Recent Biotechnological Applications of Whey: Review and Perspectives

Abstract

This paper comprehensively reviews whey, a by-product of cheese production, as a raw material for various biotechnological applications. It addresses its unique composition, the environmental impact of its inadequate disposal, and the opportunities it offers to develop high-value products in line with circular economy and sustainability principles. Using the PRISMA methodology, a systematic search was conducted in various databases (Science Direct, Scopus, and Google Scholar) with specific inclusion and exclusion criteria. Studies from the last five years were considered, focusing on food applications, the production of bioproducts (such as lactic acid, biopolymers, bioethanol, biomass, and enzymes), and the use of whey as a culture medium for the expression of recombinant proteins. It is concluded that the use of whey in biotechnological applications mitigates the environmental impact associated with its disposal and represents an economic and sustainable alternative for the industrial production of bioproducts. The integration of pretreatment technologies, experimental designs, and improvements in producing strains brings these processes closer to competitive conditions in the industry, opening new perspectives for innovation in the fermentation sector.

1. Introduction

Whey is produced in cheesemaking when the casein in milk is coagulated; whey remains after curd removal is finished [1]. It is characterized by a unique composition that includes water, lactose, proteins, minerals, and vitamins; the specific composition can differ based on such variables as the type of cheese produced, the production processes used, and the source of the milk [2]. It is initially rich in approximately 55% of milk nutrients and serves as a healthy resource [1]. The two primary forms of cheese whey produced are sweet and acid; both have varying characteristics and pose distinct challenges and opportunities for processing and utilization. Sweet whey from hard cheeses, such as Swiss or Cheddar, have a higher pH (higher than 5.6) due to rennet coagulation, while acid whey from soft cheeses, such as cottage cheese, has a lower pH since it is coagulated by acid [3,4,5,6]. Proteins constitute one of the most essential components of bovine milk, representing approximately 3–3.5% of its composition. Two large fractions can be distinguished: casein, which represents approximately 80% of the total proteins, and the remaining 20% which is made up of whey proteins, among which β-lactoglobulin and α-lactalbumin stand out, as they have a high bioavailability for humans [7]. During the cheesemaking process, the coagulation of casein leads to the formation of rennet, leaving the soluble protein fraction in the whey. This makes whey especially rich in bioactive peptides, which may have antioxidant, antimicrobial, and antihypertensive functions [8]. Among carbohydrates, lactose is the most abundant, providing sustained energy and promoting calcium absorption. Whey contains high concentrations of lactose, making it an excellent medium for fermentation processes and for producing concentrated dairy products [9]. Regarding lipids, bovine milk contains a complex mixture of triglycerides, phospholipids, cholesterol, and fatty acids. The fatty acids present include both saturated and unsaturated types, providing energy and essential fatty acids (such as omega-3 and omega-6) which are crucial for cellular functions and cardiovascular health in humans [10]. Although most of the fat is retained in the rennet during cheese making, whey may contain small amounts of lipids and emulsifiers that enhance the bioavailability of certain nutrients and act as carriers of fat-soluble compounds [11]. Minerals include calcium, phosphorus, potassium, magnesium, sodium, and zinc, among others, which play critical roles in regulating osmotic balance, muscle function, and nerve transmission and as cofactors of various metabolic enzymes. In whey, they remain soluble and available for absorption [12]. In addition to minerals, bovine milk is rich in both fat-soluble (A, D, E, and K) and water-soluble (B2, B12, folic acid, and niacin) vitamins, which together contribute to the development of offspring and to the nutritional contribution to humans who consume it [10].

The food industry has identified whey as a versatile functional ingredient. Its soluble proteins, primarily β-lactoglobulin and α-lactalbumin, possess excellent emulsifying and foaming properties. These proteins stabilize emulsions in processed products, such as ice creams, dressings, and margarines, and generate foams in desserts and baked goods, enhancing texture and consistency. Dehydrated whey (whey powder), rich in lactose, proteins, and minerals, serves as an additive to enrich various foods. Whey powder increases the protein and nutritional content of dairy derivatives or bakery products, contributing creaminess and improving the moisture retention in doughs [13]. Whey proteins, including bovine immunoglobulins and lactoferrin, exhibit bioactive properties in health. Consuming whey provides immunoglobulins that can bolster the immune system. Lactoferrin and other whey glycoproteins display antimicrobial and antiviral properties, aiding host defenses against infections. Additionally, specific cysteine-rich peptides from whey elevate intracellular glutathione levels, protecting against oxidative damage and potentially inhibiting tumor cell proliferation. Both in vitro and in vivo studies have demonstrated the anti-neoplastic effects of whey proteins, such as delaying tumor growth in animal models. However, human clinical trials are preliminary, necessitating larger-scale studies to confirm these antitumor effects [14]. Traditionally, some whey is used as animal feed (e.g., fresh whey is supplied to pigs) or spread on agricultural fields as raw liquid fertilizer. However, current production volumes far exceed the capacity of these disposal methods. The improper disposal of millions of liters of whey can cause the eutrophication of surface waters, unpleasant odors, and attraction of pests, creating serious environmental and public health issues [15]. It is considered a principal by-product of cheese manufacturing and is a major environmental and economic issue due to its voluminous and organic load character. For decades, whey was regarded as a low-value by-product; however, its indiscriminate discharge poses significant environmental challenges. Globally, approximately 178.5 billion liters of whey is produced annually as a by-product of cheesemaking, given that around 85% of processed milk is converted into whey. This waste, with a high Biochemical Oxygen Demand (BOD) of up to 40,000 mg/L, can cause eutrophication if disposed of untreated, leading to excessive algal growth and negatively impacting aquatic ecosystems. The lack of infrastructure for proper management in some regions, particularly in developing countries, exacerbates this environmental problem, underscoring the need for sustainable solutions for its use [16]. Approximately 9–10 L of whey is produced per kg of cheese, representing a substantial volume that dairy industries must manage responsibly. In response to stringent environmental regulations and the nutrient loss associated with whey disposal, research efforts have intensified to valorize this by-product. Whey is now recognized as a potential source of functional ingredients and bioactive compounds for food and biomedical applications rather than a waste product [17]. Growing environmental awareness and stricter regulations (e.g., rules prohibiting the discharge of dairy effluents without treatment) have driven the search for sustainable whey solutions. Currently, its valorization is promoted within the concept of the dairy biorefinery, transforming whey from waste into a raw material for various production processes. This approach aligns with circular economy principles and sustainable development goals by minimizing waste and reintroducing nutrients and energy into the production cycle. Various methods have been formulated to valorize cheese whey into useful products; they consist of various technologies ranging from physicochemical treatments to microbial processes. The traditional applications of whey include using whey in animal feed and in the production of lactose, whey protein concentrates, and whey protein isolates. It has also been employed in agriculture to fertilize and irrigate the land; nevertheless, this is constrained by its salinity and the potential for offensive odors, which can negatively impact soil quality and plant growth [18]. Specifically, through microbial fermentation, whey can be converted into various products, including ethanol, lactic acid, and single-cell protein, which have applications in the food, feed, and chemical industries [19]. The utilization of microbial consortia in whey fermentation has gained attention for its potential to enhance the efficiency and versatility of bioconversion processes. Anaerobic digestion, a biological process in which microorganisms break down organic matter without the presence of oxygen, can also convert whey into biogas—a renewable energy source composed primarily of methane and carbon dioxide. The implementation of anaerobic digestion for whey treatment not only reduces the environmental impact of whey disposal but also provides a sustainable energy source that can be used for heating, electricity generation, or as a transportation fuel [20]. Moreover, cheese whey can be processed into fertilizers through aerobic digestion and microalgal-biomass-harvesting digestates, along with the recovery of phosphorus and nitrogen [18]. The development of novel bioprocessing technologies, such as immobilized cell bioreactors and membrane bioreactors, has further improved the efficiency and productivity of whey fermentation processes. Immobilized cell and enzyme technology has also been applied to whey bioconversion processes to enhance the economic viability of these processes. These bioreactors offer advantages such as high cell densities, enhanced mass transfer rates, and improved product recovery, making them attractive options for industrial-scale whey valorization [19]. On the other hand, recombinant protein production has become elemental for modern biotechnology, enabling the large-scale synthesis of proteins for diverse applications, ranging from therapeutic drug development to industrial biocatalysis [21]. The selection of an appropriate host organism is a priority for efficient recombinant protein production, influencing the protein yield, post-translational modifications, and overall process economics. In this regard, cheese whey could potentially be used as a growth medium for the host organisms used in recombinant protein production. Its rich composition of lactose, proteins, and other nutrients might provide a suitable environment for cultivating certain microorganisms or cell cultures [22].

