3.1 Second cheese whey in the production of food and beverages

Given the increasing consumer awareness regarding the importance of maintaining a healthy and sustainable diet, both the scientific community and the agri-food industry are actively engaged in developing innovative, high-quality foods. These are designed to incorporate elements such as prebiotics, probiotics, microbial metabolites, and bioactive compounds (Terpou et al. 2019; Cunningham et al. 2021) while upholding the sustainability of the production process. In this context, the potential of SCW as a possible ingredient for the production of food and beverages has been explored by several authors (Table 2).

Table 2 Studies related to the utilization of SCW for the production of food, beverages, microbial starters, single cell proteins, single cell oils, and bioactive metabolites

Pontonio et al. (2021) demonstrated the potential of fermenting SCW to produce bioactive peptides with angiotensin-I-converting enzyme (ACE) inhibitory properties to enhance the health promoting effect of foods like ricotta cheese. For this purpose, pasteurized SCW was ultrafiltered and the resulting protein-rich retentate was inoculated with a selected strain of Lactobacillus helveticus, known for proteolytic activity. The fermented retentate contained 3230 ± 25 mg L−1 peptides and, after spray-drying, it was utilized in different proportions (1–5%) to fortify ricotta cheese. Samples obtained with 5% of the spray-dried fermented retentate contained 30 mg of bioactive peptides per 100 g serving and exhibited a nine-fold increase in ACE-inhibitory activity compared to the control produced without fortification. Minor modifications in terms of hardness, chewiness, acidity, odour, and taste were observed while an improvement in flavour persistence and savouriness was noted. Moreover, according to these authors, the fortification process for ricotta cheese is easily scalable, thus enabling its application for the valorisation of SCW.

Similarly, Garcia et al. (2022), based on the evidence that whey cheeses is suitable to deliver probiotic strains (Madureira et al. 2005), proposed the utilization of SCW in probiotic goat whey cheese making, thus highlighting its potential for enhancing both the nutritional and health-promoting aspects of dairy products. To achieve this, goat whey was added with 5% freeze dried diafiltered SCW and 5% of commercial probiotic cultures including Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus, along with thyme essential oil and sodium citrate. Probiotics viability at 21 days of storage of the resulting cheese reached an average of 1.7×108 CFU g−1 and was therefore largely above the minimum required for a probiotic product (107 CFU g−1).

Additionally, Borges et al. (2020) explored the use of SCW as a fat replacer in reduced-fat washed curd cheese. For this purpose, 5% of ultrafiltered concentrated SCW, buttermilk and CW were added to milk and their impact on cheese colour, texture and sensorial properties was evaluated. The experimental cheeses obtained with SCW and CW received lower scores for both texture and taste in comparison to those obtained with buttermilk thus suggesting that further works need to be done to evaluate the fat replacer properties of these by-products.

Furthermore, Pires et al. (2023) showcased the utilization of concentrated SCW in the production of probiotic ice creams. In this case SCW was ultrafiltered, pasteurized, homogenised, and added with sucrose, inulin, citric acid, and xanthan gum, before inoculation either with thermophilic yogurt starter culture, kefir culture or a mixture of probiotic cultures. Fermentation was carried out to pH 4.6 and the resulting ice creams were stored in a freezer at – 21 °C for 120 days. Although with significant differences among the products in terms of hardness, viscosity and meltdown rates, all of them were well-received by a consumer panel and retained their probiotic characteristics for up to 2 months.

For the development of a probiotic beverage, Maragkoudakis et al. (2016) inoculated pasteurized SCW with mixed cultures including Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lb. acidophilus. These bacteria lowered SCW pH to 4.2 and reached viable cell counts of about 8–9×108 CFU mL−1 thus suggesting that pasteurized SCW can be employed of the production of a fermented beverage rich in potentially beneficial bacteria. Similarly, Tirloni et al. (2020) inoculated SCW with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. The fermented SCW (pH 4.0) was then supplemented with 13.75% (w/v) pasteurized fruit purees along with pectin, potassium sorbate, sodium citrate and stored into 125-mL bottles at 4 and 12 °C. Under these conditions, the fermented SCW remained microbiologically stable for up to 14 days thus supporting the potential of SCW in the production of fermented beverages.

