Whey

Whey can be defined as the liquid milk derivative obtained during the production of cheese, casein, or similar products by separation from the curd following the milk's coagulation, and/or products derived from milk. The coagulation process primarily involves the activity of enzymes, especially those of the rennet type (Codex Alimentarius, 1995) and the whey can present in liquid, concentrated or powder forms. Approximately 80–90% of milk processed in cheese manufacturing facilities becomes whey (Buchanan et al., Reference Buchanan, Martindale, Romeih and Hebishy2023). Globally, the annual production of whey is estimated to be around 206 million tonnes (Tebbouche et al., Reference Tebbouche, Aziza, Abada, Chergui, Amrane and Hellal2024), with a yearly growth rate of approximately 1-2% (Buchanan et al., Reference Buchanan, Martindale, Romeih and Hebishy2023).

In terms of classification, sweet whey originates from enzymatic milk coagulation employing enzymes of microbial, vegetable or animal origin (such as chymosin), and presents a pH between 6.0 and 6.8. Acid whey is obtained during cheese making after acid-induced milk coagulation using lactic, acetic, or citric acids, or lactic yeast, and presents a pH below 6.0 (Brazil, 2020a). The two coagulation processes yield whey types with distinct compositions, as presented in Table 1 (Papademas and Kotsaki, Reference Papademas and Kotsaki2019). Acid whey exhibits lower lactose concentration due to the fermentation process that converts a fraction of lactose into lactic acid during the formation of the curd. Conversely, acid whey contains higher levels of calcium and phosphorus compared to sweet whey, attributed to the solubilization of the calcium-phosphorus complex in casein micelles at acidic pH (Alves et al., Reference Alves, De Oliveira Moreira, Rodrigues Júnior, Martins Mc de, Tuler Perrone and Fernandes2014).

In Brazil, production is mainly represented by sweet whey, derived from cheese manufacturing through enzymatic coagulation, since cheeses such as Mozzarella, Prato and Minas Frescal are the most commercialized varieties in the country (Santos et al., Reference Santos, Maranduba, de Almeida Neto and Rodrigues2017; Trindade et al., Reference Trindade, Soares, Scudding, Guimarães, Esmerino, Freitas, Pimentel, Silva, Souza and Almada2019). According to Trindade et al. (Reference Trindade, Soares, Scudding, Guimarães, Esmerino, Freitas, Pimentel, Silva, Souza and Almada2019), who applied a questionnaire survey to 100 dairy industries from Southeast and South Brazil (most of them subjected to Federal Inspection System), these industries use whey mainly for production of dairy beverages (60%), ricotta (20%), whey concentrate (15%), and milk blends (5%), while 27% discard the whey in the effluent treatment system or use for animal feed. Hebishy et al. (Reference Hebishy, Yerlikaya, Mahony, Akpinar and Saygili2023) performed a systematic literature review and identified and examined 17 competitive factors that influence the production and use of whey by the Brazilian dairy industry. Four of these factors were referenced by more than ten authors: nutritional composition of products improvement, reduction of the environmental impact, sensorial aspects of products improvement and new production technologies.

Whey can also be obtained through microfiltration systems (Kelly, Reference Kelly, Deeth and Bansal2019; Reig et al., Reference Reig, Vecino and Cortina2021). Membrane technology is a key processing tool in agro-food industries for the treatment of food products, by-products and food waste (Castro-Muñoz et al., Reference Castro-Muñoz, Boczkaj, Gontarek, Cassano and Fíla2020; Reig et al., Reference Reig, Vecino and Cortina2021). The global market for microfiltration membranes reached approximately USD 3.9 billion in 2022 and should reach USD 6 billion by 2027, with a compound annual growth rate of 8.8% during the forecast period of 2022-2027 (BCC Research, 2023). Membrane techniques possess the ability to physically separate fat and proteins from lactose and mineral salts, including monovalent and divalent ions (Rice et al., Reference Rice, Kentish, Vivekanand, O'Connor, Stevens and Barber2005; Baldasso et al., Reference Baldasso, Silvestre, Silveira, Vanin, Cardozo and Tessaro2022). The resulting solution, known as permeate, contains particles smaller in size than the membrane pores and typically comprises around 4.5% lactose. On the other hand, substances of larger size, such as proteins and fats, are retained by the membrane, forming a solution referred to as concentrate (Rice et al., Reference Rice, Kentish, Vivekanand, O'Connor, Stevens and Barber2005; Bhushan and Etzel, Reference Bhushan and Etzel2009; Baldasso et al., Reference Baldasso, Silvestre, Silveira, Vanin, Cardozo and Tessaro2022).

