Antioxidant and ACE-Inhibitory Activity of Common Bean Whey Fortified Yoghurt with Assessed by in vitro Static Gastrointestinal Digestion

Yogurt supplemented with plant source proteins are received increased attention but few studies investigated effect of plant source proteins supplementation on protein digestibility and releasing of bioactive peptides. In this study, a residue rich in protein derived from common beans starch production, Common Bean Whey (CBW), was applied in development of novel yogurt. CBW from four major common beans varieties, namely white, black, kidney and cranberry were utilized. The novel yogurts were subjected to in vitro Gastro-Intestinal Simulation (GIS) digestion and protein digestibility, antioxidant activity and Angiotensin I Converting Enzyme (ACE) inhibitory property were studied. Compared to the control, Common Bean Whey Fortifi ed Yogurt (CBWFY), particularly black bean whey was the predominate stable sample to protein degradation in the gastric and intestinal phases. Peptide content and SDS-PAGE revealed that CBWFY samples exhibited mostly lower hydrolysis grades in gastric and commence of duodenal phases. High antioxidant and ACE inhibitory activities results were attributed to CBWFY, in which signifi cantly (p < 0.05) higher value was observed in kidney bean whey digesta at120min phase of intestinal digestion. For the fi rst time, the outcomes of this investigation demonstrated the infl uence of four varieties of common beans whey supplementation on protein hydrolysis kinetics, digestive degree, antioxidant activity and ACE inhibitory properties on yogurt. Graphical Abstract Research Article Antioxidant and ACEInhibitory Activity of Common Bean Whey Fortifi ed Yoghurt with Assessed by in vitro Static Gastrointestinal Digestion Ahmadullah Zahir1, Zhuang Shen1, Xin Rui1*, Jing Huang1, Enayatullah Hamdard2 and Mingsheng Dong1* 1College of Food Science and Technology, Nanjing Agricultural University, Jiangsu Province, P R China 2College of Animal Science and Technology, Nanjing Agricultural University, 1 Weigang Road, Jiangsu Province, P R China Received: 06 February, 2020 Accepted: 02 March, 2020 Published: 04 March, 2020 *Corresponding author: Xin Rui, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang Road, Nanjing, Jiangsu, P.R. China, Tel: +86 15651661026; Fax: +86 25 84399090; E-mail: Mingsheng Dong, College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang Road, Nanjing, Jiangsu, P.R. China, Tel: +86 25 84396989, Fax: +86 25 84399090; E-mail:


Introduction
The growing role of developed and functional foods on the health benefi ts has experienced rapid market growth in recent years. This growth stimulated attention in the consuming of probiotic containing novel foods [1,2]. Common bean (Phaseolus vulgaris), fi rstly originated in Central and South America and extended to other regions of the globe, is broadly consumed in many regions [3,4].Common bean is an important group belonging to pulses along with lentil, pea and chickpea. Because of high protein content (18.5-32%) and low expense, pluses are regarded as "meat of the poor" and are a good source of plant proteins referring to huge parts of global population, especially in developing countries. Proteins of common bean are rich in lysine in comparison to cereal proteins. Furthermore, pulses contain a balanced nutritional ingredient which included considerable quantities of dietary fi ber (14.6-26.3%) and low in fat (2-5%) [5][6][7]. Considering the composition of common bean, they contain several bioactive substances including lectins, phytates, oligosaccharides, enzyme inhibitors, and phenolic compounds that performs important metabolic roles in animals and humans [8][9][10]. There is growing evidence that pulses ingredients can helping in decreasing the predicted complications of severe sickness for example, cardiovascular disease, diabetes, certain forms of cancer, hypertension, obesity, osteoporosis, constipation and gastrointestinal infections, improvement of lactose metabolism, suppression of Helicobacter pylori infection, and also health benefi cial effects of probiotics including antimicrobial activities [11][12][13].
Common bean is the source of high quality starch in China. A large number of by-products (Common Bean Whey, CBW) are produced in starch production, and CBW is usually discarded.
However, CBW is rich in protein, lipid, ash and also had antioxidant activity and ACE inhibitory properties.
Yogurt is a cultured dairy product with a complex gel sequence that contains protein, polysaccharides and lipids in its composition. Usually, yogurt is made by fermenting cow's milk using a symbiotic culture Lactobacillus delbruekii subsp. bulgaricus and Streptococcus thermophillus under managed environmental conditions and temperature. The function of these starter bacteria attributed to milk acid production and the synthesis of aromatic compounds [14][15][16][17].
Lately, investigations concentrated their directions on the combination of soy protein, dairy protein, and LAB have been performed [18,19]. Various biological activities through in vivo and in vitro investigations exhibited that, the health benefi ts related with the consumption of yogurt and pulses ingredients are demonstrated greatly, these activities including antiinfl ammatory, antigenicity, antimutagenic, anticarcinogenic and angiotensin I-converting enzyme-inhibitory activity infl uences [20][21][22]. In addition, it also exhibited that regular consumption of yogurt supplemented with pulses ingredients improves the digestive function, enhance cardiovascular health and immune system [23]. However, incorporation of pulses ingredients with proteins produce a complex resulting alterations in the function, structure and nutritional attributes and digestibility of proteins. According to the investigation by [17], the interactions between different pulses ingredients particularly Soy Protein Isolate (SPI) and dairy proteins caused an increase of lysine, leucine, isoleucine, methionine and threonine as well as enhanced essential amino acids bioaccessibility. Furthermore, the effect of fermentation pH has been studied on protein-tea polyphenols combination under GIS digestion with fermented soymilk curd [20]. Results revealed, that through in vitro digestion at pH 5.45.7, interaction of soymilk curd with tea polyphenols was effective regarding protein hydrolysis and digestibility. For clearly understanding under simulated functional environments of ingested food in human digestive system, an in vitro digestion scheme is an operative and certifi ed method [20]. As reported by author, pepsin-treated protein hydrolysis of black bean thorough in vitro digestion demonstrated high degree of hydrolysis compared to alcalase digestion [24]. In addition, by year 2020, the world population increases in both Africa and Asia and a larger proportion is expected, there will be demanded necessities for expanding food security and protein sources, among different pulses common bean will be take a signifi cant and considerable part [25]. Thus, it is necessary to seek pattern for supplying common bean ingredients for fortifi cation dairy products as a component of fi nal novel foods. Therefore, in current study, a fi ltered supernatant part of common beans slurry as starch extraction residue named Common Beans Whey (CBW), were used for supplementation of cow milk to produce novel Common Beans Whey Fortifi ed Yogurt (CBWFY).
However, the protein digestibility of CBWFY fermented by LAB is not well understood so far. According to our study, no investigation has been conducted to address the infl uence of CBW fortifi cation and fermentation by LAB strains on the protein digestibility and bioaccessibility of the CBWFY up to date. Therefore, in the present study, CBWFY and control were subjected into gastro intestinal digestion model. The protein bioaccessibility by digestion process of CBWFY and control were operated via analyzing the peptide content, electrophoresis, evaluation antioxidant activities and ACE-inhibitory property.
Outcomes of the current study will facilitate in developing the preliminary knowledge for the exploring the digestive attributes, antioxidant and ACE inhibitory activity in static stimulated digestion system of CBWFY developed novel foods.

Preparation of common bean whey-fortifi ed yoghurt (CBWFY)
A certifi ed quality of common bean seeds was selected and proceeded with required procedure until ready for homogenizing, these steps were included, rinsing, then soaking with 6-folds distilled water at ambient temperature for approximately 12h, hereafter drained, dispersed and fi nally the slurry was produced using homogenizer BE601AB, Midea, China. 200-mesh screen cloth was used for fi ltration of slurry to eliminate the insoluble okara, and then to remove any precipitated starch, stored at 4ºC for about 12h. Predicated starch was removed from the system. The supernatant, Common Bean Whey (CBW), was then carefully decanted and collected. At boiling temperature, CBW was sterilized for 5min and prepared for inoculation. 75% of Cow Milk (CM) was added to CBW to make CBW -CM mixture with 25% CBW content. were rated with 1-9, continuous scale (0 = no perception; 9 = strong perception). Each sample was carried out in triplicate.

In vitro GIS digestion
Standardized static in vitro digestion protocol with slight changes was used to performed static in vitro gastrointestinal digestion (SIVD) of control and CBWFY [26]. Briefl y, to perform buccal digestion, 3.5mL of control and CBWFY with SSF (1:1, v/v) was mixed and placed in a shaking water bath (SWB series, Biobase, Shandong, China) for 2min at 37°C with 55rpm. The subsequent gastric digestion was initiated by addition SGF at ratio 1:1 to the buccal sample and then the simulated gastric digestion was performed for 1h by shaking in water bath with 55rpm at 37°C. The pH of gastric digestion was adapted to 7.0 and at same ratio (1:1, v/v) of SIF was added to the gastric digesta. The digestion process was performed for 2h with 150 rpm at 37°C. During the digestion, eight samples were obtained from control and CBWFY, at all phases; including before digestion (P0), after oral digestion (P1), gastric digestion (P2) at 5min, 30min, and 60min and intestinal digestion (P3) at 5min, 30min and 120min were performed. To inactivate the enzyme, subsequently all samples were boiled for 5min.

