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Insights into the proteomic profile of newly harvested corn and metagenomic analysis of the broiler intestinal microbiota

Insights into the proteomic profile of newly harvested corn and metagenomic analysis of the... Background: The use of newly harvested corn in feed causes wet droppings in broilers and increased feed cost which was termed as “new season grain problem”. The present study was conducted to evaluate the proteomic profile of newly harvested corn and the subsequent influence on intestinal microbiol community for broiler chickens. Methods: Newly harvested corn stored for either half a month (HM) or two months (TM) was used, and the pasting properties, total soluble sugars, and proteomic analysis technology was used to explore the influence of storage on natural aging corn properties. Additionally, seventy-two 7-day-old Ross 308 male broiler chicken were fed with different stored corn. Apparent metabolizable energy (AME), digesta viscosity, intestinal morphology and microbiota were examined to explore the influence of feed corn storage on broiler chickens. Results: Pasting properties in the TM corn exhibited decreased viscoelastic properties. Proteomic studies found a total of 26 proteins that were differentially expressed between the two treatment groups. Proteins involved in starch and polysaccharides biosynthesis were upregulated in TM compared with HM. Chickens fed on TM diet had higher relative energy utilization compared to the HM birds. With increased corn storage, the relative digesta viscosity decreased significantly (P ≤ 0.05). The total number of goblet cells and lymphocytes was lower in chickens fed the TM diet. The microbiota data showed that the TM chickens had decreased abundance of diarrheal bacteria such as Hungatella hathewayi and Bacteroides fragilis, and increased butyrate-producing bacteria such as Alistipes compared to the HM chickens. Conclusions: Storage of newly harvested corn induced the synthetic reaction of large molecules and changed the solubility of starch and protein with increasing soluble sugars and decreasing pasting properties that may improve the fermentation of intestinal microbiota, improve the energy utilization and protect gut health without the risk of diarrhea. Keywords: Broilers, Corn, Metagenome, Proteome, Storage * Correspondence: yuanjm@cau.edu.cn State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, 100193 Beijing, China Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 2 of 15 Introduction fed to broiler chickens and how they affect performance Corn is the most abundantly grown cereal grain and has and health are unclear. high nutritional value, especially for poultry and swine. We herein first employed a combined high-throughput As corn production is seasonal, corn grains need to be label-free comparative proteomics and metagenomics to stored throughout the year to provide sufficient supplies study the biological mechanisms that underlie the rela- for feed industries. However, there is an asynchrony of tionship between the composition of natural aging corn supply and demand that leads to a large number of and the diversity of the complex intestinal microbial newly harvested corn being processed as animal feed community of broiler chickens. during each harvest season. The use of newly harvested corn in poultry diets is especially problematic because it Materials and methods decreases the apparent metabolizable energy (AME) and Plant material feed conversion ratio and causes wet droppings and in- One newly harvested mixed corn variety corn (ZhengDan creased feed cost [1–3]. Nutritionists have termed this 958) was sourced from Hebei Province. The corn was sown problem the “new season grain problem” [4]. in March 2015 and harvested in September 2015. After nat- Newly harvested grain should be stored for a period of ural drying to 14% moisture content, intact corn grains several weeks or months to enhance its nutritional value were stored from September to November in a warehouse [2]. During storage, respiratory and metabolic events located at the China Agricultural University Poultry Experi- continue, and changes in internal factors, such as starch, mental Base without air conditioning to simulate typical endogenous enzymes and carbonyl compounds in cell grain storage conditions. The newly harvested corn was walls and external factors occur [5, 6]. Among these fac- sampled after storage for half a month (HM) or two tors, endogenous proteolytic enzymes play a central role months (TM), frozen immediately in liquid nitrogen and in regulating the synthesis and decomposition of carbo- stored at − 80 °C until further analysis. At the same time, hydrates and proteins [7, 8]. broilers fed the corn stored for different period of the time Proteomics is an important tool that can be used to were used to further examine the differences between dif- determine the biological roles and functions of individ- ferent storage times of corn. To eliminate the influence of ual proteins that govern grain seed quality and allow for the environment and animals, the same variety of corn the systematic analysis of complex cellular mechanisms stored for one year with the same moisture content was [9]. Reports of corn proteome expression during the arti- treated as the control in the broiler trial. ficial aging period indicate that artificial aging would in- crease proteases and breakdown stored proteins, and Determination of pasting properties, total soluble sugar, impair metabolism and energy supplies [10, 11]. How- glucose and fructose ever, the physicochemical properties of naturally aging The corn samples were analyzed for pasting properties corn have yet to be elucidated. Our objective was to de- and soluble sugar. The pasting properties of corn mix- lineate the proteomic mechanisms that underlie differ- tures stored for different times and suspended in dis- ences between corn stored for different time. tilled water were determined by a Rapid Visco-Analyzer There is a clear link between bird performance and (model RVA-4C, Newport Scientific Pty. Ltd., Warrie- gut microbiota composition [12]. The intestinal micro- wood, Australia) and estimated by the method of biota plays an important role in maintaining normal gut Achayuthakan and Suphantharika [17]. Total soluble function and contributes to the development of func- sugars were estimated by the phenol-sulfuric acid tional gastrointestinal symptoms by modulating the sig- method of Dubois et al. [18], using sucrose as the stand- naling pathways of hosts [13]. We have previously ard. Glucose and fructose were determined by the HPLC shown that using newly harvested corn leads to over- method of Wilson et al. [19]. feeding and increased ileum lesions and injuries in broiler chickens [3]. Intestinal injury is associated with diarrhea and impaired gut resistance to pathogens [14]. Label-free proteomic analysis process Leonard et al. [15] reported that grain (rice, oats, barley Protein extraction and digestion and corn) protein is one of the most common triggers of The HM and TM samples were thawed at − 80 °C and enterocolitis syndrome. Additionally, non-digestible then ground and hereafter, 100 mg of each sample was polysaccharides in corn can be broken down by mem- weighed. Then 400 μL SDT lysis buffer (4% SDS, 100 bers of the intestinal microbiota, producing monosac- mmol/L Tris-Hcl, 1 mmol/L DTT, pH 7.6) was added to charides and short-chain fatty acids (SCFAs), leading to the sample, followed by solubilization for 20s with a tis- unbalanced energy status [16]. Intestinal bacterial com- sue homogenizer; this was repeated 5 times. After 20 position changes and their subsequent metabolic min ultrasonic treatment, the mixture was centrifuged changes when a diet containing newly harvested corn is for 30 min at 10,000 × g at 4 °C, and the supernatant was Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 3 of 15 saved for subsequent experiments. The BCA assay was differentially expressed proteins was performed using a used for protein quantification. mixture model-based method [22]. The significance of Protein digestion (200 μL for each sample) was per- the differentially expressed proteins between the samples formed using the FASP procedure described by Wis- was examined with the cutoff values P ≤ 0.05 and FDR ≤ niewski [20]. Briefly, the detergent DTT, and other 0.05. low molecular weight components were removed using 200 μL UA buffer (8 mol/L Urea, 150 mmol/L Bioinformatics analysis Tris-HCl, pH 8.0) and facilitated by centrifugation Differentially expressed proteins (DEPs) were sub- (14,000 × g × 15 min). Then, the 100 μLIAA (50 jected to Core Expression Analysis. In this study, the mmol/L IAA in UA) was added in UA buffer, and DEPs between HM and TM was undertaken. The the mixture was oscillated for 1 min at 600 r/min and ratio values in the datasets were converted to fold allowed to settle for 30 min in the dark at room change values, where the negative inverse was taken temperature. The filter was washed with UA buffer for values between 0 and 1. The sequence data for (100 μL) and 100 μLofNH HCO buffer twice. Sub- the selected differentially expressed proteins were 4 3 sequently, 40 μL trypsin buffer (4 μL Trypsin in 40 μL retrieved from the UniProtKB database in batches and NH HCO ) was added. The mixture was oscillated for FASTA format. Following the annotation and annota- 4 3 1 min at 600 r/min and allowed to settle for 16–18 h tion augmentation steps, the studied proteins were at 37 °C, and the resulting peptides were collected. blasted against KEGG GENES (plants) to retrieve the The filtrate was desalinized with C18 SD Extraction KOs and were subsequently mapped to pathways in Disk Cartridge and quantified at OD280. KEGG. LC-MS/MS analysis Animals and experimental design Approximately 2 μL product of protein digestion was The experiment was assigned to two periods following used for LC-MS/MS analysis and separated using a the corn storage time. During each period, seventy- nanoliter HPLC Ultimate 3000 system (Thermo Fisher two 7-day-old male broiler chickens (Ross 308) were Scientific, Louis, MO, USA). The mobile phase A was obtained from a commercial hatchery and grown for 0.1% formic acid solution, and the mobile phase B 14 d. In total, 144 Ross 308 male birds were selected. was 0.1% formic acid solution with 80% acetonitrile. Birds were randomly assigned to one of two dietary The chromatographic column C18 trap column (C18 treatments (HM vs control or TM vs control) (during 3 μm 0.10 × 20 mm) was balanced with 95% A solu- each time thirty-six birds fed with control corn, this tion. The sample was loaded onto the C18 trap col- process repeated twice). There were 6 replicates per umn through an automatic sampler and separated by treatment and 6 birds per