Ultrafiltration is one of the most prevalent techniques employed in producing whey protein concentrate (WPC) and whey protein isolate (WPI). In this procedure, low molecular weight components like water, salts and lactose permeate the membrane, while proteins, due to their larger molecular weight and size, are retained (Kravtsov et al., Reference Kravtsov, Kulikova, Anisimov, Evdokimov and Khramtsov2021). While the dairy industry initially adopted this technology to find solutions for whey utilization (Price, Reference Price, Deeth and Bansal2019), its use remains relatively nascent in Brazil. In addition to ultrafiltration, a variety of advanced techniques are used for whey processing, such as reverse osmosis, drying, chromatographic methods, ion exchange, electrodialysis, fermentation and chemical processes, offering a spectrum of pathways for creating transformed products (Kilara and Vaghela, Reference Kilara, Vaghela and Yada2018; Kelly, Reference Kelly, Deeth and Bansal2019). Ohmic Heating has been considered as a potential technology for sweet whey processing, as it provides fast and uniform heating, minimizing the generation of off-flavor compounds and providing minor changes in nutrients, when compared to the traditional heat treatment (Costa et al., Reference Costa, Cappato, Ferreira, Pires, Moraes, Esmerino, Silva, Neto, Tavares and Freitas2018).

Whey contains a wide range of components, some holding significant nutritional value and biological activities that are intensifying interest in exploring this co-product (Kilara and Vaghela, Reference Kilara, Vaghela and Yada2018). High whey protein fractions, in particular, have drawn attention for their attractive properties, leading to their integration into food formulations. Indeed, the attraction for whey has gained ground within the dairy industry (Ballatore et al., Reference Ballatore, Bettiol M, Vanden Braber, Aminahuel, Rossi, Petroselli, Erra-Balsells, Cavaglieri and Montenegro2020; Tsermoula et al., Reference Tsermoula, Khakimov, Nielsen and Engelsen2021; Elkot et al., Reference Elkot, Elmahdy, El-Sawah, Alghamdia, Alhag, Al-Shahari, AL-Farga and Ismail2024). Ingredients derived from whey exhibit the most rapid market growth among all dairy ingredients, with the market valued at $53.8 billion in 2019 and projected to reach $81.4 billion by 2025 (Tsermoula et al., Reference Tsermoula, Khakimov, Nielsen and Engelsen2021). An important technological innovation in the milk production chain involves the conversion of whey into a valuable input. Producers are increasingly considering environmental responsibility, which is gaining prominence in the industry. This shift not only introduces a fresh income source for the industrial sector but also repurposes whey into a raw material to produce other items, primarily due to its high content of lactose and protein. This transformation enhances the economic efficiency of the dairy chain (Lavelli and Beccalli, Reference Lavelli and Beccalli2022; Al-Tayawi et al., Reference Al-Tayawi, Sisay, Beszédes and Kertész2023).

Contributions from consumer demand for healthier, innovative, safer and more convenient products have fueled the expansion of dairy beverage production (Guiné et al., Reference Guiné, Florença, Barroca and Anjos2020). In this way, dairy beverages are an alternative for the use of whey due to its cost-effectiveness, ease of processing, and compatibility with existing industry equipment (Alves et al., Reference Alves, Floriano, Voltarelli, de Rensis, Pimentel, Costa and Vianna2020; Kurnick et al., Reference Kurnick, Michellim, Yada, Junior and Tribst2024). Further alternatives encompass the manufacture of ricotta, protein isolates and the production of WP, that can be used in different formulations (Pires et al., Reference Pires, Marnotes, Rubio, Garcia and Pereira2021). There has been also a growing emphasis on the manufacturing of dried dairy powders, to create new products and enhance their shelf life. These powdered forms are applied in diverse product categories, including confectionery, infant formula, sports dietary supplements and supplements for health recovery (O'Donoghue and Murphy, Reference O'Donoghue and Murphy2023). For example, the increase in infant formula consumption drives a global need for lactose, thereby amplifying interest in whey as a primary source of this sugar (Tuler Perrone et al., Reference Tuler Perrone, Schuck, Caldas Pereira Silveira and Fernandes de Carvalho A2016).