This review provides a critical overview of recent knowledge on the biotechnological applications of whey, with a focus on its valorization to produce various bioproducts. It aims to combat the environmental issues associated with whey disposal and covers its transformation into lactic acid, biopolymers, bioethanol, enzymes, and other products. The novelty of this work lies in its integration of recent optimization strategies, specifically pretreatment technologies and microbial strain engineering, to enhance bioproduct yields. Furthermore, it investigates the promising application of whey as a cost-effective alternative to IPTG in recombinant protein production, thereby contributing to sustainable and circular economy practices.

2. Materials and Methods

A systematic search was carried out based on the Prisma method following the guidelines published by Molins and Serrano (2019) [23] with the following search criteria:

• Reference framework: PerSPECTiF

The PerSPECTiF model is a tool for structuring research questions in systematic reviews, particularly in applied and social sciences. Its name is an acronym that stands for Population (group or phenomenon under study), Context (environment where the phenomenon occurs), Perspective (point of view of those involved), Exposure (intervention or technology analyzed), Comparison (alternatives or methods compared), Time (study period), and Outcomes (expected findings). This model facilitates the formulation of precise questions, enhances the search for relevant literature, and aids in evaluating the quality of selected studies, being especially useful in interdisciplinary research (see

Table 1. Databases and documents used in this review.

Question Number	Science Direct (Advanced Search)	Scopus	Google Scholar
Q1	5118	231	7228
Q2	864	5	528
Q3	1565	118	5344
Total	7547	354	13,100

).

The following is a breakdown according to PerSPECTiF:

• Population (P): biotechnological processes for the valorization of whey.
• Setting (S): biotechnology and energy industry.
• Perspective (P): researchers, biotechnologists, and industrialists in the energy and bioproducts sector.
• Exposure (E): use of whey in producing biofuels and bioproducts (e.g., organic acids, biopolymers, metabolites, etc.) and recombinant proteins.
• Comparison (C): comparison with other residual substrates or conventional carbon sources.
• Time (T): last 5 years (to capture the most recent trends).
• Findings (F): technological, economic, and environmental feasibility of using whey in these processes.

The research questions are as follows:

Q1: What are the most recent applications of whey in developing innovative foods, considering its impact on nutritional value, technological functionality, and consumer acceptance over the last five years?

Q2: What are the most recent applications of whey in producing biofuels and other biotechnological products, excluding recombinant proteins, considering their technological, economic, and environmental viability over the last five years?

Q3: What are the most recent uses of whey as a substrate in producing recombinant proteins, considering its technical and economic viability in industrial biotechnological environments over the last five years?

Search equations: Whey and food industry; whey and pollution; whey and innovative foods; whey and biotechnological products not recombinant proteins; lactose and IPTG and inducer; whey and recombinant protein production; whey and culture media.

The inclusion criteria are as follows:

• Documents that report innovations in the use of whey for food applications in the last 5 years;
• Documents that report applications in the use of whey for biotechnological applications in the production of metabolites at an industrial level in the last 5 years;
• Documents that report the use of whey as an alternative substrate for expressing recombinant proteins within the last 5 years;
• Documents that report information on whey in general and for food use, regardless of the publication date;
• Documents that report the use of IPTG and lactose as inducers of recombinant proteins, as well as the interchangeability between the two, regardless of the year of publication.

The exclusion criteria are as follows:

• Duplicate documents in databases;
• Documents related to whey that do not have food or biotechnological applications;
• Documents that report the use of whey for research related to medical applications, with tissues or human beings;
• Documents older than or equal to 10 years about biotechnological applications of whey.

Table 1 summarizes the results of this systematic search. After applying the methodology, one hundred seven documents were screened and used to prepare this work.

3. Biotechnological Applications of Whey

From an economic and environmental point of view, whey for biotechnological applications stands out for its low cost and the reuse of an industrial by-product, contributing to the process’s sustainability. In addition, its availability in large volumes facilitates the scalability of production processes at an industrial level. The transformation of whey into bioproducts reduces the environmental impact associated with the disposal of this by-product. It offers an economic alternative for industries that require affordable and renewable substrates [24]. In this context, various lines of research have been aimed at optimizing fermentative and enzymatic processes for the conversion of lactose and other whey components into compounds such as lactic acid, biopolymers, bioethanol, biosurfactants, and secondary metabolites [25]. Lactic acid is one of the most researched bioproducts from whey. This compound has applications in the food and pharmaceutical industries and in the production of bioplastics, such as polyhydroxybutyrate [26]. See Table 2.

