercial wheat varieties (Ningchun4, Ningchun50, and Westonia) via backcrossing with marker-assisted selection. Advanced translocation lines were confirmed through chromosome in situ hybridization and genotyping by target sequencing using the wheat 40 K system. Bread-making quality was found to be improved in the two types of advanced translocation lines compared to the corresponding recurrent parents. Furthermore, 1SlS·1BL translocation lines displayed better bread-making quality than 1BS·1SlL translocation lines in each genetic background. Further analysis revealed that high molecular weight glutenin subunit (HMW-GS) contents and expression levels of genes encoding low molecular weight glutenin subunits (LMW-GSs) were increased in 1SlS·1BL translocation lines. Gliadin and gluten-related transcription factors were also upregulated in the grains of the two types of advanced translocation lines compared to the recurrent parents. This study clarifies the impacts of specific glutenin subunits on bread-making quality and provides novel germplasm resources for further improvement of wheat quality through molecular breeding.

Ultrasonic-assisted resting of Tartary buckwheat dough: Study on its effect and mechanism.

Utilizing natural hypoglycemic ingredients in staple foods is a safe and effective way to improve diabetes. High Tartary buckwheat noodles have garnered research interest due to their hypoglycemic properties. However, increasing the Tartary buckwheat content poses challenges in noodle processing and affects their edible quality. Effective resting is a critical link to improve the processing performance of noodle and edible quality of noodle. Therefore, research was conducted on ultrasound assisted resting of Tartary buckwheat dough (TBD) to explore its feasibility and mechanism in improving the quality of Tartary buckwheat noodle. The results indicated that ultrasound treatment effectively promoted the migration of weakly-bound water towards strongly-bound water, thereby enhancing the gluten protein network structure and increasing the α-helix and ß-sheet contents significantly (p < 0.05). Furthermore, Texture analysis indicated decreased hardness and adhesion, and increased elasticity and stretching distance in the final noodles. Ultrasound-assisted maturation pre-treatment shortens TBD's dough's resting time and improves noodle quality, according to this study.

Celiac disease: mechanisms and emerging therapeutics.

Celiac disease (CeD) is a widespread, gluten-induced, autoimmune disorder that lacks any medicinal therapy. Towards the goal of developing non-dietary treatments for CeD, research has focused on elucidating its molecular and cellular etiology. A model of pathogenesis has emerged centered on interactions between three molecular families: specific class II MHC proteins on antigen-presenting cells (APCs), deamidated gluten-derived peptides, and T cell receptors (TCRs) on inflammatory CD4+ T cells. Growing evidence suggests that this pathogenic axis can be pharmacologically targeted to protect patients from some of the adverse effects of dietary gluten. Further studies have revealed the existence of additional host and environmental contributors to disease initiation and tissue damage. This review summarizes our current understanding of CeD pathogenesis and how it is being harnessed for therapeutic design and development.

Proteome Changes Resulting from Malting in Hordein-Reduced Barley Lines.

Hordeum vulgare L., commonly known as barley, is primarily used for animal feed and malting. The major storage proteins in barley are hordeins, known triggers of celiac disease (CD). Here, sequential window acquisition of all theoretical mass spectra (SWATH)-MS proteomics was employed to investigate the proteome profile of grain and malt samples from the malting barley cultivar Sloop and single-, double-, and triple hordein-reduced lines bred in a Sloop background. Using a discovery proteomics approach, 2688 and 3034 proteins were detected from the grain and malt samples, respectively. By utilizing label-free relative quantitation through SWATH-MS, a total of 2654 proteins have been quantified from grain and malt. The comparative analyses between the barley grain and malt samples revealed that the C-hordein-reduced lines have a more significant impact on proteome level changes due to malting than B- and D-hordein-reduced lines. Upregulated proteins in C-hordein-reduced lines were primarily involved in the tricarboxylic acid cycle and fatty acid peroxidation processes to provide more energy for seed germination during malting. By applying proteomics approaches after malting in hordein-reduced barley lines, we uncovered additional changes in the proteome driven by the genetic background that were not apparent in the sound grain. Our findings offer valuable insights for barley breeders and maltsters seeking to understand and optimize the performance of gluten-free grains in malt products.

Wheat gluten amyloid fibrils: Conditions, mechanism, characterization, application, and future perspectives.

