Discharge of whey proteins is still a current practice by small cheese producers. The development of low-cost alternatives for recovery of these proteins is fundamental for small producers who cannot apply expensive techniques. The present study investigated the complex coacervation technique as a cheap technology to recover proteins from sweet whey using carboxymethylcellulose, and the coacervate used as an ingredient in the formulation of probiotic fermented milk. The nutritional properties of whey-carboxymethylcellulose coacervates (WP-CMC) were evaluated in trials with animals (rats) using casein as a reference. All these parameters—the coefficient of feed efficiency (CEA), protein digestibility-corrected amino acid score (PDCAAS), and net protein ratio (NPR), as well as weight gain—were determined to evaluate protein quality. A sensory acceptance test was applied to evaluate the sensory characteristics of the product. The complex coacervation technique recovered 86% of the protein from sweet whey. No significant (p > 0.05) differences were observed in the biological tests for both groups (WP-CMC and Casein groups) when NPR (4.98 to 5.04), digestibility (92.35 to 90.64), and CEA (0.40 to 0.42) were evaluated. Probiotic fermented milk beverage containing WP-CMC (0.78%) and guar gum (0.68%) presented good acceptability as determined by sensory evaluation. WP-CMC can be considered an ingredient with high nutritional and biological value that could be applied in probiotic fermented milk as an alternative to small producers to allocate the residual whey from cheese manufacture.
Whey is a residue from artisanal cheese production, and contains high organic load and hard biodegradability [
Beside its high biological value, sweet whey has high water content (0.9 g protein L−1). The high volume of whey from cheese processing is still a problem for small industries and producers [
The complex coacervation technique is a simple method to recover whey proteins and to reduce the environmental impacts [
Krzeminski et al. [
A combination of probiotics and whey protein concentrate (WPC) for use in yogurt production improved the growth and survival of the probiotic microorganisms L. acidophilus (LAC 4, Rhodia) and B. longum (BL, Rhodia), especially the former [
Probiotics are defined as live microorganisms that, when administrated in adequate amounts, confer health benefits on the host [
Literature reports have demonstrated the physiological properties and technological applicability of whey proteins and whey proteins coacervated with polysaccharides. However, no reports are found on the nutritional quality and amino acids bioavailability of the polysaccharide coacervate proteins. The present study aims to evaluate both the nutritional and technological aspects of whey protein recovered by coacervation technique and its application in probiotic fermented milk.
Sweet skimmed whey was obtained from a small-scale dairy (Espírito Santo do Pinhal, São Paulo, Brazil). Sodium carboxymethylcellulose (CMC) was obtained from Walocel CRT™ 40000 PV (Dow Chemical Company, Brazil). Commercial casein was obtained from Naarden Agro Products. L-cysteine and choline bitartrate were purchased from Sigma (St. Louis, MO, USA). Bifidobacterium animalis supsp. lactis Bb-12 was kindly provided by Chr-Hansen/Brazil and Streptococcus thermophilus TA 40 by Danisco/Brazil.
WP-CMC coacervate was obtained according to a previously described procedure [
An aqueous CMC solution (0.3% w/v) was stored overnight at 7˚C to ensure complete hydration of the polymer to promote further interaction with whey proteins in solution. Initially, skimmed whey (WP) (adjusted to pH 3.0 using citric acid 10%) was mixed with the CMC solution (0.3% w/v), also adjusted to pH 3.0, at a WP:CMC ratio of 1:1 (v/v) and centrifuged at 2150 × g (SORVALL® RC-26 PLUS industrial scale centrifuge) at 22˚C. The coacervates (WP-CMC) were resuspended at pH 6.0 with sodium carbonate 1 mol・L−1, and freeze-dried before use to evaluate their biological value and application as an ingredient in fermented milk.
