Evaluation of Proprietary MDZenPro Formulation by Zenherb Labs in Mediating Protein Digestion under INFOGEST In-vitro Simulated Gastrointestinal Conditions

(1) The study has evaluated and reported the efficiency of a proprietary formulation named ‘MDZenPro’ in digesting proteins using an in-vitro simulated digestion. model. (2) MDZenPro was found to enhance the degree of hydrolysis of the proteins. This was supported by the results of SDS-PAGE. (3) The results of the study confirm the application of MDZenPro in aiding digestion. Abstract Protein breakdown by endogenous enzymes in the gastrointestinal (GI) tract results in generation of peptides and amino acids that act as building blocks in essential biological functions. Exogenous proteases possess immense potential as digestive enzyme supplements that can assist protein digestion in the GI system. Plant proteases, in addition to their promising activity, are considered to be safe in nature. The present study evaluated the potential application of a plant protease based proprietary formulation MDZenPro in digesting raw whey protein, whey protein isolate and plant protein under INFOGEST simulated GI conditions. The gastric and GI digested protein products were analyzed for determining the degree of hydrolysis. The protein profiles were evaluated using SDS-PAGE. The results of the degree of hydrolysis study revealed that MDZenPro facilitated gastric and GI digestion of proteins. This increase in degree of hydrolysis was noted to be higher than that observed in proteins that were not treated with MDZenPro. The SDS-PAGE profile further supported these findings wherein, the MDZenPro treated protein samples displayed low molecular weight fragmented peptides in contrast to the profile of undigested proteins. The present study thus highlights the promising application of ‘MDZenPro’ as an effective supplement for protein digestion. enzymes proteases, amylases and lipases that in the digestion of biomolecules such as proteins, carbohydrates and lipids respectively The naturally occurring digestive enzymes viz. gastric pepsin, trypsin and pancreatic lipase degrade proteins, lipids and carbohydrates and facilitate the absorption of nutrients.


Introduction
In recent times, digestive enzyme supplements involved in augmentation of gastrointestinal (GI) digestion have gained immense popularity. Such digestive supplements mainly consist of exogenous International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160, Volume: 4, Issue: 4, July-August 2022 enzymes namely proteases, amylases and lipases that aid in the digestion of biomolecules such as proteins, carbohydrates and lipids respectively (Ianiro et al. 2016; Meghwanshi et al. 2020). The naturally occurring digestive enzymes viz. gastric pepsin, trypsin and pancreatic lipase degrade proteins, lipids and carbohydrates and facilitate the absorption of nutrients.
Protein, in particular, is considered to be an essential biomolecule as it increases thermogenesis and satiety, provides nutrition, maintains muscle mass, positive net protein balance, lean body mass and healthy immune system (Arentson-Lantz et al. 2015; Kårlund et al. 2019). Proteases catalyze the breakdown of consumed proteins into amino acids and small peptides in the GI tract and, thereby increase their bioavailability (Wang et al. 2020). Denaturation of proteins by acid and their hydrolysis by gastric pepsin and pancreatic proteases are necessary to facilitate their bioavailability in the human body (Amigo et al. 2020). These digestive proteases are extremely important as the products obtained after protein digestion are involved in vital processes of cell growth and hormonal signaling among others (Ceuleers et al. 2016). However, the activity of such digestive proteases varies between individuals and are often insufficient in digesting proteins due to underlying problems that could be chronic or acute.
It has been reported that high protein intake can inhibit endogenous protease activity and result in incomplete digestion (Dallas et al. 2017). When such undigested proteins reach the colon, they are fermented by the colonic bacteria, which then produce toxic metabolites through putrefaction. This can further cause intestinal inflammation and other deleterious diseases such as colorectal cancer (Dallas et al. 2017; Kaur et a. 2017). Due to this, there has been an increase in the demand of external digestive protease enzyme supplements that can complement the functions of such naturally occurring proteases and consequently improve a consumer's digestive capacity and nutrient absorption (Oben et al. 2008). Previous studies have also suggested that protein intake accompanied with exogenous digestive protease is a better measure for protein digestion and absorption as compared to consuming protein hydrolysates (Jadhav et al. 2021). Intake of protein hydrolysates have also been reported to negatively affect the synthesis and secretion of pancreatic protease in digestive system (Kinouchi et al. 2012). ). The first stage consisted of the simulated gastric protein digestion process. In this, 5 mL of protein sample of 50 mg/mL concentration was added to 3.9 mL simulated gastric fluid and 5 μL of 0.3 mol/L CaCl 2 and, the pH was adjusted to 3. This was followed by addition of pepsin to the solution such that its final concentration in the solution was 200 U/mL. Next, distilled water was added to adjust the volume to 10 mL. The solution was incubated at 37°C for 2 h under shaking conditions at 150 rpm. The samples were analyzed after incubation. The samples thus obtained were used as 'gastric digested sample'. Further, 7 mL of simulated intestinal fluid and 40 μL of 0.3 mol/L CaCl 2 were added to these digested samples and the pH was adjusted to 7. Pancreatin suspension was added such that its final concentration in the reaction mixture was 10 U/mL. The reaction mixture volume was adjusted to 20 mL using distilled water and further incubated for 2 h at 37° C in shaking incubator (150 rpm). The enzyme activity was stopped by placing the solution in a boiling water bath for 5 min. This reaction mixture was further centrifuged and, the supernatant obtained was used as 'gastro-intestinal digested sample'. Gastric and GI digested samples were also obtained using MDZenPro as an enzyme source in the above-mentioned method. Additionally, WPC was subjected to simulated GI digestion using a commercially available protease. The control sample was obtained without adding any enzyme. These samples were further analyzed for determining the degree of hydrolysis. The profile of the digested products was studied using SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Determination of Degree of Hydrolysis
OPA reagent (175 μL) was added to 25 μL of appropriately diluted sample (25 mg/mL) in a microtiter plate and, the reaction mixture was incubated at 27 ± 2° C for 2 min. The absorbance of the solution was recorded at 340 nm wavelength. Standard curve was plotted using serine (3.125-100 μg/mL). The slope of the standard curve was analyzed for determining the serine equivalent free amino groups in the sample. The degree of hydrolysis was determined using the formula given below:

Degree of hydrolysis (%) =
Free amino groups in the sample Free amino groups in the acid hydrolyzed sample ×100 (The acid hydrolyzed sample was prepared by hydrolyzing the protein samples with acid. The total free amino group present in this hydrolyzed sample was determined by OPA assay.)

SDS-PAGE of In-vitro Digested Protein Samples
The digested protein products obtained from in-vitro digestion of whey and plant proteins using gastric enzymes and external enzymes along with the control were run on 10% SDS-PAGE. The gel was stained using Coomassie Brilliant Blue to analyze the presence of digested protein bands.

Results and Discussion Analysis of Degree of Hydrolysis by OPA Method
MDZenPro was studied to determine its ability to augment the in-vitro gastric and GI digestion of whey protein and plant protein samples. Degree of hydrolysis for WPC, WPI and PP under simulated GI conditions were found to be 8.3%, 14.4% and 6.6% respectively. It was observed that the degree of hydrolysis of WPC, WPI and plant proteins that were treated with the MDZenPro was higher as compared to the protein samples that were not treated with the enzymes (Figure 1 and Figure 2). The untreated set showed a protein digestion of less than 10%. Hence, when compared to an untreated set, the set with MDZenPro showed approximately 59% faster digestibility. Protein sample WPI treated with MDZenPro showed maximum degree of hydrolysis after both gastric digestion (15.77%) and GI digestion (57.75%). Whey protein is one of the most popular protein supplements available in the market owing to its amino acid profile. In the present study, the degree of hydrolysis of WPC samples after treatment with a commercially available protease and MDZenPro under simulated GI conditions were found to be comparable, thereby validating the high efficiency of MDZenPro (Table 1).
Fruits such as papaya, pineapple, figs and kiwifruit are known to be rich sources of proteases (papain, bromelain, ficin and actinidin respectively) that are able to breakdown 'hard to digest' proteins such as gluten, casein and gelatin and enhance upper GI tract protein digestion (Kaur et

Analysis of Protein Hydrolysis using SDS-PAGE
The profile of undigested and digested proteins (WPC, WPI and plant protein) were studied using SDS-PAGE. The SDS-PAGE profile pattern confirmed that upon simulated gastric and GI digestion, the protein samples treated with MDZenPro were hydrolysed into fragmented peptides (Figure 3). The WPC protein profiles obtained after hydrolysis with MDZenPro and commercial protease were comparable ( Figure 4). These peptides possessed lower molecular weight as compared to the undigested protein samples (Figure 3 and Figure 4). The fragmentation of the protein samples after treatment with exogenous MDZenPro product further substantiates the digestion of protein samples.

Conclusion
The results of the degree of hydrolysis and electrophoretic analysis in the present study indicate that MDZenPro has a positive effect on protein digestion under simulated GI conditions. The unique feature is the presence of plant protease which provides a distinct advantage to the MDZenPro formulation since, it is a component derived from naturally safe sources unlike its animal and microbial counterparts. Thus, MDZenPro possesses a promising commercial application as a digestive aid for augmenting the breakdown of proteins.

Authors Contributions
Mr. Mihir Gadani contributed towards the concept designing for the ingredient. Ms. Ratna Upadhyay was involved in the development of the ingredient. Dr. Supriya Raut did the product formulation using the ingredient.