replicate. Feed (pelleted) a chromatographic column (C18 1.9 μm 0.15 × 120 and water were provided ad libitum. The light regi- mm) at the flow rate of 600 nL/min. The gradient men was 23 L:1D and the room temperature was 28– elution procedure was as described in Additional file 30 °C. All the corn samples used to formulate basal 1:Table S1 broiler diets that met broiler recommendations of NY/T33–2004 (Table S2). Maxquant label-free quantification analysis The 6 resulting raw LC-MS/MS files were imported to Sample collection and DNA extraction the MaxQuant software (1.3.0.5) and Proteome Discov- The experimental layout is shown in Fig. 1. At 14 days of erer 2.0 (Thermo Fisher Scientific, Bremen, Germany) age, a metabolic experiment was conducted, and 3 ran- for database inquiry and LFQ label-free quantification domly selected chickens (remaining birds were still fed analysis. The database was corn_160605.fasta; enzyme: with basal diets) per cage were moved to the new cages trypsin; max missed cleavage: 2; fixed modifications: car- and fed with either HM, TM or control metabolic diets bamidomethyl (C); variable modifications: oxidation(M), (Table S3) for 3 d. Total excreta outputs and feed intakes acetyl (Protein N-term); peptide Mass Tolerance: ±15 were recorded from 15 to 18-day post hatching. Excreta ppm; Fragment mass tolerance: 20 mm; peptide confi- were dried in a forced-air oven at 60 °C for 48 h and the dence: high; peptide length: > 4. The cutoff for the global gross energy of excreta and metabolic diets were deter- false discovery rate (FDR) in peptide and protein identi- mined using an adiabatic bomb calorimeter to determine fication was 0.01. Label-free quantification was per- apparent metabolizable energy (AME). Feed and extract formed using MaxQuant as previously described [21]. nitrogen contents were determined by a macro Kjeldahl Intensity-based absolute quantification (iBAQ) in Max- method to get nitrogen retention for the calculation of Quant was performed to quantify protein abundance for nitrogen-corrected AME (AME ). The calculation equa- the identified peptides. An FDR estimation for tions are as follows: Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 4 of 15 Fig. 1 Experimental layout. Seventy two birds were divided into 2 groups, namely, HM and TM. On d 14, half birds in per treatment were selected to do metabolic experiment for three days. The left parts were still fed with experimental diets. On d 21, non-metabolic birds were killed to get samples for metagenome analysis (n =3) Metagenome analysis process AME ðÞ MJ=kg¼ðÞ Gross energy intake - Gross energy excretion diet =Feed intake DNA library construction and sequencing DNA libraries were generated using NEBNext® Ultra AMEn ðÞ MJ=kg¼ AME - 34:99 diet TM DNA Library Prep Kit for Illumina (NEB, USA) fol- ðÞ Nitrogen intake - Nitrogen excretion =1000 lowing manufacturer’s recommendations. Briefly, the DNA sample was fragmented by sonication to a size of At 21-day post-hatching, the 3 remaining birds in each 350 bp, then end-polished, A-tailed, and ligated with the cage were euthanized by injection with 5% pentobarbital full-length adaptor for Illumina sequencing. PCR prod- sodium. Immediately following euthanasia, the abdom- ucts were purified (AMPure XP system) and libraries inal cavity was opened and the duodenal contents, ap- were analysed for size distribution by Agilent2100 Bioa- proximately 3 cm lengths of duodenum were removed nalyzer and quantified using real-time PCR. The cluster- for gut morphological measurements. For viscosity mea- ing of the index-coded samples was performed on a cBot surements, jejunal digesta were collected and diluted Cluster Generation System. Then, the library prepara- with (1:1) distilled water and homogenized for 20 min at tions were sequenced on an Illumina HiSeq platform room temperature before centrifugation. The viscosity and paired-end reads were generated. was measured according to the method of Piel et al. [23]. Gene catalogue construction The cecum was collected within 5 min of euthanasia, We performed de novo assembly and gene prediction immediately placed in cryogenic vials, snap-frozen in li- for the high quality reads of 6 samples in stage I using quid nitrogen and stored at − 80 °C until DNA extrac- SOAP denovo v1.06 [24] and GeneMark v2.7 [25], re- tion. Total genomic DNA was isolated from 220 mg of spectively. All predicted genes were aligned pairwise frozen cecal contents using the QIAmp DNA Stoll Kit using BLAT and genes, of which over 90% of their (Qiagen GmbH, Hilden, Germany). The DNA concen- length can be aligned to another one with more than tration and purity were determined using a NanoDrop 95% identity (no gaps allowed), were removed as redun- 2000 spectrophotometer (Thermo Fisher Scientific, Wil- dancies, resulting in a non-redundant gene catalogue mington, DE, USA). Three samples per treatment were comprising of 659,733 genes. This gene catalogue from selected for further analysis. our cecal samples was further combined with the previ- ously constructed CD-HIT gene catalogue [26], by re- Morphological examination moving redundancies in the same manner. At last, we Intestinal samples were immersed in formaldehyde, be- obtained an updated gene catalogue that contains fore being fixed in Bouin’s solution and embedded in 362,337 genes. paraffin. The length of the intestinal villi and the depth of the intestinal crypt were measured with a linear scaled Taxonomic assignment of genes graticule. The number of goblet cells and lymphocytes Taxonomic assignment of the predicted genes was per- /μm area of the villus and crypts was measured by 25 formed using DIAMOND [27]. In our analysis, we col- squared graticules. lected the reference microbial genomes from Non- Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 5 of 15 Redundant (NR) of NCBI (v 2014-10-19), and then quantifications of 1984 proteins were divided into two aligned all genes onto the reference genomes. For each categories: proteins with quantitative ratios over 1.5 and gene, the highest scoring hit(s) above these two thresh- P ≤ 0.05 were considered upregulated, while proteins olds was chosen for the genus assignment. For the taxo- with quantitative ratios ≤1/1.5 (0.67) and P ≤ 0.05 were nomic assignment at the phylum level, the 65% identity considered downregulated. Venn diagram analysis was used instead. showed that 222 proteins were influenced by storage time, among these proteins (Fig. S1). Functional annotation The functional annotation and abundance analysis of Differentially expressed proteins in corn stored for KEGG (Kyoto Encyclopedia of Genes and Genomes), different times and carbohydrate enzyme (CAZy) (version: 2014.11.25) We identified a total of 26 proteins that were differen- were performed using BLASTP (e-value ≤ 1e-5). Based tially expressed, among which only 2 diminished propor- on the species abundance and functional abundance, the tionally to HM corn, while 24 proteins seemed to be abundance cluster analysis, and sample cluster analysis positively influenced by the increase in storage time were performed. (Table 2). Peptidyl-prolylcis-transisomerase and jasmonate-induced protein were downregulated in TM Statistical analysis compared with HM. The other identified proteins were LEfSe analysis uses the Kruskal-Wallis rank sum test to upregulated in TM compared with HM. Among these detect significantly different abundances and performs proteins, GRMZM2G162359_P01, GRMZM2G008216_ LDA scores to estimate the effect size (threshold: > 2). P02 and GRMZM5G825695_P01 proteins have no Statistical analyses were performed using SPSS software, known functions and no annotations could be found to version 16.0 (SPSS Inc., Chicago, IL, USA). The data assign or predict their functions. were subjected to an analysis of variance (ANOVA), the means were compared using Student’s t-tests, and the Functional annotation of identified proteins differences were considered significant at P ≤ 0.05. GO terms were assigned to the 222 differentially expressed proteins. Figure 3 shows GO annotations for Results proteins that were differentially expressed in HM and Corn properties TM. The identified proteins cover a wide range of cellu- The pasting characteristics of corn stored at different lar processes, molecular functions and cellular compo- times determined by the RVA are summarized in nents and can be classified into 22, 8, and 7 categories in Table 1. Storage time resulted in significant (P ≤ 0.05) these broad groups, respectively. The differentially decreases in peak viscosity, final viscosity, and setback expressed proteins in the biological functions category viscosity and an increase in pasting temperature. At the were mainly associated with cellular ketone metabolic, same time, a significant change in soluble sugars was ob- carboxylic acid metabolic and cellular amino acid bio- served during storage of corn for the two periods synthetic processes. The largest group within the mo- (Fig. 2A). Lower total concentrations (P ≤ 0.05) of glu- lecular function category was cofactor binding. The cose and fructose were found in corn stored for two cellular component functions mainly belong to the cyto- months (Fig. 2B). plasm and mitochondrion. KEGG results showed for the most differentially expressed proteins suggest that these Proteome variation of corn in response to different proteins are involved in 13 pathways (Fig. S2). storage times By merging all identified protein lists including those from two time points and three biological replicates, Bird properties affected by corn storage 2499 proteins were identified and 1984 proteins were The relative AME and AMEn values of each batch are quantified from all samples. For further analysis, the shown in Fig. 4. Both AME and AMEn were significantly TM/HM ratios were log transformed. Relative higher (P ≤ 0.05) in HM than in TM. Table 1 Pasting properties of different storage time corn Sample Peak viscosity RVA Breakdown RVA Final viscosity RVA Setback RVA Pasting temperature, °C HM 1796 ± 42.2 166 ± 21.4 3087 ± 123.0 1457 ± 71.6 77.6 ± 0.13 TM 1602 ± 68.6 99 ± 32.9 2711 ± 34.5 1208 ± 10.6 78.1 ± 0.13 P-value 0.014 0.162 0.007 0.004 0.036 Mean ± standard deviation HM, half months stored corn; TM, two months stored corn Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 6 of 15 Fig. 2 Storage effects on total soluble sugars (A) and total concentrations of glucose and fructose (B) of corn (mean ± SD, triplicate samples). (HM, half month stored corn; TM, two months stored corn) With increased corn storage, the relative digesta vis- Taxonomic characterization of the gut microbiota cosity decreased significantly (P ≤ 0.05) (Fig. 5A). The A total of 1,870,386 ORFs were predicted. Among these total number of goblet cells and lymphocytes was lower ORFs, 82.06%, 78.41%, 71.55%, 69.39%, 53.90%, 