Protein‐enriched fiber vegan meal promote satiety and suppress food intake in rats

Abstract Dietary preferences were closely associated with the pathogenesis of numbers of metabolic disorders, in particularly, obesity. Dietary fiber was shown to be capable of preventing weight gain and excessive food intake mainly through stimulating chewing and saliva secretion, and promote satiety signals. In this study, we characterized the "Vitamin World® Vegan Meal" Formula of Feihe, a novel protein‐enriched fiber dietary supplement contained potato protease inhibitor II (PI2) that developed. And we demonstrated that this particular fiber formula was effective in preventing weight gain, increasing satiety signals, and reducing food intake in rats in a dosage‐dependent manner. Our study provides lines of evidence and would further bolster the use of this nutritious vegan meal in regulating satiety and food intake in clinics.

Dietary fiber usually absorbs water and subsequently expands in the stomach to further increase the sense of satiety. The viscosity system is enhanced after fiber hydration, which slows stomach emptying and lengthens the sense of satiety. Epidemiological studies have showed that person who eats more fibers will tentatively be slimmer than those who eat fewer fibers (Tucker & Thomas, 2009). This finding further substantiates a negative correlation between eating dietary fibers and weight gain. Other than dietary fiber, potato extracts proteinase inhibitor II (PI2) enhances satiety by promoting endogenous release of cholecystokinin (CCK), a major satiety peptide produced by our body (Ku et al., 2016;Peters et al., 2011;Zhu, Lasrado, & Hu, 2017). Classic studies showed that CCK is a powerful endogenous peptide, which can prompt organs such as stomach and brain, to produce the sensation of satiety (de Graaf, Blom, Smeets, Stafleu, & Hendriks, 2004).
In addition, studies have shown that high protein diet is also effective in weight control due to strong satiety when digesting proteins compared with other macronutrients (Deibert, Schmidt-TrucksaessA, Frey, Landmann, & Berg, 2004;Mikkelsen, Toubro, & Astrup, 2000). in rats, which is developed based on the satiety mechanism of oat fiber, potato extract PI2, and soybean protein isolate. Moreover, the formula was also compared with commercial competition of meal replacement powder to provide the evidence of the weight loss, food intake, and seven physiological indicators of satiety in rats, in order to develop the ideal product for weight control.

| Animals and diets
A total of 141 male Sprague Dawley (SD) rats with initial body weight of 180-200 g were provided by the Beijing Vital River Laboratory Animal Technology Co., Ltd. These rats were housed individually in standard cages that allowed recording of food intake. The cages were placed in a temperature-and humidity-controlled room (25 ± 2℃, 55% ± 2% humidity) with a 12-hr/12-hr light/dark cycle (light off at 8:00 p.m.). Rats were given free access to water and food.
The animals were acclimatized for one week with the control diet fed prior to the experiment and fasted 12 hr before gavage with free access to water. SD rats were used as they are reported to have a good consistency in satiety evaluation. All the experiment protocols were approved in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.

| Pilot study
In pilot study, 36 rats were randomly divided to six supplement treatment groups (6 rats per group). All diets were based on the normal chow (control diet). The blank control groups (B1, B2) were fed control diet with water. One pair of positive control groups P3 and P4 were fed the control diet with meal replacement powder (0.278 g/ ml, Commercial competition). Another pair of positive control groups P5 and P6 were fed the control diet with potato extract (3.33 mg/ml, Slendesta ® , Kemin Industries). Each rat in every group was given the test supplement 1.5 ml per day.
The pilot experiment was to investigate the time point (0.5, 1, 2, 4 hr) at which serum parameters changed significantly among Ghrelin, GLP-1, GIP, CCK, PYY, LEP, and ADP after giving blank and positive control supplement, during which food intake was determined daily and body weights were measured. On the first day, after intragastric administration, blood samples of B1, P3, and P5 were collected at 0.5 hr later, while blood samples of B2, P4, and P6 were collected at 1 hr later and the weight of the feeds in each group was measured. On the fourth day after 2 days of blank feeding, blood samples of B1, P3, and P5 were collected at 2 hr after intragastric administration, while blood samples of B2, P4, and P6 were collected 4 hr after intragastric administration and the weight of the feeds in each group was measured. The blood samples were stored in −80℃ for ELISA.

