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. 2023 Jan 30;32(3):381–388. doi: 10.1007/s10068-023-01248-7

Comparison of the effects of commercial whey protein and native whey protein on muscle strength and muscle protein synthesis in rats

Jiyun Kim 1, Eun Woo Jeong 1, Youjin Baek 1, Gwang-woong Go 1,✉, Hyeon Gyu Lee 1,✉
PMCID: PMC9905349  PMID: 36778088

Abstract

Commercial whey protein (CWP) is generally produced in the cheese making process with heat treatment. Recently, native whey protein (NWP) can be obtained through microfiltration without heat treatment. The difference in physicochemical properties of CWP and NWP was confirmed in previous studies; however, in vivo research on the effect on muscle strength and protein synthesis is still lacking. In this study, rats were orally administered 1.56 g protein/kg body weight of lyophilized beverages containing CWP and NWP for 8 weeks. The biological value and net protein utilization in the NWP were significantly higher than in the CWP. Moreover, NWP increased muscle mass and grip strength compared to CWP. NWP also increased the phosphorylation of the mammalian target of rapamycin and ribosomal protein S6 kinase, pivotal proteins for muscle protein synthesis. These results suggest that NWP enhance muscle strength and protein synthesis more effectively than CWP.

Keywords: Whey protein, Microfiltration, Protein quality, Muscle strength, Muscle protein synthesis

Introduction

Skeletal muscle accounts for approximately 40% of total body weight and performs functions that are critical to life, including movement, organ function, and maintaining glucose levels (Lopes et al., 1982). Muscle mass decreases and atrophy develops when the equilibrium between muscle protein production and breakdown is interrupted (Baehr et al., 2017). Sarcopenia is currently described as a prolonged state of muscular atrophy and loss of muscle strength. Sarcopenia is an age-related disorder that has been associated to health and physical deterioration, sickness and injury, and even mortality in the elderly (Dao et al., 2020). To prevent diseases associated with muscular atrophy, it is necessary to consume high quality protein rich food, such as whey protein (Devries and Phillips, 2015).

Whey is the protein, mineral, and lactose-rich soluble fraction of milk that is separated from casein during the production of rennet coagulate cheese, rennet casein, or acid casein (Singh and Havea, 2003). Whey proteins, including β-lactoglobulin, α-lactalbumin, bovine serum albumin (BSA), and immunoglobulins, constitute 20% of milk protein (Raikos, 2010). Whey protein contains more branched-chain amino acids (BCAA), particularly leucine (Tang and Phillips, 2009), than other high-quality proteins, and its quick digestion elevates blood amino acid levels instantly (Dangin et al., 2001; West et al., 2011). Several studies have shown that whey protein stimulates protein synthesis more than other protein sources (Dijk et al., 2018) because of its high leucine content and rapid aminoacidemia (Norton et al., 2009; West et al., 2011). In another study, muscle strength was higher in rats fed an increased proportion of whey protein in milk than those with a higher proportion of casein (Jeong et al., 2022). These advantages show that whey proteins having various effects are widely utilized in food applications. Investigations on the characteristics and functionality of whey proteins prepared by different methods are being actively conducted.

Commercial whey protein (CWP) is made when cheese or casein is manufactured and is powdered by spray drying (Chegini and Taheri, 2013). These processes modify their natural form, resulting in altered function and reduced biological activity (de la Fuente et al., 2002). Whey protein can also be isolated using microfiltration and is called native whey protein (NWP). NWP contains plenty of whey proteins with small amounts of casein and has physicochemical and functional advantages over cheese whey (Evans et al., 2009). In addition, NWP is less heat-denatured than cheese whey protein because it can be manufactured without heat treatment (Heino et al., 2007). According to a previous study, the solubility, emulsification capacity, gel strength, and foam stability of NWP were better than that of CWP (Heino et al., 2007). It was concluded that the higher heat load of industrial whey protein would have had a negative effect because of the higher rate of whey protein denaturation. Depending on the heating conditions, heat treatments causes conformational changes in the structure of whey proteins, which results in changes to the protein functionality. Therefore, NWP with low thermal denaturation are expected to have more advantages than commercially available whey proteins.

Although the positive effects of CWP on muscle strength and muscle protein synthesis have been identified, studies on in vivo muscle strength and muscle protein synthesis of NWP are lacking. We hypothesized that the less heat-denatured NWP would improve muscle strength and synthesis than the CWP. Therefore, we evaluated the protein quality of CWP and NWP and compared their effects on body composition, muscle strength, and muscle protein synthesis.