3.1. Production of Lactic Acid, Starter Cultures, and Potential Probiotics

Between 2020 and 2025, several studies have optimized lactic acid fermentation using bacterial and fungal strains, taking advantage of the high concentration of lactose in whey [27]. Although the use of whey for the growth of lactic acid bacteria for producing starter cultures or potential probiotics seems obvious, improvements in technologies, such as microencapsulation and lyophilization, for whey are still being developed. Pinilla et al. (2025) [28] evaluated the circular use of simple whey as a culture medium and microencapsulation material for lactic acid bacteria (LAB). For this purpose, Lacticasei bacillus paracasei ItalPN16 was grown in a one-liter bioreactor at different controlled pHs using plain serum, and the sugar consumption, biomass yield, and protein hydrolysis were monitored. As a result, bacterial counts higher than 10 Log₁₀ CFU/mL and a consumption of around 30% of the serum sugars were obtained at the pH values of 4.5, 5.5, and 6.5. Protein analysis revealed different proteolysis patterns as a function of pH, resulting in an increased immunoglobulin (IgG) hydrolysis at a pH of 6 and an increase in free amino acids that could be incorporated into another process for use as a by-product. After bacterial drying, the range of encapsulation efficiencies was 72–90% for spray-dried (SD) powders and 82–99% for freeze-dried (FD) powders. Furthermore, The Fourier transform infrared spectroscopy (FTIR) analysis showed specific variations in the hydrophilic interactions in the -OH region of the microcapsules, depending on the drying method. Microencapsulated L. paracasei using fermented whey at a pH of 6 as the wall material showed remarkable tolerance to simulated gastrointestinal conditions and may have a potential probiotic activity. Regarding powder stability, the freeze-drying method proved more effective in protecting L. paracasei during storage at 4 °C and 25 °C. Therefore, the authors conclude that using simple whey as a culture medium in pH-controlled fermentation, followed by microencapsulation by freeze-drying, proved to be a sustainable, scalable, and economical alternative to produce commercial lactobacilli cultures. See Table 2.

3.2. Production of Bioplastics and Biopolymers

The interest in bioplastics has grown significantly in the last decade, and whey has emerged as a promising source for producing biodegradable polymers. Recent studies have explored the production of polysaccharides and poly-hydroxyalkanoates (PHAs) from whey, using microorganisms such as Cupriavidus necator, Bacillus megaterium, Bacillus cereus, and recombinant E. coli, among many other models [29]. Works published in the last 5 years have shown that the fermentation of whey and other waste products, appropriately pretreated, can be directed toward the synthesis of PHAs with physical and mechanical characteristics suitable for diverse applications, such as biodegradable packaging or even medical applications [30]. Jia et al. (2024) [26] reported the production of polyhydroxybutyrate (PHB) from agro-food processing waste. The focus of this work was a comprehensive evaluation of the substrate utilization, cell growth, and PHB inclusion in recombinant E. coli during the fermentation of various mixtures of acidic whey and hydrolysate obtained from fiber waste. They concluded that replacing acidic whey with water during the enzymatic hydrolysis of pretreated fiber waste and subsequently using it for fermentation resulted in the highest PHB yield of 5.2 g/L, with a PHB inclusion rate of 45.4%. Furthermore, the inherent lactic acid content in acidic whey eliminates the need to add acetic acid to adjust pH levels during hydrolysis, thereby saving freshwater and acid. An example is provided by Hou et al. (2021) [31] who sought to produce biodegradable polyhydroxybutyrate (PHB) sustainably and investigated the effects of metabolic and process-limiting factors during the bioconversion of acidic whey (AW) into PHB. Recombinant Escherichia coli LSBJ achieved high PHB yields using lactose and lactic acid as a carbon source. A PHB accumulation up to 85% was achieved during the growth on synthetic AW. Growth on raw AW yielded the highest PHB of 4 g/L and exhibited a high substrate utilization efficiency of 95%. In particular, lactate/lactose and C/N ratios affected the metabolic flux and PHB yields. Maintaining the fermentation pH enhanced the PHB production. Furthermore, additives of inorganic nitrogen sources, minerals, and trace metals promoted the PHB production from AW. The study enhances the understanding of AW utilization factors and demonstrates the high yields of PHB using recombinant E. coli, which could be leveraged to design a sustainable process. The use of whey not only reduces the cost of the substrate but also enables the utilization of residual nutrients that promote the accumulation of biopolymers within the cells. Furthermore, optimizing culture conditions and using controlled feeding strategies have been shown to increase the PHA yield, bringing the technology closer to a competitive level with processes based on traditional sources. See Table 2.

3.3. Biofuel Production

Bioethanol is a renewable biofuel that has received wide attention in the circular economy framework. Although traditionally obtained from energy crops, whey has been explored as an alternative substrate for ethanol production through alcoholic fermentation. In this case, the presence of lactose in whey represents a challenge, since not all fermenting microorganisms utilize it efficiently. However, recent publications have addressed this limitation by selecting or modifying strains through metabolic engineering capable of fermenting lactose [32]. Ma et al. (2024) [33] reported an innovative solution involving modifying the metabolic pathways of Clostridium saccharoperbutylacetonicum to convert all carbon sources in acidic whey into sustainable biofuels and biochemicals. By introducing several heterologous metabolic pathways related to lactose, galactose, and lactate metabolisms, the finally optimized strain, LM-09, exhibited an exceptional performance by producing 15.1 g/L of butanol with a yield of 0.33 g/g and a selectivity of 89.9%. Through the further overexpression of the alcohol acyl transferase, 2.7 g/L of butyl acetate was generated along with 6.4 g/L of butanol, resulting in a combined yield of 0.37 g/g. The findings demonstrate an innovative bioprocess that enhances the biotransformation of renewable feedstocks, thus promoting the economic viability and environmental sustainability of biotechnological applications using whey as a substrate. On the other hand, the use of yeasts for second-generation bioethanol production has also been explored. Cunha et al. (2021) [34] engineered industrial strains of Saccharomyces cerevisiae with an enhanced thermotolerance and stress resistance for the cell surface expression of cellulolytic enzymes. Furthermore, they also showed that β-galactosidase enables lactose consumption, resulting in high ethanol titers (>50 g/L) from the simultaneous use of cheese whey and pretreated corn cob as substrates. The multi-feedstock valorization approach, coupled with this lactose-consuming cellulolytic yeast, reduced material costs by 60% with a 2.5-fold increase in annual ethanol production, thus contributing to the establishment of economically viable ethanol processes. In particular, the biofuel production using whey and agro-industrial waste has developed extensively in the last five years, so if you want to delve deeper into the topic, the work of Goyal et al. (2023) [25] is recommended. See Table 2.

