The impact of the following on beef digestibility was determined by static
For years, the field of meat science has prioritised research on meat as a standalone entity, with the aim of describing its table and manufacturing qualities, composition and nutrient density. Much less is known about its attributes in the context of whole meals where meat is just one of the several ingredients or in meat-rich foods that are designed with specific functionality or consumers in mind. Incentives for understanding how meat performs in complex matrices are increasing due to recent growth of the “meal kit” industry and demand for all in one meal solutions at retail outlets. Information about this central ingredient would assist in the design of meals to optimise processing and packaging requirements and to deliver better eating experiences and nutrition.
A key functionality of meat, whether eaten alone or in food combinations, is its digestibility. This has relevance to consumers across a wide range of demographics, physiologies, and lifestyles. Indeed, all desirable nutritional benefits of meat hinge first on adequate digestion. This is not a fixed attribute, as it is influenced by intrinsic and extrinsic factors such as animal rearing method [
In this paper, we describe a linked series of experiments on intrinsic and extrinsic factors that can affect the
All animals were sourced from commercial or research farms in New Zealand, where feeding systems are typically free-range grazing on ryegrass/clover pastures with occasional supplements of conserved forages. The genetic background of the animals was primarily Friesian for the dairy-based livestock classes and Angus × Hereford for prime beef.
For these experiments, cows were end-of-service cull dairy cattle aged 6 years. Bulls were noncastrated male dairy cattle aged 18–24 months. Calves were unweaned male dairy cattle aged 4–14 days, which is considered “veal” in some markets. Prime beef steers and heifers were castrated male and unmated female beef cattle aged 24–30 months. All animals were slaughtered and processed at licensed commercial abattoirs.
To investigate the effect of age of cattle, legs from seven cows, seven bulls, and seven calves were collected. The legs were held for 24 h at 8°C–10°C to achieve rigor and then kept in a chiller (−1.5°C) over the weekend. For each leg, the semimembranosus (SM) muscle was dissected and the postrigor pH measured. A 100 g sample was minced using a food processor, and the remaining muscle was frozen at −30°C. Pooled samples of cow, bull, and calf were prepared by mixing 10 g of the mince from each of the seven animals. Subsamples of the 21 individual muscles and the three pooled samples were set aside for protein determination, and the remainders were frozen at −30°C. The pooled and individual samples (
To investigate the effect of rigor state,
To investigate the effect of ultimate pH, seven of the LD were selected based on their ultimate pH to provide a pH range of 5.6 to 6.9. Two were low pH (i.e. ≤5.8), two were intermediate pH (pH 5.81–6.2), and three were high pH (>6.2). For each of these, all seven timepoints (six prerigor and one postrigor) were used (
To investigate the effect of particle size, the two LD with low ultimate pH (i.e. ≤5.8) were selected and their 50 min prerigor and 48 h postrigor timepoints were used. These were either ground to powder using the SPEX Freezer/Mill® or smashed coarsely with mortar and pestle just prior to digestibility measurements.
To investigate the effect of muscle type or meat cut, the rhomboideus, infraspinatus, supraspinatus, and extensors/flexors from five prime heifers were collected. Each muscle/cut from each animal was separately minced through a 3 mm plate and thoroughly homogenised, then subsampled, and stored at −30°C until digestibility measurements.
To compare organ versus muscle meats, heart, kidney, spleen, and liver meat and muscle meat (M. semitendinosus, eye of the round) from five prime steers were collected, and the surface fat was removed. The tissues were minced through a 3-mm plate and stored at −30°C until digestibility measurements.
To understand digestibility of meat in the context of a meal, we first investigated which nonmeat (cereal or vegetable) accompaniments are most commonly served with red meat meals. An internet search of restaurants was carried out. A total of 101 and 120 restaurants in New Zealand and Australia, respectively, were selected that had a web presence, published their menus online, and served at least one dish in which accompaniments were cooked with a red meat or served alongside. Eligible meats were beef, lamb, calf veal, goat, venison, kangaroo, wallaby, and rabbit. The restaurants were distributed across the countries. In Australia, for instance, menus were studied from twenty restaurants in each of six cities: Brisbane (Queensland), Adelaide (South Australia), Melbourne (Victoria), Perth (Western Australia), Sydney (New South Wales), and Hobart (Tasmania). From the results of the survey, the most common accompaniments were tallied from the frequencies of their appearance in the menus (Figure
Experiment 5: summary of results from the online survey of accompaniments to meat, as described on menus in New Zealand and Australia restaurants.
