CHEMICAL ENGINEERING TRANSACTIONS

VOL. 79, 2020

A publication of

The Italian Association

of Chemical Engineering
Online at www.cetjournal.it

Guest Editors: Enrico Bardone, Antonio Marzocchella, Marco Bravi
Copyright © 2020, AIDIC Servizi S.r.l.
ISBN 978-88-95608-77-8; ISSN 2283-9216

Physicochemical Characteristics of Milk By-Products

Roberta B. de Menesesa, Leonardo F. Macielb, Maria H. M. da Rocha-Leãoc,
Carlos A. Conte-Juniora*
a
Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ), 21941-909, Rio de Janeiro, RJ, Brazil.

b
Faculdade de Farmácia, Universidade Federal da Bahia (UFBA), 40170-115, Salvador, BA, Brazil.

c
Escola de Química, Universidade Federal do Rio de Janeiro (UFRJ), 21941-909, Rio de Janeiro, RJ, Brazil.

carlosconte@id.uff.br

The physicochemical characteristics of milk by-product should be assessed by dairy industry to evaluate the
potential of different types of wheys to become ingredients. The aim of this study was to evaluate the
physicochemical characteristics of the main dairy industry residues: cheese whey, ricotta whey, and butter
whey. Whole milk was also evaluated as reference. Analyses of proximate composition, determination of
Ca2++Mg2+, pH, titratable acidity, water activity, and instrumental color, all in triplicate, were performed. The
results of proximate composition, and titratable acidity showed significant increases (moisture and titratable
acidity) and reductions (ashes, lipids, proteins, carbohydrates, energy value, pH, and Ca2++Mg2+) between the
different types of wheys and milk. The lipid and caloric reduction are considered relevant to dairy industry,
mainly to be used in products with high level of fat harmful to health. There was no significant difference
between the values for water activity. Furthermore, the color showed difference in which the milk was the
whitest sample. Principal component analysis explained 94.40% of the total variance of the data. PC1
separated butter whey and ricotta whey from milk and cheese whey. In view of the results, many
physicochemical attributes of the different dairy by-products studied were interesting, therefore the application
of these types of wheys in the development of other products, partially or even entirely, can be suggested.
Despite the similarity of butter whey to milk, the indication of the most appropriate whey to be used will depend
on the product and the aim of its application.

1. Introduction
The conversion of milk into other products (cheese, ricotta, butter, etc.) generates a large amount of by-
products that are eliminated by discharging into streams, without or with prior treatment in many degrees.
Treatment is rarely enough to prevent severe and serious environmental impact. The cheese whey was
classified as 150 times more polluting than ordinary sewage (The lancet, 1922). The damage is mainly due to
the pollution caused by both the large amount of types of wheys generated and its chemical properties (high
organic matter content, i.e. high chemical (COD) and biochemistry (BOD) oxygen demand). Lactose and lactic
acid are the main sources of pollution due to their high oxygen absorption capacity. The aquatic biota, more
precisely fishes, are suffocated by the water in which the types of wheys quickly remove the dissolved oxygen
(Rivas et al., 2010).
The solutions presented so far, however, are unfortunately not so clear. The use of whey as animal feed, while
feasible in theory and to some extent in practice, seems to be limited by commercial reasons. Previous
storage of whey in tanks prior to disposal causes the fermentation of lactose, and the production of lactic acid,
butyric acid and other organic acids, almost as detrimental as the lactose. Lately, many studies are being
developed using lactose by-products in the production of alcohol (bio-ethanol). Reports of whey treatment on
land showed that the soil had become "sick" (putrefaction). Evidence of biological methods of treatment by
addition of specific bacteria (including pig manure) has yet to show satisfactory effects (The lancet, 1922).
Although there is little information on the composition of the different lactic types of wheys, they are known to
have bioactive substances to human health and low fat presence in general. More specifically, cheese wheys
have proteins and peptides of high biological value regulating various physiological functions in human body

