Eur Food Res Technol DOI 10.1007/s00217-017-2897-z
SHORT COMMUNICATION
TGF beta2 concentration in dairy products: the effect of processing on its concentration Fernanda Lopes da Silva1 · Antônio Fernandes de Carvalho1 · Moisés Simeão1 · Cleuber Raimundo da Silva1 · Guilherme Mendes da Silva1 · Pierre Schuck2 · Italo Tuler Perrone1
Received: 15 February 2017 / Revised: 27 March 2017 / Accepted: 14 April 2017 © Springer-Verlag Berlin Heidelberg 2017
Abstract This work aims to study the effect of processing on the concentration of TGF-β2, evaluating pasteurization, membrane separation, as well as vacuum evaporation and spray drying. The TGF-β content in each sample of milk taken after each processing step has been quantified by ELISA kit. Furthermore, chemical composition analysis has been performed. TGF-β2 has shown a strong correlation (0.95) with the protein content in the samples. There is no influence of pasteurization on the concentration of TGF-β2, although pasteurization affects the distribution of TGF-β2 in the whey protein fractions and casein, which can be observed in the reduction of TGF-β2 content in whey as heat treatment is applied. This trend is unlike that of microfiltration, where it has been observed that TGF-β2 was equally distributed between casein and whey proteins. The concentration obtained by vacuum evaporation showed a concentration factor less than 1 for TGF-β2 once the constituents other than TGF-β2 became more concentrated. However, spray drying showed values greater than 1, demonstrating that TGF-β2 is concentrated in greater quantities than other solid constituents of the samples. Keywords Growth factor · Heat treatment · Vacuum evaporation · Spray drying · Microfiltration
* Italo Tuler Perrone
[email protected] 1
Food Technology Department, Federal University of Viçosa– UFV, Viçosa, Minas Gerais 36570900, Brazil
2
INRA/AGROCAMPUS OUEST, UMR1253 Science et Technologie du Lait et de l’Œuf, 65 rue de Saint Brieuc, 35042 Rennes Cedex, France
Introduction Milk and bovine colostrum contain a number of growth factors, hormones and cytokines. The distribution of these molecules is unclear since they can all be involved in cell proliferation and differentiation [1]. The most abundant growth factors present in the colostrum and bovine milk are growth factors: insulin-like (IGF) and beta transforming growth factors (TGF-β), some members of the family of epidermal growth factor (EGF) and basic fibroblast (bFGF) or fibroblast (FGF) growth factors [2, 3]. The TGF-β family plays an important role in embryogenesis, tissue repair, bone and cartilage formation and control of the immune system. TGF-β2 is the predominant form of TGF family members found in dairy products [4]. The approaches developed for extraction and concentration of growth factors from milk are based on the mass, charge or binding affinity of these factors [4]. Large-scale processes developed for extraction and concentration of such growth factors from whey are based on cation exchange chromatography. For example, Francis et al. [5] developed a mixture of growth factors using a cation exchange extraction process. Moreover, Maubois et al. [6] proposed a combination of acidification and heat treatment to prepare a protein fraction with a higher concentration of TGF-β2 by means of microfiltration using 0.1 μm pore membranes. The application of the heat treatment affected the separation and concentration of TGF-β in milk protein fractions in relation to casein and whey proteins but did not affect the concentration in milk [7], and when using pasteurization of milk for separating the whey, the yield of growth factor in the whey protein concentrate decreased [8]. Rocafi et al. [9] separated casein by acid separation and then concentrated the whey using ultrafiltration and diafiltration. After this, they used
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different thermal treatments and pH values for studying TGF-β fractions in whey proteins. They then proposed that the best way to concentrate TGF is from whey using unpasteurized milk and by pasteurizing the whey, giving a yield of 2.6 pg mg−1 of protein. Ollikainen et al. [8] observed that if milk is pasteurized and the whey is used to concentrate growth factors, less than 10% of TGF is concentrated, but if unpasteurized milk is used, it is possible to recover 40% of TGF from whey. Despite the beneficial effects of TGF-β2 described in the literature and the potential for further industrial application (development of new products), there are few studies on the effect of food technologies on the transference and activation of TGF-β2. The effect of processing and formulation on food are widely studied [10–14] and is one of the key steps for the development of new products. In this study, the concentration of TGF-β2 was 3.5 μg g−1 protein after using membranes for the separation of whey proteins. The authors observed that if pasteurization is used before the separation, it is possible to obtain a TGF-βrich casein fraction. This casein fraction has successfully been used in children with Crohn’s disease [15]. The aim of this study was to characterize the influence of processing on the concentration of TGF-β2 in bovine milk. The effect of microfiltration (no heat treatment); hightemperature and short-time pasteurization (HTST, 72 °C for 15 s); vat pasteurization (LTHM, 65 °C for 30 min); vacuum evaporation; and spray drying on the concentration of TGF-β2 has been evaluated. Finally, the objective was to relate the TGF-β2 content with the protein content in each of these steps and evaluate the best route for the production of dried dairy powders with the highest TGFβ2 concentration.