Whey powder production

According to Božanić et al. (Reference Božanić, Barukčić, Jakopović and Tratnik2014), the main industrial processing of whey is drying to produce WP, which is 70% of the annual production of whey. As a main advantage, this process does not produce residues that should be treated, but on the other hand it demands a high initial investment to purchase the equipment, and consume a lot of energy during production. Furthermore, WP has a relatively low selling price when compared to WPC.

When fluid whey is obtained from other companies (smaller cheese manufacturers, for example), fluid whey may be subjected to pasteurization when arrives at the powder processing facility. Then, it is subjected to a sequence of procedures involving evaporation, lactose crystallization and drying, as shown in Fig. 1. The membrane separation process can also be used. These steps are designed to prevent microbial growth, by eliminating or deactivating microorganisms (Ziuzina et al., Reference Ziuzina, Los, Bourke and Fetsch2018). To preserve the freshly acquired whey and control microbial growth until heat treatment, it should be immediately refrigerated at 4 ºC. The reduction in temperature causes an extension of the latency time of microorganisms already present in the whey (Kilara, Reference Kilara, Chandan, Kilara and Shah2015). Hence, it is crucial to emphasize that during the steps involving heat treatment, the binomial time and temperature needs to be carefully respected.

Figure 1.

Main steps in whey powder production.

Concentration of pasteurized fluid whey solids is achieved through vacuum evaporation, which incurs a significantly lower energy cost per kilogram of evaporated water compared to spray drying. This procedure allows concentration levels of milk solids ranging from 50% to 60% (m/m), carried out within temperatures of 45 to 70 ºC (Carić et al., Reference Carić, Akkerman, Milanović, Kentish, Tamime and Tamime2009; Schuck, Reference Schuck and Corredig2009). However, the temperature range used in the evaporators is not sufficient to ensure microbiological safety, and it is necessary to apply prior pasteurization to the fluid whey. Once concentrated, the solution proceeds to the crystallizer where the lactose undergoes crystallization. This stage aims to reduce the hygroscopicity of the powdered product, thus avoiding technological problems such as product adherence and agglomeration (Schuck, Reference Schuck and Corredig2009). From a microbial perspective, the lactose crystallization process plays a critical role in the processing of powdered whey, as the product remains at room temperature for extended periods (4 to 24 hours) under constant agitation at temperatures close to 25 ºC (Simeão et al., Reference Simeão, DA Silva, Stephani, de Oliveira, Schuck, de Carvalho and Perrone2018). After crystallization, spray drying is conducted with inlet air temperatures ranging from approximately 165 to 180 ºC and outlet air temperatures around 85 to 90 ºC. The fresh powder leaves the spray dryer at around 85 to 90 ºC, is cooled down to reach approximately 25 to 28 ºC and is then packaged. The spray drying phase significantly impacts both the yield and quality of WP. Challenges include the potential loss of powder yield due to high lactic acid and galactose content, leading to particle adhesion, and an elevated amorphous lactose content causing clumping during storage, impacting overall powder quality and shelf life (Ozel et al., Reference Ozel, McClements, Arikan, Kaner and Oztop2022).

Numerous surfaces come into contact with the product throughout WP processing, including equipment, utensils, and accessories. Consequently, the risk of pathogen cross-contamination cannot be disregarded (Martin et al., Reference Martin, de Oliveira E Silva, da Fonseca, Morales, Souza Pamplona Silva, Miquelluti and Porto2016; Andritsos and Mataragas, Reference Andritsos and Mataragas2023). To avoid this, the industry employs a strategy of cleaning and sanitization of all equipment and utensils that come into contact with the product during processing. This hygiene procedure can be performed with the use of chemicals, such as detergents and sanitizers (Martin et al., Reference Martin, de Oliveira E Silva, da Fonseca, Morales, Souza Pamplona Silva, Miquelluti and Porto2016; Wang et al., Reference Wang, Puri, Demirci, Demirci, Feng and Krishnamurthy2020).