Other authors combined uninoculated SCW and fruit juices to obtain unfermented SCW-based beverages (de Souza Silva et al. 2022). For the microbiological stabilization of SCW a thermal treatment at 100 °C for 15 minutes was carried out, and three formulations, containing different proportions of SCW, passion fruit pulp and sucrose, were produced, heated at 85 °C for 30 s and stored at 7 °C. Through these processes an isotonic beverage composed by 30% SCW, 12% passion fruit pulp, 5% sucrose was developed. This exhibited good sensory acceptance and showed higher phenolic content and antioxidant activity compared to commercially available isotonic beverages. While further in vivo studies are required to assess the impact of these beverages on hydro-electrolytic replenishment and sports performance, their findings suggest the potential usefulness of unfermented SCW as an ingredient in athlete supplements. Interestingly, according to Rizzolo and Cortellino (2017, 2018), by varying the type of fruit juice used in the formulation it is possible to modulate the levels of antioxidants, sugars, and organic acids present in SCW-based beverages. Such flexibility in formulation allows for the creation of a diverse range of products with varying nutritional and flavour profiles, catering to different consumer preferences and dietary needs.

More recently, Kurnick et al. (2024) explored the possibility of utilizing sheep and goat CW and SCW to produce unfermented (containing Lactobacillus casei as bioprotectant) and fermented beverages (with a commercial yogurt starter culture). Also in this case, CW and SCW were thermally treated (75 °C for 5 min) and added with sugars, flavouring agents and stabilizers. SCW fermented beverages suffered of lower consumers acceptance and physical instability during storage, due to the effect of acidic pH on proteins, their denaturation level and solubility.

Another possibility regards the utilization of SCW to partly replace water and sugar in the production of a sweet milk stout and a salty and acidified Gose style beer. Different amount of SCW (37% for sweet milk stout and 16% for Goose style beer) were mixed with water for grain mashing and the resulting worts (25 L) were inoculated with Saccharomyces cerevisiae for the sweet milk stout and with a mixed culture including S. cerevisiae and Lachancea thermotolerans for the Gose-style beer. Since these two yeasts do not ferment lactose, the Goose style wort was treated with lactase. After fermentation, the sweet milk stout contained 1.6 % (w/w) residual lactose and a calcium level of 14.3 mg 100 g−1, about the double of the content reported in other stouts. The Goose style beer showed no residual lactose and a calcium level of 7.06 mg 100 g−1, at the high end for many beers. Sensory attributes of the two beers were in line with that reported for other beers in the market. According to these results, the use of SCW in beer manufacturing serves to add sugars, replace up to 37% of the water used in the brewhouse, and increase beer mineral content with no marked detrimental effects on the sensory attribute of the final products. This opens new ways towards more sustainable productions in the dairy and brewing industries (Pasta et al. 2024)

Based on all these findings, SCW emerges as a promising ingredient for the production of food and beverages, and the approaches presented open avenues for reducing waste in the dairy industry while meeting the demands of consumers seeking healthier and more sustainable dietary options. Nevertheless, the processes described have ample room for technological improvement, and several issues still need to be addressed. The first is related to the perishability of SCW. So far, thermal treatments (i.e., pasteurization) have been employed to ensure the safety of SCW-based products, while the application of less invasive emerging technologies such as High Pressure Processing (HPP), cold plasma, and others could be advisable to ensure microbial inactivation and preserve the nutritional quality of the final product. Moreover, further work needs to be done to improve both the physical properties and sensory profile of SCW-based products. One of the main problems related to the production of SCW-based fermented beverages concerns their structural properties and the formation of precipitates during storage. Thus, the effects of protein content and integrity and/or their interaction with stabilizers under acidic pH should be thoroughly evaluated. Regarding the sensory properties, SCW is characterized by high concentrations of free fatty acids, esters, and ketones (Bergamaschi et al. 2018), which are associated with rancid odour descriptors thus requiring the utilization of flavouring agents in the manufacturing of SCW-containing products. Finally, while the production processes described have been implemented at the laboratory scale, their feasibility at the industrial scale needs to be assessed.