49.20% in chickens fed the TM diet (Fig. 5B). There were no sig- and 41.02% had annotations at the kingdom, phylum, nificant differences between treatments in crypt and villi class, order, family, genus and species levels, respectively. (Fig. S3). Unclassified ORFs accounted for 17.94% of unigenes, representing novel rhizosphere taxa. The abundance was Table 2 Proteins identified as differentially expressed from Label-free LC-MS/MS Accession number Protein description TM/HM ratio P-value KO GRMZM5G866758_P02 Acetyl-CoA acetyltransferase, cytosolic 1.75 0.002 GRMZM2G174883_P01 Legumin 1 1.67 0.012 K03671 GRMZM2G055434_P01 Early nodulin 1.94 0.013 GRMZM2G023347_P01 Prefoldin subunit 1.50 0.015 GRMZM2G010762_P01 Early nodulin-like protein 1.54 0.018 K00700 GRMZM2G020423_P01 Jasmonate-induced protein 0.61 0.019 K00626 GRMZM2G016958_P01 IAA-amino acid hydrolase 2.07 0.019 K01507 GRMZM2G041881_P01 Nascent polypeptide-associated complex subunit beta 4.27 0.020 GRMZM2G059353_P01 Non-green plastid inner envelope membrane protein 1.81 0.021 GRMZM2G162359_P01 Uncharacterized protein 1.97 0.021 K13448 GRMZM2G473463_P01 Mitochondrial import inner membrane translocase subunit 1.57 0.026 GRMZM2G105712_P05 60S acidic ribosomal protein 1.81 0.029 K07304 GRMZM2G397044_P02 Peptidyl-prolyl cis-trans isomerase 0.34 0.031 GRMZM2G163406_P01 Dirigent protein 2.45 0.033 GRMZM2G032628_P01 1,4-alpha-glucan-branching enzyme 2 1.95 0.034 K03232 GRMZM2G097457_P01 Pyruvate, phosphate dikinase 1.56 0.036 GRMZM2G164714_P02 Phosphoenolpyruvate carboxylase family protein 1.57 0.037 GRMZM2G071433_P01 Plasma membrane associated protein 1.60 0.038 K03016 GRMZM2G008216_P02 Uncharacterized protein 1.67 0.040 GRMZM2G445905_P03 Cellulose synthase 1.62 0.045 GRMZM5G825695_P01 Uncharacterized protein 1.85 0.045 K10999 GRMZM2G439201_P02 Elongation factor 1-beta 2 2.09 0.045 GRMZM2G026470_P01 Soluble inorganic pyrophosphatase 1 1.54 0.046 K01006 GRMZM2G306345_P01 Pyruvate orthophosphate dikinase1 1.61 0.047 GRMZM2G085260_P01 Desiccation-related protein 1.68 0.049 K02943 GRMZM2G046520_P01 Dirigent protein 2.18 0.050 K01703 HM, half months stored corn; TM, two months stored corn Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 7 of 15 Fig. 3 The proportion of the differentially expressed proteins categorized by function. A: Gene Ontology analysis for biological processes; B: Gene Ontology analysis for molecular function; C: Gene Ontology analysis for molecular function compared between groups, number of significantly chan- Firmicutes, Bacteroidetes, and Proteobacteria followed ged genes are showed in a Venn diagram. When com- by Actinobacteria, Tenericutes, Fusobacteria and Spiro- pared to HM corn treatment, a total of 97,141 genes chaetes. Bacteroidetes, Proteobacteria and Tenericutes were significantly changed in the TM groups, and were significantly enriched in stored corn subjects, while 110,678 genes were changed in the HM groups (Fig. S4). the relative abundance of Fusobacteria was significantly We aligned the reads to a catalog, and the majority of increased in newly harvested corn (P ≤ 0.05) (Fig. 6A). aligned reads were bacterial and dominated by the phyla Clostridium, Eubacterium, Bacteroides and Blatutia were Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 8 of 15 second axes variation, which accounted for 47.1% and 7.4%, respectively. In Fig. 7B, we highlight 7 species that were significantly correlated with one or both of the first two RDA axes. Among these, Acidaminococcus sp. CAG.917 was strongly associated with AME while Alis- tipes sp. CAG.157, Bacteroides dorei, Bacteroides unifor- mis and Oscillibacter ruminantium were correlated with AMEn. LEfSe analysis The LEfSe test detected differences in the relative abun- Fig. 4 Storage effects on relative AME and AMEn to control groups of 17 d broiler chickens (n = 6). (* P ≤ 0.05, ** P ≤ 0.01. HM, half dances of bacterial taxa across samples (Fig. 8). Among month stored corn; TM, two months stored corn) the genes from the species level, Alistipes and Firmicutes bacterium, Bacillus, Mycoplasma, Alistipes inops, Barne- siella intestinihominis, Barnesiella and Ruminococcus the most abundant genera in our treatments. Compared were significantly enriched in the TM treatment, while with the newly harvested corn, stored corn significantly Eubacterium desmoians, Hungatella hathewayi, Hunga- increased 8 kinds of genera bacterium such as Allstipes, tella, Lachnociostridum, Bacteroides fragilis, Fusobacter- Butyricicoccus and Mycoplasma (Fig. 6B). Species and ium and Eubacterium were significantly enriched in the genome level abundances were also calculated to deter- HM treatment (LDA > 2, P < 0.05). mine the composition of the gut microbiota (Fig. S5), we compared the composition HM and TM communities Functional analysis and bacterial metabolic processes and observed increases in Faecalibacterium prausnitzii, The function of bacterial assemblages associated with Barnesiella intestinihominis, Bacteroides dorei and Fir- different storage times of corn was cataloged using level micutes bacterium CAG:475 in TM compared with HM. 3 KEGG orthologs. Several KEGG level 2 modules were There were decreases in the abundance of Bacteroides different between the two treatments. HM treatment sig- fragilis, Subdoligranulum variabile, Ruminococcus tor- nificantly improved the metabolism of terpenoids and ques, Eubacterium sp. ER2, Clostridium sp. CAG:678 polyketides and amino acid metabolism, while TM had and Ruminococcaceae bacterium D16 in TM compared beneficially influenced metabolism of other amino acids, with HM. folding, sorting and degradation, glycan biosynthesis and metabolism and transport and catabolism and immune AME and AMEn are correlated with microbiota system (P ≤ 0.05, Fig. 9). At KEGG level 3, compared The global RDA model that selected AME and AMEn as with HM treatment, TM was enriched for carbohydrate the meaningful explanatory variables was significant metabolism (e.g., amino sugar and nucleotide sugar me- (P ≤ 0.05) (Fig. 7). The overall variation in species com- tabolism and pyruvate metabolism) (Fig. 10A), glycan position was attributed to these explanatory variables, of biosynthesis and metabolism (e.g., glycan degradation) which the majority were explained by the first and (Fig. 10B) and the immune system (e.g., RIG-I-like Fig. 5 Storage effects on relative digesta viscosity (A) and lymphocytes and goblet cells number in duodenum (n = 6). (* P ≤ 0.05, ** P ≤ 0.01. HM, half month stored corn; TM, two months stored corn) Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 9 of 15 Discussion The effect of storage on corn properties Compared to freshly harvested grain, a change in protein digestibility was reported by Paraginski et al. [5] and Ramchandran et al. [28], potentially due to protein and starch solubility. One of the most sensitive indices of the aging in grain is the change in pasting properties [29, 30] which is related to the granule size and soluble ma- terials of starch [31]. As storage duration increased, peak viscosity, final viscosity and setback decreased which were in agreement with previous studies [32, 33]. This is attributed to the formation of a complex between amyl- ose and lipids and thereby restriction of granular swelling. Dietary sugars (glucose and fructose) that escape ab- sorption in the mammalian intestine and reach the microbiota disrupt colonization by beneficial microbes [34]. In the current study we observed an increase in total soluble sugars and reductions in glucose and fruc- tose in the stored corn which were in concordance with the findings of Paraginski [5]. We observed that higher oligosaccharide and lower monosaccharide contents in stored corn could benefit gut health [35]. Differentially expressed proteins We studied the effect of corn storage and carbohydrate composition as storage is known to exhibit strong effects on starch biosynthesis [36]. Li et al. [37] reported that starch retrogradation is mainly caused by the aggrega- tion of amylose and occurs during the early stage of storage. 1,4-α-glucan-branching enzyme activity cata- lyzes ADP-glucose into amylopectin, inhibiting starch retrogradation [37, 38]. The upregulation of the 1,4-α- glucan-branching enzyme in TM suggested a decrease in the amount of amylose and an increase in amylopectin which could increase the water-insolubility of starch and protein [39]. During storage, respiratory and metabolic Fig. 6 Relative abundance of annotated phylum (A) and significant events continue. Pyruvate, phosphate dikinase and phos- different genus (B) in cecal microbiota of broilers. (HM, half month phoenolpyruvate carboxylase family proteins play key stored corn; TM, two months stored corn) * P ≤ 0.05 roles in CO transport and fixation, respectively [40]. Higher activities of these two enzymes under TM sug- gested a higher synthetic ratio of starch when carbon receptor signaling pathway) (Fig. 10C). Interestingly, we gain exceeds the carbon demands for growth (accumula- also found a different pathway for bacterial invasion of tion) [41]. epithelial cells which could putatively increase the ex- In addition to storage, the upregulation of cellulose pression of SipD and SipB in the Salmonella infections synthase, a catalytic subunit could catalyze secondary pathway, and increased the IpaB expression in the Shi- cell wall biogenesis and improve the ability to defend gella infections pathway in HM (Fig. 11A.B). The against bacteria and fungi. The significant upregulation CAZymes analysis showed that longer corn storage of dirigent proteins, which are thought to play important times resulted in higher levels of the following subsys- roles in plant secondary metabolism and are the first in- tems when compared with the shorter storage treat- termediates for lignan biosynthesis, was observed in ments; cellulose_synthase, chitin_oligosaccharide stored corn [42]. Lignans are known to form building synthase, chitin_synthase, xylanase, arabinanase and glu- blocks for the formation of lignin in the plant cell wall cosaminidase (Table S4). and are metabolized by intestinal bacteria and exhibit Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 10 of 15 Fig. 7 Bacterial abundance RDA correlation biplots by selected explanatory variables. The sites and explanatory variables (A) and species (B) plots are presented separately for clarity; they are divided from the same RDA model. H: half moths storage corn; T: two months storage corn strong antioxidant and bioactive properties such as en- with storage. Protein oxidation (carbonylation) and re- zymes and protein synthesis, cell proliferation, growth duced translation always occur during aging [45]. factor action and cell differentiation [43]. The aging process affects storage proteins, proteins re- Functional annotation of identified proteins lated to cell growth and