DOI: 10.3303/CET2079007

Paper Received: 10 October 2019; Revised: 4 December 2019; Accepted: 2 March 2020
Please cite this article as: Meneses R., Maciel L., Rocha-Leao M.H.M., Conte-Junior C.A., 2020, Physicochemical Characteristics of Milk By-
products, Chemical Engineering Transactions, 79, 37-42 DOI:10.3303/CET2079007

such as blood pressure, response glycemia, inflammatory processes, and stimulation of immune system
(Athira et al., 2015). In addition, ricotta whey has high lactose content (Sansonetti et al., 2009), and butter
whey has a little less lactose and presence of phospholipids that play an important role in many metabolic
processes in human organisms, including, according to Kuchta-Noctor (2016), in vitro anticancer properties.
Among the alternatives of reutilization that minimize environmental aggression, the application of different
dairy types of wheys in human feeding seems quite feasible. However, the development of novel food
products proves to be increasingly challenging as consumers' expectations for these products are both
palatable and healthy. At the same time, the industries’ economy would be affected by the reduction of raw
material costs (Sansonetti et al, 2009). In this context, given the scarce information and a better understanding
of its nutritional and technological potential in terms of reuse, the aim of the present study was to evaluate the
physicochemical properties of cheese whey, ricotta whey, and butter whey.

2. Materials and methods
2.1 Whole milk and milk by-products acquisition

The by-products were obtained from whole cow's milk by rennet cheese (addition and action of 7% rennet
(HA-LA®) and heating at 45±1ºC), ricotta (addition and action of 2% acetic acid (maratá®) with heating at
95±1ºC), and butter (action of centrifugation at 25±2ºC) production of an agro-industry located at Federal
Institute of Alagoas (Satuba/AL/Brazil) (Figure 1). The frozen samples were sent in thermal boxes, from up to
12 hours, to the Food Technology Laboratory of the School of Pharmacy at Federal University of Bahia
(Salvador/BA/Brazil).

Figure 1: Milk by-products acquisition.

2.2 Proximate composition determination

Moisture, ashes, dietary fibers, and proteins were determined by using the methods of the Association of
Official Analytical Chemists (AOAC, 2012). The lipid fraction was determined by the Bligh and Dyer method
(Bligh and Dyer, 1959). Additionally, total carbohydrate (Nifext fraction) was calculated by the difference of the
measured values for moisture, ashes, dietary fibers, proteins, and lipids. The energetic content was
determined based on the Atwater coefficient of proteins, lipids, and carbohydrates as 4 kcal.g

–1, 9 kcal.g–1,
and 4 kcal.g–1, respectively (FAO, 2003).

2.3 pH and titratable acidity

The pH was measured directly using a digital pH-meter (model DM-23 Digimed; Digicrom Analítica Ltda. -
Brazil) previously calibrated with pH 4.0 and 7.0 buffer solutions. The titratable acidity was measured by
titration with 0.1 N NaOH and results were expressed as % lactic acid, according to the AOAC methods
(AOAC, 2012).

2.4 Water activity

The water activity (Aw) was obtained by direct measurement in an Aqua-Lab digital thermohygrometer (model
AquaLab Lite; Decagon Devices Inc. - USA) at a controlled temperature of 25 ± 2ºC.

2.5 Calcium (Ca) and Magnesium (Mg)

The Calcium (Ca) and Magnesium (Mg) determination was done by titration with EDTA (Ethylenediamine
Tetraacetic Acid) solution according to methodology described by Bird et al. (1961).

2.6 Instrumental color

The color of the samples was determined by instrumental measurement using a Minolta colorimeter (Chroma
Meter, CR-400 - Japan), where measurements were expressed in L* (transition from black 0 to white 100), a*
(transition from green -a* to red +a*), and b* (transition from blue -b* to yellow +b*). The colorimeter was
calibrated with illuminant D65 and the standard observer was 2°.

2.7 Statistical analysis

All analyses were performed in triplicate and the statistical analyses were performed using the software
Statistica 7.0 (StatSoft - USA). One-way analysis of variance (One-way Anova) and Tukey HSD test, at a
significance level of 0.05, were used to define the difference between each whey and milk. Principal
Component Analysis (PCA) was performed with all obtained data to overview the parameters that were
affected by the different types of wheys and milk.