3.2 Second cheese whey for the production of microbial starters, single cell proteins, single cell oils, and bioactive molecules

The utilization of by-products and wastes of the agri-food industry for biobased productions represents a key strategy for their valorisation within the framework of circular economy and sustainable development. In line with this approach, several authors have explored the practicability of utilizing SCW or deproteinized whey as substrates for cultivating algae, yeast, and bacteria for the production of starters, biomass enriched in proteins (single cell proteins, SCP), oils (single-cell oils, SCO) or other valuable metabolites.

In the context of microbial starters production, Rabaioli Rama et al. (2020) investigated the possibility of using unsupplemented SCW and CW for the growth and spray drying encapsulation of Lactobacillus paracasei ATR6. After culturing the selected strain in pasteurized SCW and CW (65 °C for 30 min) in a laboratory-scale bioreactor, they found that L. paracasei ATR6 exhibited a significantly higher increase in cell density when grown in SCW compared to CW while showing comparable survival rates after spray drying on both media (>78%). The potential of SCW as a substrate for the propagation of LAB cultures was confirmed by Chessa et al. (2020) and Naziri et al. (2023). In particular, Chessa et al. (2020) proposed SCW for the propagation of mixed starter cultures for Pecorino Romano PDO cheese. At first, by utilizing commercial powder SCW rehydrated in water at different proportions they observed that halving the concentration of SCW did not affect the propagation of starter cultures, thus indicating that the nutrients in SCW are more than sufficient to support LAB growth. Then they proved that SCW is suitable for maintaining the biodiversity and technological characteristics of mixed starter cultures including termophilic lactobacilli and cocci, enterococci and mesophilic lactobacilli. Similarly, Naziri et al. (2023) after having observed that Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. jakobsenii, Lactobacillus leichmannii, and Lactobacillus crispatus, constitute the dominant microflora in SCW derived from the manufacturing of Halloumi whey cheese, proved that LAB growth in unsupplemented SCW and SCW supplemented with skim milk was comparable to that on MRS medium, reaching approximately 108 CFUmL−1. Additionally, SCW supplemented with skim milk improved LAB freeze-drying tolerance (with survival rates ranging from 75 to 91% depending on the species) and viability (> 60%) over a three-month storage period at 4 °C in vacuum-packed powders.

Interestingly, although lactose utilization is not common in yeast (Kurtzman 2011), Gottardi et al. (2023) showed that Yarrowia lipolytica strains may be cultured on dairy by-products including SCW. In particular, they observed that 18 out of 20 Y. lypolytica isolates grew on unpasteurized SCW reaching cell densities ranging from 1×106 to 8 ×107 cell ml−1 within 72 h. Interestingly, due to their proteolytic and lipolytic activities, Y. lipolytica strains released a variety of volatile compounds that were compatible with cheese flavours. Therefore, SCW was indicated as suitable substrate to produce Y. lypolitica cultures to be used as co-starters and/or food adjuncts in cheese making (Gottardi et al. 2023).