division, and cell defense [44]. In the current study, the molecular function of most During storage, grain seeds could mobilize the storage proteins was related to the activity of peptidase. Pepti- substances in a timely and efficient manner to obtain a dases are enzymes that hydrolyze peptide bonds in pro- sufficient carbon source and energy supply for germin- teins and peptides and release amino acids, peptides, and ation. The upregulation of Legumin-1 in stored corn proteins from larger peptides and proteins [46]. From suggested that germination activity could be improved the cellular process analysis, storage duration had a major impact on protein homeostasis and induced pro- teins involved in metabolic processes (mainly the protein catabolic process and amino acid biosynthetic process). For dried stored corn seed, free amino acids in grain in- creased with storage and there is a decrease in the lower molecular weight peptides and an increase in the higher molecular weight peptides [39]. The texture changes in stored grains are the consequence of modifications by of carbohydrate polymers by component polysaccharides via hydrolytic enzyme activity. The chemical and prote- omic analysis results suggest that storage probably in- duced an increase in large molecular weight starch and protein and produced modifications in solubility. This could lead to lower starch pasting properties and higher Fig. 8 Species differentially represented between HM and TM Fig. 9 The significant different functions of the cecal microbiota of samples identified by linear discriminant analysis coupled with effect the broilers. Statistics of the number of annotated genes at KEGG size (LEfSe) (LDA > 2, P < 0.05). (HM (green), half month stored corn; metabolic pathway level two. (HM, half month stored corn; TM, two TM (red), two months stored corn) months stored corn) Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 11 of 15 Fig. 10 Comparison of gene pathways of the cecal microbiota of broilers annotated genes at KEGG pathways at level three of carbohydrate metabolism (A), glycan biosynthesis and metabolism (B) and immune system (C). (HM, half month stored corn; TM, two months stored corn). * P ≤ 0.05 concentrations of beneficial oligosaccharides which indicated that the number of goblet cells was decreased could affect animal health. under the TM corn treatment. Goblet cell function re- vealed the metabolic and inflammatory phenotypes to Whole genome shotgun metagenome analysis the intestinal microbiota. Johansson et al. [49] reported In the current study, increased storage time resulted in that more anti-nutritional factors led to epithelial cell increased AME and AMEn, is similar to the results re- apoptosis along the villi and crypts, fusion of villi and ported by Fuente et al. [47]. The intestinal flora has been more and larger goblet cells. On the other hand, the re- recently proposed to affect body weight and energy generation of epithelial goblet cells is consistent with homeostasis. The results from our RDA analysis showed Acidaminococcus colonization [50]. a strong correlation between AME and Acidaminococcus The lower enrichment of Firmicutes under the TM sp. which has an effect on growth through the fermenta- treatment was in concordance with the result of Stanley tion of glutamate [48]. Mucin is secreted by goblet cells et al. [51] who reported that a class of Firmicutes was along the villi of the epithelium. The present study has negatively correlated with poultry energy utilization. Fig. 11 Comparison of pathogenic invasion related pathways of the cecal microbiota of broilers annotated genes at KEGG pathways at level three. (HM, half month stored corn; TM, two months stored corn) Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 12 of 15 Members of the genus Bifidobacteria are the main gut energy. The higher activities of GHs found in TM were microbiota recognized as being beneficial for health. associated with the better utilization of energy such as Two months stored corn significantly upregulated the AME and AMEn and related to the production of enrichment of Bifidobacteria which impacts positively SCFAs. The distal gut microbiome provides the host the host and the target of prebiotic functional foods and with the capacity to degrade these glycans. In addition, supplements [52]. At the genus level, we found that TM many members of the microbiota possess the ability to treatment had lower enrichment in eggerthella. synthesize new polysaccharides de novo. Bacterial pro- Eggerthella have been implicated as a cause of ulcerative duction of capsular polysaccharides is associated with in- colitis, liver and anal abscesses and systemic bacteremia creased resistance to phages, complements, and [53]. antimicrobial peptides [54]. Bacteroides spp. are known Bacteroides fragilis, a gram-negative, obligately anaer- to breakdown a wide variety of otherwise indigestible obic bacterium that was negatively correlated with dietary plant polysaccharides (eg., amylose, amylopectin AME [54], the present study found that higher enrich- and pullulan) [60], while proteomic analysis showed that ment of Bacteroides fragilis in HM might reduce the higher amylopectin synthetase activity with the storage host immune response toward pathogenic bacteria by of corn might contribute to increasing the abundance of suppressing inflammatory pathways. Enterotoxin pro- Bacteroides under the TM and stimulate glycan biosyn- duction by B. fragilis was identified and subsequently thesis and metabolism. Glycosyltransferases (GTs) are found to produce severe diarrheal disease in several in- enzymes that catalyze the formation of the glycosidic tact animals [55]. Strauss et al. [56] reported that Fuso- linkage to form a glycoside and involve in the synthesis bacterium isolated from IBD patients is a bacterial of glycosphinogolipids [61]. Glycosphingolipids are species associated with inflammatory disease. From the building blocks of the plasma membrane that determine LEfSe analysis, newly harvested corn treatment led to lipid rafts and are involved in cell functions such as pro- higher relative abundance of Hungatella hathewayi, Bac- liferation, apoptosis and embryogenesis [62]. Higher en- teroides fragilis and Fusobacterium which might colonize richment in GTs potentially improved the synthesis of the gut and produce enterotoxins to induce the diarrheal glycosphingolipids for TM suggested that stored corn response and be the major reason for the “newly har- fed to broilers could improve the function of epithelial vested grain problem”. Hungatella hathewayi can cause cells. a wide spectrum of illnesses ranging from mild diarrhea Beyond digestion and metabolism, the microbiota con- to pseudomembranous colitis [57]. The TM treatment tributes to the development and maintenance of the in- seemed to enrich in butyrate-producing bacteria such as testinal epithelial barrier, development of the immune Alistipes that typically play anti-inflammatory roles. system, and competition with pathogenic microorgan- Thus, prolonging the storage time could improve gut isms. Among these systems, RIG-I-like receptor signal- health by decreasing the abundance of harmful microor- ing plays a major role in pathogen sensing of RNA viral ganisms in the intestine. infection to initiate and modulate antiviral immunity [63], and it was influenced by the treatments. Salmonella Microbial gene functional diversity infections have the capacity to modulate cellular func- The core functions of the intestinal microorganisms in- tions and induce profuse actin cytoskeleton rearrange- clude pathways associated with carbohydrate and amino ments and nuclear responses that ultimately lead to acid fermentation. It has been demonstrated that the bacterial uptake and the production of proinflammatory cecal microbiome was enriched in genes involved in cytokines [64]. Shigellae are the etiological agents of carbohydrate metabolism plus the metabolism of galact- bacillary dysentery and the acute form of diarrhea ac- ose and fructose from the stored corn. Several dietary companied by blood and mucus [65]. Newly harvested components, especially polysaccharides such as inulin, corn increases the expression of SipD and SipB in the fructooligosaccharides or xylan are not modified or Salmonella infection pathway and increases IpaB ex- absorbed in the intestine and are considered as prebiotic pression in the Shigella infections pathway. SipB and promoting the growth of beneficial bacteria [58]. These IpaB apparently bind and activate caspase-1 and result microbes essentially assist the host in deriving maximum in the stimulation of an unconventional form of pro- nutritional value from the components of the diet. Mi- grammed cell death with features of necrosis, which crobial fermentation of indigestible polysaccharides re- was in accordance with the intestinal morphological sults in the production of SCFAs which are linked to the results. Consequently, a higher possibility of Salmon- immune system and enterocyte development [59]. ella and Shigellae infections could potentially occur in Glycoside hydrolases (GHs) are essential enzymes re- broiler chickens fed a diet made from newly harvested quired for the breakdown of polysaccharides. Polysac- corn, which could result in diarrhea and intestinal charide degrading enzymes contribute to metabolic epithelial cell death. Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 13 of 15 Conclusion Ethics approval and consent to participate All procedures complied with the Beijing Regulations for Laboratory Animals, Natural aging of newly harvested corn is a complex and the study was approved by The Laboratory Animal Ethical Committee of process. We used proteomics to identify 26 proteins that China Agricultural University (study approval reference number could be responsible for the synthesis of large molecules AW60701202–2-1). All methods were carried out in accordance with relevant guidelines and regulations (supplemental material “ethics approval”). and change the solubility of starch and protein, as well as soluble sugars and decrease pasting properties. Com- Author details pared with newly harvested corn, two-month-stored College of Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China. State Key Laboratory of corn appears to have a decreased abundance of harmful Animal Nutrition, College of Animal Science and Technology, China bacteria such as Hungatella hathewayi and Bacteroides 3 Agricultural University, 100193 Beijing, China. NutriGenomics Laboratory, fragilis, with an enrichment of butyrate-producing bac- Department of Poultry Science, University of Georgia, Athens, GA 30602, USA. teria such as Alistipes in the guts of broiler chickens. 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Insights into the proteomic profile of newly harvested corn and metagenomic analysis of the broiler intestinal microbiota