3. Result and discussion
3.1 Proximate composition determination

The nutritional profile is presented in Table 1. Higher moisture values were found for milk processing effluents
in comparison to whole milk. This characteristic can explain the lower content found in the other nutrients
(ashes, lipids, proteins, carbohydrates) and energetic value, where all of them were diluted. Moreover, butter
whey showed protein, lipid, and ash levels more similar to milk than the other types of wheys. There is little
information on the composition of dairy effluents, nevertheless the results of the present study were
considerably close to those found by other research that also characterized different types of wheys, such as
the ricotta whey (Bald et al., 2014, cheese whey (Bald et al., 2014) and butter whey (Morin et al., 2006).
Composition range of the different types of wheys in relation to milk was as follow: increased the moisture
(7.74% (butter whey), 10.29% (cheese whey), and 11.40% (ricotta whey)) and reduced ash (8.42% (butter
whey), 49.47% (ricotta whey), and 51.58% (cheese whey)), lipids (81.59% (butter whey), 91.11% (cheese
whey), and 99.36% (ricotta whey)), proteins (7.32% (butter whey), 77.13% (cheese whey), and 84.45% (ricotta
whey)), carbohydrates (36.65% (cheese whey), 42.40% (ricotta whey), and 47.12% (butter whey)), and energy
content (53.37% (butter whey), 65.09% (cheese whey), and 72.17% (ricotta whey)) (Table 1). None of the
samples had fibers in their composition. Moreover, the most significant change was in the lipid fraction.
Another interesting observation was the reduction of the energy value, which is considered advantageous for
providing health benefits (WHO, 2003).

Table 1: Proximate composition, energetic content, water activity, pH, titratable acidity, instrumental color, and
Ca2++Mg2+ of by-products and milk.