Another possible application of SCW regards its utilization for SCP productions. SCP are unicellular proteins produced by microbial fermentation of waste substrates and utilized as supplements in human and animal nutrition for their high nutritional profile. In this context, Schultz et al. (2006) attempted the bioconversion of deproteinized sweet and sour whey concentrates into yeast SCP. For such application the yeast Kluyveromyces marxianus CBS 6566 was selected. Having been recognized the QPS and GRAS status K. marxianus is considered safe for human and animal consumption. Moreover, it metabolizes lactose with no ethanol production thus channelling carbon flow through the production of biomass. The selected strain was inoculated in 100 L stirred bioreactor filled with 13.5 or 55 L filter sterilized deproteinized whey containing 140 g L−1 lactose and supplemented with trace elements, vitamins and ammonium sulphate. Growth was carried out at 30 °C under controlled pH and aeration. In these conditions biomass production reached 50 and 65 g L−1 with yields of 0.52 and 0.48, and a COD reduction of 90 and 83% in sweet and sour deproteinized whey concentrates, respectively. The amino acid composition indicated that when produced in sweet and sour deproteinized whey, K. marxianus SCP contain amounts of valine (6.89–7.5 g 100 g protein−1), leucine (7.62–7.74 g 100 g protein−1), isoleucine (5.07–5.48 g 100 g protein−1) and threonine (7.45–6.94 g 100 g protein−1) that are higher than those reported in World Health Organization (WHO) guidelines for SCPs.

Lactose utilizing yeasts have been proposed also for the valorisation of dairy by-products through the fermentative production of SCO, a biodiesel source compatible with the majority of traditional diesel engines. Taskin et al. (2014) showed that when grown in SCW at laboratory scale, Y. lipolytica produced 7.4 g L−1 biomass containing 58% total lipids of which 80.54% monounsaturated fatty acids. Better results were obtained by Carota et al. (2017) with Cryptococcus curvatus NRRL Y-1511 and C. laurentii UCD 68-201. When grown in SCW these strains were capable of producing 6.83 g L−1 and 5.06 g L−1 biomass, while determining COD reductions of 86.7 and 77.9%, respectively. Notably, laboratory scale bioreactor cultivation of C. laurentii led to significant increases in biomass production (up to 14.37 g L−1) and lipids productivity (9.93 g L−1 at 60 h). Moreover, the fatty acid methyl ester (FAME) composition under bioreactor conditions was characterized by a predominance of palmitic, oleic, and linoleic fatty acids and closely resembled that of Jatropha oil, a well-established feedstock in biodiesel production (Carota et al. 2017). More recently, Vasilakis et al. (2022) showed that fed-batch cultivation of C. curvatus ATCC20509 with whey lactose pulse resulted in the production of 38.1 g L−1 cell biomass and 21.7 g L−1 total lipids. Notably, the lipids were found to be rich in unsaturated fatty acids, particularly oleic acid (up to 14.3 g 100 g−1 total FA) and, based on their composition, this yeast could be considered a possible candidate for the synthesis of second-generation biodiesel.

