Whey Protein Research Articles

Antioxidant activity enhancement of icariside II through complexation with whey protein concentrate

Antioxidant activity enhancement of icariside II through complexation with whey protein concentrate

Evaluation of whey protein coating containing free and Pickering emulsion forms of Trachyspermum copticum L. essential oil on quality of refrigerated beef

AbstractThe purpose of the present research was to prepare a novel biodegradable coating of whey protein (whey) and essential oil of Trachyspermum copticum L. (forms of free (EO) and Pickering emulsion (NEO)) to improve the shelf life of beef. In this study, various microbiological, chemical, and sensory analyses were performed. The results showed that after 18 days, the highest and lowest microbiological counts were related to control samples and whey‐NEO treatments, respectively. The total viable count (TVC) was 8.2 and 6.8 log CFU/g, the total psychrotrophic count (TPC) was 8.5 and 6.7 log CFU/g, and the lactic acid bacteria (LAB) count was 7.7 and 6.5 log CFU/g for control and whey‐NEO treatments, respectively. Chemical analysis of whey‐NEO treatment and control sample were 6.3 and 7.5 for pH, 9.8 and 13.9 meq/kg for PV (peroxide value), 5.45 and 8.85 mg/kg for TBARS (thiobarbituric acid reactive substances), and 25.1 and 39.4 mgN/100 g for TVB‐N (total volatile basic nitrogen), respectively. The best result was associated with the whey‐NEO treatment; consequently, coatings with whey and EO Pickering emulsion should be considered as a potential active coating in the meat industry.

Process scale-up simulation and techno-economic assessment of ethanol fermentation from cheese whey.

Production of cheese whey in the EU exceeded 55 million tons in 2022, resulting in lactose-rich effluents that pose significant environmental challenges. To address this issue, the present study investigated cheese-whey treatment via membrane filtration and the utilization of its components as fermentation feedstock. A simulation model was developed for an industrial-scale facility located in Italy's Apulia region, designed to process 539m3/day of untreated cheese-whey. The model integrated experimental data from ethanolic fermentation using a selected strain of Kluyveromyces marxianus in lactose-supplemented media, along with relevant published data. The simulation was divided into three different sections. The first section focused on cheese-whey pretreatment through membrane filtration, enabling the recovery of 56%w/w whey protein concentrate, process water recirculation, and lactose concentration. In the second section, the recovered lactose was directed towards fermentation and downstream anhydrous ethanol production. The third section encompassed anaerobic digestion of organic residue, sludge handling, and combined heat and power production. Moreover, three different scenarios were produced based on ethanol yield on lactose (YE/L), biomass yield on lactose, and final lactose concentration in the medium. A techno-economic assessment based on the collected data was performed as well as a sensitivity analysis focused on economic parameters, encompassing considerations on cheese-whey by assessing its economical impact as a credit for the simulated facility, dictated by a gate fee, or as a cost by considering it a raw material. The techno-economic analysis revealed different minimum ethanol selling prices across the three scenarios. The best performance was obtained in the scenario presenting a YE/L = 0.45g/g, with a minimum selling price of 1.43 €/kg. Finally, sensitivity analysis highlighted the model's dependence on the price or credit associated with cheese-whey handling. This work highlighted the importance of policy implementation in this kind of study, demonstrating how a gate fee approach applied to cheese-whey procurement positively impacted the final minimum selling price for ethanol across all scenarios. Additionally, considerations should be made about the implementation of the simulated process as a plug-in addition in to existing processes dealing with dairy products or handling multiple biomasses to produce ethanol.

Designing the Properties of Probiotic Kefir with Increased Whey Protein Content

Designing the Properties of Probiotic Kefir with Increased Whey Protein Content

Formation and characterization of cold-set whey protein gels induced by L-ascorbic acid/calcium for their encapsulation and release

