3.1. Visceral Fat Reduction Effects
EWP is classified as a high-quality protein source, and when fed to rats their whole-body protein content was found to be higher when compared to those fed casein. The EWP intake was, thus, shown to increase body protein and decrease total body fat levels [
9]. In contrast, when fed the EWP, the triglyceride and leptin concentrations in the rat tail vein plasma were found to be significantly lower in comparison with those fed casein, indicating that EWP could reduce visceral fat levels [
9]. Consequently, the effects of EWP on the visceral fat in rats was further investigated and compared with casein. The results indicated that the EWP could significantly reduce white adipose tissue weight and white adipocyte area levels. EWP consumption was also found to result in significantly higher gastrocnemius muscle weight in comparison with casein [
9]. This indicates that egg white is a good source of protein that can be utilized to increase body protein, i.e., muscle mass. Since muscle tissue is the primary site of fat oxidation [
27], it was hypothesized that the consumption of egg whites may reduce both body fat and visceral fat levels.
Unheated egg whites have been used in previous studies, and it was reported that the net protein utilization rates of EWP are comparable between unheated and heated egg whites [
7]. Consequently, it was hypothesized that heated egg whites would reduce visceral fat levels in a similar manner to unheated egg whites. The effects of heated egg whites (boiled egg: 95 °C for 10 min) on visceral fat were, thus, compared to unheated egg whites and LE, with casein being used as a negative control. Although the unheated egg whites and LE reduced the visceral fat levels when compared to casein, the heated egg whites did not [
20]. These results indicate that there may be mechanisms other than the provided hypothesis, namely that egg whites reduce visceral fat levels by increasing muscle mass. A summary of the known visceral fat reduction mechanisms of EWP is provided in
Figure 2.
Figure 2.
Mechanisms for the visceral fat-lowering effects of egg white protein [
9,
21,
28,
29,
30].
Figure 2.
Mechanisms for the visceral fat-lowering effects of egg white protein [
9,
21,
28,
29,
30].
An allergy study found that the ovalbumin in EWP is not easily degraded by pepsin in unheated egg whites but is easily degraded in heated egg whites [
31]. Therefore, if ovalbumin in LE is less likely to be degraded by pepsin, the digestibility of pepsin may be involved in the visceral fat-reducing mechanism. Therefore, when we evaluated the pepsin degradability of ovalbumin in LE, it was found that the ovalbumin in LE was not easily degraded by pepsin [
21].
Pepsin is a gastric digestive enzyme, indicating that the main factor in the visceral fat-reducing effect of EWP may be occurring in the gastrointestinal tract. The triglyceride absorption into the lymph of rats undergoing thoracic lymph duct cannulation surgery was also reduced [
28]. It was shown that the suppression of lipid absorption by ovalbumin in unheated egg whites may have reduced the visceral fat level. In an in vitro study by Handa et al. (1999), ovalbumin was reported to bind to fatty acids [
32]. This indicated that ovalbumin, which is not easily affected by pepsin, can bind to fatty acids degraded by lipase in the digestive tract, thereby suppressing fatty acid absorption.
However, while the suppression of lipid absorption by EWP is approximately 20% higher than that by casein [
28], the visceral fat reduction effect is 30% less than that of casein [
9]. Despite the differences in experimental conditions, these tests were conducted by feeding diets containing 20% casein or EWP, and the visceral fat reductions by EWP could not be explained solely by the suppression of lipid absorption. Other mechanisms for reducing visceral fat using EWP include increased β-oxidation in the muscle and liver, as well as a reduction in the visceral adipocyte area [
9].
The enhancement of β-oxidation in the liver was reported in a study in which rats were fed diets containing 20% casein or EWP. The results showed that EWP consumption resulted in significantly higher activity levels for carnitine palmitoyltransferase and acyl-CoA oxidase, which are involved in β-oxidation in the liver, when compared to casein [
9].
The enhancement of β-oxidation in the muscle was identified in a similar study in which rats were a fed diet containing 20% casein or EWP, and the EWP diet was found to increase gastrocnemius muscle mass and showed higher levels of enzyme activities related to β-oxidation in the muscle when compared to casein [
33].
