Study on Cholesterol-Lowering Activity of Whey Protein-Derived Peptides

Bioactive peptides refer to peptide species with diverse compositions and arrangements formed by amide condensation of natural amino acids. The biological activities of polypeptides are mainly determined by the composition and sequence of amino acids[1]. Bioactive peptides can serve as alternatives to drugs and preservatives[2], and some peptides exhibit favorable therapeutic effects on cardiovascular diseases, metabolic disorders and infectious diseases[3]. Bioactive peptides can only exert physiological activities under specific conditions[4]. Enzymatic hydrolysis is one of the fastest, safest and most controllable technologies for the preparation of bioactive peptides[5]. Therefore, enzymatic hydrolysis was adopted in this study to obtain cholesterol-lowering bioactive peptides.

Cholesterol, also known as cholesterin, exists in lipoproteins in blood. Studies have indicated that the incidence of cardiovascular diseases is closely correlated with human serum cholesterol levels[6]. At present, cholesterol degradation in serum mainly relies on pharmaceutical drugs. Although medicines act rapidly and effectively, they bring severe side effects and are costly[7]. Whey protein is rich in easily digestible essential amino acids, as well as trace amounts of lactoferrin, glycomacropeptides, growth factors, lactoperoxidase and other components[8]. It possesses high nutritional value and unique health-promoting functions[9]. After hydrolysis by specific enzymes, numerous bioactive sequences are released from whey protein to form bioactive peptides, which exert multiple functions including cholesterol lowering, ACE inhibition, antiviral activity, immune regulation, neuromodulation and bacteriostasis[10-12].

Animal experiments can be used to determine the in vivo effects of whey protein peptides on cholesterol levels. The research conducted by Brix[13] demonstrated that dietary supplementation with whey protein significantly reduced serum cholesterol levels in experimental animals, which is beneficial for the prevention of arteriosclerosis. Nagaoka isolated and purified hydrolysates of β-lactoglobulin and obtained peptide fractions with high cholesterol-lowering activity. Animal verification experiments confirmed that the acquired peptides could effectively decrease serum cholesterol concentrations in animals. Gao Xuefei et al.[14] optimized the enzymatic hydrolysis conditions of whey protein and obtained whey protein-derived bioactive peptides with molecular weights ranging from 2000 to 12000 capable of inhibiting HMG-CoA reductase activity.

Current researches mostly focus on the preparation and functional evaluation of antihypertensive peptides from whey protein hydrolysates, while studies on cholesterol-lowering whey protein peptides with greater research significance remain limited. Accordingly, whey protein concentrate 80 (WPC80) was hydrolyzed via enzymatic hydrolysis in this experiment, and the resulting polypeptides were purified to obtain polypeptides with cholesterol-lowering activity. This research provides important theoretical foundations and practical references for the development of novel microecological preparations and functional foods.

Abstract

To prepare highly efficient cholesterol-lowering whey protein peptides, whey protein concentrate (WPC80) was hydrolyzed by proteases. Ultrafiltration concentration as well as cation and anion exchange resin purification were carried out. The effects of active peptides on serum total cholesterol, triglyceride, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol levels in mice were investigated.

Results and Analysis

2.1 Standard Curves

2.1.1 Standard Curve for Degree of Hydrolysis

Using glycine as the standard substance, the regression equation between absorbance value (y) and free amino group content (x) was established as: y = 0.04292x + 0.00166 (R² = 0.99734). The curve showed good linearity and could be applied to calculate the degree of hydrolysis of whey protein.

2.1.2 Standard Curve for Cholesterol Content

The regression equation between absorbance (y) and cholesterol content (x) was y = 0.01645x − 0.01751 (R² = 0.99718). Satisfactory linearity was observed, which could be used for the quantification of cholesterol.