Amyloid fibrils have excellent structural characteristics, such as a high aspect ratio, excellent stiffness, and a wide availability of functional groups on the surface. More studies are now focusing on the formation of amyloid fibrils using food proteins. Protein fibrillation is now becoming recognized as a promising strategy for enhancing the function of food proteins and expanding their range of applications. Wheat gluten is rich in glutamine (Q), hydrophobic amino acids, and the α-helix structure with high ß-sheet tendency. These characteristics make it very easy for wheat gluten to form amyloid fibrils. The conditions, formation mechanism, characterization methods, and application of amyloid fibrils formed by wheat gluten are summarized in this review. Further exploration of amyloid fibrils formed by wheat gluten will reveal how they can play a significant role in food, biology, and other fields, especially in medicine.

Dietary wheat gluten induces astro- and microgliosis in the hypothalamus of male mice.

Gluten, which is found in cereals such as wheat, rye and barley, makes up a major dietary component in most western nations, and has been shown to promote body mass gain and peripheral inflammation in mice. In the current study, we investigated the impact of gluten on central inflammation that is typically associated with diet-induced obesity. While we found no effect of gluten when added to a low-fat diet (LFD), male mice fed high fat diet (HFD) enriched with gluten increased body mass and adiposity compared with mice fed HFD without gluten. We furthermore found that gluten, when added to the LFD, increases circulating C-reactive protein levels. Gluten regardless of whether it was added to LFD or HFD led to a profound increase in the number of microglia and astrocytes in the arcuate nucleus of the hypothalamus, as detected by immunohistochemistry for ionised calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP), respectively. In mice fed LFD, gluten mimicked the immunogenic effects of HFD exposure and when added to HFD led to a further increase in the number of immunoreactive cells. Taken together, our results confirm a moderate obesogenic effect of gluten when fed to mice exposed to HFD and for the first-time report gluten-induced astro- and microgliosis suggesting the development of hypothalamic injury in rodents.

Understanding the Molecular Basis for Enhanced Glutenase Activity of Actinidin using Structural Bioinformatics.

BACKGROUND: Management of gluten intolerance is currently possible only by consumption of a gluten-free diet (GFD) for a lifetime. The scientific community has been searching for alternatives to GFD, like the inclusion of natural proteases with meals or pre-treatment of gluten-containing foods with glutenases. Actinidin from kiwifruit has shown considerable promise in digesting immunogenic gliadin peptides compared to other plant-derived cysteine proteases. METHODS: In this study, we aimed to understand the structural basis for the elevated protease action of actinidin against gliadin peptides by using an in silico approach. RESULTS: Docking experiments revealed key differences between the binding of gliadin peptide to actinidin and papain, which may be responsible for their differential digestive action. CONCLUSION: Sequence comparison of different plant cysteine proteases highlights amino acid residues surrounding the active site pocket of actinidin that are unique to this molecule and hence likely to contribute to its digestive properties.

Molecular Characterization and Marker Development of the HMW-GS Gene from Thinopyrum elongatum for Improving Wheat Quality.

The quality of wheat primarily depends on its storage protein quality, especially in regards to gluten content and high-molecular-weight glutenin subunits (HMW-GS). The number of HMW-GS alleles is limited in bread wheat (Triticum aestivum L.), whereas it is abundant in wheat relatives. Therefore, HMW-GS alleles from wheat relatives could provide a potential for improving quality in wheat breeding. Thinopyrum elongatum (EE) is one of the relatives of wheat. The E genome is closely related to the ABD genome in wheat; therefore, Th. elongatum is often used as an excellent exogenous gene donor for wheat genetic improvement. In this study, the high-molecular glutenin subunit gene was cloned and sequenced from Th. elongatum. A specific molecular marker for identifying the Glu-1Ey subunit gene was developed and applied to detected wheat-Th. elongatum alien introgression lines. Quality analysis indicated that the substitution and addition lines containing Th. elongatum alleles significantly (p < 0.05) increased grain protein content by 3.76% to 5.11%, wet-gluten content by 6.55% to 8.73%, flour 8-MW by 0.25% to 6.35%, and bread volume value by 33.77 mL to 246.50 mL, in comparing it with Chinese Spring. The GMP content and lactic acid SRC showed significant positive correlations with flour processing quality and might be used as indicators for wheat quality. The results were expected to provide a novel route for improving processing quality in wheat quality breeding.

Comparing the standardized amino acid digestibility of an alternative protein source with commercially available protein-based ingredients using the precision-fed cecectomized rooster assay.