Observed Parameters Ricotta Whey Butter Whey Cheese Whey Whole Milk

Moisture (%) 94.47±0.31a 91.36±0.51c 93.53±0.46b 84.80±0.55d

Ashes (%) 0.48±0.03c 0.87±0.02b 0.46±0.01c 0.95±0.04a

Lipids (%) 0.02±0.00d 0.58±0.10b 0.28±0.03c 3.15±0.21a

Proteins (%) 0.51±0.04d 3.04±0.00b 0.76±0.01c 3.28±0.00a

Carbohydrates (%) 4.51±0.17c 4.14±0.11d 4.96±0.27b 7.83±0.32a

Energetic content (kcal/100g) 20.26±1.36d 33.94±2.38b 25.41±1.75c 72.79±1.44a

Water activity (Aw) 0.70±0.01a 0.70±0.00a 0.70±0.00a 0.71±0.01a

pH 4.41±0.07d 4.86±0.09c 6.36±0.05b 6.62±0.06a

Titratable acidity (in lactic acid) 0.52±0.01a 0.39±0.08b 0.21±0.01c 0.19±0.01c

L* 31.29±0.90d 81.49±0.71b 41.37±0.83c 86.34±0.42a

a* -11.55±0.25c -7.02±0.12a -9.48±0.25b -7.25±0.17a

b* 2.15±0.58c 20.43±1.13a 11.67±1.09b 21.67±0.18a

Ca2++Mg2+ (mg/100mL) 28.11 ± 3.42d 99.57 ± 3.01b 43.34 ± 2.03c 124.17 ± 5.37a

3.2 pH and titratable acidity

According to the results presented in Table 1, the cheese whey presented a pH close to the milk, while the
ricotta whey and the butter whey presented a much different value. Regarding titratable acidity, the cheese
whey presented a value similar to milk whereas ricotta whey showed the highest one. On the other hand, the
values of titratable acidity followed contrary behavior observed for pH. The variation of pH can be explained by
the existence of several acidic compounds in whey like lactic acid, fatty acids, etc. Some studies have showed
that the average pH values of bovine whey can range from 6.03 to 6.71 (Bald et al., 2014; Macwan et al.,
2016), which corroborates the results found in the present study. Naturally, due to its production process,
ricotta whey’s pH was more acidic (mean of 4.41) compared to butter whey, cheese whey, and milk. This pH
was also more acidic than the average presented by Bald et al. (2014) with a value of 5.44 when analyzing
ricotta whey. This variation might be associated with the different types of cheese whey used in the production
of ricotta, implying different pHs in their wheys.
Taking into consideration the average found in this study (4.8), the butter whey analyzed by Antunes et al.
(2012) obtained a pH of 4.4. However, both values are in accordance with the U.S. Food and Drug
Administration (FDA), which determines a pH ranging from 4.4 to 4.8. In the present study, the result of pH for
whole milk was close to neutrality, in agreement to what is found the literature for milk (Walstra et al., 2006).

3.3 Water activity

As seen in Table 1, although the different milk by-products have more moisture than milk, there was no
significant difference for the water activity (Aw) between them, indicating that the increase occurred for the
contents of bound water and not for free water (available). This is a remarkable achievement because it is
clear the importance of water activity in food quality, especially regarding the representation of water
availability for the development of microorganisms and the occurrence of spoilage reactions (Rahman and
Labuza, 2007).

3.4 Calcium (Ca) and Magnesium (Mg)

Table 1 shows the Ca
2++Mg2+ values of milk and its residues. The milk had the highest concentration of

Ca2++Mg2+ followed by butter whey, cheese whey, and ricotta whey, respectively. Considering the obtaining
process of the evaluated dairy types of wheys, butter whey, cheese whey and ricotta whey have reduced
ashes contents, including Ca2++Mg2+, compared to whole milk (Morin, Pouliot & Jiménez-Flores, 2006; Bald et
al., 2014; Qiu et al., 2015; Cortellino & Rizzolo, 2018).
Franzoi et al. (2018) (123.35 mg/100mL), and Martino et al. (2001) (102.00 mg/100mL) found similar results to
what is displayed herein for Ca2++Mg2+ in cow's milk. Taking into account the different influences on food
composition, Jensen et al. (2012) analyzed the composition of milk from different cow breeds: Jersey (158,59
mg/100mL) and Holstein-Friesian (122.55 mg/100mL). Besides that, Haug et al. (2015) indicate that the milk
composition of cows varies in different regions of Norway: north (137.00 mg/100ml), south (133.90 mg/100ml),
east (131.90 mg/100ml), and west (132.70 mg/100mL).
The value for cheese whey of the present research was lower, but similar to what was found by Martino et al.
(2001) (54.80 mg/100mL). In contrast, Franzoi et al. (2018) found a much lower content (28.87 mg/100mL). It
is worth to remember that this variation occurs naturally and, in addition to the factors mentioned above, it is
mainly related to the type of cheese and/or cheese processing from which the whey originates.

3.5 Instrumental color

For instrumental color, the lightness refers to the proximity of white or black. As observed in Table 1, the milk
was the lightest one (highest value of L*) when compared to the others, followed by butter, cheese, and ricotta
wheys, respectively. The white color of milk results from the presence of colloidal particles, such as milk fat
globules and casein micelles, capable of scattering light in the visible spectrum (García-Pérez et al., 2005),
which explains the high L* value of the milk in contrast with the other types of wheys.
Furthermore, it was observed lower greenness (a*) and yellowness (b*) value of the cheese whey and ricotta
whey comparing with butter whey and whole milk (which did not have significant difference between them).
This fact is linked to lipid fraction (Table 1) in the same order of types of wheys (Qiu et al., 2015).
Considering that color is one of the most important quality attributes that influences consumer’s choices and is
directly linked to the appearance of dairy products (Aidoo et al., 2017), ricotta whey and cheese whey may
affect the color of the product which they can be added.
The literature does not present instrumental color data for dairy by-products, however, other characterization
studies of pasteurized whole milk presented similar performance: Schaffer et al. (1992) with L* (86.10), a* (-
2.10), and b* (7.80) and Bonacina et al. (2017) with L* (mean 89.37), a* (mean -7.76), and b* (mean 19.79).