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Abstract

Background: The use of newly harvested corn in feed causes wet droppings in broilers and increased feed cost which was termed as “new season grain problem”. The present study was conducted to evaluate the proteomic profile of newly harvested corn and the subsequent influence on intestinal microbiol community for broiler chickens. Methods: Newly harvested corn stored for either half a month (HM) or two months (TM) was used, and the pasting properties, total soluble sugars, and proteomic analysis technology was used to explore the influence of storage on natural aging corn properties. Additionally, seventy-two 7-day-old Ross 308 male broiler chicken were fed with different stored corn. Apparent metabolizable energy (AME), digesta viscosity, intestinal morphology and microbiota were examined to explore the influence of feed corn storage on broiler chickens. Results: Pasting properties in the TM corn exhibited decreased viscoelastic properties. Proteomic studies found a total of 26 proteins that were differentially expressed between the two treatment groups. Proteins involved in starch and polysaccharides biosynthesis were upregulated in TM compared with HM. Chickens fed on TM diet had higher relative energy utilization compared to the HM birds. With increased corn storage, the relative digesta viscosity decreased significantly (P ≤ 0.05). The total number of goblet cells and lymphocytes was lower in chickens fed the TM diet. The microbiota data showed that the TM chickens had decreased abundance of diarrheal bacteria such as Hungatella hathewayi and Bacteroides fragilis, and increased butyrate-producing bacteria such as Alistipes compared to the HM chickens. Conclusions: Storage of newly harvested corn induced the synthetic reaction of large molecules and changed the solubility of starch and protein with increasing soluble sugars and decreasing pasting properties that may improve the fermentation of intestinal microbiota, improve the energy utilization and protect gut health without the risk of diarrhea. Keywords: Broilers, Corn, Metagenome, Proteome, Storage * Correspondence: yuanjm@cau.edu.cn State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, 100193 Beijing, China Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 2 of 15 Introduction fed to broiler chickens and how they affect performance Corn is the most abundantly grown cereal grain and has and health are unclear. high nutritional value, especially for poultry and swine. We herein first employed a combined high-throughput As corn production is seasonal, corn grains need to be label-free comparative proteomics and metagenomics to stored throughout the year to provide sufficient supplies study the biological mechanisms that underlie the rela- for feed industries. However, there is an asynchrony of tionship between the composition of natural aging corn supply and demand that leads to a large number of and the diversity of the complex intestinal microbial newly harvested corn being processed as animal feed community of broiler chickens. during each harvest season. The use of newly harvested corn in poultry diets is especially problematic because it Materials and methods decreases the apparent metabolizable energy (AME) and Plant material feed conversion ratio and causes wet droppings and in- One newly harvested mixed corn variety corn (ZhengDan creased feed cost [1–3]. Nutritionists have termed this 958) was sourced from Hebei Province. The corn was sown problem the “new season grain problem” [4]. in March 2015 and harvested in September 2015. After nat- Newly harvested grain should be stored for a period of ural drying to 14% moisture content, intact corn grains several weeks or months to enhance its nutritional value were stored from September to November in a warehouse [2]. During storage, respiratory and metabolic events located at the China Agricultural University Poultry Experi- continue, and changes in internal factors, such as starch, mental Base without air conditioning to simulate typical endogenous enzymes and carbonyl compounds in cell grain storage conditions. The newly harvested corn was walls and external factors occur [5, 6]. Among these fac- sampled after storage for half a month (HM) or two tors, endogenous proteolytic enzymes play a central role months (TM), frozen immediately in liquid nitrogen and in regulating the synthesis and decomposition of carbo- stored at − 80 °C until further analysis. At the same time, hydrates and proteins [7, 8]. broilers fed the corn stored for different period of the time Proteomics is an important tool that can be used to were used to further examine the differences between dif- determine the biological roles and functions of individ- ferent storage times of corn. To eliminate the influence of ual proteins that govern grain seed quality and allow for the environment and animals, the same variety of corn the systematic analysis of complex cellular mechanisms stored for one year with the same moisture content was [9]. Reports of corn proteome expression during the arti- treated as the control in the broiler trial. ficial aging period indicate that artificial aging would in- crease proteases and breakdown stored proteins, and Determination of pasting properties, total soluble sugar, impair metabolism and energy supplies [10, 11]. How- glucose and fructose ever, the physicochemical properties of naturally aging The corn samples were analyzed for pasting properties corn have yet to be elucidated. Our objective was to de- and soluble sugar. The pasting properties of corn mix- lineate the proteomic mechanisms that underlie differ- tures stored for different times and suspended in dis- ences between corn stored for different time. tilled water were determined by a Rapid Visco-Analyzer There is a clear link between bird performance and (model RVA-4C, Newport Scientific Pty. Ltd., Warrie- gut microbiota composition [12]. The intestinal micro- wood, Australia) and estimated by the method of biota plays an important role in maintaining normal gut Achayuthakan and Suphantharika [17]. Total soluble function and contributes to the development of func- sugars were estimated by the phenol-sulfuric acid tional gastrointestinal symptoms by modulating the sig- method of Dubois et al. [18], using sucrose as the stand- naling pathways of hosts [13]. We have previously ard. Glucose and fructose were determined by the HPLC shown that using newly harvested corn leads to over- method of Wilson et al. [19]. feeding and increased ileum lesions and injuries in broiler chickens [3]. Intestinal injury is associated with diarrhea and impaired gut resistance to pathogens [14]. Label-free proteomic analysis process Leonard et al. [15] reported that grain (rice, oats, barley Protein extraction and digestion and corn) protein is one of the most common triggers of The HM and TM samples were thawed at − 80 °C and enterocolitis syndrome. Additionally, non-digestible then ground and hereafter, 100 mg of each sample was polysaccharides in corn can be broken down by mem- weighed. Then 400 μL SDT lysis buffer (4% SDS, 100 bers of the intestinal microbiota, producing monosac- mmol/L Tris-Hcl, 1 mmol/L DTT, pH 7.6) was added to charides and short-chain fatty acids (SCFAs), leading to the sample, followed by solubilization for 20s with a tis- unbalanced energy status [16]. Intestinal bacterial com- sue homogenizer; this was repeated 5 times. After 20 position changes and their subsequent metabolic min ultrasonic treatment, the mixture was centrifuged changes when a diet containing newly harvested corn is for 30 min at 10,000 × g at 4 °C, and the supernatant was Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 3 of 15 saved for subsequent experiments. The BCA assay was differentially expressed proteins was performed using a used for protein quantification. mixture model-based method [22]. The significance of Protein digestion (200 μL for each sample) was per- the differentially expressed proteins between the samples formed using the FASP procedure described by Wis- was examined with the cutoff values P ≤ 0.05 and FDR ≤ niewski [20]. Briefly, the detergent DTT, and other 0.05. low molecular weight components were removed using 200 μL UA buffer (8 mol/L Urea, 150 mmol/L Bioinformatics analysis Tris-HCl, pH 8.0) and facilitated by centrifugation Differentially expressed proteins (DEPs) were sub- (14,000 × g × 15 min). Then, the 100 μLIAA (50 jected to Core Expression Analysis. In this study, the mmol/L IAA in UA) was added in UA buffer, and DEPs between HM and TM was undertaken. The the mixture was oscillated for 1 min at 600 r/min and ratio values in the datasets were converted to fold allowed to settle for 30 min in the dark at room change values, where the negative inverse was taken temperature. The filter was washed with UA buffer for values between 0 and 1. The sequence data for (100 μL) and 100 μLofNH HCO buffer twice. Sub- the selected differentially expressed proteins were 4 3 sequently, 40 μL trypsin buffer (4 μL Trypsin in 40 μL retrieved from the UniProtKB database in batches and NH HCO ) was added. The mixture was oscillated for FASTA format. Following the annotation and annota- 4 3 1 min at 600 r/min and allowed to settle for 16–18 h tion augmentation steps, the studied proteins were at 37 °C, and the resulting peptides were collected. blasted against KEGG GENES (plants) to retrieve the The filtrate was desalinized with C18 SD Extraction KOs and were subsequently mapped to pathways in Disk Cartridge and quantified at OD280. KEGG. LC-MS/MS analysis Animals and experimental design Approximately 2 μL product of protein digestion was The experiment was assigned to two periods following used for LC-MS/MS analysis and separated using a the corn storage time. During each period, seventy- nanoliter HPLC Ultimate 3000 system (Thermo Fisher two 7-day-old male broiler chickens (Ross 308) were Scientific, Louis, MO, USA). The mobile phase A was obtained from a commercial hatchery and grown for 0.1% formic acid solution, and the mobile phase B 14 d. In total, 144 Ross 308 male birds were selected. was 0.1% formic acid solution with 80% acetonitrile. Birds were randomly assigned to one of two dietary The chromatographic column C18 trap column (C18 treatments (HM vs control or TM vs control) (during 3 μm 0.10 × 20 mm) was balanced with 95% A solu- each time thirty-six birds fed with control corn, this tion. The sample was loaded onto the C18 trap col- process repeated twice). There were 6 replicates per umn through an automatic sampler and separated by treatment and 6 birds per replicate. Feed (pelleted) a chromatographic column (C18 1.9 μm 0.15 × 120 and water were provided ad libitum. The light regi- mm) at the flow rate of 600 nL/min. The