Materials and methods

Sample preparation

Maeil Dairies Co. Ltd. (Pyeongtaek, Korea) provided all whey protein isolates that were used as treatments in the experiment. Skim milk was microfiltered (TetraPak Filtration Solutions, Silkeborg, Denmark) using a spiral-wound membrane (Synder filtration, CA, USA; 0.1 µm) made of polyvinylidene fluoride with a concentration factor (CF) of CF4 (inlet pressure of 50 kPa and outlet pressure of 100 kPa). The permeate of skim milk was ultrafiltered using a spiral membrane (Alfa Laval, Lund, Sweden; 0.05 μm) with a CF6 (inlet pressure of 50 kPa and outlet pressure of 150 kPa) to concentrate protein and remove lactose. Diafiltration (DF) was used to increase the purity of the whey protein. Another treatment was prepared by mixing whey protein isolate and lactose according to the protein and carbohydrate contents of the sample prepared using MF. All protein treatments were lyophilized and powdered.

CWP was obtained during the manufacturing process of cheddar cheese. The production process involved pasteurizing raw whole milk at 72 °C for 16 s in a plate heat exchanger. After adding cheese starter to the pasteurized milk, the liquid was heated to 30–38 °C to separate the casein and obtain the whey. The whey was then heated to 50 °C, and concentrated by ultrafiltration. The whey was processed into a powdered form by spray-drying, with a temperature of 160–200 °C at the inlet and 89–101 °C at the outlet. In contrast, microfiltration and DF separated whey proteins without a starter at temperatures below 10 °C. In addition, freeze-drying was performed to powder whey protein.

Animals and experimental design

The Animal Experimental Ethics Committee of Hanyang University approved all experimental procedures (approval number HY-IACUC-21-0041). Eighteen male Sprague–Dawley rats (70 ± 20 g, 3-week-old) were acquired from Orient Bio (Seongnam, Korea) and maintained under regulated conditions of ambient temperature (22 ± 1 °C), relative humidity (50 ± 10%), and a 12 h light–dark cycle. The rats were fed chow and tap water ad libitum during the acclimation. The rats were randomly separated into three groups (n = 6) after the adaptation period: (1) CWP, (2) NWP, and (3) nitrogen-free whey protein (N-free). The N-free group was used for protein quality evaluation, so the rest of the experiments were not carried out. All powdered whey proteins were diluted with 0.9% sodium chloride (NaCl) and administered orally (10 mL/kg body weight) in the dosage of 1.56 g protein/kg body weight for 8 weeks. The dose for rats was determined based on that of an adult with a body weight of 60 kg consuming a whey protein beverage containing 15 g protein (0.25 g protein/kg body weight of humans) as a protein supplement. The nutritional composition of the samples is shown in Table 1. The N-free group was administered as much as 10 mL/kg body weight of 0.9% NaCl to equalize the oral administration conditions with other groups.

Table 1.

Nutritional compositions of powdered protein

CWP NWP
Carbohydrate (g/100 g) 62.70 56.70
Protein (g/100 g) 29.00 28.90
Fat (g/100 g) 0.00 0.00
Minerals (g/100 g) 0.31 8.47
Moisture (g/100 g) 7.99 5.93
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CWP commercial whey protein, NWP native whey protein

Growth performance

Every week, body weight and food consumption were examined at the same time. Body weight gain was calculated using the difference between the initial and subsequent measured weights. The feed efficiency ratio was calculated by dividing total feed intake by weight growth.

Protein quality evaluation

After 14 days of oral administration of the protein treatments, rats were individually placed in metabolic cage for 5 days. Feces and urine were collected to determine total nitrogen (N) content. To evaluate dietary protein quality, true digestibility (TD), biological value (BV), and net protein utilization (NPU) were calculated using Eqs. (1), (2), and (3) as follows (Proll et al., 1998):

TD%=IN-FN-FeNIN×100, 1
BV%=IN-FN+UN-FeN-UeNIN-FN-FeN×100, 2
NPU%=TD×BV100, 3

where IN is the ingested nitrogen, FN the fecal nitrogen, FeN the fecal endogenous nitrogen, UN the urinary nitrogen and UeN the urinary endogenous nitrogen.