Mushrooms (button, Mushrooms: peeled, stalks trimmed, sliced, and blended in a food processor Onions: peeled, chopped, and blended in a food processor Potatoes: peeled, chopped, and blended in a food processor Rice: ground to powder in a Waring blender Tomatoes: chopped and blended in a food processor (skin on) Pumpkin: removed skin, deseeded, and blended in a food processor
The pooled samples from Experiment 1 of minced SM from either cow, bull, or calf were each combined with each of the accompaniments (3 × 6 = 18 treatment combinations) in a 1 : 2 w/w ratio of meat and accompaniment. Unaccompanied meats served as controls. The various meats were weighed into 100 ml Schott bottles to give the equivalent of 875 mg protein (3.7 to 4.5 g of raw meat) and cereal/vegetable added at 2 × weight of meat. Total volume (including the volume of water contributed by the vegetables) was adjusted to 25 ml with deionised water. The mixture was homogenised using an IKA Ultra-Turrax® (13,500 rpm for 30 sec) and the shaft rinsed back into the Schott bottle with 5 ml of deionised water. The resulting slurry was placed on a laboratory shaker in the refrigerator overnight (4°C).
The following day, the slurries were cooked in a waterbath (100°C) for 30 min and then homogenised using the Ultra-Turrax to break up the lumps formed during cooking. Concentrated HCl was added to bring the acid content to 0.1 M. The slurries were diluted with 0.1 M HCL to a volume slightly less than that required to achieve a protein concentration of 23 mg/ml, then the pH was adjusted with 6 N NaOH to pH 1.9, and the total volume was topped up to reach 23 mg/ml.
Ultimate pH of the meats used in Experiments 1 and 2 was measured using a Hanna pH meter (#HI99163) as previously described [
The protein content of muscles and organs in Experiments 1 and 4 was determined from total N using AOAC methods by a commercial analytical service (Eurofins Ltd, Hamilton, NZ).
Proteolytic enzymes in the accompaniment foods of Experiment 5 may contribute to the efficiency of
Casein zymography was carried out on the extracts according to manufacturer’s instruction. Four volumes of each extract were mixed with 1 volume of 5X sample loading buffer (50% glycerol, 10% SDS, 0.1% bromophenol blue and 150 mM Tris-HCl; pH 6.8). Gels were 4–16% Novex Zymogram Blue Casein prestained gels (Thermo Fisher Scientific). The extracts (30
The digestibility of meat proteins was determined as previously described by Farouk et al. [
For most muscle and organ meats, approximately 4.5 g of a sample was weighed into a 100 ml glass Schott bottle, sealed, and placed in a boiling waterbath for 15 min with intermittent mixing by swirling. The cooked sample was allowed to cool, covered with 34 ml of 0.1 M HCl, and then homogenised using the Ultra-Turrax for 1 min. The pH was adjusted to 1.9 with 6 N NaOH and made up to 36 ml with deionised water. Note that special attention was paid to the prerigor meat samples from Experiment 2 because these were susceptible to spontaneous glycolysis unless kept frozen until the moment of cooking. For these, approximately 4.5 g of a sample was weighed into a cold Schott bottle and immediately doused with 34 ml of boiling water and then placed in the boiling waterbath for 120 sec. The cooked sample was allowed to cool and its concentration brought to 0.1 M with concentrated HCl and then homogenised using the Ultra-Turrax. The pH was adjusted to 1.9 with 6 N NaOH and made up to 36 ml with deionised water. For the meats combined with accompaniments from Experiment 5, the prepared slurries were already cooked, so these were used as is in their Schott bottles.
Samples were incubated in a shaking waterbath at 37 ± 0.2°C for 15 min. Two ml of pepsin solution (Sigma P6887, 1.575 mg/ml; enzyme:substrate ratio 1 : 280 in 0.1 M HCL, equivalent to 12.5 U/mg protein) was added to start the proteolysis. Controls were prepared using cooked meat and 0.1 M HCL without pepsin. An aliquot of 500
Pancreatin (Sigma P8096, 2.2 ml of 4 mg/ml, enzyme : substrate ratio 1 : 100 w/w in 0.1 M phosphate buffer, pH 8) was added to the Schott bottle and digestion allowed to proceed for further 2 h, with aliquots withdrawn at intervals. Enzyme activity in each aliquot was quenched by lowering the pH to 2 by the addition of 6 M HCl. Laemmli loading buffer protocol was then followed as above.
