Confrontation of the “Dual Tracer” Indirect Method With Direct Ileal Sampling for Indispensable Amino Acid Digestibility of Sunflower Isolate in Humans

Objectives The direct assessment of ileal samples of amino acid (AA) digestibility is invasive in humans. A less invasive but indirect method, namely « dual tracer » was recently developed. It relies on the plasma isotopic enrichment ratio of two labeled protein, a reference protein or alternately AAs labeled with 13C and the test protein labeled with 15N. This recent method has not yet been challenged against direct measurement of ileal digestibility. Methods Seven healthy volunteers were intubated with naso-ileal tube. Every 30min for 4h, they ingested sunflower biscuits containing a total of 25g of 15N intrinsically labeled sunflower protein isolate. They also ingested 60g chocolate containing a total of 400mg of a mix of 13C algal individual AAs. Ileal contents were collected continuously for 8h following the first meal and plasma was sampled every 30min for 4h and hourly between 4 and 8h. 15N and 13C indispensable amino acid (IAA) ileal digestibility were determined by measuring 15N and 13C enrichment in AAs by GC-C-IRMS and AA content by UHPLC in ileal effluent. Plasma and meal 15N and 13C IAA enrichment were measured by GC-C-IRMS. Isotopic 15N/13C ratio were determined using area under the curve value for each isotope. Results Using direct ileal sampling, average IAA ileal digestibility was: (i) 88.5 ± 5.0% for sunflower isolate (15N) with values ranged from 85.8 ± 5.1% for threonine to 91.1 ± 5.8% for methionine, and (ii) 97.6 ± 1.7% for free AAs (13C) with values ranged from 95.9 ± 2.3% for lysine to 98.8 ± 0.8% for phenylalanine. With the “dual tracer” method, digestibility of isoleucine, leucine, threonine and valine was significantly lower than with ileal determination (from 7.9% for threonine to 24.3% for leucine), Methionine and phenylalanine values were aberrant (over 100%) For lysine, the difference between the two methods was not statistically different (4.7%, p = 0.49). Conclusions With our methodological conditions, the “dual tracer” method provides physiological values for most IAA except methionine and phenylalanine. However, values were low compared to ileal digestibility (about 10%) and interindividual variability was high. This less invasive method is promising but requires methodological improvements. Funding Sources French Research National Agency (ANR), financial support of SOFIPROTEOL under the FASO Project PRODIAL.

Amino Acid Digestibility and Digestible Indispensable Amino Acid Score (DIAAS)-Like Values of Black Soldier Fly Larvae Fed Different Forms and Concentrations of Calcium Using the Precision-Fed Cecectomized Rooster Assay

Black soldier fly larvae (BSFL) is an alternative protein source for animals, including dogs and cats. Dietary calcium source is an essential nutrient for BSFL development in the pupal stage. Calcium carbonate (CaCO3) and calcium chloride (CaCl2) are common calcium sources, but differ in solubility, acid-binding capacity, and calcium concentration. A high calcium concentration in BSFL may affect how well nitrogen and amino acids (AA) are digested by animals consuming them, thereby affecting feed conversion efficiency. Our objective was to determine the effects of dietary calcium form and concentration on nutrient composition, AA digestibility, and digestible indispensable amino acid score (DIAAS)-like values of BSFL intended for use in animal feeds using the precision-fed cecectomized rooster assay. All BSFL tested in this study were harvested at 18 d after hatch. Industry standard rearing conditions were maintained and a commercial layer ration was fed to all BSFL until 11 d post-hatch. From d 11 to 18, BSFL were fed a combination of distiller’s dried grains with solubles from a distillery, bakery by-product meal, and varied calcium sources. All BSFL diets contained 0.2% calcium in the basal diet plus additional calcium in the following amounts and forms: BSFLA: 1.2% CaCl2; BSFLB: 1.2% CaCO3; BSFLC: 0.75% CaCO3; BSFLD: 0.6% CaCO3 + 0.6% CaCl2. At d 18, BSFL were washed and frozen. Prior to the rooster assay, BSFL were lyophilized and ground. 16 cecectomized roosters (4 roosters/substrate) were randomly assigned to test substrates. After 24 h of feed withdrawal, roosters were tube-fed 20 g of test substrates. Following crop intubation, excreta was collected for 48 h. Endogenous corrections for AA were made using 5 additional cecectomized roosters. All data were analyzed using a completely randomized design and the GLM procedure of SAS 9.4. Nutrient and AA digestibilities were not different among substrates. DIAAS-like values were calculated to determine protein quality according to Association of American Feed Control Officials nutrient profiles and National Research Council recommended allowances for dogs and cats. Although AA digestibilities did not differ, those containing CaCO3 generally had higher DIAAS-like reference values than the diet containing CaCl2 alone (BSFLA). Aromatic AA (Phe + Tyr) and sulfur AA (Met + Cys) were often first-limiting AA. Our results suggest that calcium source fed to BSFL did not affect AA digestibility and protein quality.