3.4. Biomass Production

An emerging approach to whey valorization is the integration of microphytic algae as biomass-producing agents. Algae, due to their ability to photosynthesize, can use organic nutrients and CO₂ to grow and provide additional benefits, such as the production of antioxidant compounds and natural pigments. The combination of whey with algae cultures has been the subject of recent research. Whey, rich in lactose and nutrients, can act as a liquid fertilizer that enriches the culture medium for algae. This is particularly relevant in hybrid systems, where microalgal biomass is produced by integrating organic and light sources. Algae, growing in the presence of whey, not only accumulate biomass but can also absorb excessive nutrients, helping to reduce the environmental impact of dairy waste. Preliminary studies have shown that Chlorella spp. and Scenedesmus obliquus can grow efficiently in media supplemented with diluted whey [35]. These hybrid cultures show an accelerated growth, increased lipid and pigment accumulation, and a carbon sequestration capacity that can be harnessed to produce biofuels and nutraceutical compounds. Chen et al. (2024) [36] explored the use of cost-effective inorganic carbon (the ionic form of CO₂) and organic carbon sources (lactose, a primary sugar in whey wastewater) to support the growth of Chlorella sorokiniana UTEX 1230 and improve the protein content by optimizing the nitrogen supply. NaHCO₃ was more effective than Na₂CO₃, resulting in a 3.4-fold increase in biomass concentration, with an initial concentration of 5 mM HCO₃⁻ compared to the basal medium. Adding 0.75% lactose led to a 4.05-fold increase in biomass concentration, with a maximum specific growth rate of 1.28/d, a maximum biomass concentration of 695.88 mg/L, and a maximum biomass productivity of 250.91 mg/L/d. Nitrogen supplementation significantly increased the protein concentration, from 16.41% (250 mg/L NaNO₃) to 52.95% (1000 mg/L NaNO₃). These results support the potential of using whey wastewater bioremediation to produce high-value proteins through microalgae cultivation. In another study, Braun et al. (2024) [37] reported the addition of whey as an alternative source of organic nutrients in the fed-batch mode for microalgae (Spirulina platensis, Chlorella homosphaera, and Scenedesmus obliquus) cultivation. Microalgae demonstrated the acceptance of serum at low concentrations, leading to increased maximum specific growth rates (μmax) and biomass concentrations ([X]max) compared to the serum-free culture in a standard medium (μmax of 0.168 d⁻¹ vs. 0.075 d⁻¹ and [X]max of 2.274 g L⁻¹ vs. 0.970 g L⁻¹, respectively, for S. platensis). Therefore, cost savings on culture media of up to 32%, 57%, and 73% were possible in cultivating S. obliquus, S. platensis, and C. homosphaera, respectively, considering the production of 1 kg of biomass. The addition of the serum presented different effects on the intracellular protein levels considering the microalgae species: an increase was observed in C. homosphaera (24.18 to 43.96 g/100 g biomass) and a decrease in S. platensis (73.75 to 44.06 g/100 g biomass) compared to controls. Scaling up with a natural light source may affect the microalgae productivity and biomass composition in a serum-supplemented medium due to the influence of irradiation. Optimizing large-scale microalgae production using agro-industrial waste can lead to significant advances in biomass production at reduced costs. These biomasses can be integrated into bioenergy, biofertilizer manufacturing, and animal feed production. By using waste as a nutritional source and optimizing the cultivation and biomass generation processes a closed cycle is established, in line with the principles of the circular bioeconomy and improving the sustainability of the process. See Table 2.

3.5. Other Metabolites of Industrial Interest

Currently, the synthesis of prebiotic lactulose is receiving increased interest and is being intensively studied due to its functional characteristics. It is also part of the valorization of whey or lactose. Lactulose can act as a prebiotic because the human digestive system does not metabolize it. Thus, it passes through the stomach and small intestine without degradation. Lactulose can also treat constipation and hepatic encephalopathy as an antiendotoxin, an antidiabetic agent to treat inflammatory bowel diseases, etc. The synthesis of lactulose can be performed by isomerization or transgalactosylation, where enzymatic isomerization shows an improved yield and selectivity. From the perspective of the reaction yield and ease of separation, the synthesis of lactulose by cellobiose 2-epimerase-catalyzed isomerization in the enzymatic membrane reactor is a promising approach. Following this strategy, such industrial preparative chromatography is the only major unit that separates lactose and lactulose [38]. Amino acids are another type of metabolite of interest in the biotechnology industry. Blandón et al. (2023) [39] reported using whey as the primary carbon source, while they used hydrolysates of Red Tilapia (Oreochromis sp.) viscera as the nitrogen source. In this context, it was possible to maximize the l-threonine production, but at the expense of the biomass production. The optimal conditions for biomass and l-threonine maximization (after 24 h) were identified and experimentally validated, resulting in biomass and l-threonine productions of 0.767 g/L and 0.406 g/L, respectively. This work has demonstrated the technical feasibility of using whey and viscera hydrolysates of Red Tilapia (Oreochromis sp.) for the l-threonine production by E. coli ATCC^® 21277TM.

Table 2. Biotechnological applications of whey in the last 5 years.

Category	Product	Microorganism	Reference
Lactic acid, starter cultures, and potential probiotics	Lactic acid	Lacticaseibacillus casei BL23	[40] (2021)
	Lactic acid, probiotics	Lacticaseibacillus paracasei ItalPN16	[28] (2025)
	Starter culture	Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. jakobsenii, Lactobacillus leichmannii and Lactobacillus crispatus	[41] (2023)
	Starter culture, probiotic	Lactiplantibacillus plantarum	[42] (2021)
	Starter culture, probiotic	Lactococcus lactis	[43] (2024)
Bioplastics and Biopolymers	Polyhydroxybutyrate (PHB)	Recombinant E. coli	[26] (2024)
	Polyhydroxyalcanoates (PHA)	Paracoccus homiensis	[44] (2022)
	Polyhydroxyalcanoates (PHA)	Several	[45] (2022)
	Biopolymers	Lactic Acid Bacteria	[46] (2023)
	Polyhydroxybutyrate (PHB)	Recombinant Escherichia coli LSBJ	[31] (2021)
Biomass and Biofuels	Biomass	Chlorella sp.	[35] (2024)
	Biomass	Yeasts	[47] (2024)
	Biomass	Chlorella sorokiniana UTEX 1230	[36] (2024)
	Biomass	Spirulina platensis, Chlorella homosphaera and Scenedesmus obliquus.	[37]
	Butyl acetate, butanol	Clostridium saccharoperbutylacetonicum	[33] (2024)
	Bioethanol	Recombinant Saccharomyces cerevisiae	[34] (2021)
	Bioethanol	Several	[32] (2022)
Enzymes	[NiFe]-hydrogenases	Cupriavidus necator, E. coli	[48] (2024)
	Endoglucanases	Recombinant Trichoderma reesei, recombinant Aspergillus carneus	[49] (2024)
	α-amylase	Recombinant Anoxybacillus karvacharensis	[50] (2024)
	β-galactosidase	Several	[51] (2022)
	α-Galactosidases	Several	[52] (2020)
	[NiFe]-hydrogenases	Escherichia coli, Ralstonia eutropha	[53] (2023)
Other metabolites of industrial interest	L-threonine	Escherichia coli ATCC® 21277	[39] (2023)
	5-Hydroxytryptophan (5-HTP)	E. coli strain C1T7-S337A/F318Y	[54] (2024)
	Lactulose	Enzymatic Bioreactor	[38] (2023)
	Recombinant hen ovalbumin and bovine β-lactoglobulin	Trichoderma reesei	[55] (2023)
	Galactooligosaccharides	Pantoea anthophila	[56] (2021)
	β-Farnesene	Recombinant Escherichia coli	[57] (2021)
	D-tagatosa	Paracoccus denitrificans	[58] (2023)

Table 2. Biotechnological applications of whey in the last 5 years.