Studies investigating the decrease in visceral fat area in rats have also shown an increase in the expression of a series of genes, including peroxisome proliferator-responsive receptor (PPAR) γ2 and adiponectin in visceral adipocytes, as well as improved insulin sensitivity [
29,
30]. The reductions in visceral fat by EWPs were also thought to be caused by these effects.
The following two possibilities were considered as explanations for the increased β-oxidation in the muscle and liver and the increased gene expression in the visceral adipocytes. The first was the possibility of a secondary effect for lipid absorption suppression. The second was the possibility of a direct effect of the EWP-derived components on the liver, muscle, and adipose tissue. For the latter, the absorption rate of the unheated EWP was reported to be over 90% [
21]. In addition, EWP was digested into peptides in the gastrointestinal tract [
34]. Therefore, the absorption of peptides derived from EWP may generate these effects. In the liver, soy-derived peptides reportedly enhance the gene expression of enzymes related to β-oxidation in a mouse model for diabetes [
35]. For white fat cells, it has been reported that soy hydrolysate enhances the gene expression of PPARγ2, albeit this has only been found for in vitro results [
36]. Based on these findings, we expected to find a peptide with similar effects in the EWP. The identification of the peptide sequence that exerts this effect in EWP will be a subject for future studies.
Reductions in visceral fat using EWP in humans have not yet been investigated. Like EWP, lactoferrin and β-conglycinin from soybeans have also been reported to reduce visceral fat [
37,
38]. Lactoferrin is reported to reduce visceral fat at 300 mg daily and β-conglycinin at 5 g per day [
37,
38]. The inhibition of lipid absorption has been reported as a mechanism for visceral fat reduction using β-conglycinin [
39], and the inhibition of lipid absorption by ovalbumin, ovotransferrin, and lysozyme has been reported as a visceral fat reduction mechanism for EWP [
28,
40]. To consume 5 g of these three components in equal measure, 8 g of LE as EWP needs to be consumed.
At the same time, the function of ovotransferrin in EWP was found to be similar to that of lactoferrin. If ovotransferrin has the same physiological activity as lactoferrin, it was hypothesized that consuming 3 g of lactic-fermented egg white per day as protein would reduce the level of visceral fat. Based on this, the minimum effective amount of lactic-fermented egg white was determined to be 3–8 g of protein per day.
Therefore, the effects of LE containing 6 or 8 g of EWP per day or 8 g of whey protein per day was evaluated on the visceral fat levels of 22 Japanese adult males (overall) with a BMI ≥ 24 and waist ≥ 85 cm for eight weeks. The results showed that the consumption of lactic-fermented egg white, which was equivalent to 8 g of EWP per day, reduced the visceral fat area when compared to that before consumption. A further analysis in subjects with a BMI > 25 showed that 8 g per day of lactic-fermented egg white as EWP significantly reduced the visceral fat area and visceral fat/subcutaneous fat ratio when compared to pre-consumption levels or when whey protein was used instead [
21]. The results indicate that 8 g of EWP per day may reduce the total visceral fat area.
Moreover, in a double-blind, placebo-controlled study, adult men and women with a visceral fat area >100 cm
2 consumed lactic-fermented egg white (8 g/day as EWP) or 8 g/day of whey protein for 12 weeks. The results showed that the lactic-fermented egg white group significantly reduced the visceral fat area and visceral fat/subcutaneous fat ratio when compared to the pre-consumption and whey protein groups (
Table 2). This suggested that the effective amount of EWP to reduce visceral fat was 8 g per day [
41].
Table 2.
Changes in the visceral fat area and ratio of visceral to subcutaneous fat areas in subjects fed LE or milk whey for 12 weeks [
41].
Table 2.
Changes in the visceral fat area and ratio of visceral to subcutaneous fat areas in subjects fed LE or milk whey for 12 weeks [
41].
| | Time (Weeks) |
---|
| | 0 | 12 |
---|
Visceral Fat area (Δcm2) | Milk whey | 0 ± 0 | 1.71 ± 4.00 |
| LE | 0 ± 0 | −8.89 ± 2.75 * |
Visceral/Subcutaneous (Δ) | Milk Whey | 0 ± 0 | 0.0260 ± 0.0187 |
| LE | 0 ± 0 | −0.0876 ± 0.0128 * |
The effective amount of EWP was determined to be 8 g, according to the amount indicated in a pilot study assessment of the minimal effective dose in a double-blind, placebo-controlled study.
