Functional foods containing bioactive compounds of whey may play an important role in prevention and treatment of obesity. The aim of this study was to investigate the prospects of the biotechnological process of coacervation of whey proteins (CWP) in chitosan and test its antiobesogenic potential.
Over the past decades, the incidence of obesity in the population has increased severely, and it has become a public health challenge. Its etiology is multifactorial, encompassing environmental, dietary, physical inactivity, and genetic factors. Obesity is a complex disease associated with a high-calorie diet, which contributes to the development of several other chronic noncommunicable diseases [
The fat tissue is not merely an energy storage organ, as it plays crucial endocrine and immune roles. White adipose tissue (WAT) is an endocrine organ secreting pro- and anti-inflammatory adipokines such as tumor necrosis factor alpha (TNF-
The main role of adipose lipolytic enzymes is to provide other tissues with FAs in case of energy demand. Triglyceride stored in the lipid droplet is first hydrolyzed by the adipose triglyceride lipase enzyme (ATGL), also known as desnutrin, releasing a diacylglycerol moiety and FA, which requires an abhydrolase domain containing 5 (ABHD-5) promoter to be activated. After hydrolysis by ATGL, diacylglycerols are then hydrolyzed sequentially by hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL), producing nonesterified fatty acids (NEFAs) and glycerol [
Whey protein (WP) has been found to be an excellent prophylactic against obesity, because of the high biological value mediated by bioactive peptides. These act as antimicrobial agents, antihypertensive, and regulators of immune function, reducing body fat as well as a variety of related beneficial mechanisms for human health. They also have additional functions; for example, they have appetite suppressant effects [
Chitosan complex coacervation with WP is composed of by-products from the processing of shrimp, crab (chitosan), and cheese, adding an environmental benefit to the product, as these by-products may be reused and not disposed of in landfill sites or released into rivers by producers [
In this study we used sweet cheese whey (SW 1108 bag 25 kg) with 1.5% fat marketed by Company Alibra-PR. Dissolving 10 g of the whey powder in 100 mL of distilled water. Chitosan was used for the coacervation medium molar mass with 75–85% degree of deacetylation and viscosity of 200–800 cps (Sigma-Aldrich 44887-7). A concentration of 0.75 mg/mL of Chitosan was used. Chitosan was dissolved in citric acid (208 mmol/L) and, after this step, added to the cheese whey in a proportion of 1 : 1 under stirring at room temperature for 1 h. The pH of the solution of chitosan and WP was adjusted to 6 with NaOH (250 mmol/L) and solubilized at room temperature (±25°C) with stirring. Solids coagulated with chitosan known as coacervate (CWP) were collected by centrifugation (1300 g).
In order to obtain on average 30% of the protein coacervate 3 L cheese whey was used. Thus, samples of CWP were obtained for their chemical analysis of total lipids, total protein, and lactose. Finally, for measurements of samples of mineral micronutrients, Ca, K, Mg, and P, of cheese whey, CWP, and Chitosan, 100
This study was approved by the Research Ethics Committee of the Universidade Federalde São Paulo, Escola Paulista de Medicina (UNIFESPEPM), as the search protocol number 0473/10. Experimental procedures are in accordance with Principles of Laboratory Animal Care formulated by the National Institutes of Health (National Institutes of Health Publication number 96-23, revised 1996).
Forty-nine male Swiss mice twelve-week-old from CEDEME (Centro de Desenvolvimento de Modelos Experimentais da Universidade Federal de São Paulo) were housed five in a cage in a standard experimental animal laboratory and kept under controlled conditions of light (12 h light-dark cycle with lights on at 6 am) and temperature (24 ± 1°C). All mice received water and food
Composition of the control diet and diet enriched with saturated fatty acids according to AIN-93. Coacervate was resuspended in 300
Components (%) | Control diet (C) | High fat diet (HF) |
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Corn starch | 72.07 | 40.87 |
Casein | 14.0 | 14.0 |
Soybean oil | 4.0 | 4.0 |
Lard | — | 31.2 |
Cellulose | 5.0 | 5.0 |
Vitamin mix | 1.0 | 1.0 |
Mineral mix | 3.5 | 3.5 |
L-cystine | 0.18 | 0.18 |
Choline bitartrate | 0.25 | 0.25 |
Butyl hydroquinone. g/kg | 0.008 | 0.008 |
Energy. kcal/kg | 3,802.8 | 5,362.8 |
Treatment by gavage | ||
Coacervate (CWP) | (C-CWP) 100 mg·kg·day | (HF-CWP) 100 mg·kg·day |
Water (W) | (C-W) 300 |
(HF-W) 300 |
Fatty acids (%) | ||
Saturated (SFA) | 17.12 | 34.13 |
Monounsaturated (MUFA) | 25.63 | 39.14 |
Polyunsaturated (PUFA) | 57.25 | 26.67 |
PUFA n3 | 4.32 | 6.37 |
PUFA n6 | 52.65 | 19.98 |
Figure
Partial composition of micronutrients by ICP and chemical macronutrients of CWP, WP, and Chitosan.