Category	Product	Microorganism	Reference
Lactic acid, starter cultures, and potential probiotics	Lactic acid	Lacticaseibacillus casei BL23	[40] (2021)
	Lactic acid, probiotics	Lacticaseibacillus paracasei ItalPN16	[28] (2025)
	Starter culture	Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. jakobsenii, Lactobacillus leichmannii and Lactobacillus crispatus	[41] (2023)
	Starter culture, probiotic	Lactiplantibacillus plantarum	[42] (2021)
	Starter culture, probiotic	Lactococcus lactis	[43] (2024)
Bioplastics and Biopolymers	Polyhydroxybutyrate (PHB)	Recombinant E. coli	[26] (2024)
	Polyhydroxyalcanoates (PHA)	Paracoccus homiensis	[44] (2022)
	Polyhydroxyalcanoates (PHA)	Several	[45] (2022)
	Biopolymers	Lactic Acid Bacteria	[46] (2023)
	Polyhydroxybutyrate (PHB)	Recombinant Escherichia coli LSBJ	[31] (2021)
Biomass and Biofuels	Biomass	Chlorella sp.	[35] (2024)
	Biomass	Yeasts	[47] (2024)
	Biomass	Chlorella sorokiniana UTEX 1230	[36] (2024)
	Biomass	Spirulina platensis, Chlorella homosphaera and Scenedesmus obliquus.	[37]
	Butyl acetate, butanol	Clostridium saccharoperbutylacetonicum	[33] (2024)
	Bioethanol	Recombinant Saccharomyces cerevisiae	[34] (2021)
	Bioethanol	Several	[32] (2022)
Enzymes	[NiFe]-hydrogenases	Cupriavidus necator, E. coli	[48] (2024)
	Endoglucanases	Recombinant Trichoderma reesei, recombinant Aspergillus carneus	[49] (2024)
	α-amylase	Recombinant Anoxybacillus karvacharensis	[50] (2024)
	β-galactosidase	Several	[51] (2022)
	α-Galactosidases	Several	[52] (2020)
	[NiFe]-hydrogenases	Escherichia coli, Ralstonia eutropha	[53] (2023)
Other metabolites of industrial interest	L-threonine	Escherichia coli ATCC® 21277	[39] (2023)
	5-Hydroxytryptophan (5-HTP)	E. coli strain C1T7-S337A/F318Y	[54] (2024)
	Lactulose	Enzymatic Bioreactor	[38] (2023)
	Recombinant hen ovalbumin and bovine β-lactoglobulin	Trichoderma reesei	[55] (2023)
	Galactooligosaccharides	Pantoea anthophila	[56] (2021)
	β-Farnesene	Recombinant Escherichia coli	[57] (2021)
	D-tagatosa	Paracoccus denitrificans	[58] (2023)

4. The IPTG Inducer

During the previous decades, the recombination engineering of microorganisms has enabled the set-up of production processes that are not feasible or highly expensive, which has benefited diverse bioprocesses to produce metabolites of industrial interest. This has been possible after many years of experimentation with Escherichia coli, the golden host microorganism, which has enabled the development of expression vectors and molecular biology strategies focused on homologous and heterologous expressions for protein synthesis. One of the best known mechanisms is the Lac Operon-based expression system. The lac operon consists of four elements: (1) the lac promoter, (2) the lac operator, (3) the repressor, and (4) the genes under the regulatory control for transcription—β-galactosidase (lacZ), a lactose permease (lacY), and a transacetylase (lacA) [59]. This regulation system was first discovered in E. coli, where it was found to function as an expression modulator to activate useful enzymes for lactose metabolism as an energy source after glucose depletion. The regulation is controlled by the lacI repressor protein bound to the lac operator, which interferes with RNA polymerase, blocking the transcription of genes codified within the lac operon. However, in the presence of allolactose, acting as an inductor, an allosteric interaction occurs with the lacI repressor, changing its binding affinity to the operator DNA region and relieving its repressive control. Once the repressor is released, the transcription can take place by the RNA polymerase to provide a further translation of the corresponding proteins [60,61]. Therefore, the lac-based system has been widely used for the recombinant expression of diverse proteins in different microorganisms, such as E. coli [62,63,64], Bacillus sp. [65,66,67], yeasts [68,69,70], and mammalian cells [71], among others. These systems have made use of the analogous lactose molecules, such as isopropyl β-D-thiogalactopyranoside (IPTG), as synthetic inducers, since they preferred a non-metabolizable additive that could maintain the induction effect for an extended period. Other analogs are ortho-nitrophenyl-β-galactoside (ONPG), methyl-1-thio-β-D-galactoside (TMG), and β-D-galactopyranosyl 1-thio-β-D-galactopyranoside (TDG), among others [64]; however, IPTG remains the universal inducer for recombinant protein expression [72,73,74] (Figure 1). Nevertheless, some disadvantages associated with the use of IPTG have been described, such as a metabolic burden, toxicity at high concentrations, and high cost, which limits its use as an inductor for the industrial production of recombinant proteins [75,76]. Conversely, metabolizable sources, such as lactose, galactose, or dairy product derivatives, are practical alternatives to replace synthetic inductors like IPTG. These natural inducers are commonly low-cost and non-toxic, and numerous works have established strategies for incorporating natural inductors to achieve high yields of the corresponding recombinant proteins. The following section will present representative examples of the production of recombinant protein expressions using lactose and waste by-products from the dairy industry, such as milk whey, cheese whey, and others.