gradient men was 23 L:1D and the room temperature was 28– elution procedure was as described in Additional file 30 °C. All the corn samples used to formulate basal 1:Table S1 broiler diets that met broiler recommendations of NY/T33–2004 (Table S2). Maxquant label-free quantification analysis The 6 resulting raw LC-MS/MS files were imported to Sample collection and DNA extraction the MaxQuant software (1.3.0.5) and Proteome Discov- The experimental layout is shown in Fig. 1. At 14 days of erer 2.0 (Thermo Fisher Scientific, Bremen, Germany) age, a metabolic experiment was conducted, and 3 ran- for database inquiry and LFQ label-free quantification domly selected chickens (remaining birds were still fed analysis. The database was corn_160605.fasta; enzyme: with basal diets) per cage were moved to the new cages trypsin; max missed cleavage: 2; fixed modifications: car- and fed with either HM, TM or control metabolic diets bamidomethyl (C); variable modifications: oxidation(M), (Table S3) for 3 d. Total excreta outputs and feed intakes acetyl (Protein N-term); peptide Mass Tolerance: ±15 were recorded from 15 to 18-day post hatching. Excreta ppm; Fragment mass tolerance: 20 mm; peptide confi- were dried in a forced-air oven at 60 °C for 48 h and the dence: high; peptide length: > 4. The cutoff for the global gross energy of excreta and metabolic diets were deter- false discovery rate (FDR) in peptide and protein identi- mined using an adiabatic bomb calorimeter to determine fication was 0.01. Label-free quantification was per- apparent metabolizable energy (AME). Feed and extract formed using MaxQuant as previously described [21]. nitrogen contents were determined by a macro Kjeldahl Intensity-based absolute quantification (iBAQ) in Max- method to get nitrogen retention for the calculation of Quant was performed to quantify protein abundance for nitrogen-corrected AME (AME ). The calculation equa- the identified peptides. An FDR estimation for tions are as follows: Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 4 of 15 Fig. 1 Experimental layout. Seventy two birds were divided into 2 groups, namely, HM and TM. On d 14, half birds in per treatment were selected to do metabolic experiment for three days. The left parts were still fed with experimental diets. On d 21, non-metabolic birds were killed to get samples for metagenome analysis (n =3) Metagenome analysis process AME ðÞ MJ=kg¼ðÞ Gross energy intake - Gross energy excretion diet =Feed intake DNA library construction and sequencing DNA libraries were generated using NEBNext® Ultra AMEn ðÞ MJ=kg¼ AME - 34:99 diet TM DNA Library Prep Kit for Illumina (NEB, USA) fol- ðÞ Nitrogen intake - Nitrogen excretion =1000 lowing manufacturer’s recommendations. Briefly, the DNA sample was fragmented by sonication to a size of At 21-day post-hatching, the 3 remaining birds in each 350 bp, then end-polished, A-tailed, and ligated with the cage were euthanized by injection with 5% pentobarbital full-length adaptor for Illumina sequencing. PCR prod- sodium. Immediately following euthanasia, the abdom- ucts were purified (AMPure XP system) and libraries inal cavity was opened and the duodenal contents, ap- were analysed for size distribution by Agilent2100 Bioa- proximately 3 cm lengths of duodenum were removed nalyzer and quantified using real-time PCR. The cluster- for gut morphological measurements. For viscosity mea- ing of the index-coded samples was performed on a cBot surements, jejunal digesta were collected and diluted Cluster Generation System. Then, the library prepara- with (1:1) distilled water and homogenized for 20 min at tions were sequenced on an Illumina HiSeq platform room temperature before centrifugation. The viscosity and paired-end reads were generated. was measured according to the method of Piel et al. [23]. Gene catalogue construction The cecum was collected within 5 min of euthanasia, We performed de novo assembly and gene prediction immediately placed in cryogenic vials, snap-frozen in li- for the high quality reads of 6 samples in stage I using quid nitrogen and stored at − 80 °C until DNA extrac- SOAP denovo v1.06 [24] and GeneMark v2.7 [25], re- tion. Total genomic DNA was isolated from 220 mg of spectively. All predicted genes were aligned pairwise frozen cecal contents using the QIAmp DNA Stoll Kit using BLAT and genes, of which over 90% of their (Qiagen GmbH, Hilden, Germany). The DNA concen- length can be aligned to another one with more than tration and purity were determined using a NanoDrop 95% identity (no gaps allowed), were removed as redun- 2000 spectrophotometer (Thermo Fisher Scientific, Wil- dancies, resulting in a non-redundant gene catalogue mington, DE, USA). Three samples per treatment were comprising of 659,733 genes. This gene catalogue from selected for further analysis. our cecal samples was further combined with the previ- ously constructed CD-HIT gene catalogue [26], by re- Morphological examination moving redundancies in the same manner. At last, we Intestinal samples were immersed in formaldehyde, be- obtained an updated gene catalogue that contains fore being fixed in Bouin’s solution and embedded in 362,337 genes. paraffin. The length of the intestinal villi and the depth of the intestinal crypt were measured with a linear scaled Taxonomic assignment of genes graticule. The number of goblet cells and lymphocytes Taxonomic assignment of the predicted genes was per- /μm area of the villus and crypts was measured by 25 formed using DIAMOND [27]. In our analysis, we col- squared graticules. lected the reference microbial genomes from Non- Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 5 of 15 Redundant (NR) of NCBI (v 2014-10-19), and then quantifications of 1984 proteins were divided into two aligned all genes onto the reference genomes. For each categories: proteins with quantitative ratios over 1.5 and gene, the highest scoring hit(s) above these two thresh- P ≤ 0.05 were considered upregulated, while proteins olds was chosen for the genus assignment. For the taxo- with quantitative ratios ≤1/1.5 (0.67) and P ≤ 0.05 were nomic assignment at the phylum level, the 65% identity considered downregulated. Venn diagram analysis was used instead. showed that 222 proteins were influenced by storage time, among these proteins (Fig. S1). Functional annotation The functional annotation and abundance analysis of Differentially expressed proteins in corn stored for KEGG (Kyoto Encyclopedia of Genes and Genomes), different times and carbohydrate enzyme (CAZy) (version: 2014.11.25) We identified a total of 26 proteins that were differen- were performed using BLASTP (e-value ≤ 1e-5). Based tially expressed, among which only 2 diminished propor- on the species abundance and functional abundance, the tionally to HM corn, while 24 proteins seemed to be abundance cluster analysis, and sample cluster analysis positively influenced by the increase in storage time were performed. (Table 2). Peptidyl-prolylcis-transisomerase and jasmonate-induced protein were downregulated in TM Statistical analysis compared with HM. The other identified proteins were LEfSe analysis uses the Kruskal-Wallis rank sum test to upregulated in TM compared with HM. Among these detect significantly different abundances and performs proteins, GRMZM2G162359_P01, GRMZM2G008216_ LDA scores to estimate the effect size (threshold: > 2). P02 and GRMZM5G825695_P01 proteins have no Statistical analyses were performed using SPSS software, known functions and no annotations could be found to version 16.0 (SPSS Inc., Chicago, IL, USA). The data assign or predict their functions. were subjected to an analysis of variance (ANOVA), the means were compared using Student’s t-tests, and the Functional annotation of identified proteins differences were considered significant at P ≤ 0.05. GO terms were assigned to the 222 differentially expressed proteins. Figure 3 shows GO annotations for Results proteins that were differentially expressed in HM and Corn properties TM. The identified proteins cover a wide range of cellu- The pasting characteristics of corn stored at different lar processes, molecular functions and cellular compo- times determined by the RVA are summarized in nents and can be classified into 22, 8, and 7 categories in Table 1. Storage time resulted in significant (P ≤ 0.05) these broad groups, respectively. The differentially decreases in peak viscosity, final viscosity, and setback expressed proteins in the biological functions category viscosity and an increase in pasting temperature. At the were mainly associated with cellular ketone metabolic, same time, a significant change in soluble sugars was ob- carboxylic acid metabolic and cellular amino acid bio- served during storage of corn for the two periods synthetic processes. The largest group within the mo- (Fig. 2A). Lower total concentrations (P ≤ 0.05) of glu- lecular function category was cofactor binding. The cose and fructose were found in corn stored for two cellular component functions mainly belong to the cyto- months (Fig. 2B). plasm and mitochondrion. KEGG results showed for the most differentially expressed proteins suggest that these Proteome variation of corn in response to different proteins are involved in 13 pathways (Fig. S2). storage times By merging all identified protein lists including those from two time points and three biological replicates, Bird properties affected by corn storage 2499 proteins were identified and 1984 proteins were The relative AME and AMEn values of each batch are quantified from all samples. For further analysis, the shown in Fig. 4. Both AME and AMEn were significantly TM/HM ratios were log transformed. Relative higher (P ≤ 0.05) in HM than in TM. Table 1 Pasting properties of different storage time corn Sample Peak viscosity RVA Breakdown RVA Final viscosity RVA Setback RVA Pasting temperature, °C HM 1796 ± 42.2 166 ± 21.4 3087 ± 123.0 1457 ± 71.6 77.6 ± 0.13 TM 1602 ± 68.6 99 ± 32.9 2711 ± 34.5 1208 ± 10.6 78.1 ± 0.13 P-value 0.014 0.162 0.007 0.004 0.036 Mean ± standard deviation HM, half months stored corn; TM, two months stored corn Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 6 of 15 Fig. 2 Storage effects on total soluble sugars (A) and total concentrations of glucose and fructose (B) of corn (mean ± SD, triplicate samples). (HM, half month stored corn; TM, two months stored corn) With increased corn storage, the relative digesta vis- Taxonomic characterization of the gut microbiota cosity decreased significantly (P ≤ 0.05) (Fig. 5A). The A total of 1,870,386 ORFs were predicted. Among these total number of goblet cells and lymphocytes was lower ORFs, 82.06%, 78.41%, 71.55%, 69.39%, 53.90%, 49.20% in chickens fed the TM diet (Fig. 5B). There were no sig- and 41.02% had annotations at the kingdom, phylum, nificant differences between treatments