Microbiological aspects relevant for whey powder production

Fluid whey is considered to be a favorable medium for microorganism growth, due to factors such as pH (6.0-6.8), high water activity (0.92%) and significant amounts of nutrients (Table 1: Pescuma et al., Reference Pescuma, de Valdez and Mozzi2015; Pires et al., Reference Pires, Marnotes, Rubio, Garcia and Pereira2021). This co-product has a diverse microbiota generally composed of both beneficial bacteria with significant technological importance, but also spoilage microorganisms or even pathogens (Andrighetto et al., Reference Andrighetto, Marcazzan and Lombardi2004; Walsh et al., Reference Walsh, Meade, Mcgill and Fanning2012; Morandi et al., Reference Morandi, Battelli, Silvetti, Goss, Cologna and Brasca2019, Reference Morandi, Cremonesi, Arioli, Stocco, Silvetti, Biscarini, Castiglioni, Greco, D'Ascanio and Mora2022). The whey microbial diversity can be influenced by factors like dairy herd health, milking procedures, environmental hygiene, milk processing and factors related to whey handling, storage, and processing (Murphy et al., Reference Murphy, Martin, Barbano and Wiedmann2016). In addition to beneficial bacteria (such as acid lactic bacteria – LAB), fluid whey can be contaminated by aerobes mesophiles, coliforms, enterobacteria and microorganisms originating from diseased animals (Mycobacterium bovis, Brucella abortus, S. aureus, Coxiella burnetii, Listeria monocytogenes, Salmonella, Streptococcus agalactiae, Streptococcus uberis, and Escherichia coli) or from biofilms attached on equipment, utensils and processing environments (Pseudomonas, Flavobacterium, Enterobacter, Cronobacter, Klebsiella, Acinetobacter, Aeromonas, Alcaligenes, Achromobacter, Corynebacterium, Microbacterium, Micrococcus) (Andrighetto et al., Reference Andrighetto, Marcazzan and Lombardi2004; Vissers and Driehuis, Reference Vissers, Driehuis and Tamime2008; Walsh et al., Reference Walsh, Meade, Mcgill and Fanning2012; Morandi et al., Reference Morandi, Battelli, Silvetti, Goss, Cologna and Brasca2019).

Table 1.

Typical composition of sweet and acid whey (Papademas and Kotsaki, Reference Papademas and Kotsaki2019)

Smaller cheese manufacturers who supply fluid whey to industries sometimes do not achieve adequate refrigeration, which may jeopardize the microbiological quality and safety of the whey. Thus, the co-product is subjected to pasteurization to inactivate pathogenic microorganisms, spoilage agents, and enzymes (Lewis and Deeth, Reference Lewis, Deeth and Tamime2008; Kilara, Reference Kilara, Chandan, Kilara and Shah2015). Most vegetative cells are typically destroyed by high-temperature short-time (HTST) treatment from 72 to 75 ºC for 15 to 20 seconds, followed by cooling to 5ºC. If whey is immediately processed through drying or evaporation, refrigeration may be omitted (Brazil, 2020a; Hebishy et al., Reference Hebishy, Yerlikaya, Mahony, Akpinar and Saygili2023). However, some microorganisms may persist in the whey, such those from the groups of thermoduric and thermophilic, represented mainly by Alcaligenes, Microbacterium, Micrococcus, Enterococcus, Streptococcus, Lactobacillus and Corynebacterium, as well as spore-forming bacteria such Bacillus, Geobacillus and Clostridium, which has relevance for the quality and safety of dairy products (Touch and Deeth, Reference Touch, Deeth and Tamime2008). Despite not being able to multiply in dried dairy products, such bacteria can stay viable in dried products for long periods and upon reconstitution they may grow or their respective spores can germinate (Pal et al., Reference Pal, Alemu, Mulu, Karanfil, Parmar and Nayak2016) representing risks for the consumers, mainly for infants.