| Formal experiment
The The formal experiment was to investigate the satiety of the VW-VM Formula compared with commercial competition of meal replacement powder and potato extract PI2 from body weight, food intake, and serum parameters. The grouping and dosing of experiment are listed in Table 1. All of the 90 rats were acclimatized for one week with the control diet and weighed one day before the experiment. Each group began gavage at 7:00 a.m. every morning and collected the blood sample 1 hr later for 5 days. After 7 days of gavage weighed the rats separately and recorded the weight of the feed.

| Statistical analysis
Data were analyzed by Student's t test and one-way ANOVA test by using StatView Statistics software program (Brainpower). Date were examined to ensure patterns of constant variance and a normal distribution. Results that were not fit to these conditions would be transformed to proper data using logarithms or square roots. The data were presented as means ± SEM, differences were considered significant if p < .05, and significant differences were represented as * for p < .05, ** for p < .01, and *** for p < .001.

| VW-VM-A containing PI2 enhances satiety in rats by regulating serum PYY, Ghrelin, GLP-1, CCK, and GIP
The pilot study investigated the changes of CCK, PYY, GIP, LEP, GLP-1, ADP, and Ghrelin levels in rat serum samples on 4 time points (0.5, 1, 2, and 4 hr) after gavage with sterile distilled water (blank control), potato extract PI2 (positive control 1), and commercial meal replacement powder (positive control 2). Our experiments were performed for five consecutive days, and data were averaged for all days for comparison but listed individually for each day in Table 2. We found that after 1 hr of gavage, all positive control groups could significantly enhance satiety revealed by the increased level of satiety peptides in serum, except LEP.
The levels of PYY in serum after 2 hr of gavage and ADP in serum after 4 hr of gavage were also increased obviously. In addition, the serum level of CCK and ADP were elevated more by potato extract PI2 treatment than competing goods treatment, while the effect of the competing goods was more obvious on increasing GLP-1 in serum. These In a summary, Formula VW-VM-A/L increased the level of GIP (n = 2) and CCK (n = 2) but showed little effect on GLP-1 (n = 2).
Whereas formula A has decreased Ghrelin level (n = 4). In addition, the effect of competing goods was reflected in PYY (n = 2) and GIP (n = 2). However, the effect of single potato extracts PI2 was not satisfactory, which mainly affected GLP-1 (n = 2). These results indicated that the most effective formula among the testing supplements on

| Dietary supplements did not affect body weight gain
After evaluating serum markers for satiety, we looked into the body weight gain in rats treated with different dietary supplements. We  Table 3).
These results suggested that Formulas A is capable of preventing excessive body weight gain in adult rats provided with normal chow.

| The effect on food intake
The total food intake of every cage was weighed continuously for 5 days after gavage at 0.5, 1, and 6 hr every day. Average daily  The result showed the blank control group had the highest food intake (Table 4). Food intake of positive control groups and Formula VW-VM groups were decreased compared with the control group at different time points (0.5, 1, and 6 hr) ( Figure 6). These results suggested that Formula VW-VM-A/H and VW-VM-B were relatively most effective in reducing food intake.

| CON CLUS IONS
In this study, we systematically evaluated the "Vitamin World ® Vegan Meal" Formula of Feihe containing dietary supplement's function in regulating satiety, body weight gain and food intake using rats as a model. By measuring variety of biological markers related with satiety, we reveled a remarkable role of Formula VW-VM-A/L and VW-VM-A/H were capable of promoting satiety by increasing PYY, CCK, GIP, and ADP as well as decreasing LEP and Ghrelin.
Interestingly, we noted that high dosage of Formula VW-VM-A/H exhibited stronger effect on increasing the level of PYY, CCK, and ADP, and decreasing LEP and Ghrelin than low dosage Formula A.
We reason that Formula A contained PI2 may promote satiety in a dosage-dependent manner. However, GIP was not further increased upon high dosage treatment compare with low dosage Formula A treatment. This particular phenotype could be due to a strong effect on other satiety-related factors which might in turn suppress the disproportionately enhanced level of GIP. In addition, we observed that Formula VW-VM-A/H and VW-VM-B successfully attenuated excessive body weight gain in rats and inhibited food intake for days.
These results were consistent and provide numbers of evident to support our hypothesis that Formula VW-VM containing dietary supplement was effective in suppressing body weight gain and food intake chronically. We therefore view Formula VW-VM-A/H as a competent dietary supplement for regulating daily food consumption in health subjects which could potentially be used as a tool for bodyweight control. Rats given Formula VW-VM-A/H gavage reduced the food intake, promoted satiety, and reduced body weight gain.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest to this research.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.