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Materials and methods This work was carried out in the INOVALEITE laboratory of the Food Technology Department at Federal University of Viçosa (UFV), Brazil. Figure 1 shows the steps that were used for the concentration of TGF-β2 from raw milk. At each step, a sample was taken for further analysis and kept frozen (−20 ± 2 °C) until the time of analysis. The powders were vacuum packed and kept away from light. Three replicates were performed. Sample preparation In the present study the raw milk was provided by the farm of the University, in which 49 cows produced the milk and they were in different stages of lactation. There were two points of milk collection for the study. The first was collected direct from the farm (RM), and the second was collected from a cooling vat (refrigerated raw milk–RRM) inside the dairy industry of the University before the pasteurization process. Microfiltration Microfiltration was performed using equipment from Tetra Pak Processing, France, by means of tangential filtration through tubular ceramic “Membralox®” membranes with 0.1 and 1.4 μm pore sizes. The 1.4 μm pore size membrane microfiltration occurred using an average permeation flux of 145 L h−1, a system with a total membrane area of 0.24 m2 and a transmembrane pressure of 0.5 bar at an average temperature for the process of 38 °C. Microfiltration with a 0.1 μm pore membrane had an average
Fig. 1 Concentration of TGF-β2 from whole raw milk, using membrane separation or heat treatment, vacuum evaporation and spray drying
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permeation flux of 35 L h−1, with a total system membrane area of 0.24 m2 and a transmembrane pressure of 0.5 bar. A concentration factor of 3 was applied to obtain microfiltered whey at an average process temperature of 45 °C. Upon removal of the microfiltered whey, casein dialysis was begun using pasteurized distilled water. This process was used to obtain microfiltered whey (MW) and casein (CS) that did not undergo heat treatment. Pasteurization and cheese production To evaluate the effect of pasteurization, two thermal treatments were used: high-temperature and short-time pasteurization (HTST, 72 °C • 15 s−1) and vat pasteurization (LTHM, 65 °C • 30 min−1). In high-temperature and shorttime pasteurization, 50 kg of raw milk was pasteurized in a plate pasteurizer (Fisher Term, Brazil), while in vat pasteurization, 100 kg of milk was pasteurized in a stainless steel jacketed tank (Biasinox, Brazil). These processes yielded pasteurized milk (PM) and vat-pasteurized milk (VPM). VPM has been used for fresh cheese production (Minas Frescal type) with the addition of 0.02 g • 100 g−1 lactic acid (Macalé, Brazil), 0.04 g • 100 g−1 calcium chloride (Macalé, Brazil) and 0.015 g • 100 g−1 rennet (Clerici, Brazil) to give whey (W) and cheese (CHE).
Composition of samples For every step of the different processes, a sample was taken, and the composition of fat, protein, ash, moisture and lactose, which was calculated by the difference, was determined. The fat content was assessed in duplicate by the Gerber volumetric method [16]. The total nitrogen content in the samples was measured in duplicate by the Kjeldahl method, and the nitrogen content multiplied by 6.38 (conversion factor) to obtain the protein equivalent values [17]. The ash content was determined in duplicate using methods described by Latimer [17]. The moisture content of the samples was measured in duplicate according to Zenebon et al. [16]. The concentration of TGF-β was determined by ELISA (enzyme-linked immunosorbent assay) (Quantikine, R&D Systems Inc., Minneapolis, MN, USA) according to the methodology proposed by the manufacturer. Statistical analysis The results were evaluated using analysis of variance (ANOVA) and Tukey tests. Analyses were performed using Excel (Excel 2010 version, Microsoft, Redmond, Washington, USA).