Protein (micro-Kjeldahl), moisture, ash and lipid contents were determined according to AOAC [
The amino acid levels were determined by high-performance liquid chromatography (HPLC) using a reverse phase column on a Shimadzu HPLC (Shimadzu Corporation, Japan) and UV detector at 254 nm. The amino acids released by acid hydrolysis (110˚C/22 h) were subjected to pre-column derivatization with phenyl isothiocyanate (PITC). Quantification was performed by internal calibration using amino butyric acid (AAAB) as standard [
After resuspension of WP-CMC in sodium carbonate to pH 6.0, protein recovery was measured by the following equation:
In order to evaluate the effect of pH on complex stability, protein solubility was determined in the supernatant at different pH values. The WP-CMC coacervate solution was prepared using distilled water (1 mg protein mL−1) and the pH value was adjusted to 2.0, 3.0, 4.0, 5.0, or 6.0, and kept under stirring for 30 min. Then, samples were centrifuged at 2150 × g (SORVALL® RC-26 PLUS industrial scale centrifuge) for 30 min at 22˚C. The supernatants were evaluated for protein content by Micro-Kjeldahl method [
Male wistar rats (n = 21) with initial weight of 56.16 ± 3.15 g were acclimated to standard housing conditions and fed on a 10% casein diet for 4 days. The 10% casein diet (
Rats were then randomized according to weight into 3 groups (7 rats/group). The groups consumed ad libitum diets containing 10% protein consisting of Group 1―Casein (Control) or Group 2―WP-CMC coacervate (WP- CMC) for 14 days. The third group (n = 7) was fed on a diet without protein during the same period (14 days) for determination of endogenous nitrogen loss. Apart from the protein content, nutrient levels of all diets were adequate for the growth of rats according to AING-95 [
The coefficient of alimentary efficiency (CAE) of the complexes was calculated according to Osborne, Mendel, & Ferry [
True protein digestibility (D%) was determined as recommended by the Food and Agriculture Organization (FAO) [
PDCAAS was estimated by calculating the score of the most limiting essential amino acid [
Initially, a sachet of freeze-dried probiotic culture Bifidobacterium animalis subsp. lactis Bb-12 (Chr-Hansen) was dissolved in 1 L of sterile whole milk, obtaining 3 × 109 colony forming units∙mL−1 (CFU∙mL−1) of viable probiotic cells. The inoculated milk was transferred to sterile tubes (10 mL) and stored at −18˚C. A culture of Streptococcus thermophilus (TA 40, kindly provided by Danisco) was previously dissolved in milk in the same way (
For manufacture of the fermented milk, commercial skim milk powder was dissolved in water at 10.00% (w/v), and 10.00% sugar, 0.78% WP-CMC coacervate, and 0.68% guar gum were added, according to the results obtained in a previous study [
The mixture was heat-treated at 85˚C for 30 min, and then cooled and inoculated with the probiotic bacteria and starter culture (2.00% B. animalis and 1.00% S. thermophilus), previously dissolved in sterilized milk, as described above. Fermentation was carried out at 45˚C for approximately 4 hours until pH 4.6 was reached. At this point, 140 μL strawberry flavoring (supplied by Symrise®) and 50 μL carmine colorant from cochineal (provided by Corantec®) were added for each 100 mL product. The beverage was homogenized under a pressure of 70 Kgf/cm2 using an FT9 Armfield homogenizer, and kept under refrigeration at 8˚C ± 1˚C.
Microbiological analyses were carried out at the beginning and at the end of the storage period (3 and 28 days, respectively), according to the procedures recommended by the American Public Health Association [
Experimental data | ||
---|---|---|
Ingredients (g) | Casein | WP-CMC |
Casein (84.41%a) | 118.47 | - |
WP-CMC (55.45%a) | - | 180.34 |
Saccharose | 100.00 | 100.00 |
Soy oil | 70.00 | 70.00 |
Fiber (cellulose) | 50.00 | 50.00 |
Mineral mix (AIN-93G) | 35.00 | 35.00 |
Vitamin mix (AIN-93G) | 10.00 | 10.00 |
L-cistine | 3.00 | 3.00 |
Choline bitartrate (41.1% C) | 2.50 | 2.50 |
Terc butyl hydroquinone | 0.014 | 0.014 |
Cornstarch | 611.01 | 549.14 |
Total | 1000 | 1000 |
Energy (kcal/g) | 3.87 | 3.62 |
aProtein percentage.