3.6 Principal component analysis (PCA)

PCA explained 94.40% of the total variance of the data (Figure 2), in which the first component (PC1)
predominated and contributed with a greater percentage of variance explanation (73.50%) than the second
component (PC2) (20.90%). PCA separated all types of wheys and milk based on physicochemical
parameters (proximal composition, pH, titratable acidity, Aw, instrumental color, and Ca2++Mg2+). PC1
separated butter whey and ricotta whey (one group) from milk and cheese whey (another group). The cheese
whey characteristics are more similar to the whole milk than butter and ricotta wheys. PC2 separated butter
whey, ricotta whey, and milk (one group) from cheese whey (another group). Butter whey and ricotta whey can
be identified by greater redness and titratable acidity. The cheese whey by greater Ca2++Mg2+, yellowness,
pH, and lightness, and the whole milk by greater amount of minerals, lipids, carbohydrates, proteins, energy
value, and water activity.

Figure 2: Physicochemical and instrumental data of different types of wheys and whole milk in the plane
defined by the first two principal components.

4. Conclusions
This study investigated the main physicochemical properties of cheese whey, ricotta whey, and butter whey. In
physicochemical aspects, the different types of wheys had a remarkable reduction of the lipid fraction and
energetic content. Although there was a difference in moisture, the water activity did not vary among samples.
The pH and titratable acidity of cheese whey were closer to milk than the other types of wheys. Moreover, the
instrumental color allowed to identify that the milk was the whitest and opaquest sample followed by butter
whey, cheese whey, and ricotta whey.
Thus, many physicochemical parameters of the different dairy types of wheys studied are interesting and the
use of cheese whey, ricotta whey, and butter whey showed to be potential ingredients in food products. The
butter whey presented the most similar characteristics to the milk, however, the choice of the most appropriate
whey to be used in food depends on the product that the whey will be added and the aim of its application.
Anyway, the reuse of these dairy by-products can be characterized as an alternative in the reduction of
environmental impact with socioeconomic importance, aiming at minimizing pollution and costs. However,
further experiments should be conducted in order to better understand the technological potential of the
application of these by-products in human food, including possible sensory changes.

Acknowledgments

The authors are grateful to the Federal Institute of Alagoas (IFAL), campus Satuba, which donated the dairy
by-products and the Federal University of Bahia (UFBA) and Federal University of Rio de Janeiro (UFRJ) that
provided the ideal infrastructure for the analyses.

References

Antunes, A.E.C., Liserre, A.M., Faria, E.V., Yotsuyanagi, K., Lerayer, A.L.S., 2012, Analise descritiva
quantitativa de buttermilk probiótico, Brazilian Journal of Food and Nutrition, 23(4), 619-30.

AOAC (Association of Official Analytical Chemists), 2012, Official methods of analysis (19.ed.), AOAC
Publishing, Gaithersburg, USA.

Athira, S., Mann, B., Saini, P., Sharma, R., Kumar, R., Singh, A.K., 2015, Production and characterisation of
whey protein hydrolysate having antioxidant activity from cheese whey, Journal of the Science of Food and
Agriculture, 95(14), 2908-2915.

Bald, J.A., Vincenzi, A., Gennari, A., Lehn, D.N., & de Souza, C.F.V., 2014, Características físico-químicas de
soros de queijo e ricota produzidos no Vale do Taquari, RS, Revista Jovens Pesquisadores, 4(3), 90-99.

Bird, E.W., Weber, J., Cox, C.P., Chen, T.C.,1961, Determination of calcium and magnesium in milk by EDTA
titration, Journal of Dairy Science, 44(6), 1036-1046.

Bligh, E.G., Dyer, W.J.,1959, A rapid method of total lipid extraction and purification, Canadian journal of
biochemistry and physiology, 37(8), 911-917.

Bonacina, M.S., Baccin, M.A., da Rosa, L.S., 2017, Sensory and instrumental characteristics of integral
pasteurized milk, Revista do Instituto de Laticínios Cândido Tostes, 72(3), 139-151.