Walsh et al. (Reference Walsh, Meade, Mcgill and Fanning2012), described the presence of thermoduric bacterial isolates in an Irish WPC process. B. licheniformis, Mic. lacticum, S. warneri, E. durans and B. subtilis were recorded as the predominant microorganisms in the process line, while Mic. phyllosphaerae, Neisseria subflava, Rothia aeria and Strep. mitis were observed in dairy produce or indeed in any food product. Most recently, the rpoB and 16S rRNA genes have emerged as tools to characterize the spore populations of psychrophilic, mesophilic, and thermophilic contaminants present in sweet whey, WPC, non-fat dry milk and acid WPs. Through this approach, researchers identified spores from at least 12 genera, 44 species, and 216 rpoB allelic types, with Bacillus licheniformis, Geobacillus spp. and Anoxybacillus spp. standing out as the predominant ones. Moreover, when comparing spore species obtained from raw materials and the final powdered products, it was observed that while certain species, like B. licheniformis, were present in both the initial materials and the finished powders, other species such as Geobacillus spp. and Anoxybacillus spp. were more commonly detected in the final powdered products (Miller et al., Reference Miller, Kent, Watterson, Boor, Martin and Wiedmann2015). By using metagenomic sequencing, McHugh et al. (Reference McHugh, Feehily, Tobin, Fenelon, Hill and Cotter2018) also identified different groups of mesophilic sporeformers (namely, Bacillus cereus, Bacillus licheniformis, Bacillus paralicheniformis, and a heterogeneous group containing Brevibacillus brevis) in WP produced over 1 year in Ireland.

Anoxybacillus sp. and Geobacillus sp., together with Bacillus sp. are recognized as dominant spores identified in dairy powder products. These Gram-positive, aerobic bacteria produce relatively heat-resistant spores that can be activated by heat shock (Yuan et al., Reference Yuan, Liu, Ren, Zhang, Zhao, Kan, Yang, Ma, Li and Zhang2012; Zain et al., Reference Zain, Flint, Bennett and Tay2016). The occurrence of such spores in WP may represent spoilage potential, but presence of B. cereus can also be considered a concern in terms of safety since such isolates might be capable of causing emetic and enterotoxin food poisoning. Research on clostridial spores in dairy powder products are scarcer, although Clostridium species were already reported in milk powders (Barash et al., Reference Barash, Hsia and Arnon2010; Buehner et al., Reference Buehner, Anand and Djira2015), and the presence of pathogenic C. botulinum and C. perfringens in milk powder is also considered a concern (Pal et al., Reference Pal, Alemu, Mulu, Karanfil, Parmar and Nayak2016). Other microorganisms potentially present in fluid whey, such as Pseudomonas, can lead to the deterioration of the raw material (Vissers and Driehuis, Reference Vissers, Driehuis and Tamime2008) or potentially cause foodborne outbreaks, as in the case of Salmonella, L. monocytogenes, C. sakazakii, and S. aureus (Ünüvar, Reference Ünüvar, Holban and Grumezescu2018). A common factor among these microorganisms is their vulnerability to heat treatments applied during fluid whey processing (Al-Holy et al., Reference Al-Holy, Al-Nabulsi, Osaili, Ayyash and Shaker2012; Necidová et al., Reference Necidová, Bursová, Haruštiaková, Bogdanovičová and Lačanin2019). However, the presence of S. aureus in fluid whey is a concern, given its ability to produce heat-resistant enterotoxins (Necidová et al., Reference Necidová, Bursová, Haruštiaková, Bogdanovičová and Lačanin2019).