Protein cold-set gels are potential carriers for the encapsulation of bioactive compounds. Meanwhile, bioactive compounds have also been used as cross-linkers or accessories for protein gelation, but their encapsulation has not been investigated during gelation for potential nutritional value. In this study, cold-set gels of whey protein isolate (WPI) were induced by L-ascorbic acid (L-AA) at pH 4.0–6.0, L-ascorbic acid plus CaCl2 at pH 3.8, or L-ascorbic calcium at pH 7.0. The range of pH for WPI gelation induced by L-AA narrowed with the decrease of WPI content from 10% to 6% (w/v). The gel networks were stabilized by non-covalent and covalent interactions, which difference was dependent on the use of cross-linkers. L-Ascorbic recoveries were above 76% in WPI hydrogels, where the highest recovery reached 98%. L-Ascorbic encapsulation efficiency ranged from 69% to 100%. Hydrogels and cyrogels varied from a smooth and dense structure to a coarse structure. L-ACa at 16 mM induced uniform and compact structure of both gels and the highest hardness and storage modulus of hydrogel. L-Ascorbic groups were effectively immobilized into WPI gels via their interaction with whey protein and calcium. L-Ascorbic loading capacity in WPI hydrogels and gel powder increased as the pH decreased and its calcium concentration increased. The release of free L-ascorbic acid from gel powder was independent on protein re-hydration. These results suggest the potential use of protein cold-set gels for the encapsulation and delivery of bioactive compounds with themselves or their combination with calcium as cross-linkers.

Fabrication and characterisation of whey–pea protein‐based emulsion gels induced by transglutaminase cross‐linking

Food industry has partially replaced animal proteins with plant‐derived alternatives, and effects of combining these two types on properties of food systems need to be elucidated. This study fabricated and characterised emulsion gels of mixed whey and pea protein, varying protein mass ratios from 100:0 to 0:100 and oil volumes from 30% to 45%, induced by transglutaminase cross‐linking. An increase in pea protein led to larger particle size and reduced surface charge in the solutions. Emulsion droplet size ranged from 0.60 to 15.17 μm, increasing with higher levels of pea protein and oil. Mixed protein gels exhibited significantly greater hardness compared to gels with individual proteins. Water‐holding capacity of pure whey protein emulsion gels exceeded 90%, while the value remained above 80% when pea protein was less than 50%. Partially replacing whey protein with pea protein may have potential applications in food products such as salad dressings and cheese.

Protein-added kefir: biochemical changes in in vitro digestion stages.

While yogurt is the leading fermented milk product, kefir is at the top of the beverage scale. Milk proteins, on the other hand, show specific functions that positively affect healthy nutrition due to the bioactive components, that they provide the necessary amino acids for growth and development. In our study, kefir, a functional product enriched with whey proteins, casein and skimmed milk powder, which are the natural components of milk, was produced. Added-protein kefir samples were applied the in vitro digestion protocol, static method. In order to observe different protein behaviors, samples were taken pre-digestion, at 120th minute and at 240th minute of digestion protocol. ACE and Antioxidant capacity determination analyzes were carried out. While ACE inhibition values were in the range of 78.63-90.30% pre-digestion, they changed in the range of 86.97-96.38% after gastrointestinal digestion. It was determined that the ACE inhibition values of the control sample remained at the lowest level at all stages of digestion and that the difference between all of samples was significant (P < 0.05). Antioxidant activity value ranging from 0.3615-0.5512 meq Ascorbic acid/μg before digestion was determined as 1.3796-1.9313 meq Ascorbic acid/μg after gastrointestinal digestion (P < 0.05). Kefir samples containing whey protein stand out with their high potential in terms of both antioxidant activity capacity and ACE inhibition activity at all stages of digestion. Considering their therapeutic effects in fermented products, it is thought that whey proteins among milk proteins will be important alternative sources to enrich the protein content in kefir production. © 2024 Society of Chemical Industry.

High-Pressure Processing of Milk and Dairy Products: Latest Update

The growing global demand for minimally processed fresh foods has driven the development of innovative food processing methods. High-pressure processing (HPP), a leading non-thermal technique, has proven to be environmentally friendly, cost-effective, and versatile across various food types. Its ability to extend shelf life and ensure microbial safety is well established, particularly in dairy products. However, further research is needed to fully understand HPP’s impact on improving the digestion of milk compounds and assessing potential safety risks. Recent studies, for example, demonstrate HPP’s efficacy in enhancing the microbial safety and nutritional value of products like cheese, yogurt, and whey protein isolate. This review explores these advancements and highlights HPP’s role in improving the physicochemical, organoleptic, and functional properties of milk and dairy products.

Study on stability of rose anthocyanin extracts and physicochemical properties of complex with whey protein isolate after spray drying.