in crypt and villi class, order, family, genus and species levels, respectively. (Fig. S3). Unclassified ORFs accounted for 17.94% of unigenes, representing novel rhizosphere taxa. The abundance was Table 2 Proteins identified as differentially expressed from Label-free LC-MS/MS Accession number Protein description TM/HM ratio P-value KO GRMZM5G866758_P02 Acetyl-CoA acetyltransferase, cytosolic 1.75 0.002 GRMZM2G174883_P01 Legumin 1 1.67 0.012 K03671 GRMZM2G055434_P01 Early nodulin 1.94 0.013 GRMZM2G023347_P01 Prefoldin subunit 1.50 0.015 GRMZM2G010762_P01 Early nodulin-like protein 1.54 0.018 K00700 GRMZM2G020423_P01 Jasmonate-induced protein 0.61 0.019 K00626 GRMZM2G016958_P01 IAA-amino acid hydrolase 2.07 0.019 K01507 GRMZM2G041881_P01 Nascent polypeptide-associated complex subunit beta 4.27 0.020 GRMZM2G059353_P01 Non-green plastid inner envelope membrane protein 1.81 0.021 GRMZM2G162359_P01 Uncharacterized protein 1.97 0.021 K13448 GRMZM2G473463_P01 Mitochondrial import inner membrane translocase subunit 1.57 0.026 GRMZM2G105712_P05 60S acidic ribosomal protein 1.81 0.029 K07304 GRMZM2G397044_P02 Peptidyl-prolyl cis-trans isomerase 0.34 0.031 GRMZM2G163406_P01 Dirigent protein 2.45 0.033 GRMZM2G032628_P01 1,4-alpha-glucan-branching enzyme 2 1.95 0.034 K03232 GRMZM2G097457_P01 Pyruvate, phosphate dikinase 1.56 0.036 GRMZM2G164714_P02 Phosphoenolpyruvate carboxylase family protein 1.57 0.037 GRMZM2G071433_P01 Plasma membrane associated protein 1.60 0.038 K03016 GRMZM2G008216_P02 Uncharacterized protein 1.67 0.040 GRMZM2G445905_P03 Cellulose synthase 1.62 0.045 GRMZM5G825695_P01 Uncharacterized protein 1.85 0.045 K10999 GRMZM2G439201_P02 Elongation factor 1-beta 2 2.09 0.045 GRMZM2G026470_P01 Soluble inorganic pyrophosphatase 1 1.54 0.046 K01006 GRMZM2G306345_P01 Pyruvate orthophosphate dikinase1 1.61 0.047 GRMZM2G085260_P01 Desiccation-related protein 1.68 0.049 K02943 GRMZM2G046520_P01 Dirigent protein 2.18 0.050 K01703 HM, half months stored corn; TM, two months stored corn Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 7 of 15 Fig. 3 The proportion of the differentially expressed proteins categorized by function. A: Gene Ontology analysis for biological processes; B: Gene Ontology analysis for molecular function; C: Gene Ontology analysis for molecular function compared between groups, number of significantly chan- Firmicutes, Bacteroidetes, and Proteobacteria followed ged genes are showed in a Venn diagram. When com- by Actinobacteria, Tenericutes, Fusobacteria and Spiro- pared to HM corn treatment, a total of 97,141 genes chaetes. Bacteroidetes, Proteobacteria and Tenericutes were significantly changed in the TM groups, and were significantly enriched in stored corn subjects, while 110,678 genes were changed in the HM groups (Fig. S4). the relative abundance of Fusobacteria was significantly We aligned the reads to a catalog, and the majority of increased in newly harvested corn (P ≤ 0.05) (Fig. 6A). aligned reads were bacterial and dominated by the phyla Clostridium, Eubacterium, Bacteroides and Blatutia were Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 8 of 15 second axes variation, which accounted for 47.1% and 7.4%, respectively. In Fig. 7B, we highlight 7 species that were significantly correlated with one or both of the first two RDA axes. Among these, Acidaminococcus sp. CAG.917 was strongly associated with AME while Alis- tipes sp. CAG.157, Bacteroides dorei, Bacteroides unifor- mis and Oscillibacter ruminantium were correlated with AMEn. LEfSe analysis The LEfSe test detected differences in the relative abun- Fig. 4 Storage effects on relative AME and AMEn to control groups of 17 d broiler chickens (n = 6). (* P ≤ 0.05, ** P ≤ 0.01. HM, half dances of bacterial taxa across samples (Fig. 8). Among month stored corn; TM, two months stored corn) the genes from the species level, Alistipes and Firmicutes bacterium, Bacillus, Mycoplasma, Alistipes inops, Barne- siella intestinihominis, Barnesiella and Ruminococcus the most abundant genera in our treatments. Compared were significantly enriched in the TM treatment, while with the newly harvested corn, stored corn significantly Eubacterium desmoians, Hungatella hathewayi, Hunga- increased 8 kinds of genera bacterium such as Allstipes, tella, Lachnociostridum, Bacteroides fragilis, Fusobacter- Butyricicoccus and Mycoplasma (Fig. 6B). Species and ium and Eubacterium were significantly enriched in the genome level abundances were also calculated to deter- HM treatment (LDA > 2, P < 0.05). mine the composition of the gut microbiota (Fig. S5), we compared the composition HM and TM communities Functional analysis and bacterial metabolic processes and observed increases in Faecalibacterium prausnitzii, The function of bacterial assemblages associated with Barnesiella intestinihominis, Bacteroides dorei and Fir- different storage times of corn was cataloged using level micutes bacterium CAG:475 in TM compared with HM. 3 KEGG orthologs. Several KEGG level 2 modules were There were decreases in the abundance of Bacteroides different between the two treatments. HM treatment sig- fragilis, Subdoligranulum variabile, Ruminococcus tor- nificantly improved the metabolism of terpenoids and ques, Eubacterium sp. ER2, Clostridium sp. CAG:678 polyketides and amino acid metabolism, while TM had and Ruminococcaceae bacterium D16 in TM compared beneficially influenced metabolism of other amino acids, with HM. folding, sorting and degradation, glycan biosynthesis and metabolism and transport and catabolism and immune AME and AMEn are correlated with microbiota system (P ≤ 0.05, Fig. 9). At KEGG level 3, compared The global RDA model that selected AME and AMEn as with HM treatment, TM was enriched for carbohydrate the meaningful explanatory variables was significant metabolism (e.g., amino sugar and nucleotide sugar me- (P ≤ 0.05) (Fig. 7). The overall variation in species com- tabolism and pyruvate metabolism) (Fig. 10A), glycan position was attributed to these explanatory variables, of biosynthesis and metabolism (e.g., glycan degradation) which the majority were explained by the first and (Fig. 10B) and the immune system (e.g., RIG-I-like Fig. 5 Storage effects on relative digesta viscosity (A) and lymphocytes and goblet cells number in duodenum (n = 6). (* P ≤ 0.05, ** P ≤ 0.01. HM, half month stored corn; TM, two months stored corn) Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 9 of 15 Discussion The effect of storage on corn properties Compared to freshly harvested grain, a change in protein digestibility was reported by Paraginski et al. [5] and Ramchandran et al. [28], potentially due to protein and starch solubility. One of the most sensitive indices of the aging in grain is the change in pasting properties [29, 30] which is related to the granule size and soluble ma- terials of starch [31]. As storage duration increased, peak viscosity, final viscosity and setback decreased which were in agreement with previous studies [32, 33]. This is attributed to the formation of a complex between amyl- ose and lipids and thereby restriction of granular swelling. Dietary sugars (glucose and fructose) that escape ab- sorption in the mammalian intestine and reach the microbiota disrupt colonization by beneficial microbes [34]. In the current study we observed an increase in total soluble sugars and reductions in glucose and fruc- tose in the stored corn which were in concordance with the findings of Paraginski [5]. We observed that higher oligosaccharide and lower monosaccharide contents in stored corn could benefit gut health [35]. Differentially expressed proteins We studied the effect of corn storage and carbohydrate composition as storage is known to exhibit strong effects on starch biosynthesis [36]. Li et al. [37] reported that starch retrogradation is mainly caused by the aggrega- tion of amylose and occurs during the early stage of storage. 1,4-α-glucan-branching enzyme activity cata- lyzes ADP-glucose into amylopectin, inhibiting starch retrogradation [37, 38]. The upregulation of the 1,4-α- glucan-branching enzyme in TM suggested a decrease in the amount of amylose and an increase in amylopectin which could increase the water-insolubility of starch and protein [39]. During storage, respiratory and metabolic Fig. 6 Relative abundance of annotated phylum (A) and significant events continue. Pyruvate, phosphate dikinase and phos- different genus (B) in cecal microbiota of broilers. (HM, half month phoenolpyruvate carboxylase family proteins play key stored corn; TM, two months stored corn) * P ≤ 0.05 roles in CO transport and fixation, respectively [40]. Higher activities of these two enzymes under TM sug- gested a higher synthetic ratio of starch when carbon receptor signaling pathway) (Fig. 10C). Interestingly, we gain exceeds the carbon demands for growth (accumula- also found a different pathway for bacterial invasion of tion) [41]. epithelial cells which could putatively increase the ex- In addition to storage, the upregulation of cellulose pression of SipD and SipB in the Salmonella infections synthase, a catalytic subunit could catalyze secondary pathway, and increased the IpaB expression in the Shi- cell wall biogenesis and improve the ability to defend gella infections pathway in HM (Fig. 11A.B). The against bacteria and fungi. The significant upregulation CAZymes analysis showed that longer corn storage of dirigent proteins, which are thought to play important times resulted in higher levels of the following subsys- roles in plant secondary metabolism and are the first in- tems when compared with the shorter storage treat- termediates for lignan biosynthesis, was observed in ments; cellulose_synthase, chitin_oligosaccharide stored corn [42]. Lignans are known to form building synthase, chitin_synthase, xylanase, arabinanase and glu- blocks for the formation of lignin in the plant cell wall cosaminidase (Table S4). and are metabolized by intestinal bacteria and exhibit Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 10 of 15 Fig. 7 Bacterial abundance RDA correlation biplots by selected explanatory variables. The sites and explanatory variables (A) and species (B) plots are presented separately for clarity; they are divided from the same RDA model. H: half moths storage corn; T: two months storage corn strong antioxidant and bioactive properties such as en- with storage. Protein oxidation (carbonylation) and re- zymes and protein synthesis, cell proliferation, growth duced translation always occur during aging [45]. factor action and cell differentiation [43]. The aging process affects storage proteins, proteins re- Functional annotation of identified proteins lated to cell growth and division, and cell defense [44]. In the current study, the molecular function of most During storage, grain seeds could mobilize the storage proteins was related