In vitro methods to investigate the hypothesis that the transit of animal products in the mouth and stomach will result in the formation of lipid oxidation end-products that will affect the chemistry and bioavailability of proteins

Mylène Delosière, Mike Boland

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Mylène Delosière, Mike Boland. In vitro methods to investigate the hypothesis that the transit of animal products in the mouth and stomach will result in the formation of lipid oxidation end-products that will affect the chemistry and bioavailability of proteins. INRAE; Riddet Institute. 2014, pp.3. *hal-05051106*

HAL Id: hal-05051106

https://hal.science/hal-05051106v1

Submitted on 29 Apr 2025

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**Short Scientific report from Mylène Gobert** (1<sup>st</sup> January-24<sup>th</sup> June 2014, New-Zealand)

Mylène Gobert, INRA, UR QuaPA, Saint-Genès-Chamanelle, France

Mike Boland, Riddet Institute, Massey University, Palmerston North, New Zealand

Written on 10th July 2014

**Purpose.** The purpose of this mission was to use *in vitro* methods to investigate the hypothesis that the transit of animal products in the mouth and stomach will result in the formation of lipid oxidation end-products that will affect the chemistry and bioavailability of proteins.

**Work carried out.** We chose to study meat products, particularly beef, which is rich in iron, known to promote oxidation processes. Work was focused on the effect on digestion of meat proteins using an *in vitro* model of human gastric digestion.

The first part of work considered proteins from beef muscles differing in their lipid content after incubation of comminuted muscle samples in air at 37°C for 30 min.

The second part of work compared proteins in samples from matched cuts of beef obtained from grain- and grass-fed animals. The two beef muscle samples, which differed in their fat content, were cooked, minced and introduced into the Human Gastric Simulator (Kong & Singh, 2010). Samples were taken at regular intervals. Protein content and electrophoretic profiles were compared for each. Evaluation of lipid oxidation level was attempted using the TBARS assay. Sample preparation for polyacrylamide gel electrophoresis (PAGE) using native gels and, separately, under denaturing reducing and non-reducing conditions, using denaturing and reducing agents (Sodium Dodecyl Sulfate (SDS), Dithiothreitol (DTT)) was carried out in order to detect protein aggregates and protein digestion.

A third part of the work was initiated on the oxidation of beef in the mouth, with trials on chewing of cooked beef using a mouth-robot (Sun C. et al., 2014).

**Main results.** The first protein analyses showed that, after incubation in air, cubes of fat beef had a lower soluble protein content than minced lean beef. We suspected lipid oxidation was occurring preferentially in the fat beef, leading to the formation of insoluble protein aggregates. Several weeks later we were unable to reproduce this effect. It is possible that the beef fatty acid content changed due to beef cattle (which are mostly pasture-fed in New Zealand) ingesting "dry" grass following dry climatic conditions in the New Zealand late summer. It is well established that under dry conditions pasture grasses have lower levels of unsaturated lipids and it is to be expected that following consumption, animal fat will reflect this.

The most instructive comparison was the one with beef from grain- vs grass-fed animals: the beef from grain- fed animals contained a very high amount of lipid compared to the beef from grass-fed animals. After the simulated digestion in the mouth and stomach, raw meat, bolus and digesta from the two different meats were compared in terms of PAGE profiles, free amino nitrogen contents and TBARS levels. Unfortunately, the TBARS assay did not work on digesta, which are a complex matrix. This assay needs to be further developed to be suitable for digesta samples.

During the digestion, native PAGE profiles (figure 1) showed the loss of high molecular weight proteins (150-200 kDa), particularly after 15 min, and appearance of smaller proteins and peptides (around 30 kDa). In these native conditions, no big differences were seen between the profiles from the beef samples.
<figure><img src="image_2.png" /><figcaption>Figure 1: Native condition PAGE profiles of proteins from digesta from grass- and grain-fed beef after 15, 30, 45 and 60 min in the Human Gastric Simulator.</figcaption></figure>

Denatured and reduced PAGE profiles (figure 2) showed the disappearance of all proteins with time. We note a disappearance of proteins around 15 kDa after 15 min. One of these proteins is likely to be myoglobin, which is major component of the sarcoplasmic protein and has a molecular weight of 17 kDa. At 30 min, there were differences between samples for more proteins detected in the profile from beef from grain-fed animals than in beef from grass-fed animals. Further analysis will give the kinetic parameters for digestion of each protein.

<figure><img src="image_3.png" /><figcaption>Figure 2: Denatured (SDS) and reduced (DTT) PAGE profiles of proteins from digesta from grass- and grain-fed beef after 15, 30, 45 and 60 min in the Human Gastric Simulator.</figcaption></figure>

Concerning beef degradation in the mouth and before introduction into the *in vitro* stomach, we tried to reproduce the role of the mouth (chewing) using basic equipment. A meat mincer with
addition of meat juice and saliva was used in order to produce a swallowable bolus compatible with
use in the Human Gastric Simulator. This bolus was compared to chewed meat obtained using the
mouth-robot. The bolus from minced meat needed less compression force than chewed meat, which
indicated that our bolus preparation conditions differed from those of beef chewed by the mouth-
robot. These results suggest that the question of sample preparation to reproduce destructuring in
the mouth may be important in the study of solid products such as cooked beef.

**Collaboration/Foreseen article.** This work is continuing with comparisons of the protein band intensities in electrophoretic profiles of each meat sample, bolus and digesta in order to clearly show differences in rates of digestion of the proteins between different meat samples. We are corresponding by e-mail in order to discuss about the valorisation. The next meeting will be held in October when Mike Boland will visit INRA in Clermont-Ferrand.

**References.**

Kong, F. and Singh, R.P. (2010). A human gastric simulator (HGS) to study food digestion in human stomach. Journal of Food Science. 75 (9), E627-E635.

Sun, C., Xu, WL., Bronlund, JE., & Morgenstern, M. (2014). Dynamics and Compliance Control of a Linkage Robot for Food Chewing. IEEE TRANSACTIONS ON INDUSTRIAL ELECTRONICS. 61(1), 377-386.