The required protein source for LE per day in only egg whites was confirmed to be 8 g of protein, based on an analysis of its protein content.
3.2. Cholesterol-Lowering Effects
Although eggs contain cholesterol, they do not necessarily increase serum cholesterol levels. This is because an individual’s genetic factors and diet must also be considered. It is known that some responders are prone to increased serum cholesterol concentrations when cholesterol is ingested [
42,
43]. Furthermore, dietary influences such as functional components that reduce cholesterol absorption, such as phytosterols, soy protein, or dietary fiber, should be considered [
44,
45,
46,
47,
48]. Chicken eggs’ functional components, specifically egg yolk phospholipids, have been reported to suppress cholesterol absorption [
49]. As for egg whites, in a study of female college students with borderline or mild hypercholesterolemia, Asato et al. reported that 23 g of EWP per day lowered their serum total cholesterol concentrations [
50]. Therefore, the authors investigated the mechanism of serum cholesterol reduction via EWP intake, and a summary of this is presented in
Table 3.
Table 3.
Effects of egg white protein on cholesterol metabolism when compared with casein [
28,
40].
Table 3.
Effects of egg white protein on cholesterol metabolism when compared with casein [
28,
40].
| | Cholesterol Level |
---|
In vivo (Rats) | Serum | ↓ |
| Hepatic | ↓ |
| Stomach | N.S. |
| Intestinal contents (solid) | ↑ |
| Intestinal contents (micelle) | ↓ |
| Intestinal mucosa 1 (upper) | N.S. |
| Intestinal mucosa 2 | ↓ |
| Intestinal mucosa 3 | N.S. |
| Intestinal mucosa 4 (lower) | N.S. |
| Lymph | ↓ |
| Fecal | ↑ |
In vitro | Micellar solubility | ↓ |
| Bile acid binding | N.S. |
| Phospholipid binding | N.S. |
| Transfer to triolein | ↓ |
| Water holding capacity | ↑ |
| Settling volume | ↑ |
| Viscosity | ↓ |
Egg white is rich in cystine, a type of sulfur-containing amino acid that reportedly may lower serum cholesterol levels [
51]; however, some reports refute this [
52]. The effects of cystine on the cholesterol-lowering effects of EWP were investigated. When rats were fed a high-cholesterol diet containing casein or EWP under conditions with or without cystine, no significant effect from the cystine was observed [
40].
Avidin in egg white binds to biotin and suppresses biotin absorption. It has been reported that a deficiency of biotin decreases the serum cholesterol concentration. The serum-cholesterol-lowering effect of EWP was compared with that of casein under conditions with or without biotin, and the biotin was not observed to have an effect [
40].
Ovomucin in egg white has been reported to reduce cholesterol absorption [
53]. Therefore, we investigated the effects of EWP on lipid absorption. When rats were fed a high-cholesterol diet containing casein or EWP, EWP increased the excretion of neutral sterols and bile acids in the feces [
40]. EWP was also found to reduce cholesterol absorption into the lymph of rats undergoing permanent lymph duct cannulation surgery. This indicated that the primary mechanism of the EWP effect was to reduce cholesterol absorption [
28].
The cholesterol absorption inhibition function of EWP was investigated. High-cholesterol diets containing casein or EWP were fed under meal-feeding conditions, and cholesterol concentrations in the stomach contents, small intestinal mucosa, and small intestinal contents were measured 2 h after consumption [
40]. The cholesterol in the stomach contents was not affected by the EWP. The cholesterol contents in the small intestinal mucosa were lower than those with the casein in the upper part of the small intestine, where cholesterol absorption actively takes place [
40]. When the small intestinal contents were separated by ultracentrifugation into the aqueous and solid phases to determine the cholesterol contents, the EWP was found to have decreased the cholesterol content in the aqueous phase and increased it in the solid phase [
40]. Since cholesterol absorption requires dissolution in bile acid micelles, the results indicated that EWP might reduce the absorption of dietary cholesterol by affecting the bile acid micelles.