Since mixotrophic growth enhances microalgal biomass productivity, Tsolcha et al. (2016) assessed the feasibility of microalgal SCO production in SCW wastewater (SCWW) obtained by diluting SCW with tap water at different proportion. By doing so they obtained SCWW growth media differing in the initial concentrations of total sugars and mineral compounds, and utilized it for the cultivation of a mixed microbial population dominated by a Choricystis-like chlorophyte, in a 4 L aerobic photobioreactor. In the presence of the highest concentration of SCW, biomass productivity reached a maximum of 228.25 mg L−1d−1 and the growth rate recorded (0.45 d−1) was among the highest reported in the literature for similar microbial populations. Similarly, the maximum oil concentration, that ranged in between 60.75 ± 3 to 120.50 ± 8 mg L−1, was obtained in SCWW containing higher concentrations of SCW. Interestingly, the fatty acids profile of the lipids accumulated was in agreement with that reported for other chlorophytes in dairy effluents and appeared suitable for biodiesel production. Moreover, the microbial population utilized determined up to 92.3% reduction of COD. Other authors assessed SCW as a substrate for the production of algal, yeast and bacterial biomass enriched in carotenoids and other valuable metabolites. In particular Ribeiro et al. (2017a) focused on the microalga Chlorella protothecoides which, due to the production of appreciable amounts of proteins, chlorophyll, lipids, and valuable carotenoids, may be suitable for nutraceuticals and food/feed supplements (Campenni’ et al. 2013). When cultured in SCW containing medium within 1 L photobioreactor C. protothecoides shifted to mixotrophic growth. Under these conditions, the growth rate increased and biomass production more than doubled compared to that produced under autotrophic conditions, reaching up to 3.6 g L−1, possibly due to lactose and mineral nutrients content in SCW. Similarly, the volumetric productivities of chlorophyll (42.17 mg L−1) and carotenoids (11.98 mg L−1) were higher than under autotrophic growth and were achieved in a shorter time, thus suggesting that the mixotrophic cultivation of C. protothecoides in SCW represents a viable strategy to economize the costs associated with microalgal biomass production while contributing to SCW disposal. Similarly, Russo et al. (2021) utilized SCW for the cultivation of the heterotrophic microalga Galdieria sulphuraria, a promising biorefinery platform for the production of pigments, antioxidants and for the removal of nitrogen, sugars and phosphorus from wastewaters (Zimermann et al. 2020; Pan et al. 2021). Microfiltered heat treated SCW was diluted in water to obtain different concentrations of reducing sugars (1.0÷2.5%). At 2% reducing sugars G. sulphuraria produced up to about 5 g L−1 biomass enriched in glycogen and polyunsaturated fatty acids, and with a protein content that was comparable to that obtained under standard conditions of cultivation. Moreover, supplementation of SCW-based medium with glucose and nitrogen significantly increased biomass production (10.65 g L−1). Also, the red yeast Rhodotorula glutinis was explored for the production of biomass and carotenoids in SCW (Ribeiro et al. 2017b). Biomass production ranged in between 2.59 and 2.81 g L−1 within 21 days batch cultivation and was comparable to that obtained in Malt Extract broth employed as a reference medium. Similarly, carotenoids production, that reached a maximum of 0.5 µg gdw−1 after 21 days in SCW, showed not significant differences in the two substrates.

Coronas et al. (2023) assessed the possibility of utilizing SCW for the cultivation of Propionibacterium freudenreichii, to produce bacterial biomass and vitamin B12. For this purpose, four isolates of P. freudenreichii, selected for lactose utilization and in vitro probiotic features such as resistance to bile salts, acid stress, osmostress and lyophilization, were cultured in unpasteurized SCW. After 72 hours incubation at 30 °C, the four isolates reached cell densities ranging from 108 to 109 CFU mL−1. Moreover, they showed a total production of cobalamin derivatives (vitamin B12) in the range 0.49–1.31 mg L−1 thus suggesting that by choosing the appropriate starter, SCW can be converted into a probioactive preparation that combines probiotic properties and bioactive compounds.

Following an important breakthrough that showed the feasibility of lactobionic acid (LBA) production in CW-based medium (Alonso et al. 2011; Goderska et al. 2015), also SCW was utilized to produce LBA. This is a complex polyhydroxy acid with a wide range of significant applications. Due to its various beneficial effects, including prebiotic, bioactive, gelling, stabilizing, and antimicrobial properties LBA is utilized in functional foods (Nielsen et al. 2009; Sarenkova et al. 2018; Cardoso et al. 2019; Sáez-Orviz et al. 2022). SCW fermentation with Pseudomonas taetrolens resulted in 34.25±2.86 g L−1 LBA with an 85% yield (De Giorgi et al. 2018).

Overall, these studies underscore the versatility and potential of SCW as a cost-effective and sustainable substrate for microbial starter production and biobased applications. While careful selection of the cultivation system and microbial strains is crucial for all these production processes, using dairy effluents as culture media offers numerous advantages. SCW not only supports the proliferation of LAB while preserving the biodiversity and technological characteristics of microbial consortia but also serves as a suitable cell protectant for long-term preservation of starter cultures. Moreover, although the feasibility of large-scale production requires further evaluation, SCW, possibly due to its high C/N ratio, appears to meet the nutritional requirements for producing cell biomass enriched in microbial oils and proteins. Additionally, it serves as a growth medium for microalgae, yeast, and bacteria, resulting in biomass production enriched in lipids, pigments, and other bioactive metabolites while leading, at the same time, to wastewater treatment.