Use of the precision-fed cecectomized rooster assay to determine standardized amino acid digestibility, true metabolizable energy content, and digestible indispensable amino acid scores of plant-based protein by-products used in canine and feline diets

Traditionally, protein by-products from oil seeds and cereal grains have been used in pet foods as sustainable, inexpensive, and protein-rich ingredients. However, the on-going demonization of soy- and corn-based ingredients continue to hinder their use in pet food and treat formulations. Ideally, the further demonstration of their protein quality and nutrient composition may encourage their favorable return as acceptable ingredients in pet foods and treats. Therefore, the objectives of this study were to determine the macronutrient composition, indispensable amino acid profile, standardized amino acid digestibility, true metabolizable energy content corrected for nitrogen (TMEn), and digestible indispensable amino acid scores (DIAAS-like) of soy flakes (SF), peanut flour (PF), soybean meal (SBM), and corn gluten meal (CGM). Standardized amino acid digestibility was assessed using the precision-fed cecectomized rooster assay. All test ingredients demonstrated a profile of highly digestible indispensable amino acids except for lysine in PF, which was lowest (P < 0.05) at 45.5%. The SBM and CGM had the highest (P > 0.05) digestibilities of indispensable amino acids. A DIAAS-like values was calculated for each ingredient using either AAFCO (2020) recommended values or NRC (2006) recommended allowances as the reference protein pattern. For adult dogs compared to AAFCO recommended values, the first-limiting amino acid was lysine for PF and CGM but it was methionine for SF and SBM. For adult cats compared to AAFCO recommended values, the first-limiting amino acid was lysine for PF and CGM but it was threonine for SF. There was no first-limiting amino acid in SBM for cats as DIAAS-like values were over 100% for all indispensable amino acids. The TMEn values were highest (P < 0.05) for PF and CGM (4.58 and 4.31 kcal/g [dry matter basis], respectively). The protein quality of these plant-based protein by-products reflect their value as nutritional ingredients for canine and feline diets. However, the prior processing of these by-products must be considered before exposing them to additional processing methods, such as extrusion. Additionally, the inclusion of complementary proteins or supplemental amino acids will be needed to meet all indispensable amino acid requirements for a nutritionally complete and balanced pet food.

Digestible Indispensable Amino Acid Scores (DIAAS) of Six Cooked Chinese Pulses

Values for the digestible indispensable amino acid score (DIAAS) of a protein are based on true ileal amino acid (AA) digestibility values obtained in adult humans or in the growing pig as an animal model. An experiment was conducted using growing pigs to determine the true ileal digestibility (TID) values of AA in six cooked Chinese pulses (kidney bean, mung bean, adzuki bean, broad beans, peas and chickpeas). Each pulse was included in a diet as the only source of crude protein (CP). An N-free diet was given to allow determination of gut endogenous AA losses. Seven growing pigs each fitted with a T-cannula at the terminal ileum were allotted to a 7 by 6 incomplete Latin square with seven diets and six 7-d periods. The true digestibility values % for the total indispensable AA were higher (p < 0.001) for broad beans (87.3 ± 2.98) and lower (p < 0.001) for kidney bean (73.3 ± 4.84) than for the other pulses. For the older child (over 3 years), adolescent and adult, the DIAAS (%) was 88 for kidney bean, 86 for mung bean, 76 for chickpeas, 68 for peas, 64 for adzuki bean and 60 for broad beans.