Proteins and peptides in the digesta collected during
For Experiment 5 which combined beef with nonmeat accompaniments, 20
To quantify the overall efficiency of
For experiments 1 and 4, we calculated the relative digestibility. This was calculated by summing the density of all bands in a gel lane >10 kDa and then normalising to (i.e., dividing by) the summed density of bands in the respective T0 lane (timepoint 0, prior to digestion activation). Analysis of variance (ANOVA) was performed on this point estimate using Genstat software (17th Edition, VSN International, 2014). Pairwise
The effects of intrinsic and extrinsic factors on the
Figure
Experiment 1: SDS PAGE showing the effect of age of cattle on the digestibility of protein in cooked semimembranosus from cow (C), bull (B), and calf (veal, V). Results of gastric and intestinal phases for the pooled samples from 7 animals per age are presented as examples.
Experiment 1: the relative digestibility (see methods) of cooked semimembranosus from cow (C), bull (B), and calf (veal, V) at 5 min and one hour of gastric phase and two hours of intestinal phase. Results for the pooled samples from 7 animals per age are presented as examples.
The time of sampling of LD muscle (prerigor, from 50 min through 200 min postmortem) had little influence on the digestibility of beef proteins, and this was not markedly affected by rigor at 48 h. Typical results for T60 of the gastric phase are shown in Figure
Experiment 2: SDS PAGE showing the effect of pre- and postrigor sampling time on the digestibility of protein in cooked M. longissimus dorsi from bull beef. Results at T60 of gastric phase for two of the 48 animals in this experiment are presented as examples.
The proteins of high ultimate pH meat digested faster than their low ultimate pH equivalent. Typical results for T0 through T60 of the gastric phase are shown in Figure
Experiment 2: SDS PAGE showing the effect of ultimate pH (pHu) on the digestibility of protein in cooked M. longissimus dorsi from bull beef. Results during the gastric phase for the sampling timepoint from two of the seven animals in this experiment are presented as examples.
The digestibility of proteins from different muscles and meat cuts from prime beef heifers was compared. Typical results for T0, T5, and T60 of the gastric phase and T240 of the intestinal phase are shown in Figure
Experiment 3: SDS PAGE showing the effect of muscle type/cut on the digestibility of protein in cooked meat from prime heifers. Result at T0, T5, and T60 of gastric phase and T240 of intestinal phase for one of the five animals in this experiment are presented as examples. L to R extensors and flexors, rhomboideus, infraspinatus, and supraspinatus.
There was little effect of particle size on the digestibility of cooked proteins in meat. Figure
Experiment 2: SDS PAGE showing the effect of mincing/particle size on the digestibility of protein in cooked M. longissimus dorsi from bull beef. Results at T0, T30, and T60 of gastric phase for the two low pHu animals in this experiment are presented as examples. FG = finely ground; CG = coarsely ground.
The structure and composition of organ meats is substantially different from muscle meat, and this has consequences for digestion. For instance, the protein content of the heart, kidney and spleen from prime steers was 10–27% less on a fresh-weigh basis (Table
Experiment 4: protein content and digestibility of bovine organ meat and muscle meat.