3.3 Second cheese whey in the formulation of animal feed

In Italy the Legislative Decree 22/97, known as the "Ronchi Decree," deems SCW a “waste” if directly used for animal feeding. Consequently, farmers intending to use it for this purpose must obtain specific authorization. On the other side SCW is categorized as by-product of animal origin with low risks when utilized in the formulation of animal feed, as per Regulation (EC) No. 1774/2002. The value of SCW in the formulation of animal feed lies in its nutritional properties (Minhalma et al. 2007; Pontonio et al. 2021). Also, LAB involved in spontaneous SCW acidification contribute notably to its dietary value. Therefore, SCW is a composite mixture with various constituents, some of which holding significant value for livestock nutrition. However, SCW is inherently very diluted, and its use is restricted by the gastric capacity of the animals to which it is fed. Consequently, SCW concentration is advised to achieve optimal results in animal nutrition.

Regarding the amount of SCW to be used for animal feed formulation no data were found in literature while more information is available on CW or CW permeate. CW permeate, obtained by membrane treatment to remove proteins and other solids (Menchik et al. 2019), is a deproteinized CW, assimilable to SCW, that contains higher amounts of lactose and milk oligosaccharides compared to other milk co-products (Barile et al. 2009; Dallas et al. 2014).

Lactose is the main nutrient for weanling pigs to achieve maximum performance during the initial period after weaning (Grinstead et al. 2000). Newborn piglets rely on milk from sows during lactation and, as a result, their digestive tracts are adapted to lactose digestion upon weaning. The supplementation of lactose in nursery feeds positively improves the growth performance of pigs, although the growth responses to lactose gradually disappears with age due to reduced lactase activity after weaning (Mahan et al. 2004; Cromwell et al. 2008; Gahan et al. 2009). When this happens, residual undigested lactose may result in excessive fermentation by gut microbiota thus aggravating enteric diseases and determining post-weaning diarrhoea (Pierce et al. 2005). Lactose also plays a role in stimulating the synthesis of B-vitamins by gut microbiota in both mammals and poultry (Atkinson et al. 1957). Furthermore, it demonstrates favourable effects on the absorption, retention, and utilization of essential minerals such as calcium, phosphorus, and magnesium (Atkinson et al. 1957). Thus, the inclusion of lactose in the diet has the potential to enhance nutrient digestibility. In cattle, this improvement may be linked to lactose's impact on ruminal fermentation and its role in increasing butyrate production which, in turn, could potentially enhance absorption capacity by promoting rumen papillae development and growth (Dirksen et al. 1985; Xu 1999; DeFrain et al. 2004, 2006). Moreover, the decrease in ruminal ammonia nitrogen (NH3–N) and milk urea nitrogen suggests the possibility of greater microbial protein synthesis in cows fed with lactose (Susmel et al. 1995; DeFrain et al. 2004). Ammonia can be utilized in ruminants by bacteria for the synthesis of amino acid required for growth. Considering that this process is energy-dependent, lactose could provide an adequate amount of energy for utilization of ammonia in the rumen. As the content of urea in milk is an indicator of the effective use of diet protein by the ruminant, a low urea milk content indicates a better utilization of diet protein, hence suggesting a positive role of lactose in ruminal protein metabolism.

Also, milk oligosaccharides have been well documented for their beneficial prebiotic effects (Bering 2018; Ramani et al. 2018) preventing intestinal dysfunction and aiding in the development of infant brain (Bode 2012; Moukarzel and Bode 2017). One possible mechanism for these positive effects could be related to the binding of milk oligosaccharides to pathogenic bacteria in the small intestine (Morrow et al 2005), thus inhibiting possible attachment of pathogenic bacteria and their toxins to enterocytes (El-Hawiet et al. 2015; Nguyen et al. 2016). Another possible mode of action could be an increase of Bifidobacterium and lactobacilli in the small intestine, resulting in the production of short-chain fatty acids used as energy sources by enterocytes (Garrido et al. 2013; Yu et al.