to the activity of peptidase. Pepti- substances in a timely and efficient manner to obtain a dases are enzymes that hydrolyze peptide bonds in pro- sufficient carbon source and energy supply for germin- teins and peptides and release amino acids, peptides, and ation. The upregulation of Legumin-1 in stored corn proteins from larger peptides and proteins [46]. From suggested that germination activity could be improved the cellular process analysis, storage duration had a major impact on protein homeostasis and induced pro- teins involved in metabolic processes (mainly the protein catabolic process and amino acid biosynthetic process). For dried stored corn seed, free amino acids in grain in- creased with storage and there is a decrease in the lower molecular weight peptides and an increase in the higher molecular weight peptides [39]. The texture changes in stored grains are the consequence of modifications by of carbohydrate polymers by component polysaccharides via hydrolytic enzyme activity. The chemical and prote- omic analysis results suggest that storage probably in- duced an increase in large molecular weight starch and protein and produced modifications in solubility. This could lead to lower starch pasting properties and higher Fig. 8 Species differentially represented between HM and TM Fig. 9 The significant different functions of the cecal microbiota of samples identified by linear discriminant analysis coupled with effect the broilers. Statistics of the number of annotated genes at KEGG size (LEfSe) (LDA > 2, P < 0.05). (HM (green), half month stored corn; metabolic pathway level two. (HM, half month stored corn; TM, two TM (red), two months stored corn) months stored corn) Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 11 of 15 Fig. 10 Comparison of gene pathways of the cecal microbiota of broilers annotated genes at KEGG pathways at level three of carbohydrate metabolism (A), glycan biosynthesis and metabolism (B) and immune system (C). (HM, half month stored corn; TM, two months stored corn). * P ≤ 0.05 concentrations of beneficial oligosaccharides which indicated that the number of goblet cells was decreased could affect animal health. under the TM corn treatment. Goblet cell function re- vealed the metabolic and inflammatory phenotypes to Whole genome shotgun metagenome analysis the intestinal microbiota. Johansson et al. [49] reported In the current study, increased storage time resulted in that more anti-nutritional factors led to epithelial cell increased AME and AMEn, is similar to the results re- apoptosis along the villi and crypts, fusion of villi and ported by Fuente et al. [47]. The intestinal flora has been more and larger goblet cells. On the other hand, the re- recently proposed to affect body weight and energy generation of epithelial goblet cells is consistent with homeostasis. The results from our RDA analysis showed Acidaminococcus colonization [50]. a strong correlation between AME and Acidaminococcus The lower enrichment of Firmicutes under the TM sp. which has an effect on growth through the fermenta- treatment was in concordance with the result of Stanley tion of glutamate [48]. Mucin is secreted by goblet cells et al. [51] who reported that a class of Firmicutes was along the villi of the epithelium. The present study has negatively correlated with poultry energy utilization. Fig. 11 Comparison of pathogenic invasion related pathways of the cecal microbiota of broilers annotated genes at KEGG pathways at level three. (HM, half month stored corn; TM, two months stored corn) Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 12 of 15 Members of the genus Bifidobacteria are the main gut energy. The higher activities of GHs found in TM were microbiota recognized as being beneficial for health. associated with the better utilization of energy such as Two months stored corn significantly upregulated the AME and AMEn and related to the production of enrichment of Bifidobacteria which impacts positively SCFAs. The distal gut microbiome provides the host the host and the target of prebiotic functional foods and with the capacity to degrade these glycans. In addition, supplements [52]. At the genus level, we found that TM many members of the microbiota possess the ability to treatment had lower enrichment in eggerthella. synthesize new polysaccharides de novo. Bacterial pro- Eggerthella have been implicated as a cause of ulcerative duction of capsular polysaccharides is associated with in- colitis, liver and anal abscesses and systemic bacteremia creased resistance to phages, complements, and [53]. antimicrobial peptides [54]. Bacteroides spp. are known Bacteroides fragilis, a gram-negative, obligately anaer- to breakdown a wide variety of otherwise indigestible obic bacterium that was negatively correlated with dietary plant polysaccharides (eg., amylose, amylopectin AME [54], the present study found that higher enrich- and pullulan) [60], while proteomic analysis showed that ment of Bacteroides fragilis in HM might reduce the higher amylopectin synthetase activity with the storage host immune response toward pathogenic bacteria by of corn might contribute to increasing the abundance of suppressing inflammatory pathways. Enterotoxin pro- Bacteroides under the TM and stimulate glycan biosyn- duction by B. fragilis was identified and subsequently thesis and metabolism. Glycosyltransferases (GTs) are found to produce severe diarrheal disease in several in- enzymes that catalyze the formation of the glycosidic tact animals [55]. Strauss et al. [56] reported that Fuso- linkage to form a glycoside and involve in the synthesis bacterium isolated from IBD patients is a bacterial of glycosphinogolipids [61]. Glycosphingolipids are species associated with inflammatory disease. From the building blocks of the plasma membrane that determine LEfSe analysis, newly harvested corn treatment led to lipid rafts and are involved in cell functions such as pro- higher relative abundance of Hungatella hathewayi, Bac- liferation, apoptosis and embryogenesis [62]. Higher en- teroides fragilis and Fusobacterium which might colonize richment in GTs potentially improved the synthesis of the gut and produce enterotoxins to induce the diarrheal glycosphingolipids for TM suggested that stored corn response and be the major reason for the “newly har- fed to broilers could improve the function of epithelial vested grain problem”. Hungatella hathewayi can cause cells. a wide spectrum of illnesses ranging from mild diarrhea Beyond digestion and metabolism, the microbiota con- to pseudomembranous colitis [57]. The TM treatment tributes to the development and maintenance of the in- seemed to enrich in butyrate-producing bacteria such as testinal epithelial barrier, development of the immune Alistipes that typically play anti-inflammatory roles. system, and competition with pathogenic microorgan- Thus, prolonging the storage time could improve gut isms. Among these systems, RIG-I-like receptor signal- health by decreasing the abundance of harmful microor- ing plays a major role in pathogen sensing of RNA viral ganisms in the intestine. infection to initiate and modulate antiviral immunity [63], and it was influenced by the treatments. Salmonella Microbial gene functional diversity infections have the capacity to modulate cellular func- The core functions of the intestinal microorganisms in- tions and induce profuse actin cytoskeleton rearrange- clude pathways associated with carbohydrate and amino ments and nuclear responses that ultimately lead to acid fermentation. It has been demonstrated that the bacterial uptake and the production of proinflammatory cecal microbiome was enriched in genes involved in cytokines [64]. Shigellae are the etiological agents of carbohydrate metabolism plus the metabolism of galact- bacillary dysentery and the acute form of diarrhea ac- ose and fructose from the stored corn. Several dietary companied by blood and mucus [65]. Newly harvested components, especially polysaccharides such as inulin, corn increases the expression of SipD and SipB in the fructooligosaccharides or xylan are not modified or Salmonella infection pathway and increases IpaB ex- absorbed in the intestine and are considered as prebiotic pression in the Shigella infections pathway. SipB and promoting the growth of beneficial bacteria [58]. These IpaB apparently bind and activate caspase-1 and result microbes essentially assist the host in deriving maximum in the stimulation of an unconventional form of pro- nutritional value from the components of the diet. Mi- grammed cell death with features of necrosis, which crobial fermentation of indigestible polysaccharides re- was in accordance with the intestinal morphological sults in the production of SCFAs which are linked to the results. Consequently, a higher possibility of Salmon- immune system and enterocyte development [59]. ella and Shigellae infections could potentially occur in Glycoside hydrolases (GHs) are essential enzymes re- broiler chickens fed a diet made from newly harvested quired for the breakdown of polysaccharides. Polysac- corn, which could result in diarrhea and intestinal charide degrading enzymes contribute to metabolic epithelial cell death. Yin et al. Journal of Animal Science and Biotechnology (2022) 13:26 Page 13 of 15 Conclusion Ethics approval and consent to participate All procedures complied with the Beijing Regulations for Laboratory Animals, Natural aging of newly harvested corn is a complex and the study was approved by The Laboratory Animal Ethical Committee of process. We used proteomics to identify 26 proteins that China Agricultural University (study approval reference number could be responsible for the synthesis of large molecules AW60701202–2-1). All methods were carried out in accordance with relevant guidelines and regulations (supplemental material “ethics approval”). and change the solubility of starch and protein, as well as soluble sugars and decrease pasting properties. Com- Author details pared with newly harvested corn, two-month-stored College of Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China. State Key Laboratory of corn appears to have a decreased abundance of harmful Animal Nutrition, College of Animal Science and Technology, China bacteria such as Hungatella hathewayi and Bacteroides 3 Agricultural University, 100193 Beijing, China. NutriGenomics Laboratory, fragilis, with an enrichment of butyrate-producing bac- Department of Poultry Science, University of Georgia, Athens, GA 30602, USA. teria such as Alistipes in the guts of broiler chickens. 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Journal

Journal of Animal Science and Biotechnology – Springer Journals

Published: Feb 9, 2022

Keywords: Broilers; Corn; Metagenome; Proteome; Storage

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