Nutritional composition | |||
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g/100 g | |||
CWP | WP | Chitosan | |
Proteins | 30 | 35 | — |
Lactose | 1.7 | 40 | — |
Lipids | 0.4 | 1.5 | — |
Ca | 0.47 | 0.55 | 0.07 |
K | 0.37 | 1.69 | 0.010 |
Mg | 0.06 | 0.11 | <0.001 |
P | 0.46 | 0.53 | 0.003 |
Na | 0.81 | 1.32 | 0.26 |
(a) Electrophoretic profile coacervate using 0.75 mg/ml of chitosan. ST: standard of different molecular weights,
For total lipid extraction, diet samples were homogenized in chloroform and methanol 2 : 1 (v/v), mixed, and incubated at room temperature for 5 min. Then, additional volumes of 1.25 mL chloroform and 1.25 mL deionized H2O were added, and finally, following being vigorously homogenized for 3 min, samples were centrifuged at 1000 rpm for 5 min at room temperature. The chloroform layer was dried under N2, and the total extract was converted into methyl esters and was analyzed in gas chromatography (GC), coupled with a flame ionizer detector (FID), (Varian GC 3900) and fatty acid profile was determined by calculating the retention time, using a pattern of fatty acids with known retention time (Supelco, 37 Components). The addition was initiated at a temperature of 170°C maintained for 1 minute and then a ramp of 2.5°C/min to a final temperature of 240°C, which was maintained for 5 minutes. The injector and detector were maintained at 250°C and 260°C, respectively. We used a column CP wax 52CB, with a thickness of 0.25 mm, internal diameter of 0.25
After 12 hours of fasting, blood was collected from the tail vein to assess basal glucose concentration. Then, a glucose (Merck) solution (1.4 g/kg) was administrated by gavage. Blood samples were collected after 15, 30, 45, 60, and 120 minutes to measure glucose concentration using a glucose analyzer (AccuCheck Roche).
At the end of the experimental period, animals were fasted for 12 h overnight prior to being sacrificed by decapitation. Trunk blood was collected and immediately centrifuged (1125 g/15 min at 4°C). Serum was separated and stored at −80°C for later biochemical and hormonal determination. The adipose tissue depots, retroperitoneal (RET), mesenteric (MES), and epididymal (EPI), were dissected, weighed, immediately frozen in liquid nitrogen, and stored at −80°C.
Serum concentrations of glucose, total cholesterol, triglycerides, and HDL-c were measured by an enzymatic colorimetric method using commercial kits (Labtest, Brazil). Concentrations of insulin and adiponectin were measured using specific enzyme-linked immunosorbent assay (ELISA) kits (Milipore and R&D Systems). LPS was determined using a commercial kit (Lonza).
Following euthanasia, mesenteric adipose tissue was removed, homogenized into a specific total protein extraction buffer [1% Triton X-100, 100 mm Tris-HCl (pH 7.4), 100 mm sodium pyrophosphate, 100 mm sodium fluoride, 10 mm EDTA, 10 mm sodium orthovanadate, 2.0 mm phenylmethylsulfonyl fluoride, and 0.1 mg aprotinin/mL], and centrifuged at 12,000 g for 30 min at 4°C. The supernatant was saved, and the protein concentration was determined using the BCA assay (Bio-Rad, Hercules, California) with bovine serum albumin (BSA) as a reference. Quantitative assessment of TNF-
After euthanasia, the mesenteric adipose tissue was dissected and homogenized in 1.0 mL of solubilization buffer at 4°C [1% Triton X-100, 100 mm Tris-HCl (pH 7.4), 100 mm sodium pyrophosphate, 100 mm sodium fluoride, 10 mm EDTA, 10 mm sodium orthovanadate, 2.0 mm phenylmethylsulfonyl fluoride, and 0.1 mg aprotinin/mL]. Insoluble material was removed by centrifugation for 30 min at 9000 g in a 70 Ti rotor (Beckman, Fullerton, CA, USA) at 4°C. The protein concentration of the supernatants was determined using the BCA assay (Bio-Rad, Hercules, CA, USA). Proteins were denatured by boiling (5 min) in a Laemmli sample buffer containing 100 mM DTT and were run on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis in a Bio-Rad miniature slab gel apparatus.