Cortellino, G., Rizzolo, A., 2018, Storage Stability of Novel Functional Drinks Based on Ricotta Cheese Whey
and Fruit Juices, Beverages, 4, 02-16.

FAO (Food and Agriculture Organization of the United Nations), 2003, Food energy: methods of analysis and
conversion factors, FAO Publishing, Rome, IT.

Franzoi, M., Niero, G., Penasa, M., Cassandro, M., de Marchi, M., 2018, Development and validation of a new
method for the quantification of soluble and micellar calcium, magnesium, and potassium in milk, Journal
of dairy science, 101(3), 1883-1888.

García‐Pérez, F.J., Lario, Y., Fernández‐López, J., Sayas, E., Pérez‐Alvarez, J.A., Sendra, E., 2005, Effect of
orange fiber addition on yogurt color during fermentation and cold storage, Color Research and
Application, 30(6), 457-463.

Haug, A., Steinnes, E., Harstad, O.M., Prestløkken, E., Schei, I., Salbu, B., 2015, Trace elements in bovine
milk from different regions in Norway, Acta Agriculturae Scandinavica, 65(2), 85-96.

Jensen, H.B., Poulsen, N.A., Andersen, K.K., Hammershøj, M., Poulsen, H.D., Larsen, L.B., 2012, Distinct
composition of bovine milk from Jersey and Holstein-Friesian cows with good, poor, or noncoagulation
properties as reflected in protein genetic variants and isoforms, Journal of Dairy Science, 95, 6905-6917.

Kuchta-Noctor, A.M., Murray, B.A., Stanton, C., Devery, R., Kelly, P.M., 2016, Anticancer Activity of Buttermilk
Against SW480 Colon Cancer Cells is Associated with Caspase-Independent Cell Death and Attenuation
of Wnt, Akt, and ERK Signaling, Nutrition and Cancer, 68(7), 1234-1246.

Macwan, S.R., Parmar, S.C., Prajapati, J.B., Aparnathi, K.D., 2016, Evaluation of Cheddar Cheese Whey and
Paneer Whey as a Growth Medium for Lactic Acid Bacteria, Journal of Pure and Applied Microbiology,
10(2), 1261-1268.

Martino, F.A.R., Sánchez, M.L.F., Sanz-Medel, A., 2001, The potential of double focusing-ICP-MS for studying
elemental distribution patterns in whole milk, skimmed milk and milk whey of different milks, Analytica
Chimica Acta, 442(2), 191-200.

Morin, P., Pouliot, Y., Jiménez-Flores, R., 2006, A comparative study of the fractionation of regular buttermilk
and whey buttermilk by microfiltration, Journal of Food Engineering, 77(3), 521-528.

Qiu, Y., Smith, T.J., Foegeding, E.A., Drake, M.A., 2015, Effects of microfiltration on whey, Journal of Dairy
Science, 98(9), 5862-5873.

Rahman, M.S. (Ed.), 2007, Handbook of food preservation, CRC press, New York, USA.
Rivas, J., Prazeres, A.R., Carvalho, F., Beltran, F., 2010, Treatment of cheese whey wastewater: combined

coagulation− flocculation and aerobic biodegradation, Journal of agricultural and food chemistry, 58(13),
7871-7877.

Sansonetti, S., Curcio, S., Calabrò, V., Iorio, G., 2009, Bio-ethanol production by fermentation of ricotta
cheese whey as an effective alternative non-vegetable source, Biomass Bioenerg, 33(12), 1687-1692.

Kneifel, W., Ulberth, F., Schaffer, E., 1992, Tristimulus colour reflectance measurement of milk and dairy
products, Lait, 72(4), 383-391.

The Lancet, 1992, River pollution by milk products, The Lancet, 200(5171), 772-773.
Walstra, P., Walstra, P., Wouters, J.T., Geurts, T.J., 2006, Dairy science and technology, CRC Press, Boca

Raton, USA.
WHO (World Health Organization), 2003, Diet, nutrition and the prevention of chronic diseases: report of a

joint WHO/FAO expert consultation, WHO Publishing, Geneva, SWZ.