Vacuum evaporation
Results and discussion
A single-stage vacuum evaporator Model 70049 (Treu®, Brazil) was used. PM was concentrated to a soluble solids value of 42 ± 2% Brix, which was measured using a digital refractometer (Biobrix®, 2WAJ-D model, Brazil), and whey and MW were concentrated to 53 ± 2% Brix, with a mean concentration time of 90 ± 20 min. The boiling temperature was 65 ± 5 °C. Finally, to facilitate drying and further concentrate the whey, crystallization to 70% rate in a double jacketed vat (Inolux, Brazil) and cooling to 30 °C was performed. This process step produced concentrated milk (CM), concentrated whey (CW) and concentrated microfiltered whey (CMW).
Chemical composition of the samples
Spray drying For CMW and CW drying, a Production Minor spray dryer (NIRO-GEA, Germany) was used with a rotary atomizer. The drying parameters included an inlet temperature of 170 ± 5 °C and an outlet temperature of 90 ± 5 °C. For CM and CS drying, a Production spray dryer by (GEANIRO, Germany) with rotary atomizer was used. The drying parameters were an inlet temperature of 180 ± 5 °C and an outlet temperature of 90 ± 5 °C.
Concentrations found for TGF-β2 in raw milk (RRM, RM) (Table 1) are in agreement with values reported in the literature, describing values from 1.0 to 7.0 ng g−1 solids [1, 4], as well as pasteurized milk (PM) and vat-pasteurized milk (VPM) [18]. There are no reports in the literature for Minas Frescal cheese (CHE), concentrated milk (CM), concentrated whey (CW), or for dried products such as milk powder (MP), whey powder (WP), microfiltered whey powder (MWP) and casein powder (CSP). The concentration of TGF-β2 is strongly related to the protein in each sample. Upon applying a Pearson linear correlation, a value of 0.95 was obtained, and this positive correlation shows that as the protein content in the sample is increased, the TGF-β2 is likewise increased. Many observations in this and other studies [8, 18, 19] suggest that the interaction between TGF-β and milk proteins could explain the distribution of this growth factor in casein fractions and whey proteins depending on the type of heat treatment the milk has undergone previously. Studies suggest that this molecule can interact with the casein micelles during pasteurization [18, 19]. The heat treatment can enhance the polymerization and non-specific
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Table 1 Results for chemical composition of the samples (TGF-β2 expressed on dry matter) (n = 3) Ash (g • 100 g−1)
Lactosea (g • 100 g−1)
3.2 ± 0.5 3.5 ± 0.1 3.5 ± 0.1
0.76 ± 0.02 0.80 ± 0.05 0.72 ± 0.14
3.26 ± 2.86 2.98 ± 1.08 2.52 ± 2.82
2.30 ± 0.52 1.65 ± 0.06 2.31 ± 0.79
2.9 ± 0.3
2.9 ± 0.0
0.81 ± 0.00
4.49 ± 2.12
1.70 ± 0.00
7.98 ± 2.49 5.79 ± 3.01 4.91 ± 3.00 10.30 ± 2.65 34.90 ± 3.68 42.72 ± 12.07 55.64 ± 9.03 48.17 ± 5.49
3.5 ± 0.9 1.0 ± 0.7 0.9 ± 0.1 9.1 ± 2.0 14.9 ± 2.2 12.4 ± 1.1 7.9 ± 3.7 5.7 ± 1.2
0.0 ± 0.0 0.4 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 15.2 ± 5.3 11.9 ± 2.2 0.7 ± 0.6 0.0 ± 0.0
1.35 ± 1.22 0.55 ± 0.01 0.46 ± 0.01 1.32 ± 0.01 3.80 ± 0.04 2.76 ± 0.02 5.41 ± 2.15 4.22 ± 0.24
3.14 ± 3.05 3.83 ± 3.17 3.59 ± 0.96 0.09 ± 0.05 2.26 ± 0.84 15.62 ± 2.94 39.79 ± 7.47 37.90 ± 3.58
2.22 ± 0.11 0.84 ± 1.17 1.77 ± 0.04 1.65 ± 0.00 0.24 ± 0.00 0.70 ± 0.07 0.39 ± 0.18 0.02 ± 0.00
96.13 ± 2.01 93.74 ± 5.62 94.12 ± 5.69
26.8 ± 5.9 11.4 ± 4.8 9.9 ± 3.0
26.0 ± 7.8 5.7 ± 2.5 0.0 ± 0.0
6.02 ± 0.08 8.42 ± 0.69 8.71 ± 0.84
37.27 ± 0.95 68.24 ± 0.13 75.50 ± 6.75
1.38 ± 0.05 0.68 ± 0.80 0.01 ± 0.00
92.38 ± 0.42
74.8 ± 6.3
0.0 ± 0.0
10.46 ± 2.09
7.08 ± 1.04
2.74 ± 0.01
Sample
Dry matter (g • 100 g−1)
Refrigerated raw milk Raw milk Pasteurized milk (72 °C • 15 s−1) Pasteurized milk (65 °C • 30 min−1) Microfiltered whey Whey Microfiltered whey Casein Cheese Concentrated milk Concentrated whey Concentrated microfiltered whey Milk powder Whey powder Microfiltered whey powder
10.37 ± 3.68 10.46 ± 4.35 10.10 ± 3.12
3.2 ± 0.0 3.2 ± 0.2 3.4 ± 0.1
11.08 ± 0.98
Casein powder
Protein (g • 100 g−1)
Fat (g • 100 g−1)
TGF-β2 (ng • g−1 DM)
Where: a Calculated data