Pingyin rose is an edible flower rich in anthocyanins. In this study, antioxidant capacity and color were used as the main evaluation indexes to investigate the effects of common physical and chemical factors on the stability of rose anthocyanin extracts (RAEs). In addition, the physicochemical properties of the whey protein isolate (WPI)-RAEs complex after spray drying were studied. Vitamin C, temperature, and some metal ions can cause different degrees of discoloration of RAEs solution. More importantly, heat treatment, as well as most metal ions and sugars, had no significant effect on the antioxidant capacity of RAEs solution (p>0.05). Moreover, compared to spray-dried pure WPI, the WPI-RAEs powder was delicate and uniform, and had higher particle size, bulk density, moisture activity, and better gel properties. The release rate of all WPI-RAEs sol/gel to RAEs reached about 89% in the intestinal digestion stage, but the WPI-RAEs interaction reduced the digestibility of protein in the intestinal digestion stage. We hope that this study can provide a theoretical basis for the development and utilization of WPI-RAEs as food ingredients.

Is there sufficient evidence to support the health benefits of including donkey milk in the diet?

Donkey milk has attracted attention due to its distinctive nutritional composition and potential health advantages, particularly because of its whey protein content, which includes lysozyme, α-lactalbumin, lactoferrin, and β-lactoglobulin and vitamin C, among other components. These elements contribute to immunoregulatory, antimicrobial, antioxidant, and anti-inflammatory properties, positioning donkey milk as a possible therapeutic option. In addition, due to the low levels of caseins, the casein-to-whey protein ratio, and the β-lactoglobulin content in donkey milk, it presents an optimal alternative for infant formula for individuals with cow's milk allergies. Moreover, research into donkey milk's potential for cancer prevention, diabetes management, and as a treatment for various diseases is ongoing, thanks to its bioactive peptides and components. Nevertheless, challenges such as its low production yield and the not fully understood mechanisms behind its potential therapeutic role necessitate more thorough investigation. This review consolidates the existing knowledge on the therapeutic possibilities of donkey milk, emphasizing its importance for human health and the need for more detailed studies to confirm its health benefits.

Two major bovine milk whey proteins induce distinct responses in IEC-6 intestinal cells

Abstract α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) are major whey proteins in bovine milk. We studied the effects of these molecules on the intestinal cell response by comparing the native form with the denatured form containing oligomers obtained by treatment with 2,2,2-trifluoroethanol (TFE). We previously reported that proteins in native and TFE-treated forms exhibited cell growth stimulation and cytotoxicity, respectively, in undifferentiated rat crypt IEC-6 and human colon Caco-2 cells. However, neither whey protein showed cytotoxicity even in the TFE-treated form in differentiated Caco-2 cells. Only undifferentiated immature intestinal cells can distinguish between these native and denatured proteins. Moreover, α-LA and β-LG exhibited different oligomer formation characteristics during the TFE treatment. In the present study, we compared the effects of native and TFE-treated whey proteins on IEC-6 cells in more detail. The native forms of both whey proteins exhibited cell proliferative effects in a concentration-dependent manner. For the TFE-treated forms, α-LA showed rapid and potent cytotoxicity, whereas β-LG altered cell responses depending on its concentration and exposure time; lower concentration/shorter exposure and higher concentration/longer exposure induced cell growth stimulation and cytotoxicity, respectively. Pre-treatment of the cell membrane with cholesterol suppressed the effects on the cell response only in TFE-treated β-LG (TFE-β-LG). In a preliminary examination using inhibitors of signal transduction, TFE-treated α-LA acted on the intrinsic apoptosis pathway via Bcl-2-associated X and p53, whereas the action of TFE-LG did not require this pathway. Tyrosine phosphorylation is necessary for the cell proliferation effect of both native whey proteins; however, native α-LA, but not native β-LG, also required activation of the pathway with selective epidermal growth factor receptor tyrosine kinase and Janus kinase 2/3. In summary, the two major bovine milk whey proteins induced similar yet discrete responses in undifferentiated intestinal cells. Even when oligomers are formed, β-LG may be much less hazardous to immature intestinal cells than α-LA.