The proteins were electrotransferred from gels to nitrocellulose membranes for ~1.30 h/4 gels at 15 V (constant) in a Bio-Rad semidry transfer apparatus. Nonspecific protein binding to the nitrocellulose was reduced by preincubation for 2 h at 22°C in blocking buffer (1% BSA, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). The nitrocellulose membranes were incubated overnight at 4°C with antibodies against hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), abhydrolase domain containing protein 5 (ABHD-5), perilipin A, phospho 5′ AMP-activated protein kinase (p-AMPK
All results are presented as mean ± standard error of the mean (SEM). Statistical significances were assessed using two-way analysis of variance (ANOVA) followed by Tukey’s
After six weeks of treatment, the hyperlipidic diet promoted an increase in the body weight when compared to the control (C-W versus HF-W). On the other hand, HF-CWP showed a lower body weight when compared to HF-W (Figure
Total mass (g), delta on the body mass gain (g/100 g body weight), and relative mass of tissue (RMT) (g/100 g body weight) of mice treated with high fat diet (HF) or control normocaloric (C) associated with gavage of coacervate (CWP) or water (W).
C-W |
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HF-CWP |
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RTM (g/100 g) | ||||
EPI |
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MES |
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RET |
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Σ adipose tissue |
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EPI: epididymal; MES: mesenteric; RET: retroperitoneal. Data submitted with an average ± EPM. bC-W versus HF-W and dHF-CWP versus HF-W. (
Evolution of the average gain in body mass (g) of mice for eight weeks of treatment with high fat diet (HF) or control normocaloric (C) associated with gavage of coacervate (CWP) or water (W). Data submitted with an average ± EPM. (b) C-W versus HF-W and (d) HF-CWP versus HF-W. (
The hyperlipid diet increased the triacylglycerol (TAG) and VLDL when compared to control group (C-W versus HF-W), while the association with coacervate reduced these parameters (HF-W versus HF-CWP). Insulin level and HOMA index were increased in the animals fed with hyperlipidic diet (C-W versus HF-W). When associated with coacervate, the hyperlipid diet promoted an increase in the adiponectin and a decrease in LPS concentrations (HF-W versus HF-CWP) (Table
Serum of triacylglycerol, total cholesterol, glucose, insulin, and adiponectin of mice treated with high fat diet (HF) or control normocaloric (C) associated with gavage of coacervate (CWP) or water (W).
Serum measurements | C-W |
C-CWP |
HF-CWP |
HF-W |
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Triacylglycerol (mg/dL) |
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Total cholesterol (mg/dL) |
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LDL (mg/dL) |
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VLDL (mg/dL) |
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HDL (mg/dL) |
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Glucose (mg/dL) |
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Insulin (ng/mL) |
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HOMA-IR |
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Adiponectin (ng/mL) |
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Lipopolysaccharides (EU/mL) |
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Data submitted with an average ± EPM. bC-W versus HF-W and dHF-CWP versus HF-W. (
The oral glucose tolerance test showed that the hyperlipidic diet promoted an increase at 15 minutes when compared to control (HF-W versus C-W). The AUC (area under the curve) analysis increased HF-W compared with C-W (Figure
OGTT and AUC (area under the curve) after eight weeks of treatment with high fat diet (HF) or normocaloric control (C) associated with gavage of coacervate (CWP) or water (W). Glycemia in time zero (basal-B), 15, 30, 60, 90, and 120 minutes after gavage of 0.2 g/100 g body weight of glucose. Data submitted with an average ± EPM. (b) C-W versus HF-W. (
There was a significant decrease in IL-10 concentrations in the animal fed with high fat diet when compared to animals fed the control diet (HF-W versus C-W). The concentration of IL-6 in C-CWP group was lower when compared to C-W group. The IL-10/TNF-
Concentrations of IL-6, TNF-
C-W |
C-CWP |
HF-CWP |
HF-W | |
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IL-6 |
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TNF-α |
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IL-10 |
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Data submitted with an average ± EPM. aC-W versus C-CWP; bC-W versus HF-W. (
Figures
Protein expression of HSL (a); ATGL (b); perilipin A (c); ABHD-5 (d); and pAMPK (e) in the mesenteric adipose tissue. The data are expressed in arbitrary units (A.U). Data submitted with an average ± EPM. (A) C-W versus C-CWP; (B) C-W versus HF-W; (C) C-CWP versus HF-CWP; and (D) HF-CWP versus HF-W. (
HSL protein expressions were reduced in C-CWP and HF-W when compared to the C-W group (Figure
A positive correlation between AUC and TAG (
Correlation between different experimental groups: (a) TAG and AUC in different experimental groups. (b) Insulin and STA (sum of adipose tissues MES, RET, and EPI). (c) Insulin and IL-10 of serum and mesenteric adipose tissue, respectively, and (d) glucose and IL-10 of serum and mesenteric adipose tissue, respectively.