interactions of TGF-β2 with other proteins as a result of its hydrophobic character. Pasteurization effect on milk, whey and casein It has been observed that the concentration of TGF-β is higher during HTST pasteurization and lower during LTHM pasteurization, although there is no significant difference (p < 0.05), showing that pasteurization does not affect the concentration of TGF-β2 in milk as observed by Ginjala and Pakkanen [7] and Elfstrand et al. [20]. The same result was observed by Akbache et al. [19], where after pasteurization (72 °C • 20 s−1), similar values were found for both raw milk (33 pg mg−1 of solids) and pasteurized milk (32 pg mg−1 of solids). The relative strength of this growth factor with heating is explained by the presence of nine disulfide bonds [1]. There was a decrease in the concentration of TGF-β2 during LTHM pasteurization compared to HTST pasteurization. In pasteurization, the main objective is the destruction of vegetative cells of microorganisms that have very large differences in the values of Z, which is the temperature required to increase the speed of the degradation reaction in relation to the nutrients by 10 times. The Z parameter is used in the optimization of thermal treatments applied in the food industry. Therefore, the treatments at higher temperatures and for shorter times, i.e., HTST, present real
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advantages over LTHM pasteurization once there is less degradation of nutrients and hence TGF-β [21]. It can also be noted that there is no significant difference (p < 0.05) when comparing the distribution of TGF-β2 in whey protein and in the casein in both treatments (pasteurization and microfiltration), although there is a major reduction of the TGF-β2 content in fractions of whey and casein proteins in pasteurization. The distribution of growth factor fractions between whey proteins and caseins is strongly dependent on active or inactive forms or TGF-β. When there is no heat treatment or at temperatures below 65 °C, the distribution of the latent or active form in whey and casein is not very specific [18]. In the present study, the TGF-β2 content measured from the microfiltered whey came from a volume of 50 kg and casein from 10 kg, that is, 20% of whey volume, and the difference found was 28.2%. The difference in values is probably due to the higher concentration of casein in relation to whey as noted by Ollikainen et al. [8]. The dialysis process added to the production of micellar casein does not decrease the concentration of TGF but only reduces the lactose content and washes off β-lactoglobulin. However, when comparing TGF-β levels present in whey or microfiltered casein to whey or casein obtained from cheese production (milk pasteurization), it is possible to notice a decrease in both of the latter. Similar results were observed by Akbache et al. [22], who compared whey produced by
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microfiltration of raw milk to whey from cheddar and mozzarella production (pasteurized milk) and found that the first had higher values of TGF-β. Other authors also have observed that the pasteurization of milk decreases the yield of growth factors in fractions of whey protein [8, 9, 19]. Effect on concentration of vacuum evaporation and spray drying Vacuum evaporation and spray drying allow for the concentration of food constituents due to the removal of the water existing therein. However, in evaporation, the water removal process is slow because there is an indirect heat exchange. On the other hand, in spray drying, there is an instantaneous water removal since the particle comes into direct contact with hot air. The effect of such processing on the concentration of TGF-β2 has not been reported in the literature. Thus, it is important to know how these processing steps affect the concentration of TGF-β2 in milk, whey, microfiltered whey and casein. Table 1 shows the TGF-β2
concentration factor in relation to dry matter in milk, whey, casein and microfiltered whey during the evaporation and drying process. The concentration factor was calculated by subtracting the values of the concentration of TGF-β2 (ng g−1 dry matter) obtained in the previous step in relation to the phase of interest. Note that the evaporation process has a factor less than 1 in all samples, which shows that the evaporation has a negative effect on the concentration of TGF-β2 since it could concentrate in larger quantities than other present solids in relation to TGF-β2. As opposed to what is observed when drying, which has values greater than 1, except for the microfiltered whey, in spray drying, TGF-β2 can be concentrated in larger quantities compared to other solid constituents. The observed differences in concentration factor in relation to the effect of evaporation and drying are due mainly to the effect of temperature. Upon concentration, various effects can be observed in the properties of milk and/or whey, such as increasing interactions and damage to the proteins caused by sudden pressure changes [23].