5. Whey as Low-Cost Media for Recombinant Protein Expression

Enzyme production is fundamental for numerous industrial applications. Traditionally, enzyme synthesis has been performed using conventional substrates and conventional fermentation processes; however, the increasing demand for sustainable and high-throughput processes has driven the search for alternatives that optimize production efficiency and input cost. In this context, whey stands out as an attractive and economical substrate. Its utilization contributes to the circular economy by transforming waste into a valuable resource and offers a nutritive medium for the growth and activity of productive microorganisms. The incorporation of precision fermentation allows for the design of robust and reproducible processes aimed at producing high-quality enzymes [77]. Filamentous fungi produce various products, such as organic acids, secondary metabolites, and industrial enzymes. In addition to enzymes, fungi frequently produce special proteins and peptides with intriguing functions, such as animal proteins for food, hydrophobins, or antimicrobial peptides. The procedure can be carried out using native or genetically modified strains to produce numerous secreted proteins or target proteins in specialized fungal hosts. Over time, it has become clear that filamentous fungi have some advantages over other recombinant protein expression systems [78]. One justification for using filamentous fungi as expression hosts for the heterologous production of target proteins is that they have strong secretory pathways that are superior to those of bacterial hosts and can process eukaryotic proteins post-translationally as efficiently as mammalian cells, including glycosylation and disulfide bond formation. Like other eukaryotes, the endoplasmic reticulum of fungi is a highly dynamic structure. The potential of filamentous fungi to produce pharmaceutically functional proteins for medicinal use has been hailed. Some organisms, such as Aspergillus and Trichoderma species, are known for producing and secreting large amounts of proteins [78]. A concrete example of such systems is found in the work of Iskandaryan et al. (2024) [48], who used a mixture of curd and cheese whey (CW) to explore two-phase growth with Cupriavidus necator H16 and E. coli in the production of hydrogenases (Hyds), enzymes valuable in biohydrogen production, demonstrating an enhanced biomass as well as Hyd activity and electrical potential (∼0.65 V) for the growth of C. necator in the CW medium with added glycerol. The residual growth medium available after the C. necator cultivation and cell removal was used for E. coli cultivation. The maximum fermentative growth of E. coli was reached after 72 h and with a H₂ yield of ∼6 mmol/L/g of dry whey after 48 h. This study demonstrates the economically viable production of biomass, hydrogenase enzymes, and H₂ using inexpensive industrially produced whey within the 3R concept. The richness of carbohydrates in whey makes it the ideal substrate for industrial production or enzymatic modification for various purposes. One of the most significant enzymes of industrial interest is amylase, which can be produced using whey as a substrate. An example is the work by Ghevondyan et al. (2024) [50], who produced recombinant α-amylase from the K1 strain of Anoxybacillus karvacharensis. The enzyme showed its maximum activity at 55 °C and a pH of 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. The Michaelis constant (Km) and the maximum reaction rate (Vmax) were determined using soluble starch as a substrate, obtaining 1.2  ±  0.19 mg mL⁻¹ and 1580.3  ±  183.7 μmol mg⁻¹ protein min⁻¹, respectively. It should be noted that the most favorable conditions for producing biomass and recombinant α-amylase were achieved by treating acidic whey with β-glucosidase for 24 h. Endoglucanases are among the enzymes of interest for lignocellulosic waste; in this field, the work of Crament et al. (2024) [49] evaluated recombinant strains of Aspergillus niger D15 expressing fungal endoglucanases (Trichoderma reesei eg1 and eg2 and Aspergillus carneus aceg) for their ability to utilize lactose as a carbon source to determine whether dairy waste could be used as feedstock for enzyme production. Recombinant strains of A. niger D15[eg1]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG produced maximum endoglucanase activities of 34, 54, and 34 U/mL, respectively, in lactose and 23, 27, and 22 U/mL in serum. Strain A. niger D15[eg2]PyrG was used to optimize the serum medium. The maximum endoglucanase activity of 46 U/mL occurred in a 10% whey medium containing 0.6% NaNO₃. The results indicate that whey can be used as a raw material for producing recombinant enzymes. Table 2 summarizes some of the biotechnological applications of whey in the last 5 years. Different biotechnological microorganisms have benefited from genetic modifications, providing an increased production of recombinant proteins. However, the expression strategy and the formulation of low-cost media must be considered to ensure the feasibility of production processes and further scalability. The use of lactose as an inducer has been previously evaluated in diverse works, demonstrating its suitability in obtaining increased expression levels in E. coli [79,80]. However, cost-effective options have been evaluated from the last 10 years, which consist of derivative products from the dairy industry. In this sense, skimmed milk has been reported as a successful replacement of IPTG for the recombinant production of FliC protein under Lac operon control in E. coli BL21. In addition, bacterial growth was supported and enhanced by the uptake of high lactose levels derived from adding skimmed milk to the media [81]. In addition, promissory results have been obtained with the cheese whey permeate (CWP) addition in fermentations with E. coli, demonstrating the benefits promoted by permeates during flask cultures. For instance, CWP has beneficial effects on cell biomass production, of at least a 1.3-fold higher biomass due to the significant availability of nutrients provided by CWP, such as lactose and micronutrients. It was also observed that permeates can relieve the cellular stress derived from the recombinant production of a toxic protein, showing a better induction behavior than IPTG [82]. Moreover, this great advantage of whey for promoting cell growth has also been observed for other than E. coli, such as Lactococcus lactis [24], Bacillus, and Clostridium species [29], as noted before in previous sections. The functional properties of whey derivatives will be explained in more detail in the following section, which will summarize the role played by whey as an additive in the fermentation media for increasing the production of recombinant proteins at a flask and bioreactor scale.

Formulation of Industrial Culture Media Using Whey for Recombinant Protein Expression Induction

As presented in this work, the importance of incorporating waste by-products, such as milk whey and cheese whey, has highlighted the importance of the circular economy and, at the same time, it has been highly beneficial in optimizing biotechnological developments. This section describes the formulation of industrial media using whey derivatives to evidence the role played by whey in batch and fed-batch cultures, aimed at demonstrating the advancements reported for the recombinant production directed to replace IPTG and other synthetic inductors.

Table 3. Reports of whey derivatives utilization as inducers for recombinant protein production in bioreactor fermentations.

Whey Concentration	Recombinant Microorganism	Productivity/Yield	Scale (Lab/Pilot/Industrial)	Reference
Cheese whey and cheese whey permeate (CWP) solution (10 g/L lactose) at bioreactor scale	Recombinant Kluyveromyces sp. β-galactosidase enzyme produced in E. coli pET-30a(+)	Approximately 26 U/mg of recombinant β-galactosidase after 16 h induction at bench bioreactor	Lab: flask and 2 L Biorreactor	[83] (2021)
80 g/L whey powder as a substrate	Engineered E. coli strain F13 with recombinant plasmid with inducible lac UV5 promoter	β-farnesene production for fuel substitution reaching 4.74 g/L	Lab: 7 L batch bioreactor	[57] (2021)
5% w/v of cheese whey powder, supplemented or not with 0.5% yeast extract (Difco) (YE) or 1.5% corn steep liquor (CSL)	ccpA mutant (BL21) of Lacticaseibacillus casei BL23	Lactic acid production obtained final values of 44.23 g L−1 for BL71	Lab: bottles (uncontrolled conditions and 5 L Bioreactor (controlled conditions)	[40] (2021)
Cheese whey permeate and ricotta whey (5 g/L of lactose) as inducers	E. coli C41(DE3) modified with expression vector pET-35b(+)	β-galactosidase fused to a cellulose-binding domain (CBD) 573.75 and 615.93 U/Lh	Lab: fed-batch cultures in 2 L Stirred Tank Bioreactors	[84] (2023)
Sweet and acid whey (SW, AW) were collected, pretreated, and filtrated at a concentration of 36 g/L and 32 g/L dry whey	Hyd mutant E. coli strains	Higher and prolonged H2 production 750–770 mL/g of initial dry whey after 96 h growth	Lab: 250 mL baffled flasks in a batch system	[53] (2023)
Crude glycerol (500 g/L), lactose solution (165 g/L) and CWP at a constant rate of 0.2 mL/min	Recombinant β-galactosidase from Marinomonas sp. ef1 (M-βGal) in E. coli	2000 kU of recombinant M-βGal were successfully produced along with 30 g of galactose accumulated in the culture medium	Lab: shaked flask and 2 L Bioreactor fed-batch, cultures with CWP are comparable with those achieved using lactose	[85] (2024)
10 g/L of whey powder was added as an inducer in 5 L batch fermentations	Recombinant E. coli BL21/CPD/GPS strain (S337A/F318Y)	5-Hydroxytryptophan (5-HTP) amino acid with a yield of 1.649 g/L	Lab: 5 L fermenter	[54] (2024)

presents a compilation of relevant works from the last 5 years related to integrating whey derivatives as powders, solutions, or permeates and supplemented as a substrate or as fermentation feeding in bioreactor cultures.