Preparation of fat‐free mulberry ice cream by using inulin and whey protein isolate as a fat substitute

AbstractThe standard ice cream consists of high calories and fat which could cause metabolic syndrome upon regular consumption. Therefore, this study developed a fat‐free ice cream using fat replacer including inulin (carbohydrate‐based) and whey protein isolate (WPI, protein‐based) at various concentrations (6%, 8%, and 10%) or their combination (1:1). Additionally, freeze‐dried mulberry fruit powder was used as a functional ingredient. The results indicated that both, inulin and WPI, function differently thereby affecting the physicochemical properties of the ice cream. The combination of inulin:WPI at a 4:4 ratio produced high‐quality standard ice cream with a higher sensory score. Further incorporation of freeze‐dried mulberry powder (2%) in a selected fat‐free ice‐cream base showed enhanced sensory, nutraceutical, and textural properties of the ice cream. All fat‐free and fat‐free mulberry ice cream had a trace amount of fat (0.06%), while the calorie was reduced to 88.9 kcal/serving compared with a full‐fat ice cream (195.7 kcal/serving). Hence, fat‐free mulberry ice cream with 74.5% of nonfat milk, 5.0% of skim milk powder, 8% of inulin:WPI (4:4), 9.5% of sugar, 0.5% of emulsifier, and 2% freeze‐dried mulberry powder could serve as the best alternative to the full‐fat ice cream.

Optimizing the Process Parameters for Carrot Cake Development Using Response Surface Methodology

Carrots (Daucus carota L.) are rich in vitamins B1, B2, B6, β-carotene, and dietary fibers, having nutritional benefits such as, antioxidant, and anticancer properties. Carrot offers a valuable nutritional boost for enhancing bakery products. Incorporating carrot into cake preparation not only adds a unique flavor and moist texture but also boosts the nutritional value with additional vitamins and fiber. The research systematically examines the effects of varying process parameters such as carrot, sweetened condensed milk, and whey protein concentrate (WPC) on specific volume, springiness, and overall acceptability of carrot cake using Central Composite Design (CCD) of Response Surface Methodology (RSM). The optimal formulation for carrot cake consisted of 70.8 g of carrot, 200 g of sweetened condensed milk, and 4.2 g of WPC. This combination resulted in a high-quality carrot cake with a specific volume of 2.58 cm³/g, a springiness measurement of 1.049 mm, and an overall acceptability rating of 8.3. This approach not only enhances product quality but also supports waste reduction by valorizing carrot, aligning with sustainable food processing practices.

Effect of non-covalently bound alkaline amino acids on the structural characterization, microstructure, and rheological properties of whey protein emulsion gel

The objective of this study was to explore the impact of L-arginine (Arg) and L-lysine (Lys) on the structural changes and rheological properties of whey protein isolate (WPI) emulsion gels through the utilization of multi-spectroscopic techniques and rheological analysis. The results indicated that the incorporation of Lys/Arg caused WPI molecules to become unfolded, thus exposing hydrophobic amino acids and altering the microenvironment of aromatic amino acids. Microstructural studies revealed that WPI emulsion gels containing 0.5% Lys or 2% Arg resulted in a more uniform and compact network structure. This phenomenon could be attributed to the capability of Lys/Arg to improve the hydrophobic interactions and hydrogen bonding between molecules during gel formation, ultimately leading to a network structure characterized by excellent rheological properties, textural properties, and water-holding capacity. These findings provide a theoretical for the development of emulsion gels with improved structural and rheological properties.

Serum metabolism-transcriptomics investigated into the immunity of whey protein isolate-galacto-oligosaccharide conjugates after dynamic high-pressure microfluidics pretreatment

The objective of this study was to investigate the immunomodulatory effects of whey protein isolate (WPI)-galacto-oligosaccharide conjugates following dynamic high-pressure microfluidics pretreatment (DHPM) in cyclophosphamide-induced immunosuppressed mice. DHPM facilitated the conjugation of WPI and galacto-oligosaccharide, and inhibited the generation of fluorescent advanced glycation end products (AGEs) and pentosidine. The conjugates demonstrated a significant immune recovery effect on CTX-induced immunosuppressed mice, as evidenced by the enhancement of IgG antibody levels (from 3.5 to 4.1) and the reduction of the levels of immunosuppressive effector factors TGF-β (from 148.1 to 111.2) and IFN-γ (from 34.4 to 17.9). Furthermore, the conjugates exhibited a notable ability to repair histological lesion in the spleen of CTX-induced immunosuppressed mice. Spleen transcriptomics revealed that the Marco, Klrc3 and Cd209b genes were associated with the immune enhancement activity of the conjugates. Metabolomic analysis identified arginine biosynthesis, sphingolipid metabolism, alanine, aspartate and glutamate metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis as key pathways in the immune enhancement activity of the conjugates. Metabolomics combined with transcriptomics indicated the importance of macrophage activation in the restoration of immunosuppressed mice’s immunity by the conjugates. Therefore, the improvement in immunity observed with WPI-galacto-oligosaccharide conjugates may be related to the activation of macrophages.