Body composition

The rats were fasted for 12 h and anesthetized with ketamine (100 mg/kg) and xylazine (15 mg/kg) at the end of the experiment. The body compositions such as fat in tissue (%) and lean mass (g) were analyzed using dual-energy X-ray absorptiometry (InAlyzer, Medikors, Inc., Seoul, Korea) before the sacrifice. After sacrifice, the plantaris and soleus muscles in the hind leg of the rats were immediately excised, and their weights were measured.

Grip strength test

To analyze the effect on muscular strength at week 4 and week 8 of the treatment period, grip strength was assessed using a grip strength meter (Bioseb, Largo, FL, USA). Rats were placed on a T-bar with their forepaws on it and gently pulled backward until they let go of their grip. The results are expressed in grams as the average of three median values.

Plasma leucine concentrations

After seven weeks of oral administration, the rats were food-deprived overnight. Then dynamic profiles of the amino acid release from protein were measured via jugular vein blood at 0 min before protein treatment administration and again at 30, 60, 90, and 120 min after protein treatment administration. Blood was collected in a heparin treated tubes and centrifuged at 4 °C for 15 min at 2,000 × g to obtain plasma. Plasma was deproteinized with a 10% solution of 5-sulfosalicylic acid (4/1 v/v) for 1 h, then centrifugated at 12,000 × g for 5 min at 4 °C. The plasma leucine concentration was determined by a high-performance liquid chromatography analysis using a Dionex Ultimate 3000 (Thermo Dionex, Waltham, MA, USA) instrument equipped with an Agilent 1260 infinity FL detector (Agilent, USA). The analytical column used was an Inno C18 column (4.6 mm × 150 mm, 5 µm) (Youngjin Biochrom, Seongnam, Korea). The mobile phase consisted of (a) pH 7, 40 mM sodium phosphate; and (b) water:methanol:acetonitrile (10:45:45 v/v). (a) Linear gradient was followed to 5% (b) as the beginning conditions, then 5% (b) at 3 min, 55% (b) at 25 min, 90% (b) at 31 min, and 5% (b) at 34.5 min. The injection volume was 0.5 L, and the flow velocity was 1.5 mL/min. The UV detector wavelength was 338 nm.

Western blot assay

A lysis buffer was used to homogenize and lyse the plantaris muscle tissue. The supernatants were collected after centrifuging the lysates. The bicinchoninic acid assay was used to determine the protein concentrations (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, IL, USA). On a 10% polyacrylamide gel, the same amount of protein was loaded and transferred to a polyvinylidene fluoride membrane. After blocking with 5% BSA, the membrane was incubated with a primary antibody (diluted as 1:1000 with 5% BSA) overnight. The mechanistic target of rapamycin (mTOR), S6 Kinase 1 (S6K1), ribosomal protein S6 (S6), phospho-mTOR (Ser2448), phospho-S6K1 (Thr389), phospho-S6 (Ser240/244), and β-actin antibodies were purchased from Cell Signaling (MA, USA). The membranes were then rinsed and treated each with a secondary antibody for 1 h at room temperature (Anti-rabbit IgG, HRP-linked Antibody, Bio-Rad). Protein expression was measured using Image Lab software (Bio-Rad, Hercules, CA, USA). Phosphorylation values were determined by dividing phosphorylated protein expression by total protein expression that was non-phosphorylated.

Statistical analysis

All data are presented as the mean ± standard error of the mean (SEM). Differences between the groups were evaluated by Student’s t-test. Plasma leucine levels were analyzed by two-way ANOVA followed by Bonferroni post. GraphPad Prism version 9 (GraphPad Software, La Jolla, CA, USA) was used for all statistical analyses, and the significance was determined at p < 0.05.

Results and discussion

Animal growth performance

Table 2 shows the final body weight, weight gain, total feed intake, and feed efficiency ratio. There were no significant differences between the two groups in final body weight, weight gain, total feed intake, and feed efficiency ratio. These results are consistent with a previous study that showed no difference in food intake, growth rate, and feed conversion ratio when skim milk-based diets of various pre-heating conditions were fed (Moughan et al., 1989). In addition, raw milk showed no difference was found in body weight, food intake, and feed efficiency ratio from pasteurized (72 °C for 15 s) and boiled (30 s) milk (Efigênia et al., 1997), and similar results were obtained for bottled sterile formula and other formulas (Sarriá et al., 2000). These findings could be related to the carbohydrate and protein contents of the whey powder. Therefore, there was no difference in growth performance according to heat denaturation of proteins.