Numerous procedures for isolation and recovery of WP have been investigated and reported [
The emergence of food compounds with health benefits may eventually become a good strategy to improve public health. In recent years, functional food has attracted the attention from scientific community, consumers, and food manufacturers. The list of nutraceuticals compounds (vitamins, probiotics, bioactive peptides, and antioxidants among others) is extensive, and scientific evidence seems to increasingly support the concept of health promotion through food ingredients [
Functional foods are usually marketed as food containing ingredients technologically manipulated to perform a benefit for health [
It is now well established that excessive consumption of saturated fat is related to the development of dyslipidemias [
There is strong evidence of an immunomodulatory role of WP [
The IL-10 is a pleiotropic cytokine that controls inflammatory processes by eliminating the proinflammatory cytokines production such as IL-1, IL-6, and IL-8, and TNF-
In this sense, there is an immunomodulatory mechanism underlying CWP, most likely the IL-10 cytokine, which has a homeostatic metabolic effect in the mesenteric adipose tissue. Our result suggests that IL-10 may be a positive regulator of insulin sensitivity and increased glucose uptake. This mechanism can protect the adipose tissue against insulin resistance. Although the precise origin of the unchecked inflammatory response in obesity is still unclear, it is well known that in obesity the overproduction of proinflammatory cytokines affects metabolism. For example, TNF-
Regarding the composition of the CWP, we may highlight the presence of
Evidence suggests that eating WP causes the decrease in calorie intake, increased basal energy expenditure, and modulates insulin sensitivity and glucose homeostasis, leading to changes in lipid metabolism in adipose tissue, liver, and muscle [
A study conducted by Gaidhu et al. [
Lipolysis does seem to play a crucial physiological role by recruiting a source of energy mobilized in times of stress and/or energy deprivation. Moreover, the very significant reduction in lipolysis is clearly harmful, as demonstrated in the clinical domain by the syndromes resulting from deficiencies in the lipolytic apparatus [
Another interesting finding was the significantly higher protein expression of perilipin A (52%) in HF-W group, which also refers to larger deposits of triglycerides, since this protein is primarily anchored around the droplets of neutral lipids in adipocytes. This is in line with studies showing that increased protein expression perilipin A leads to increased storage of triglycerides by reducing its hydrolytic rate. [
Finally, there is plenty of evidence suggesting that the intake of WP may lower consumption of calories, increase baseline energy expenditure, and improve insulin sensitivity and glucose homeostasis, thus leading to changes in lipid metabolism in adipose tissue, liver, and muscle [
CWP were able to promote nutritional and physiological improvements in HF-CWP group, such as reduction in body mass and decreased serum lipid levels followed by decreased serum insulin and LPS. In addition, intervention with CWP resulted in higher adiponectin contents and attenuated processes that would lead to glucose intolerance. Therefore, CWP could play a beneficial role, in some way, in modulating lipolysis in animals treated with hyperlipidic diet.
Abhydrolase domain containing 5
Area under the curve
Coacervate
Control diet plus coacervate
Control diet plus tap water
High fat diet plus coacervate
High fat diet plus tap water
Hormone-sensitive lipase
Lipopolysaccharide
Monoglyceride lipase
Nonesterified fatty acid
Phospho 5′ AMP-activated protein kinase
Triacylglycerol
Oral glucose tolerance test
Triglyceride lipase enzyme
Whey protein.
The authors declare that they have no competing interests.
All authors read and approved the final paper.
The authors thank the staff of the LAMEROA Laboratory of USP for analytical assistance of diets. This work was supported by FAPESP (Grants no. 2009/53801-5), CAPES, and CNPQ.