A. Jonker

Molecular basis of protein structure in proanthocyanidin and anthocyanin-enhanced Lc-transgenic alfalfa in relation to nutritive value using synchrotron-radiation FTIR microspectroscopy: A novel approach

To date there has been very little application of synchrotron radiation-based Fourier transform infrared microspectroscopy (SRFTIRM) to the study of molecular structures in plant forage in relation to livestock digestive behavior and nutrient availability. Protein inherent structure, among other factors such as protein matrix, affects nutritive quality, fermentation and degradation behavior in both humans and animals. The relative percentage of protein secondary structure influences protein value. A high percentage of beta-sheets usually reduce the access of gastrointestinal digestive enzymes to the protein. Reduced accessibility results in poor digestibility and as a result, low protein value. The objective of this study was to use SRFTIRM to compare protein molecular structure of alfalfa plant tissues transformed with the maize Lc regulatory gene with non-transgenic alfalfa protein within cellular and subcellular dimensions and to quantify protein inherent structure profiles using Gaussian and Lorentzian methods of multi-component peak modeling. Protein molecular structure revealed by this method included alpha-helices, beta-sheets and other structures such as beta-turns and random coils. Hierarchical cluster analysis and principal component analysis of the synchrotron data, as well as accurate spectral analysis based on curve fitting, showed that transgenic alfalfa contained a relatively lower (P<0.05) percentage of the model-fitted alpha-helices (29 vs. 34) and model-fitted beta-sheets (22 vs. 27) and a higher (P<0.05) percentage of other model-fitted structures (49 vs. 39). Transgenic alfalfa protein displayed no difference (P>0.05) in the ratio of alpha-helices to beta-sheets (average: 1.4) and higher (P<0.05) ratios of alpha-helices to others (0.7 vs. 0.9) and beta-sheets to others (0.5 vs. 0.8) than the non-transgenic alfalfa protein. The transgenic protein structures also exhibited no difference (P>0.05) in the vibrational intensity of protein amide I (average of 24) and amide II areas (average of 10) and their ratio (average of 2.4) compared with non-transgenic alfalfa. Cluster analysis and principal component analysis showed no significant differences between the two genotypes in the broad molecular fingerprint region, amides I and II regions, and the carbohydrate molecular region, indicating they are highly related to each other. The results suggest that transgenic Lc-alfalfa leaves contain similar proteins to non-transgenic alfalfa (because amide I and II intensities were identical), but a subtle difference in protein molecular structure after freeze drying. Further study is needed to understand the relationship between these structural profiles and biological features such as protein nutrient availability, protein bypass and digestive behavior of livestock fed with this type of forage.

Metabolic characteristics of proteins and biomolecular spectroscopic profiles in different batches of feedstock (wheat) and their co-products (wheat distillers dried grains with solubles) from the same bioethanol processing plant.

The objectives of this study were (1) to reveal the metabolic characteristics of proteins in different batches of feedstock (wheat) for bioethanol production and their co-products (wheat distillers dried grains with solubles, wDDGS) from the same bioethanol processing plant, and (2) to characterize biomolecular spectral profile associated with nutrient digestion in the rumen and intestine of dairy cattle. The metabolic characteristics of proteins were determined using the DVE/OEB system (where DVE=total truly absorbed protein supply, and OEB=degraded balance of protein) based on chemical profiles and rumen and intestinal digestion data from dairy cattle. The biomolecular spectral characteristics were investigated by using the molecular spectroscopy technique attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FT/IR). Multivariate molecular spectral analyses-agglomerative hierarchical cluster analysis (AHCA), and principal component analysis (PCA)-were conducted to identify the spectral differences in biomolecular inherent structure among the wheat and wDDGS batches. The results showed that (1) the metabolic characteristics of proteins in the wheat and wDDGS from the same bioethanol processing plant were significantly affected by batch, with total truly absorbed protein supply (DVE value) ranging from 101 to 116 g/kg of dry matter (DM) in wheat and from 153 to 182 g/kg of DM in wDDGS; (2) the degraded balance of protein (OEB value) in the wDDGS (but not the wheat) from the same bioethanol processing plant was significantly affected by batch, with the OEB value ranging from -19 to -26 g/kg of DM in the wheat and from 145 to 181 g/kg of DM in the wDDGS; and (3) the biomolecular spectral analyses with AHCA and PCA revealed biomolecular spectral profiles and differences among the wheat and wDDGS samples.