Attributes | Organ meats | Beef | SED |
| |||
---|---|---|---|---|---|---|---|
Heart | Kidney | Spleen | Liver | ||||
Protein (%) | 18.43ab | 16.47a | 17.33a | 20.27bc | 22.73c | 1.21 | 0.001 |
RD @ 5 min | 54.68a | 73.90b | 45.01a | 84.06b | 56.34a | 6.11 | 0.001 |
RD @ 1 h | 79.86ab | 82.53ab | 75.55a | 86.43b | 75.84ab | 5.30 | 0.05 |
RD @ 4 h | 95.33ab | 91.98a | 94.50ab | 96.32b | 95.09ab | 3.10 | 0.006 |
RD = relative digestibility; SED = standard error of difference; means in the same row bearing the same superscripts are not different (
The online survey of menus from New Zealand and Australia restaurants revealed that the most common accompaniments served with red meat were potato, onion, mushroom, tomato, rice, noodle, bean, and carrot (Figure
SDS PAGE separation of proteins and peptides in the digesta of beef cooked with the top five accompaniments plus pumpkin showed that meats from all three age categories of animals (4-day-old calf, 18- to 24-month-old bull, or 6-year-old cow) were most digestible when cooked with mushroom, whereas digestion was least efficient when the meats were cooked with rice and potatoes. Based on relative digestibility calculation and averaging over all animal ages, the rank order of protein digestibility was found to be mushroom > pumpkin > onion = tomato > rice > potato. Figure
Experiment 5: SDS PAGE showing the effect of vegetable accompaniments in a cooked “meal” containing semimembranosus from cow, bull, or calf on the digestibility of total proteins. Results of gastric and intestinal phases for the meal containing pooled bull meat from Experiment 1 with and without mushroom are presented as examples.
Experiment 5: density tracings of SDS PAGE showing the effect of vegetable accompaniments in a cooked “meal” containing semimembranosus from cow, bull, or calf on the digestibility of total proteins. Results at T0 of gastric phase and T240 of intestinal phase for meals containing pooled bull meat from Experiment 1 with six accompaniments are presented as examples.
Enhanced digestion from cooking with mushroom (and pumpkin) could be due to the presence of endogenous proteolytic enzymes in these vegetables that were not present in the other accompaniments as observed in the results (zymograms bands visible on gels but too faint when photographed, thus not included) of the zymogram gel separation of enzymes extracted from the six accompaniments which showed faint protease activity seen for pumpkin and mushroom and not for the other accompaniments.
Meat is usually considered to be the skeletal muscle of animals, along with any attached fat, connective tissue, blood and blood vessels, and may also include organ tissues such as the liver, heart, kidney, and intestines [
We previously determined the effects of ultimate pH, ageing, and cooking on beef digestibility and suggested that chefs could exploit the attributes to tailor the choice of muscle and preparation to the requirements of customers [
Age of livestock at slaughter is a significant determinant of meat functionality and eating quality, and it may also affect digestibility. Experiment 1 with three ages of beef demonstrated that when the relative digestibility of all the proteins resolved in Figure
Sustainable production of animals as a source of food demands that we make full use of every carcass. Unlocking the potential of the less familiar cuts and promoting their inherent benefits is an important role for nutritional research. Experiment 4 demonstrated that beef organ meats/offals such as liver and kidney were more digestible than muscle meat from the same carcass (Table
When meat is served at a meal, its accompaniments are usually chosen to provide a balance of nutrition or for culinary and gustatory purposes. Well-informed combining can also produce beneficial biochemical synergies. For instance, consuming orange juice that contains ascorbic and citric acids will enhance the bioavailability of ferric iron in plant foods [
For Experiment 5, we decided that the “best” accompaniments to study were those in wide and common use. A survey of meal designs in restaurants provided insight and objective measures, although perhaps biased towards luxury and indulgence eating. The top accompaniments were potato, onion, mushroom, tomato, and rice (Figure
Although the present study was not designed to determine the effect of cooking
Within the parameters of the present study, beef was observed to be more digestible or digested faster when it came from an older animal, at prerigor, and when it had high ultimate pH or contained less collagen content. Some beef organ meats were more digestible than beef muscle. Digestibility improved when meat was cooked with vegetables that contain proteolytic enzymes and diminished slightly with carbohydrate-rich or starchy foods such as rice and potatoes.
These results help to support a rational basis for the design of prepared meals where meat protein is a central ingredient, such as ready meals, institutional catering, and novel product categories of foods. Formulating around digestion functionality creates opportunities for speciality foods suited to infants and elder consumers. These are novel, value-adding ways to make the nutrition of meat more widely available and to stimulate sustainable utilisation of the entire carcass.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.
This work was supported by grants from governmental contestable funding pools (NZ Ministry of Science and Innovation and later the NZ Ministry of Business, Innovation and Employment) and the AgResearch Strategic Science Investment Fund (contract A19119). The authors are grateful for valuable assistance in the field and laboratory from the AgResearch technical staff Kevin Taukiri and Ancy Thomas and from the University of Waikato student interns Oliver Cook, Hannah VanderWoude, and Georgia Clements, and from the French intern Fanny Badée.