2.2 Selection of Hydrolytic Enzymes

The hydrolysis effects of five enzymes on whey protein are illustrated in Figure 3. The order of cholesterol micelle inhibition rate was: trypsin + neutral protease > trypsin + alkaline protease > trypsin > alkaline protease > neutral protease. The ranking of degree of hydrolysis was consistent with that of inhibition rate. Existing research[19] revealed that bioactive peptides reduce blood cholesterol concentration by decreasing cholesterol solubility in bile solutions. The degree of hydrolysis reflects the hydrolytic capacity and hydrolysis extent of different enzymes towards whey protein. Consequently, the combination of trypsin and neutral protease was selected for the preparation of cholesterol-lowering whey protein peptides.

2.3 Enzyme Ratio

As shown in Figure 4, the solubility and inhibition rate increased markedly with the rising dosage of trypsin. When the mass ratio of trypsin to neutral protease reached 4:1, the inhibition rate and degree of hydrolysis peaked at 19.83% and 8.33%, respectively. Further increasing the proportion of trypsin failed to promote the reaction obviously; instead, it hindered the further hydrolysis of hydrolysates by neutral protease, reduced the generation of cholesterol-lowering active peptides and lowered both inhibition rate and hydrolysis degree. Therefore, the optimal enzyme mass ratio of trypsin to neutral protease was determined as 4:1 for subsequent experiments.

2.4 Study on Ultrafiltration Concentration of Cholesterol-Lowering Peptides

At 45 °C and 0.20 MPa, pre-prepared 7% hydrolyzed peptide solution was subjected to ultrafiltration. The permeate and retentate were collected separately, and their inhibition rates were determined and compared with the original hydrolysate. The results are shown in Table 3.

As presented in Table 3, the inhibition rate of the ultrafiltrated solution reached 29.71%, higher than that of the initial solution (19.83%). In contrast, the retentate exhibited the lowest cholesterol micelle solubility inhibition rate of only 5.12%, indicating a low proportion of cholesterol-lowering small molecular peptides retained in the retentate. It can be concluded that ultrafiltration serves as an effective method for separating and concentrating small-molecule polypeptides, which further demonstrates the irreplaceable role of ultrafiltration in separation and concentration of active peptides.

2.5 Purification and Desalination of Cholesterol-Lowering Peptides Using Cation and Anion Exchange Resins

Desalination rate and peptide recovery rate were taken as evaluation indicators to determine the optimal flow rate for desalination via ion exchange resins.

2.5.1 Effects of Flow Rate of Cation Exchange Resin on Desalination Indicators

Cation-anion exchange method was adopted in this study to remove cations and anions (e.g. Na⁺, Cl⁻) from sample solutions. This approach generally achieves favorable peptide recovery and imposes minor impacts on the physiological functions of peptides. Moreover, desalination using cation and anion exchange resins has been widely applied in the separation and purification of active peptides. For instance, Zhang Zhongyi et al.[20] utilized ion exchange resins for desalination of soybean peptides and verified its excellent performance in separating and purifying bioactive peptides. In addition, ion exchange resins are reusable and maintain high desalination efficiency after repeated cycles, reducing experimental costs.

Figure 5 shows that the desalination rate declined from 90.68% to 78.58% with increasing flow velocity. The main reason is that higher flow rates shorten the contact time between salt ions and resins, leading to incomplete ion exchange and direct elution of partial salts, thus decreasing desalination efficiency. By contrast, peptide recovery rate gradually increased from 64.31% to 86.57% as flow rate rose. When the flow rate ranged from 9 BV/h to 11 BV/h, the growth of peptide recovery slowed down, only rising slightly from 86.53% to 86.57%. Meanwhile, the desalination rate at 9 BV/h was higher than that at 11 BV/h. Accordingly, 9 BV/h was selected as the optimal elution flow rate for cation exchange resin.

2.5.2 Effects of Flow Rate of Anion Exchange Resin on Desalination Indicators

It can be seen from Figure 6 that desalination rate decreased moderately from 64.45% to 55.42% with increased flow velocity, and the variation became gentle within the flow rate range of 9–11 BV/h. The peptide recovery rate continuously rose from 61.54% to 82.43% and maintained an upward trend. Taking both indicators into comprehensive consideration, the elution flow rate for anion exchange resin was set as 11 BV/h.