± 1˚C for 24 h [
The viability of the B. animalis culture was determined using MRS Agar (Oxoid) supplemented with 0.5% L-cysteine hydrochloride at 10.00%, 1.00% lithium chloride at 10.00% and 0.50% dicloxacillin at 0.10%.
Descriptive sensory analysis was carried out after 3 days of storage at 6˚C, by a group of 35 panelists (mean age 24 years) at the Sensory Laboratory of the Institute of Food Technology (ITAL/Campinas, Brazil). The research project was approved by the Ethics Committee for research in human beings of PUC-Campinas (n. 992/07), and all participants signed a term of consent. The overall acceptance was evaluated by the attributes appearance, consistency, taste, and overall impression, using a nine-point hedonic scale (9 = extremely like, 5 = neither like nor dislike, and 1 = extremely dislike). Sample were identified by a three random number code and served in plastic cups accompanied by natural mineral water for palate cleansing. The test was conducted in individual booths with fluorescent lighting and equipped with computerized Compusense Five version 4.8 for data collection and analysis.
Analyses were performed in triplicate. Data were expressed as mean standard deviations (SD), and compared by analysis of variance (ANOVA) and Tukey’s test. Statistical analysis was performed using the STATISTICA 12.0® software package for Windows (StatSoft, Inc., Tulsa, OK, USA). Differences were considered statistically significant at p < 0.05.
The chemical composition of the skimmed whey and the amino acid composition of the coacervate (WP-CMC) are described in
As expected, proteins and lactose were the predominant components in WP-CMC and skimmed whey, respectively. As previously observed by Capitani et al. [
The recovery of sweet whey proteins by complex coacervation was effective, since 86% proteins were recovered as protein-binding CMC polymers (pH 3.5), when compared to the initial composition.
The coacervate proteins remained insoluble in the range of pH 3.0 to pH 6.0, as shown in
All groups fed on the protein diets, except the non-protein group, presented a positive and linear tendency towards weight gain during the experimental period (14 days). No significant differences (p > 0.05) were observed in weight gain, diet intake, and energy ingestion between the groups (Casein and WP-CMC Groups) (
Although similar growth and weight gain values were found for different protein sources in the experimental groups, a lower weight gain was observed in the WP-CMC (~8.7%) during 14 days, when compared to the Casein group. The Casein group consumed 1.43 g diet・day−1, while the animals of the WP-CMC group consumed
Component (g∙100g−1) | Skimmed whey | WP-CMCa | |||
---|---|---|---|---|---|
Ash | 9.31 ± 0.05 | 1.98 ± 0.96 | |||
Protein | 13.05 ± 0.43 | 57.23 ± 0.32 | |||
Lipids | 0.89 ± 0.05 | 9.42 ± 0.00 | |||
Lactose | 76.73 ± 0.32 | 10.6 ± 0.30 | |||
Amino acid (AA) (mg AA g∙protein−1) | Requirement patternb | Casein | WP-CMC | ||
His | 19.0 | 35.1 ± 0.4 | 19.5 ± 0.1 | ||
Ile | 28.0 | 55.6 ± 0.1 | 51.2 ± 0.1 | ||
Leu | 66.0 | 119.9 ± 0.5 | 117.1 ± 0.8 | ||
Lys | 58.0 | 90.6 ± 0.7 | 82.1 ± 0.5 | ||
Met + Cys | 25.0 | 40.3 ± 0.4 | 40.6 ± 0.6 | ||
Phe + Tyr | 63.0 | 135.1 ± 1.0 | 70.7 ± 0.5 | ||
Thr | 34.0 | 52.9 ± 0.1 | 53.7 ± 0.3 | ||
Trp | 11.0 | 11.7 ± 0.2 | 13.0 ± 0.7 | ||
Val | 35.0 | 75.5 ± 0.6 | 58.5 ± 0.2 | ||
aWP-CMC = whey-carboxymethylcellulose coacervate; bReference WHO [
Diets | ||
---|---|---|
Parameter | Casein | WP-CMC |
Weight gain (g) | 48.21 ± 4.20 | 43.75 ± 6.28 |
Total food intake (g) | 120.15 ± 12.96 | 103.46 ± 14.70 |
Total protein intake (g) | 12.07 ± 1.30 | 10.88 ± 1.53 |
NPRa | 5.01 ± 0.26 | 5.04 ± 0.30 |
True Digestibility (% D) | 92.35 ± 1.01 | 90.64 ± 2.68 |
Food Efficiency (% EF) | 0.40 | 0.42 |
PDCAASc (%) | 0.98 | 0.93 |
aNPR: Net Protein Ratio; cPDCCAS: Protein Digestibility Corrected Amino Acid Score.