Characterization of Peptide Profiles and the Hypoallergenic and High Antioxidant Activity of Whey Protein Hydrolysate Prepared Using Different Hydrolysis Modes.

Food proteins and peptides are generally considered a source of dietary antioxidants. The aim of this study was to investigate the antioxidant activity, allergenicity, and peptide profiles of whey protein hydrolysates (WPHs) using different hydrolysis methods. The results demonstrated that the degrees of hydrolysis of the hydrolysates with one-step (O-AD) and two-step (T-AD) methods reached 16.25% and 17.64%, respectively. The size exclusion chromatography results showed that the O-AD had a higher content of >5 and <0.3 kDa, and the distribution of peptide profiles for the two hydrolysates was significantly different. Furthermore, 5 bioactive peptides and 15 allergenic peptides were identified using peptidomics. The peptide profiles and the composition of the master proteins of the O-AD and T-AD were different. The DPPH and ABTS radical scavenging abilities of WPHs were measured, and hydrolysates were found to exhibit a strong radical scavenging ability after being treated using different hydrolysis methods. An enzyme-linked immunosorbent assay showed that the sensitization of WPHs was significantly reduced. This study may provide useful information regarding the antioxidant properties and allergenicity of WPHs.

Comparative efficacy of amino acid availability and peptidomic analysis of alternative proteins from different sources under dynamic in vitro protein digestion

The increasing global demand for animal protein highlights the critical necessity of exploring alternative protein sources. Accessing the nutritional characteristics of emerging alternative proteins is vital for food processing and consumer acceptance. This study investigated the amino acid availability and peptidomics profiles of plant- (soy), fungal- (filamentous fungus Fusarium venenatum), microalgae- (Nannochloropsis oculata), and insect-based (black soldier fly) proteins using an advanced dynamic in vitro digestion system, with whey protein as a reference. After gastric digestion, soy and mycelial proteins, despite displaying distinct microstructures, exhibited slightly higher protein digestibility levels of 28.79% and 26.36%, respectively, compared to whey protein (22.39%). However, following small intestinal digestion, whey protein (61.31%) significantly outperformed alternative proteins. Mycelial protein (43.71%) exhibited digestibility similar to soy protein (46.12%), markedly surpassing both microalgae protein (33.35%) and insect protein (26.58%). Notably, insect protein achieved an amino acid availability nearly approaching that of whey protein, underscoring its substantial proteolysis capability. Furthermore, microalgae protein produced the highest percentage and number of bioactive peptides under simulated digestion, suggesting potential health benefits such as angiotensin I-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitor activities, which could mitigate the risk of hypertension and Type 2 diabetes. Alternative proteins from different sources demonstrate diverse digestive mechanisms and proteolysis profiles, thereby bolstering their potential applications as nutritious food ingredients in the future.

Fiber complex‐stabilized high‐internal‐phase emulsion for allicin encapsulation: microstructure, stability, and thermal‐responsive properties