The mechanism of cholesterol absorption inhibition by EWP was evaluated in vitro. Since EWP reaches the small intestine after pepsin digestion in the stomach, experiments were performed using pepsin-digested egg white. The solubility of the cholesterol in the bile acid micelles of the EWP pepsin hydrolysates was lower than that of the casein–pepsin hydrolysates [
28]. This effect was maintained in the pepsin–pancreatin hydrolysate, although the effect was slightly weakened [
28].
The components in the bile acid micelles that were affected by the EWP pepsin hydrolysate were examined, and the hydrolysate was not bound to the bile acids or phospholipids. In addition, the EWP pepsine hydrolysate was found to have a higher water holding capacity, settling volume, and relative viscosity than the casein–pepsin hydrolysate, suggesting that the physicochemical properties of the EWP pepsine hydrolysate inhibited the solubility of the cholesterol in the bile acid micelles. The release of cholesterol monomers from the bile acid micelles was also found to be suppressed [
28].
EWP mainly comprises ovalbumin, ovomucoid, ovotransferrin, and lysozyme [
54]. The solubility of cholesterol by pepsin hydrolysate in bile acid micelles was examined, and ovalbumin, ovotransferrin, and lysozyme pepsine hydrolysate were found to be effective in the micellar cholesterol solubility [
28]. In addition, reconstituted EWP pepsin–hydrolysate residue, which was reconstituted from these four components, inhibited the solubility of cholesterol in the bile acid micelles as much as the EWP, suggesting that ovalbumin, ovotransferrin, and lysozyme were the components involved [
28]. Furthermore, because of the high ovalbumin content among these components, ovalbumin was assumed to be the main component of the cholesterol-lowering effects in the EWP.
Furthermore, a study on hamsters demonstrated that heated EWP does not lower the serum cholesterol concentrations [
55]. This suggests that the cholesterol-lowering effect of EWP, like the visceral fat reduction effect, may be due to unheated egg whites or LE. Although ovomucin reportedly inhibits cholesterol absorption [
47], EWP contains only a small amount of ovomucin, and its contribution to the serum-cholesterol-lowering effect of EWP seems to be low. At this stage, the synthesis and catabolism of cholesterol in the liver and the effects of EWP in small intestinal absorptive cells have not yet been investigated, but are expected to be a focus of future research.
The effective dose of EWP by which to lower serum cholesterol levels in humans is approximately 23 g per day, but whether a lower amount would be effective is currently unknown. To test this, LE were fed at a rate of 4 g (control group), 6 g, or 8 g per day as EWP to borderline or mildly hypercholesterolemic subjects for eight weeks. The results showed that the consumption of 8 g of EWP per day significantly reduced the serum total cholesterol and LDL-cholesterol concentrations when compared to the control subjects. In addition, there was no significant difference in serum total cholesterol and LDL-cholesterol concentrations when 6 g of lactic-fermented EWP per day was consumed when compared to the control subjects (
Table 4) [
56]. This suggests that the minimal effective dose of EWP to reduce serum total cholesterol and LDL-cholesterol concentrations is 8 g/day.
Table 4.
Changes in the serum total cholesterol and LDL-cholesterol levels in subjects fed LE (as 4 g, 6 g, or 8 g of EWP) for 8 weeks [
56].
Table 4.
Changes in the serum total cholesterol and LDL-cholesterol levels in subjects fed LE (as 4 g, 6 g, or 8 g of EWP) for 8 weeks [
56].
| Egg White Protein | Time (Weeks) |
---|
Intake/Day | 0 | 4 | 8 |
---|
Total Cholesterol (Δmg/dL) | 4 g | 0 ± 0 | −4.46 ± 3.36 | 3.11 ± 3.38 |
6 g | 0 ± 0 | −7.42 ± 3.76 | −5.97 ± 3.53 |
8 g | 0 ± 0 | −11.3 ± 3.63 | −11.0 ± 3.74 * |
LDL-Cholesterol (Δmg/dL) | 4 g | 0 ± 0 | −7.61 ± 2.98 | −2.07 ± 2.91 |
6 g | 0 ± 0 | −8.00 ± 3.51 | −9.48 ± 3.12 |
8 g | 0 ± 0 | −10.0 ± 3.00 | −13.7 ± 3.74* |