Table 2.

Growth performance of the rats administered with commercial whey protein or native whey protein for 8 weeks

CWP NWP
Final body weight (g) 410.6 ± 10.7 415.4 ± 2.7
Weight gain (g) 330.9 ± 9.9 337.0 ± 1.7
Total feed intake (g) 928.6 ± 9.6 940.9 ± 3.7
Feed efficiency ratio 0.356 ± 0.008 0.358 ± 0.003
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All data are expressed as mean ± SEM

CWP commercial whey protein, NWP native whey protein

Protein quality

The quality evaluation of dietary protein is shown in Table 3. There were no differences in nitrogen intake and fecal nitrogen content between groups. However, the nitrogen content in urine was 21% higher in the CWP group than in the NWP group (p < 0.05). Despite no difference in TD, the NWP group had substantially greater BV and NPU than the CWP group (p < 0.05). Among industrially heated dairy products, one study showed that spray-dried milk had significantly lower BV and NPU than other whey products (Lacroix et al., 2008). Another previous study found that gastric digestion of the heated whey protein concentrate was delayed compared to the unheated whey protein due to the formation of whey protein aggregates and increased particle size (Stender et al., 2018). These results may be caused by a blocking of the ε-amino groups of lysine residues, which inhibits the action of digestive enzymes (Hellwig et al., 2014). Therefore, NWP without heat treatment during manufacturing had better protein retention than CWP.

Table 3.

Evaluation of protein quality in rats administered with commercial whey protein or native whey protein for 8 weeks

CWP NWP
Nitrogen intake (g) 2.84 ± 0.07 3.00 ± 0.06
Fecal nitrogen (g) 0.11 ± 0.03 0.12 ± 0.00
Urinary nitrogen (g) 0.46 ± 0.04 0.38 ± 0.00*
True digestibility (%) 97.13 ± 0.53 97.31 ± 0.13
Biological value (%) 92.81 ± 1.35 95.94 ± 0.12*
Net protein utilization (%) 90.17 ± 0.28 93.13 ± 0.27*
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All data are expressed as mean ± SEM

CWP commercial whey protein, NWP native whey protein

*Significant differences where p < 0.05

Body composition analysis

The body composition and muscle tissue weight of each group are shown in Fig. 1. The fat content (%) in the tissue of the two groups was identical (Fig. 1A), but lean mass (g) was 5.4% higher in the NWP group than in the CWP group (p < 0.05, Fig. 1B). The soleus and plantaris muscles comprise the representative skeletal muscles of the hind limbs. The NWP group measured an average 15.65% heavier soleus (Fig. 1C) and 7.3% heavier plantaris (Fig. 1D) than CWP group (p < 0.05). Muscle mass is associated with high protein reserves in the diet (Laleg et al., 2019), and protein digestibility is an independent regulator of postprandial protein retention (Dangin et al., 2001). Similar to our findings, another study measured a higher lean mass and elevated nitrogen digestibility and utilization rate of NWP when compared to soy protein (Wróblewska et al., 2018). Several studies have shown that poor or unbalanced dietary protein in animals and human increases nitrogen loss and limits protein synthesis due to inefficient use of essential amino acids (Ha and Zemel, 2003). In this study, the protein retention of the NWP was higher than that of the CWP, and resulted in higher muscle mass.

Fig. 1.

Fig. 1

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Body composition and relative skeletal muscle weight in rats administered with commercial whey protein or native whey protein for 8 weeks. (A) Fat in tissue, (B) lean mass, (C) relative soleus muscle weight, (D) relative plantaris muscle weight, and (E) representative image of dual-energy X-ray absorptiometry. All data are expressed as mean ± SEM. *Significant differences where p < 0.05. CWP commercial whey protein, NWP native whey protein, BW body weight

Muscle strength

To investigate the effect of CWP and NWP on muscle strength, grip tests were performed at weeks 4 and 8 of the experiment. In week 4, there was no significant difference between the groups (CWP: 492.4 ± 10.5 g, NWP: 485.7 ± 1.2 g, Fig. 2A), however the grip strength of both groups improved in week 8. Notably, the grip strength of NWP (588.0 ± 10.2 g) was 8% higher than CWP (540.6 ± 6.2 g) in the week 8 (p < 0.05). These findings suggest that NWP is more effective for muscle strength than CWP, along with an increase in muscle mass. Similarly, many studies have found a positive correlation between muscle mass and strength (Chen et al., 2013). The basis for supporting these benefits can be found in the properties of microfiltration in which the microfiltration preserves proteins better, in particular, highly bioavailable leucine content (Hamarsland et al., 2019).