1.23 g diet・day−1. It is well known that polysaccharides can modify food texture and thus enhance product viscosity. The dissociation of WP-CMC during the digestion process, for example, can lead to polymer release. Therefore, the digestion process (at ~pH 2.0) may have contributed to the partial separation of the polymers, and the CMC, partially soluble or in the coacervated form may have contributed to satiety, reducing slightly diet intake in this group (WP-CMC).
However, future studies on adult animals should be carried out to evaluate others parameters such as blood glucose and cholesterol. This design would provide greater support for the discussion of the benefits of using whey proteins-CMC coacervate for weight gain and satiety, in addition to developing special food formulations.
High true digestibility (D) and net protein ratio (NPR) values were observed for the protein in the coacervate products and casein, with no significantly (p > 0.05) differences between groups (
No statistical differences were observed in PDCCAS values (p > 0.05) for all groups (
It should be emphasized that complex coacervation is a process that principally involves electrostatic interactions, which are considered to be chemically weak [
Microbiological analyses were carried out at the beginning and at the end of fermented milk storage (28 days). Counts lower than 3 NMP∙mL−1 for coliforms at 35˚C and coliforms at 45˚C were obtained in the beginning and at the end of the storage period. For mold and yeast counts, values lower than 10 UFC∙mL−1 were also observed in the fermented milk. Thus, the product presented appropriate microbiological quality according to the Brazilian legislation [
For a probiotic product to be marketed as presenting health-promoting benefits, it must present viable minimum probiotics count from 108 to 109 CFU for a daily-consumption sized portion of the product [
The sensory evaluation of the probiotic fermented milk presented scores corresponding to “moderately like” for all attributes. Among the criteria used by consumers, the highest score (7.3 ± 1.1) was obtained for the attribute appearance, followed by consistency (6.2 ± 1.2), and overall impression (6.0 ± 1.6) on a 9-point hedonic scale. The beverage formulation could be altered according to the target public, especially with regard to flavor, since this attribute received the lowest score (5.0 ± 1.1). The use of coacervate allowed the preparation of a probiotic beverage, with protein of high biological value and good acceptability.
Protein precipitation from sweet whey using complex coacervation with carboxymethylcellulose is an alternative to recover soluble proteins, being a rapid, efficient, low cost, and clean technique that allows small dairy producers to reuse whey from cheese manufacture. It is advantageous from the environmental point of view, since there is no discard, besides the enrichment of a fermented dairy beverage with a protein of high biological value, as observed in the experimental test. In addition, adequate probiotics viability in the probiotic fermented milk containing WP-CMC was observed. This study evidenced the potential of whey protein-carboxymethyl- cellulose coacervate as a low-cost strategy to recover sweet whey, which could be used to produce new ingredients for application in probiotic fermented dairy products.
The authors acknowledge financial support from Fundação de Amparo à Pesquisa de São Paulo (FAPESP) and CNPq for the scientific initiation scholarship.