According to Schwabe et al. (Reference Schwabe, Notermans, Boot, Tatini and Krämer1990), the thermal inactivation of SEs varies based on the food matrix and pH. Assessing the impact of heat treatment on enterotoxin activity is challenging, as it depends on the type of enterotoxin and the concentration in the food matrix. European law establishes criteria for coagulase-positive staphylococci cheeses, milk powder and whey powder. If the counts exceed 10⁵ cfu/g, the batch must be tested for the presence of SEs, which must be absent in 25 g of the products (European Commission, 2005). Brazilian Ministry of Agriculture and Livestock Production (in Portuguese, Ministério da Agricultura e Pecuária) also establishes microbiological criteria for WP production, including the counting of viable mesophilic aerobes (≤ 10⁵ cfu/g), total coliforms at 30-35 ºC (≤ 100 cfu/g), thermotolerant coliforms at 45 ºC (≤ 10 cfu/g), coagulase-positive staphylococci (≤ 100 cfu/g), Salmonella spp. (absence/25 g) (Brazil, 2020a). In addition, once available for commercialization, WP must meet the microbiological standards established by National Health Surveillance Agency (in Portuguese: Agência Nacional de Vigilância Sanitária: Brazil, 2022) which determines the counting of viable mesophilic aerobes (≤ 10⁵ cfu/g), coagulase-positive staphylococci (≤ 100 cfu/g), and absence of Salmonella spp., Enterobacteriaceae and SEs.

The potential risk of S. aureus and SEs in whey and whey powder

Powdered milk is not sterile and has the potential to harbor diverse bacteria, including toxin producing pathogens (Bogdanovičová et al., Reference Bogdanovičová, Necidová, Haruštiaková and Janštová2017). S. aureus is a Gram-positive bacteria, mesophilic cocci with a multiplication temperature in the range of 7 to 48 ºC. The general survive features of this bacteria are pH limits between 4.2 and 9.3, minimum water activity of 0.85, and ability to survive in environments with up to 25% NaCl concentration. These immobile bacteria exhibit a diameter ranging from 0.5 to 1.5 µm and do not produce spores. They can be found in isolation, pairs, short chains, or clusters (Bennett et al., Reference Bennett, Hait, Tallent, Labbé and García2013; Bhunia, Reference Bhunia and Bhunia2018). S. aureus is a pathogen of great concern in food due to the ability of certain strains to produce heat-stable SEs that, once ingested, can lead to symptoms of gastrointestinal disorders, such as vomiting, nausea and abdominal cramps (Bennett et al., Reference Bennett, Hait, Tallent, Labbé and García2013; Landgraf and Destro, Reference Landgraf, Destro, Morris and Potter2013). Despite notable improvements in food safety procedures, SEs still figure as one of the main causes of foodborne outbreaks worldwide (Grispoldi et al., Reference Grispoldi, Karama, Armani, Hadjicharalambous and Cenci-Goga2021; Hu et al., Reference Hu, Li, Fang and Ono2021).

From 2013 to 2022, S. aureus was listed as the main agent of reported outbreaks of food poisoning in Brazil, ranking third among pathogenic bacteria, behind E. coli and Salmonella spp. During this period, the Health Surveillance Secretariat reported a total of 6,523 outbreaks of foodborne diseases, affecting 107,513 individuals and resulting in 112 fatalities. Of these outbreaks, in 1,571 (24.08%) it was possible to specify the causative agents and Staphylococcus spp. was identified as the etiological agent in 170 (10.8%) outbreaks (Brazil, 2023). In a more detailed report, from 2016 to 2019, 2,504 outbreaks of foodborne diseases were identified, and information was present regarding the type of food involved for 894 cases (35.7%), with milk and dairy products responsible for 81 (9.06%) of these cases (Brazil, 2020b). However, the actual number could be significantly higher due to the underreporting of isolated cases.

Food poisoning caused by S. aureus typically occurs shortly after the ingestion of food contaminated with preformed toxins at concentration above 1 ng/g of food (Landgraf and Destro, Reference Landgraf, Destro, Morris and Potter2013; Bhunia, Reference Bhunia and Bhunia2018). In a study conducted by Bogdanovičová et al. (Reference Bogdanovičová, Necidová, Haruštiaková and Janštová2017), the possibility of survival and growth of S. aureus in milk powder after its reconstitution was evaluated. Powdered milk was inoculated with 10² to 10⁵ cfu/g of enterotoxigenic S. aureus and the reconstituted milk was stored for 48 h at 4, 15 and 25 ºC. The multiplication of S. aureus as well as the production of SEA, SEB and SEC enterotoxins was regularly detected, and at the higher temperature and inoculum concentration, pathogen growth and enterotoxin detection were greater (Bogdanovičová et al., Reference Bogdanovičová, Necidová, Haruštiaková and Janštová2017), demonstrating the risk that WP contamination could pose to consumers. The presence of staphylococci in a WP plant was examined and S. aureus was identified, originating from three powder residue samples from the working floor near filling devices. On the other hand, S. aureus was not present in air samples, but S. saprophyticus and S. xylosus were isolated. In addition, before the study was started, it was discovered that the WP contained a small number of S. aureus of a specific phage type, distinct from any isolates detected within the production line or its environment. Eight months later, S. aureus isolates from a site previously found to be contaminated, were of different phage types (Kleiss et al., Reference Kleiss, van Schothorst, Cordier and Untermann1994).