As observed in Table 3, the general trend of research is focused on the recombinant protein expression in E. coli, where the production response in the bioreactor is widely characterized. The main observable effects are the increase in biomass production yields since whey-derived products are easily metabolized by cells, promoting the suitable replication of recombinant plasmids and increasing related product yields. This advantage is highlighted in Lacticaseibacillus casei strains, where the supplementation with 5% w/v of cheese whey powder in bioreactor media optimized the use of carbon and energy sources for the increased production of lactic acid compared to the traditional MRS medium [40]. Souza et al. (2021) [86] present an alternative strategy for the heterologous expression of the l-arabinose isomerase (l-AI) enzyme from Enterococcus faecium DBFIQ E36 in recombinant Escherichia coli, using residual whey lactose as an inducer in an autoinduction system. Traditionally, the induction of the protein expression in E. coli is performed with isopropyl β-D-1-thiogalactopyranoside (IPTG), but its high cost and toxicity limit its industrial viability. This work compares commercial lactose, whey lactose, and IPTG in the expression of l-AI, observing that the enzymatic activity is higher when residual lactose is used. The enzyme was purified and characterized, showing optimal catalytic activity at a pH of 5.6 and 50 °C, suitable for converting D-galactose into D-tagatose, a sweetener with functional properties. The results highlight the economic and environmental viability of using dairy by-products to produce enzymes with applications in biotechnology and functional foods. β-Farnesene can replace petroleum products as a specialty fuel to solve the global energy crisis, but its production by recombinant Escherichia coli using glucose and IPTG is expensive. An alternative is the bifunctional use of whey powder as a substrate and inducer for β-farnesene production in genetically modified Escherichia coli since it can replace both components due to its lactose content. This allowed cell growth and, at the same time, it was transformed into allolactose, which activated the β-farnesene production. The β-farnesene yield reached 2.41 g/L in shaker flasks, 65.1% higher than glucose and IPTG. In a 7 L bioreactor, the production increased to 4.74 g/L, 197% of the yield in the shaker flasks. These results demonstrate that whey powder is a viable and economical alternative for the synthesis of β-farnesene, which expands its applications and reduces costs in biofuel production [57]. For the recombinant expression of β-galactosidase in Escherichia coli. Mobayed et al. (2021) [83] compared cheese whey and its permeate with the traditional inducer IPTG in two heterologous E. coli strains (BL21(DE3) and Rosetta(DE3)), measuring the enzymatic activity and protein production. The results showed that the induction with the whey permeate (10 g/L lactose) achieved a specific β-galactosidase activity higher than that obtained with IPTG, reaching approximately 46 U/mg in shake flasks and 26 U/mg in a bioreactor. The enzyme B-gal42, derived from the strain Pantoea anthophila isolated from Tejano and belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used to synthesize galactooligosaccharides (GOSs) from lactose or whey. The crude, cell-free enzyme extracts showed a high stability and were used in reactions to produce GOS. In the presence of 400 g/L of lactose, the maximum GOS yield reached 40% (w/w), with a conversion of 86%, as determined by HPAEC-PAD. This enzyme showed a marked preference for the formation of GOS with β(1 → 6) and β(1 → 3) galactosyl bonds. When comparing the synthesis of GOS using whey and pure lactose, both at a concentration of 300 g/L, a yield of 38% and a conversion of 60% of lactose were obtained in both cases, with an identical product profile according to HPAEC-PAD [56].

6. Discussion

Furthermore, one of the most notable advances has been the use of lactose, naturally present in whey, as an inducer in systems based on the lac operon. Traditionally, synthetic inducers, such as IPTG, have been used to activate the expression of heterologous genes; however, the use of lactose or whey derivatives allows for comparable induction with the added advantage of reducing the toxicity and cost associated with these compounds [72]. Recent studies have demonstrated that incorporating whey derivatives into culture media not only enhances the protein expression but also promotes better stability and reproducibility in fermentation. Formulations have been designed to integrate cheese whey, whey permeates, or whey powder as carbon sources and expression inducers [20,22,31,38,39,48,49,52,53,87]. These formulations have led to higher yields in terms of biomass and enzyme activity compared to traditional IPTG-induced media [71,73,74,75,81]. The use of dairy waste has facilitated the implementation of autoinduction systems, where the progressive presence of lactose in the medium activates recombinant gene expression without the need for additional interventions. This approach simplifies the process and minimizes batch-to-batch variability. Although most research has focused on E. coli, advances have been reported in eukaryotic systems, such as some filamentous fungi and yeast, where the use of whey serves not only as an inducer but also improves cell growth and the stability of the recombinant product [49,52]. Despite these advances, the use of whey as a culture medium presents challenges that require optimization. Variability in whey composition, influenced by factors such as the type of cheese produced and processing conditions, can impact the reproducibility of results [28,72,88]. Therefore, it is essential to establish pretreatment and standardization protocols for the by-product to ensure optimal culture conditions. Furthermore, the integration of whey into industrial fermentation processes necessitates adjustments to parameters such as pH, nutrient concentration, and feeding strategy to optimize the protein expression and minimize potential metabolic inhibitions [37,49,88]. Growing interest in sustainability and the circular economy is driving the search for solutions that make the most of industrial waste. In this context, the use of whey to produce heterologous proteins not only reduces the environmental impact associated with its disposal but also presents new opportunities for the bioproduction of enzymes and other high-value products. With advances in genetic engineering and the development of optimized strains, whey-based systems are poised to become a robust and scalable platform for producing bioproducts. The integration of real-time monitoring technologies and co-fermentation strategies will further optimize these processes, consolidating the role of whey as a strategic resource in modern biotechnology. As we have summarized in this manuscript, the advantages of using whey-based by-products are demonstrated. Nevertheless, specific parameters, such as scalability, cost-effectiveness, safety, and efficiency, are still thoroughly evaluated. This is quite important, since whey-based sources are cost-effective and valorized to establish bioprocesses. However, issues such as the variable properties of whey by-products, microbiological aspects necessary to ensure safety, and feasible processing for further applications, among others, must be considered (Figure 2). For instance, various whey processing techniques enable the production of diverse value-added products, including whey powder, proteins, lactose hydrolysates, and whey protein concentrates, each with specific requirements [89]. For example, several challenges remain related to the production of whey powder, as production stages can compromise the final yields and quality, making suitable storage and processing operations difficult [90]. The whey powder quality is particularly determinant, since high moisture provides ideal conditions for the proliferation of pathogens and spore-forming bacteria from the genera Bacillus, Geobacillus, Anoxybacillus, Brevibacillus, Paenibacillus, and Clostridium [91]. Therefore, suitable facilities for the adequate processing of dairy products, including whey pretreatment, filtration, and protein concentration, are necessary to maintain sterile conditions and preserve the high quality of the end products.