Dietary intake of amino acids and vitamins compared to NRC requirements in obese cats undergoing energy restriction for weight loss

Background This study aimed to determine if obese cats undergoing energy restriction for weight loss would meet the National Research Council’s (NRC) indispensable amino acid and vitamin recommendations when fed a purpose-formulated diet. Thirty cats were placed into one of two groups; obese (BCS 8 to 9/9; n = 16) and lean (BCS 4 to 5/9; n = 14) and included in a non-randomized retrospective observational study. Cats were fed a veterinary weight loss food during a 4-week period of weight maintenance. Obese cats (O-MAINT) refers to obese cats during this period, L-MAINT to lean cats. After this initial 4-week period, the lean cats finished the study at this time and the 16 obese cats continued and were energy restricted for a 10-week period (O-RESTRICT). Analysis for dietary concentrations of indispensable amino acid and vitamin contents were performed. Daily food intakes were used to determine minimum, maximum and average daily intakes of individual nutrients for all three groups and compared against NRC 2006 minimum requirements (MR), adequate intakes (AI) and recommended allowances (RA) for adult cats. Results Over 10 weeks, O-RESTRICT cats lost 672 g ± 303 g, representing a weight loss rate of 0.94 ± 0.28% per week. Daily intake of the majority of indispensable amino acids and vitamins was greater than the NRC 2006 recommended allowance (RA per kg ideal body weight ^0.67), except for arginine, choline, crude protein, phenylalanine plus tyrosine and threonine. All O-RESTRICT cats had minimum, average, and maximum arginine intakes less than the NRC AI. Minimum daily intake of choline was below NRC RA for all O-RESTRICT cats and below NRC MR for two. All, except one, O-RESTRICT cats had a maximum and average choline intake below RA. Conclusions All cats remained clinically healthy and showed no clinical signs of deficiency. Dietary choline and arginine requirements of obese cats as well as health risks associated with low dietary intake during energy restriction warrant further investigation.

PSIX-2 Amino acid digestibility and digestible indispensable amino acid scores of a few protein isolates and concentrates derived from cereal grains, plant proteins, or dairy proteins

The objective of this experiment was to determine standardized ileal digestibility (SID) of amino acid (AA) and digestible indispensable amino acid scores (DIAAS) for brown rice protein concentrate (BRPC), pea protein concentrate (PPC), two rapeseed protein isolates (RSP-1 and RSP-2), soy protein isolate (SPI), and whey protein isolate (WPI). Seven cannulated barrows (36.51 ± 1.61 kg) were randomly allotted to a 7 × 7 Latin square design and fed a diet containing each protein as the sole source of AA. A nitrogen-free diet was also used. The DIAAS was calculated for children from 6-mo to 3-yr and persons &gt;3-yr. Data were analyzed using PROC MIXED of SAS in a randomized complete block design with pigs as the experimental unit. Among proteins, WPI had the greatest (P &lt; 0.05) SID of all indispensable AA (IAA), except Lys and Thr, and RSP-1 had the least (P &lt; 0.05) SID of most IAA. The SID of all AA, except Leu and Met, did not differ between RSP-2 and SPI, and the SID of all IAA, except Arg and Lys, was greater (P &lt; 0.05) for RSP-2 than for PPC and BRPC. For DIAAS (Table 1), RSP-2 was the only protein with a DIAAS greater than 100 for children 6-mo to 3-yr, and both RSP-2 and WPI had a DIAAS greater than 100 for persons &gt;3-yr. For both reference patterns, the DIAAS for RSP-2 and WPI was greater (P &lt; 0.05) than for SPI. The DIAAS for RSP-1 was less (P &lt; 0.05) than for SPI and greater (P &lt; 0.05) than for PPC, which had a DIAAS greater (P &lt; 0.05) than BRPC. In conclusion, RSP-2 resulted in an excellent quality protein for persons &gt;6-mo, and WPI is an excellent quality protein for persons &gt;3-yr.