Nutrient profile and availability of co-products from bioethanol processing

Bioethanol production in North America has led to the production of considerable quantities of different co-products. Variation in nutrient profiles as well as nutrient availability among these co-products may lead to the formulation of imbalanced diets that may adversely affect animal performance. This study aimed to compare three types of dried distillers grains with solubles [100% wheat DDGS (WDDGS); DDGS blend1 (BDDGS1, corn to wheat ratio 30:70); DDGS blend2 (BDDGS2, corn to wheat ratio 50:50)] and their different batches within DDGS type with regard to: (i) protein and carbohydrate sub-fractions based on Cornell Net Carbohydrate and Protein System (CNCPS); (ii) calculated energy values; and (iii) rumen degradation of dry matter (DDM), organic matter (DOM), crude protein (DCP), neutral detergent fibre (DNDF) and starch (Dstarch) at 36 and 72 h of ruminal incubations. Wheat DDGS had a lower intermediately (PB2, 136.4 vs. 187.4 g/kg DM) and a higher slowly degradable true protein (PB3, 142.2 vs.105.3 g/kg DM) than BDDGS1, but similar to those of BDDGS2. Sugar (CA4) was higher, whereas starch (PB1) and digestible fibre (PB3) were lower in WDDGS than in BDDGS1 and BDDGS2. All carbohydrate sub-fractions determined differed significantly between the two batches of BDDGS2. The BDDGS2u2003had the highest calculated energy values (TDN, DE(3×) , ME(3×) , NEL(3×) , NE(m) and NE(g) ) among the three DDGS types. The energy values were slightly different between the batches of the three DDGS types. At all incubation times, wheat DDGS had a significantly higher (p < 0.05) DDM, DOM, DCP and DNDF than both DDGS blends. Differences were observed between different batches within DDGS types with regard to in situ rumen degradation of DM, OM, CP, NDF and starch. In conclusion, differences were observed in protein and carbohydrate sub-fractions and in situ ruminal degradation of DM, OM, CP, NDF and starch among the three DDGS types and different batches within DDGS type. This indicates that the nutrients supplied to ruminants may not only differ among different types of DDGS but it may also differ among different batches within DDGS type.

Non-destructive analysis of the conformational differences among feedstock sources and their corresponding co-products from bioethanol production with molecular spectroscopy

The objective of this study was to determine the possibility of using molecular spectroscopy with multivariate technique as a fast method to detect the source effects among original feedstock sources of wheat and their corresponding co-products, wheat DDGS, from bioethanol production. Different sources of the bioethanol feedstock and their corresponding bioethanol co-products, three samples per source, were collected from the same newly-built bioethanol plant with current bioethanol processing technology. Multivariate molecular spectral analyses were carried out using agglomerative hierarchical cluster analysis (AHCA) and principal component analysis (PCA). The molecular spectral data of different feedstock sources and their corresponding co-products were compared at four different regions of ca. 1800-1725 cm(-1) (carbonyl CO ester, mainly related to lipid structure conformation), ca. 1725-1482 cm(-1) (amide I and amide II region mainly related to protein structure conformation), ca. 1482-1180 cm(-1) (mainly associated with structural carbohydrate) and ca. 1180-800 cm(-1) (mainly related to carbohydrates) in complex plant-based system. The results showed that the molecular spectroscopy with multivariate technique could reveal the structural differences among the bioethanol feedstock sources and among their corresponding co-products. The AHCA and PCA analyses were able to distinguish the molecular structure differences associated with chemical functional groups among the different sources of the feedstock and their corresponding co-products. The molecular spectral differences indicated the differences in functional, biomolecular and biopolymer groups which were confirmed by wet chemical analysis. These biomolecular and biopolymer structural differences were associated with chemical and nutrient profiles and nutrient utilization and availability. Molecular spectral analyses had the potential to identify molecular structure difference among bioethanol feedstock sources and their corresponding co-products.

Protein molecular structures in alfalfa hay cut at three stages of maturity and in the afternoon and morning and relationship with nutrient availability in ruminants

BACKGROUNDnMolecular structures in feed protein influence its digestive behavior, availability and utilization. From a nutritive point of view, stage of maturity and cutting time are important factors affecting nutrient profiles and availability of alfalfa (Medicago sativa L.) hay in ruminants. The objectives of this study were to determine protein molecular structures by Fourier transform infrared spectroscopy (FTIR), and their relationship with nutrient profiles and availability in ruminants of alfalfa hay cut at early bud, late bud and early flower stages and in afternoon and morning.nnnRESULTSnWith advancing maturity, molecular structure ratios of α-helix:β-sheets decreased, while amide I:amide II increased (P ≤ 0.05). Alfalfa cutting in afternoon versus morning increased protein structure α-helix:β-sheets and α-helix:others ratios (P < 0.05) and tended to decrease the proportion of β-sheets (Pu2009=u20090.09). Positive correlations were found for α-helix:β-sheet ratio (R ≥ 0.60; P < 0.05) with intermediately degradable protein (PB2) and ruminal degradability and intestinal protein supply, and all these parameters correlated negatively with amide I:amide II ratio (R ≤ -0.62; P < 0.05).nnnCONCLUSIONnProtein molecular structures in alfalfa hay changed with advancing maturity and during the day and these protein structures affected predicted nutrient availability of alfalfa hay in ruminants.