AbstractBACKGROUNDStimuli‐responsive emulsions have garnered significant attention for their ability to enhance sensory qualities and control the release of encapsulated nutrient in emulsion‐based products. However, the characteristics of synthetic materials of fabricating stimuli‐responsive emulsions have been a crucial limitation in the food industry. Regulating the behavior of molecules at the interface could potentially achieve the desired stimuli‐responsive behavior, but currently there is limited information available.RESULTSHigh‐internal‐phase emulsions (HIPEs) were fabricated for the encapsulation of allicin, stabilized by a complex of 20 g kg−1 whey protein amyloid fibrils (WPF) and 20 g kg−1 glycyrrhizin fibers (GA). The intermolecular interactions between WPF and GA in the fiber complexes were predominantly governed by hydrophobic and electrostatic forces. These complexes adsorbed and stacked around the oil droplets, forming a protective interfacial film that enhanced droplet stability. An increased proportion of WPF (WPF = 3:1 or 4:1) surrounding the oil droplets enhanced the accelerated storage stability of HIPEs, with instability indexes approaching 0.2. Additionally, HIPEs displayed a temperature‐dependent modulus, with the emulsion stabilized by a WPF ratio of 3:1 showing the highest modulus at 85 °C. The encapsulation efficiency of allicin in HIPEs ranged from 88.69 ± 6.62% to 101 ± 1.37% at 25 °C, and from 31.95 ± 1.92% to 78.69 ± 4.63% after incubation at 85 °C for 8 h. The release profile of allicin from the HIPEs exhibited thermal responsiveness, depending on the interfacial content of GA.CONCLUSIONThese findings indicated that the thermal‐responsive properties of HIPEs can be strategically engineered by manipulating their interfacial characteristics. © 2024 Society of Chemical Industry.

Combining Ultra-Turrax and ultrasonic homogenization to achieve higher vitamin E encapsulation efficiency in spray drying

Vitamin E, a soluble antioxidant widely used in the food and pharmaceutical industries, is rich in tocopherols and phytosterols. Since vitamin E molecules are highly sensitive to oxidation, encapsulation is a viable and effective technique for preservation of the properties of Vitamin E and improving its stability during storage, maintaining the nutritional value. In this work, the aim was to encapsulate concentrated vitamin E using a combination of Ultra-Turrax and ultrasonication to achieve higher encapsulation efficiency in spray drying. In the first stage, the vitamin E oil was encapsulated employing only Ultra-Turrax homogenization, with subsequent optimization of spray drying. The coating materials used were maltodextrin and whey protein isolate. Optimization of the spray drying step evaluated the effects of the drying air temperature (T) and the feed flow rate (Q), to obtain better yields and a high-quality product. In the second stage, the use of ultrasonication in an additional homogenization step was evaluated, aiming to further improve the encapsulation process. The results showed that the best drying conditions (first stage) were T = 180 °C and Q = 0.6 L/h, which provided the highest yield (67.73%) and high encapsulation efficiency (73.73%). The microspheres produced had similar properties, with mean diameters ranging from 0.64 to 12.99 μm. In the second stage of the investigation, the application of ultrasonication immediately after the Ultra-Turrax homogenization enabled the encapsulation efficiency to be increased to 94.05%, with a yield of 57.54%, using an ultrasonication time of only 7 min. This showed that addition of the ultrasonic homogenization step to the process greatly improved the encapsulation efficiency and could be used to produce vitamin E-enriched powder microcapsules by spray drying, with application in the food industry.

Proteomic and N-glycomic comparison of synthetic and bovine whey proteins and their effect on human gut microbiomes.

Advances in food production systems and customer acceptance have led to the commercial launch of dietary proteins produced via modern biotechnological approaches as alternatives to traditional agricultural sources. At the same time, a deeper understanding of how dietary components interact with the gut microbiome has highlighted the importance of understanding the nuances underpinning diet-microbiome interactions. Novel food proteins with distinct post-translational modifications resulting from their respective production systems have not been characterized, nor how they may differ from their traditionally produced counterparts. To address this, we have characterized the protein composition and N-glycome of a yeast-synthesized whey protein ingredient isolated from commercially available ice cream and compared this novel ingredient to whey protein powder isolate derived from bovine milk. We found that despite strong similarities in protein composition, the N-glycome significantly differs between these protein sources, reflecting the biosynthetic machinery of the production systems. Further, the composition profile and diversity of proteins found in the synthetic whey protein were lower relative to bovine whey protein, despite both being predominantly composed of β-lactoglobulin. Finally, to understand whether these differences in N-glycome profiles affected the human gut microbiome, we tested these proteins in an in vitro fecal fermentation model. We found that the two whey protein sources generated significant differences among three distinct microbial compositions, which we hypothesize is a product of differences in N-glycan composition and degradation by these representative microbial communities. This work highlights the need to understand how differences in novel biotechnological systems affect the bioactivity of these proteins, and how these differences impact the human gut microbiome.