Variations in active peptide inhibition rate are listed in Table 4. After desalination and purification by anion exchange resin, the cholesterol-lowering bioactivity significantly increased from 29.71% to 47.82% (P＜0.05), further proving that desalination with cation and anion exchange resins achieves favorable effects on polypeptide separation and purification.

2.6 Determination of Components of Cholesterol-Lowering Whey Protein Polypeptides

2.6.1 Establishment of Standard Curve for Polypeptide Quantification

As shown in Figure 7, the regression equation between absorbance (y) and glutathione concentration (x) was y = 0.03826x + 0.01205 (R² = 0.99593). Good linearity was obtained for the quantification of polypeptides in active peptide samples.

2.6.2 Component Analysis of Cholesterol-Lowering Whey Protein Polypeptides

WPC80 was hydrolyzed under the above optimized conditions, followed by concentration and desalination of hydrolysates. The cholesterol-lowering active peptides were freeze-dried, and the contents of peptide, moisture and ash were measured. The results are summarized in Table 5.

Table 5 indicated that after concentration and desalination of whey protein hydrolysates, the total content of cholesterol-lowering active peptides reached 80.24% ± 0.97%, moisture content was 4.23% ± 0.18%, and ash content was 0.88% ± 0.06%. The freeze-dried cholesterol-lowering peptides contained low inorganic residues (mainly inorganic salts) and high peptide content, which verified the satisfactory efficiency of enzymatic hydrolysis, concentration and desalination procedures established in this experiment.

2.7 In Vivo Verification Experiment of Cholesterol-Lowering Peptides

At the end of the 4-week feeding trial, serum lipid indicators of mice in three groups were determined, and the results are presented in Table 6.

According to Table 6, the serum total cholesterol (TC) concentration of mice in Group C was markedly lower than those in Group A and Group B, whereas no significant difference was observed between Group A and Group B (P>0.05). For serum triglyceride (TG), Group C exhibited the lowest level, followed by Group A, and Group B had the highest TG content, with significant differences detected between every two groups (P<0.05). The ranking of high-density lipoprotein cholesterol (HDL-C) content was Group A > Group C > Group B, without significant intergroup differences (P>0.05). Group C possessed the lowest low-density lipoprotein cholesterol (LDL-C) level; LDL-C concentration of Group A was slightly lower than Group B, and no significant difference existed between Group A and Group B (P>0.05).

Serum triglyceride levels in healthy humans are affected by both endogenous synthesis and metabolism of dietary components. The serum TG concentration of mice in Group C was significantly lower (P<0.05) than that in Group A and Group B, demonstrating that the prepared enzymatically hydrolyzed whey protein peptides exert prominent effects on reducing serum TG. Namely, the active polypeptides fed to Group C contain fractions with excellent physiological activity capable of lowering triglycerides. One possible mechanism is that the obtained active peptides directly participate in the digestion, absorption or transport of dietary TG and reduce circulating TG levels. Another possibility is that these peptides interfere with endogenous TG synthesis in mice, resulting in decreased serum TG concentration. It is also probable that the polypeptides act on both physiological pathways simultaneously to reduce overall serum TG levels. Regardless of the exact metabolic pathway involved, whey protein peptides possess remarkable cholesterol-lowering capacity. If these polypeptides can be further isolated and prepared stably to modulate digestion, absorption or biosynthesis of triglycerides in healthy organisms, they will provide a promising strategy to overcome the drawbacks of lipid-lowering drugs with severe side effects in modern medicine.

Conclusions

In this study, whey protein was hydrolyzed by the compound enzymes of trypsin and neutral protease at a mass ratio of 4:1. The cholesterol micelle inhibition rate of crude hydrolysate reached 19.83%. After ultrafiltration concentration and purification via cation-anion exchange resins, the cholesterol inhibition rate of active peptides increased to 47.82%, and the total peptide content was 80.24%. In vivo animal experiments verified the significant cholesterol-lowering physiological activity of whey protein-derived peptides. Further research will focus on amino acid sequencing of the purified cholesterol-lowering active peptides. This work provides theoretical basis and technical support for developing novel microecological preparations and functional health foods.

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