Wang et al. (Reference Wang, Meng, Zhang, Zhou, Zhang, Yang, Xi and Xia2012) evaluated samples of powdered infant formula milk and showed that 11.2% of them were positive for enterotoxigenic S. aureus. Even if these enterotoxigenic S. aureus-contaminated infant foods are reconstituted at low temperatures and subsequently stored at room temperature for extended durations, they still pose a health risk to children. Asao et al. (Reference Asao, Kumeda, Kawai, Shibata, Oda, Haruki, Nakazawa and Kozaki2003) documented a significant outbreak in Japan linked to the ingestion of reconstituted milk tainted with staphylococcal enterotoxin A. On the other hand, Cho et al. (Reference Cho, Kim, Kim, Park, Kwon J, Kim, Lee and Rhee2018) conducted a study of the microbial composition of powdered infant formula along the manufacturing processes and no presumptive colony for Cronobacter spp., Salmonella spp., and S. aureus was detected. B. cereus was the only detected bacteria, which also represents a concern.

In general, SEs exhibit resistance to freezing, drying and heat treatments, such as 100 ºC, 110 ºC, and 121 ºC for 3 min. This suggests that conventional thermal processing techniques might not be sufficient to entirely eliminate the threat posed by S. aureus in milk and dairy products (Guanhong et al., Reference Guanhong, Zonghong, Yao, Xu, Ting and Xin2023). Necidová et al. (Reference Necidová, Janštová and Karpíšková2012) demonstrated that the multiplication rates of S. aureus and the production of SEs in brain heart infusion culture medium and milk sources differ significantly. The culture medium often showed higher S. aureus counts and increased SE production compared to the food matrix. This raises the question of how the food matrix might impact the thermal stability and inactivation of enterotoxins. To investigate this further, Necidová et al. (Reference Necidová, Bursová, Haruštiaková, Bogdanovičová and Lačanin2019) assessed the effect of heat treatment on the activity of SEs of type A, B, and C in milk. A total of 38 S. aureus strains capable of producing SEA, SEB, or SEC were subjected to heat treatment at 100, 110 and 121 ºC for 3 min. Prior to heat treatment, all samples were positive for SE production. However, after heat treatment, positive rates of decrease were obtained, being 36.8%, 34.2%, and 31.6% at 100, 110 and 121ºC, respectively. The reduction in positive samples differed according to the type of enterotoxin, with SEA being detected in higher amounts before and after heat treatment. Notably, the SE concentrations decreased significantly after heat treatment, leading to the conclusion that the success of SE inactivation depends on the initial concentration present before heat treatment.

Among the foods that cause outbreaks and staphylococcal intoxications, raw milk, pasteurized milk and cheese are noteworthy, with S. aureus frequently associated in epidemiological investigations (Kümmel et al., Reference Kümmel, Stessl, Gonano, Walcher, Bereuter, Fricker, Grunert, Wagner and Ehling-Schulz2016; Hu et al., Reference Hu, Wang, Fang, Okamura, Ono and Fetsch2018; Gajewska et al., Reference Gajewska, Chajęcka-Wierzchowska and Zadernowska2022; Zhang et al., Reference Zhang, Wang, Jin, Li, Zhang, Shi and Zhao2022). Considering that the fluid whey used in WP production predominantly originates from cheese manufacturing, where S. aureus and SE are recurrent, and considering that such microbiological criteria are applied to WP, monitoring S. aureus and associated SEs must be performed in WP, in order to avoid food poisoning outbreaks involving food that include WP in their formulations.