Using single-cell-based proteins in pet foods is of interest, but little testing has been done. Therefore, our objective was to determine the amino acid (AA) digestibilities, assess protein quality of a novel microbial protein (MP) (FeedKind), and compare it with other protein-based ingredients using the precision-fed cecectomized rooster assay. Test ingredients included: MP, chicken meal (CM), corn gluten meal (CGM), pea protein (PP), and black soldier fly larvae. Thirty cecectomized roosters (n = 6/ingredient) were randomly assigned to test ingredients. After 24 h of feed withdrawal, roosters were tube-fed 15 g test ingredient and 15 g corn, and then excreta were collected for 48 h. Endogenous AA corrections were made using additional roosters. Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality according to Association of American Feed Control Officials (AAFCO), The European Pet Food Industry Federation, and National Research Council reference values for growing and adult dogs and cats. Data were analyzed using the Mixed Models procedure of SAS 9.4, with P ≤ 0.05 being significant. All reactive lysine:total lysine ratios, an indicator of heat damage, were higher than 0.9, except for CM (0.86). Digestibility of indispensable and dispensable AA were >85% and >80% for MP, respectively, with indispensable AA digestibilities being >80% for all other ingredients. In general, CGM had the highest, while CM had the lowest AA digestibilities. Two exceptions were lysine and tryptophan. Lysine digestibility for MP was higher than that of all other ingredients, while tryptophan digestibility for MP was higher than that of CM, CGM, and PP. Threonine digestibility was highest for CGM and MP. Valine digestibility was highest for CGM, PP, and MP. DIAAS-like calculations identified limiting AA of each ingredient and depended on the reference used and life stage and species of animal. Using AAFCO guidelines, all DIAAS-like values for MP were >100 suggesting that it could be used as the sole source of protein in adult dog and cat diets; only methionine had DIAAS-like values <100 for growing kittens. For dogs, limiting AA was most commonly methionine, threonine, and tryptophan in the other protein sources. For cats, limiting AA was most commonly lysine and methionine. Lysine was severely limited in CGM across all life stages considered. Further research in dogs and cats is necessary, but our data suggest that the MP tested has high AA digestibilities and is a high-quality protein source that may be useful in pet foods.

Single-cell-based proteins are of interest for use in pet foods, but little testing has been done. The objective of this experiment was to compare the amino acid (AA) digestibilities and protein quality of a novel microbial protein (MP) (FeedKind) with chicken meal (CM), corn gluten meal (CGM), pea protein (PP), and black soldier fly larvae ingredients using the precision-fed cecectomized rooster assay. Cecectomized roosters were tube-fed the test ingredients and excreta were collected. All reactive lysine:total lysine ratios, an indicator of heat damage, were higher than 0.9, except for CM. Digestibility of indispensable and dispensable AA were >85% and >80% for MP, respectively, with indispensable AA digestibilities being >80% for all other ingredients. In general, CGM had the highest, while CM had the lowest AA digestibilities. Lysine and tryptophan were exceptions, being highest for MP. Threonine and valine digestibilities were also high for MP. Digestible indispensable AA score-like values identified limiting AA of each ingredient. Limiting AA was most commonly methionine, threonine, and tryptophan for dogs and lysine and methionine for cats. Our data suggest that the MP tested has high AA digestibilities and is a high-quality protein source that may be useful in pet foods.

Effects of FODMAPs and Gluten on Gut Microbiota and Their Association with the Metabolome in Irritable Bowel Syndrome: A Double-Blind, Randomized, Cross-Over Intervention Study.

BACKGROUND: A mechanistic understanding of the effects of dietary treatment in irritable bowel syndrome (IBS) is lacking. Our aim was therefore to investigate how fermentable oligo- di-, monosaccharides, and polyols (FODMAPs) and gluten affected gut microbiota and circulating metabolite profiles, as well as to investigate potential links between gut microbiota, metabolites, and IBS symptoms. METHODS: We used data from a double-blind, randomized, crossover study with week-long provocations of FODMAPs, gluten, and placebo in participants with IBS. To study the effects of the provocations on fecal microbiota, fecal and plasma short-chain fatty acids, the untargeted plasma metabolome, and IBS symptoms, we used Random Forest, linear mixed model and Spearman correlation analysis. RESULTS: FODMAPs increased fecal saccharolytic bacteria, plasma phenolic-derived metabolites, 3-indolepropionate, and decreased isobutyrate and bile acids. Gluten decreased fecal isovalerate and altered carnitine derivatives, CoA, and fatty acids in plasma. For FODMAPs, modest correlations were observed between microbiota and phenolic-derived metabolites and 3-indolepropionate, previously associated with improved metabolic health, and reduced inflammation. Correlations between molecular data and IBS symptoms were weak. CONCLUSIONS: FODMAPs, but not gluten, altered microbiota composition and correlated with phenolic-derived metabolites and 3-indolepropionate, with only weak associations with IBS symptoms. Thus, the minor effect of FODMAPs on IBS symptoms must be weighed against the effect on microbiota and metabolites related to positive health factors.