Fig. 2 Routes of production of dried dairy powders, which shows the concentration factors at each stage and a value for the initial concentration of TGF-β2 in raw milk. Units expressed in ng of TGF-β2 per g of product (ng g−1)
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These effects are more pronounced in evaporation since the products are subject to high temperatures (approximately 65 ± 5 °C) for long periods of time (approximately 2 h), unlike drying, where the particle is subject to a high temperature for a short time, minimizing thermal damage to the product. The most thermal damage is observed in microfiltered whey, which has a value close to 0 in the process of concentration and a value significantly below the other proteins during spray drying. This greater sensitivity of microfiltered whey to temperature changes is probably because the first heat treatment occurs in the vacuum evaporator, where there has been to previous thermal treatment, so the proteins are in their native form and then begin to denature. Similar to the microfiltered, whey there are no micelles of casein; thus, proteins begin to form clusters, which leads to precipitation in the vacuum evaporator during the experiment. Consequently, this resulted in a lower concentration factor because the concentration of TGF-β is strongly dependent on the protein content. Figure 2 shows the four possible routes for production of dried dairy powders: the production of casein powder (Route 1), the production of milk powder (Route 2), the production of whey powder (Route 3) and the production of microfiltered whey powder (Route 4). In this Figure, the concentration factors calculated in the preceding item as well as some transmission component factors for the microfiltration and pasteurization steps, which were calculated in the same way as concentration factors, are presented. A value of 2.0 ng g−1 was assigned for the TGFβ2 concentration in raw milk, a value used in all routes. The total yield (calculated from the beginning) is determined by the product of the concentration factors in each step. As an example, for Route 1 the total yield was 1.64 (the product of 1.34 times 0.74 times 1.66). By plotting the concentrations along each route, it is possible to note that starting from milk with the same concentration of TGF-β2, the best route for the production of a dried dairy powder with high TGF-β2 concentration is Route 1, using micellar casein powder without application of heat treatment and using only the drying step, because this route has achieved a micelle casein powder with 3.3 ng g−1 TGF-β2. This is followed by the production of milk powder (Route 2) with 1.2 ng g−1 TGF-β, though this route applies heat treatment, vacuum evaporation and drying. Again, it becomes evident that the production of a dried dairy powder with a high TGFβ2 content cannot be obtained once fractionation of whey proteins occurs, as evidenced in producing whey powder (Route 3) and microfiltered whey powder (Route 4), which both have lower levels of TGF-β2.
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Conclusion TGF-β has a strong correlation (0.95) with the sample protein content, which explains the difference in the distribution of TGF-β2 in whey protein and casein when a prior heat treatment is applied, because changes that occur in the protein will directly affect TGF-β2. Pasteurization does not affect the concentration of TGF-β2 in milk, as opposed to whey and casein, because pasteurization reduces the proportion of TGF-β2 present in whey. However, when heat treatment is not applied, TGF-β2 is equally distributed between whey and casein. Vacuum evaporation adversely affects the concentration of TGFβ2 since it has a concentration factor less than 1 compared to the concentration in the remaining constituents of the sample. On the other hand, spray drying positively affects the concentration of TGF-β2 since it is concentrated in a higher proportion compared to the other solid constituents of each sample. The best way to obtain a dried dairy powder with a high concentration of TGF-β2 is through micellar casein powder production using milk that has not been thermally pretreated and has only been used in spray drying as a medium for the concentration of constituents. Acknowledgements This work was supported by the National Counsel of Technological and Scientific Development–CNPq, by the Coordination for the Improvement of Higher Education Personnel— CAPES and by FAPEMIG. Compliance with ethical standards Conflict of interest The authors has not any kind conflict of interest. Compliance with ethics requirements The authors has not any kind compliance with ethics requirement statements.
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