S. aureus can multiply within temperatures ranging from 7 to 48ºC, with optimum growth at 35-37 ºC, demanding adequate refrigeration when the whey is subjected to storage before processing in order to inhibit its multiplication and subsequent production of SEs (Hu et al., Reference Hu, Wang, Fang, Okamura, Ono and Fetsch2018). In a study performed by Halpin-Dohnalek and Marth (Reference Halpin-Dohnalek and Marth1989), fresh cheddar cheese whey was inoculated with S. aureus at 10⁶ cfu/ml and held at 4, 25 and 37 ºC for 48 h; the number of staphylococci increased in whey at 25 and 37 ºC and decreased or remained constant in whey at 4 ºC, revealing the importance of low temperature before the processing of this co-product.

In addition to avoiding growth of S. aureus in the whey, if the concentrated mixture becomes recontaminated by S. aureus at any post-pasteurization phase, the conditions of crystallization can inadvertently lead to an increase in the pathogen population and potentially production of SEs, although studies are needed to clearly demonstrate this. This is important because S. aureus is well known for its ability to produce biofilms and persist within food processing facilities, which may allow cross-contamination to foods and turn sanitization procedures ineffective, as chemicals may not reach inside the biofilm at necessary concentrations (Lee et al., Reference Lee, Mangolin, Gonçalves, Neeff, Silva, Cruz and Oliveira2014; Idrees et al., Reference Idrees, Sawant, Karodia and Rahman2021). Toté et al. (Reference Toté, Horemans, Berghe, Maes and Cos2010) evaluated the inhibitory effect of different biocides on the biofilm of S. aureus. Among those analyzed, sodium hypochlorite and peracetic acid were the most effective in reducing the viability of both the biofilm matrix and the viable mass. Furthermore, Meira et al. (Reference Meira, de Medeiros Barbosa, Aguiar Athayde, de Siqueira-júnior and de Souza2012) explored the adhesion, kinetics of detachment, and development of biofilms by S. aureus isolates from food service surfaces on both stainless steel and polypropylene surfaces, as well as the effectiveness of peracetic acid (30 mg/L) and sodium hypochlorite (250 mg/L) in removing bacterial cells from the preformed biofilm matrix. The isolates showed high ability to adhere and form biofilm on the tested surfaces when immersed in a food-based broth at 7 and 28 °C. While both disinfectants reduced the number of adhered cells, they were not entirely successful in completely removing the viable cells of S. aureus forming a mature biofilm.

The removal of biofilms from within the crystallizer holds special significance, as adhered S. aureus cells can detach and reintroduce contamination to the concentrated mixture. Once the lactose crystallization stage is completed, the product is dehydrated using spray drying, a process that involves the dispersion of the mixture into small droplets under a stream of hot air at 160 to 200 ºC (da Silva et al., Reference da Silva, Martins, Silveira, Simeão, Mendes, ÍT, Schuck and de Carvalho2017). The extensive surface area of the droplets and the elevated temperatures of the drying air result in rapid dehydration (Schuck, Reference Schuck and Corredig2009). Despite the high temperature of the drying air, the droplets themselves reach relatively low temperatures (around 40 ºC). Consequently, spray drying is not a procedure that can be associated with the thermal elimination of microorganisms. Therefore, the microbial population within the concentrate containing crystallized lactose may not be substantially reduced, remaining potentially viable in powder form. In addition, thermoresistant SEs can persist despite the spray drying process (Tatini, Reference Tatini1976; Emiliano et al., Reference Emiliano, Martins, Freitas, Carvalho and Perrone2022). In this way, the microbial quality of the initial fluid whey (raw material) and the management of recontamination risks during processing are essential to ensure the safety of WP.

The microbiological quality of WPs is intricately linked to the microbial content initially present in the raw material, as well as to the possibility of contamination during and processing. This review highlights the need for more research to assess the microbiological integrity of WP, especially in Brazil, where the production of WP has been growing in recent years (Lopes et al., Reference Lopes, Guimarães and Ribeiro2023). Given the increasing use of WP as an ingredient for various food products and its global economic significance, continuous surveillance for the presence of spoilage microbiota and potential pathogens, including S. aureus and associated SEs, is indispensable to prevent potential outbreaks linked to the consumption of products containing WP in their formulation.