Fermentation, degradation and microbial nitrogen partitioning for three forage colour phenotypes within anthocyanidin‐accumulating Lc‐alfalfa progeny

BACKGROUNDnAlfalfa has the disadvantage of having a rapid initial rate of protein degradation, which results in pasture bloat, low efficiency of protein utilisation and excessive nitrogen (N) pollution into the environment for cattle. Introducing a gene that stimulates the accumulation of monomeric/polymeric anthocyanidins might reduce the ruminal protein degradation rate (by fixing protein and/or direct interaction with microbes) and additionally reduce methane emission. The objectives of this study were to evaluate in vitro fermentation, degradation and microbial N partitioning of three forage colour phenotypes (green, light purple-green (LPG) and purple-green (PG)) within newly developed Lc-progeny and to compare them with those of parental green non-transgenic (NT) alfalfa.nnnRESULTSnPG-Lc accumulated more anthocyanidin compared with Green-Lc (P < 0.05), with LPG-Lc intermediate. Volatile fatty acids and potentially degradable dry matter (DM) and N were similar among the four phenotypes. Gas, methane and ammonia accumulation rates were slower for the two purple-Lc phenotypes compared with NT-alfalfa (P < 0.05), while Green-Lc was intermediate. Effective degradable DM and N were lower in the three Lc-phenotypes (P < 0.05) compared with NT-alfalfa. Anthocyanidin concentration was negatively correlated (P < 0.05) with gas and methane production rates and effective degradability of DM and N.nnnCONCLUSIONnThe Lc-alfalfa phenotypes accumulated anthocyanidin. Fermentation and degradation parameters indicated a reduced rate of fermentation and effective degradability for both purple anthocyanidin-accumulating Lc-alfalfa phenotypes compared with NT-alfalfa.

Assessing protein availability of different bioethanol coproducts in dairy cattle.

Bioethanol production has led to the production of considerable quantities of different coproducts. Variation in nutrient profiles as well as nutrient availability among these coproducts may lead to an imbalance in the formulation of diets. The objectives of this study were to fractionate protein and carbohydrates by an in situ approach, to determine ruminal availability of nutrients for microbial protein synthesis and to determine protein availability to dairy cattle for three types of dried distillers grains with solubles (DDGS; 100% wheat DDGS (WDDGS); DDGS blend1 (BDDGS1, corn to wheat ratio 30 : 70); DDGS blend2 (BDDGS2, corn to wheat ratio 50 : 50)) and for different batches within DDGS type using the 2010 DVE/OEB protein evaluation system. The results indicated that all DDGS types are quantitatively good sources of true protein digested and absorbed in the small intestine (DVE values; 177, 184 and 170 g/kg dry matter (DM) for WDDGS, BDDGS1 and BDDGS2, respectively). Rumen degraded protein balances (OEB) values were 159, 82, 65 g/kg DM in WDDGS, BDDGS1 and BDDGS2, respectively. Despite the differences in ruminal availability of nutrients among the different batches of DDGS, the DVE values only differed between the batches of BDDGS1 (194 v. 176 g/kg DM). In conclusion, when DDGS is included in the rations of dairy cattle, variation in its protein value due to factors such as DDGS batch should be taken into consideration.

Carbohydrate and Lipid Spectroscopic Molecular Structures of Different Alfalfa Hay and Their Relationship with Nutrient Availability in Ruminants

Objective This study was conducted to determine molecular structures related to carbohydrates and lipid in alfalfa hay cut at early bud, late bud and early flower and in the afternoon and next morning using Fourier transform infrared spectroscopy (FT/IR) and to determine their relationship with alfalfa hay nutrient profile and availability in ruminants. Methods Chemical composition analysis, carbohydrate fractionation, in situ ruminal degradability, and DVE/OEB model were used to measure nutrient profile and availability of alfalfa hay. Univariate analysis, hierarchical cluster analysis (CLA) and principal components analysis (PCA) were conducted to identify FT/IR spectra differences. Results The FT/IR non-structural carbohydrate (NSCHO) to total carbohydrates and NSCHO to structural carbohydrate ratios decreased (p<0.05), while lignin to NSCHO and lipid CH3 symmetric to CH2 symmetric ratios increased with advancing maturity (p<0.05). The FT/IR spectra related to structural carbohydrates, lignin and lipids were distinguished for alfalfa hay at three maturities by PCA and CLA, while FT/IR molecular structures related to carbohydrates and lipids were similar between alfalfa hay cut in the morning and afternoon when analyzed by PCA and CLA analysis. Positive correlations were found for FT/IR NSCHO to total carbohydrate and NSCHO to structural carbohydrate ratios with non-fiber carbohydrate (by wet chemistry), ruminal fast and intermediately degradable carbohydrate fractions and total ruminal degradability of carbohydrates and predicted intestinal nutrient availability in dairy cows (r≥0.60; p<0.05) whereas FT/IR lignin to NSCHO and CH3 to CH2 symmetric stretching ratio had negative correlation with predicted ruminal and intestinal nutrient availability of alfalfa hay in dairy cows (r≥−0.60; p<0.05). Conclusion FT/IR carbohydrate and lipid molecular structures in alfalfa hay changed with advancing maturity from early bud to early flower, but not during the day, and these molecular structures correlated with predicted nutrient supply of alfalfa hay in ruminants.