Fig. 2.

Fig. 2

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(A) Grip strength in rats given commercial whey protein or native whey protein for 8 weeks at weeks 4 and 8. (B) Plasma leucine concentrations in rats administered with commercial whey protein or native whey protein. All data are expressed as mean ± SEM. *Significant differences where p < 0.05. CWP commercial whey protein, NWP native whey protein, ∆ the grip strength differential between weeks 4 and 8

The effect of NWP on blood leucine concentrations was examined (Fig. 2B). There were significant differences in time but no differences between the treatment (CWP or NWP) groups and time × treatment interactions. These results are consistent with the previous studies in which no differences were found in the postprandial plasma amino acid concentration of mini pigs administered either CWP or NWP (Welch-Jernigan et al., 2019). In addition, clinical studies of serum amino acid concentrations did not show significant differences in the postprandial dynamics of ultra-heat treated (UHT) milk compared with milk pasteurized using MF (Lacroix et al., 2008). In another study, the plasma leucine concentration was highest at 30 min after oral administration and decreased from 60 min in both groups (Norton et al., 2009). Heat denaturation modulates whey protein coagulation and digestion kinetics but does not affect amino acid absorption in vivo (Wang et al., 2018; Welch-Jernigan et al., 2019). In this study, the leucine concentration in the blood was not confirmed as evidence of enhanced muscle mass and strength.

Muscle protein synthesis

Essential proteins that are involved in skeletal muscle protein synthesis were studied. mTOR plays an important role in protein synthesis in skeletal muscle through phosphorylation changes of downstream S6K1 in rats (Bodine et al., 2001) and mice (Akasaki et al., 2014). Previous studies have shown that modulation of the protein kinase B/rapamycin (AKT/mTOR) signaling pathway may increase skeletal muscle mass and performance in rats (Yoo et al., 2021) or mice (Akasaki et al., 2014).

In the current study, the phosphorylation of mTOR, S6K1, and S6 were analyzed (Fig. 3). The CWP group had significantly higher phosphorylation of mTOR and S6K1 than the NWP group (p < 0.05). However, the phosphorylation of S6 between the CWP and NWP groups was not significantly different. These findings support the NWP group's higher lean mass, muscular weight, and grip strength than the CWP group. Likewise, previous research has shown that consuming natural whey protein dramatically increases protein synthesis compared to heat-treated whey protein (Hewlings and Kalman, 2020). Another study discovered Sprague–Dawley rats with higher lean mass and gastrocnemius weight exhibited higher S6K phosphorylation (Norton et al., 2017). Thus, the enhanced mTOR signaling pathway in skeletal muscle stands as a key molecular mechanism that underlies physical phenotypes such as increased muscle mass and strength.

Fig. 3.

Fig. 3

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Effect of commercial whey protein and native whey protein on muscle protein synthesis. All data are expressed as mean ± SEM. *Significant differences where p < 0.05. CWP commercial whey protein, NWP native whey protein

In conclusion, this study investigated the effects of CWP and NWP on protein quality, muscle strength, and muscle protein synthesis. The NWP group showed considerably higher BV and NPU than the CWP group. Strikingly, the lean mass and the relative weight of the soleus and plantaris were higher in the NWP group than in the CWP group. The grip test for muscle strength was improved in the NWP group than in the CWP group at week 8. What’s more, the phosphorylation of mTOR and S6K1, the molecular markers of skeletal muscle protein synthesis, was increased in the NWP group compared with those in the CWP group. Taken together, these results suggest that NWP with minimal heat denaturation has higher protein bioavailability and improves muscle strength and muscle protein synthesis more effectively than CWP with heat treatment during the manufacturing process.

Acknowledgements

This work was carried out with the support of Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Innovative Food Product and Natural Food Material Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (119017-03).

Declarations

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Jiyun Kim, Email: kjy0601@hanyang.ac.kr.

Eun Woo Jeong, Email: bravoadria@hanyang.ac.kr.

Youjin Baek, Email: jyyj161126@hanyang.ac.kr.

Gwang-woong Go, Email: gwgo1015@hanyang.ac.kr.

Hyeon Gyu Lee, Email: hyeonlee@hanyang.ac.kr.

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