In this context, process scale-up and simulations are recommended to be performed before implementation to evaluate process capabilities (volumes), economic viability, processing stage requirements (equipment size), and their impact on process optimization and efficiency, thereby determining the product quality and final selling price [92].

As a sample of the industrial application of these technologies,

Table 4. Technology companies that currently utilize whey as a substrate to produce high-value biotechnological products.

Industry	Company Description	URL
Davisco Foods International	Recognized as one of the leading producers of whey-derived ingredients, such as whey protein concentrates and isolates, used in both functional foods and biotechnological applications.	https://daviscofood.com/ (accessed on 30 March 2025)
Hilmar Cheese Company	Specializing in the transformation of whey into high-quality protein products, the company utilizes the by-product of cheesemaking to produce ingredients for the nutrition and food industries.	https://www.hilmar.com/ (accessed on 30 March 2025)
Fonterra Co-operative Group	This dairy giant uses whey to produce a wide range of ingredients, including derivatives used in functional formulations and fermentation processes in the biotechnology industry.	https://www.fonterra.com/nz/en.html (accessed on 30 March 2025)
Glanbia Nutritionals	Develops and supplies dairy-based ingredients, including whey derivatives, used in both food applications and fermentation and bioprocessing processes.	https://www.glanbianutritionals.com/es-es (accessed on 30 March 2025)
DuPont Nutrition & Biosciences (formerly Danisco)	It uses whey derivatives in the design of culture media and in fermentation processes to produce enzymes, cultures, and other biotechnological products. It has also used whey derivatives as an economical and sustainable medium in recombinant expression processes, facilitating the production of enzymes and other biotechnological products.	https://www.iff.com/ (accessed on 30 March 2025)
Kerry Group	This company develops ingredient solutions for the food industry, leveraging whey components to produce proteins and other derivatives that provide functionality and nutritional value. Kerry has developed ingredient solutions based on dairy products. Its biotechnology strategies include processes that utilize whey to produce bioactive proteins and optimize the expression of recombinant proteins, leveraging the nutritional benefits of whey.	https://www.kerry.com/ (accessed on 30 March 2025)
Agropur Cooperative	In addition to its dairy products, Agropur has implemented processes to valorize whey, transforming it into ingredients used in the food industry and in sustainable biotechnology applications.	https://www.agropur.com/fr (accessed on 30 March 2025)
Lactalis Ingredients	As part of the Lactalis Group, this division specializes in the production of dairy-derived ingredients, including those derived from whey, which are utilized in various industrial applications.	https://www.lactalisingredients.com/ (accessed on 30 March 2025)
Fermentec	This company specializes in fermentation technologies and has developed processes that utilize dairy by-products, such as whey, for the production of biofuels, thereby contributing to the valorization of industrial waste.	https://fermentec.com.br/ (accessed on 30 March 2025)

shows some companies that, at the time of writing this document, are already implementing whey as a substrate for biotechnological applications.

These companies are representative examples of how whey is harnessed to transform a traditional by-product into high-value ingredients and bioproducts, integrating circular economy and sustainability strategies into their processes. The potential for utilizing whey remains significant, with applications in emerging technologies such as precision fermentation, cell culture, and regenerative medicine.

Table 5. Emerging technologies and research areas where whey still offers potential.

Technology/Research Area	Description/Potential	References
Integrated Dairy Biorefinery	Integrate the conversion of whey into multiple high-value products, including bioethanol, bioplastics, bioactives, enzymes, and biomass, in a biorefinery that simultaneously exploits all its fractions.	[93,94,95]
Precision Fermentation and Metabolic Engineering	Optimize the expression of recombinant proteins and specific metabolites through strain engineering (using bacteria, yeasts, or fungi) to more efficiently utilize lactose in whey. Research and scale-up the production of biopolymers (PHB, PHA) from whey, using modified microorganisms and optimized fermentation processes to compete with traditional sources.	[96,97]
Co-cultures and Hybrid Systems with Microalgae	Develop co-culture systems where whey serves as a nutrient source for microalgae, combining biomass production with bioremediation and the generation of bioactive compounds or biofuels.	[36,37,98,99]
Enzymes and Bioactives via Enzymatic Hydrolysis	Develop enzymatic processes, including immobilized systems, to produce bioactive peptides and other functional compounds from whey with applications in the food and pharmaceutical industries.	[100,101]
Continuous Fermentation Cells and Autoinduction Systems	Optimize high-performance fermenters using autoinduction systems based on lactose, reducing costs and improving scalability in the expression of recombinant proteins.	[33,102,103]
Cell Cultures and Regenerative Medicine	Explore the use of whey components as a substrate in in vitro protein synthesis systems, enabling rapid and controlled protein production without the need for living cells.	[104,105,106]
Nanotechnology and Nutrient Encapsulation	Apply micro- and nanocapsule techniques to protect and gradually release whey’s bioactive compounds, enhancing their stability and efficacy in therapeutic and nutraceutical applications.	[107,108]

summarizes potential applications in new research areas and emerging technologies.

7. Conclusions

This review demonstrates that whey has established itself as a versatile and promising raw material in the biotechnology field, with applications that transcend the production of recombinant proteins. Whey represents a waste valorization strategy that contributes to sustainability and cost reduction in the production of bioproducts. The integration of pretreatment technologies, experimental designs, and microorganism engineering has made it possible to overcome challenges associated with substrate variability and conversion efficiency, bringing these processes closer to competitive conditions on an industrial scale. Likewise, adopting co-fermentation strategies and applying real-time monitoring systems have improved the stability and reproducibility of the processes, opening the door to new applications and product diversification. Prospects point toward greater integration between the dairy industry and the biotechnology sector, process standardization, and the exploration of new whey-derived products, which could significantly advance the circular economy and sustainable production. In summary, the use of whey in biotechnological applications for producing bioproducts represents a convergence of scientific and technological advances that favor the transformation of waste into a valuable resource. This approach, supported by recent research, not only enhances economic and productive efficiency but also contributes to reducing the environmental impact, offering an innovative solution to the current challenges faced by industry and society. With the continued advances in bioprocessing technologies and the growing demand for sustainable processes, it is foreseeable that whey will expand and consolidate as a key strategy in future biotechnology.