Peptide content of digested samples
Peptide content of control and CBWFY at all phases of GIS digestion were determined by using OPA assay. For preparation the OPA solution, the previous method reported by [20] was used. Before measurement, obtained samples from different phases of digestion were centrifuged and fi ltered with at 10kDa cutoff tubes (Millipore, USA). For determination of peptide content, the fi ltered parts were obtained and prepared. 2mL of OPA solution and 50μL of fi ltered samples were taken and mixed. At room temperature, the mixed solution incubated for two min and at 340nm by using (U-4100, Hitachi Ltd., Japan) the absorbance was measured. The test was carried out in triplicate.

Electrophoresis
Protein profi le of control and CBWFY samples, at all phases of GIS digestion were carried out by conducting SDS-PAGE. The experiment was performed following the previous reported protocol [20]. Stacking and separating gel with 4% and 12% concentration were used, respectively. Before electrophoresis, obtained samples from various digestion phases, were warmed in boiling temperature for 5min and in each line 20μL of sample was loaded. The electrophoresis was performed by using Bio-Rad instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA), voltage 60V was used for stacking gel and 120V for separating gel. A protein marker with molecular weight ranged   Vitamin C was used as a positive control.

Evaluation of antioxidant activities
Hydroxyl radical scavenging ability: The hydroxyl radical scavenging capacity of control and CBWFY test samples were conducted following the previous method described by [28] with some modifi cations. In brief, 1mL of digested sample was blended into 1mL FeSO 4 (9mM), and then 1mL H 2 O 2 (8.8mM) was added into the mixture and blended thoroughly.
Immediately, 1mL of freshly prepared salicylic acid-ethanol solution (9mM) was mixed and blended comprehensively. The mixed solution was stored for 60min at 37°C, and at 510nm the absorbance of blended sample was measured. Final values were illustrated as μg VCE/g d.w.

ACE inhibitory activity of digested samples
The ACE property of test samples were performed using previous protocol reported by [29]. In brief, for preparation of 10 μl of samples, 50μl of 2.17mM HHL, and 10μl of ACE (1.55 mU), borate buffer (100mM, including 300mM NaCl, pH 8.3) was used and mixed to commence reaction. For consecutive period the reaction was carried out for 30min at 37°C. The reaction was ended by addition of 85μl of 1 M HCl and subsequently, 20μl of sample was inserted into a ZORBAX Eclipse. Plus, C18 reversed-phase analytical column (4.60 × 250mm, 5μm particle size, Agilent). 50% (v/v) methanol in water with 0.1% Trifl uoroacetic acid (TFA), was used to wash the samples for 15min at a fl ow rate of 0.7mL min-1. The elution was managed at 228nm. The absorbance of HA peak was calculated. Control samples were prepared without the addition of CBWFY and control digested samples, while blank samples were prepared without addition of enzyme. For calculation the ACE inhibitory activity following equation was used: ACE inhibitory activity (%) = [(Ac -As) / (Ac -Ab)] x 100 Where Ac stand as absorbance for the control which 10 μl borate buffer was poured in substitution of sample, while Ab stand for the blank absorbance without ACE and As stand for test sample absorbance.

Statistical analysis
The statistical analysis was subjected by variance (ANOVA) using Duncan's multiple comparison tests and differences were considered to be signifi cant at (p < 0.05) level using IBM SPSS Statistic software.

Proximate composition of CBW
As shown in Table 1, regarding protein content, no signifi cant (p < 0.05) difference was recorded between CM and CBW, whereas signifi cantly (p < 0.05) lower value of lipid contents were obatined for samples CBW. This indicated that the CBW is appropriate supplementary ingerdeints for development of novel food. Signifi cantly (p < 0.05) higher value of moisture and ash contents were attributed to CBW than CM, except lower value of ash content was obtained with white bean whey.  cross linking of proteins was occurred, thus more bands were appeared. According to the studies [33], in cow milk protein the  S1 -and -CNs, the bound possess phosphate groups effects several functional attributes to the cow milk proteins, such as their digestion process, bioaccessibility and immunogenicity.

Peptide content and electrophoresis under GIS digestion
The author also mentioned that, CNs containing divalent bound phosphate groups were corresponding for the reduction digestibility of this protein, which is supporting our observation, since 75% of CBWFY is cow milk. These four yogurts showed more intensives bands at P0 ( Figure 2B-E), implying that the main portions were not degraded in the gels.
After the buccal and gastric digestion P1 and P2 (P2-5, P2-30, P2-60), the predominant bands could be observed in all CBWFY samples ( Figure 2B-E), which were predicted to be aggregation between cow milk protein (casein) and 7S vicilin. This might be probably due to the aggregations of proteins make bands resistance to enzymatic hydrolyse. However, the intensity of bands corresponding to ,  and  casein of all sample decreased throughout the oral and gastric digestion, but in three samples were still visible at initial phase of duodenal digestion (P3-5, Figure 2C-E). This diminution in the band intensity could be due to the dilution of stimulated to the saliva and breakdown by enzymes and stimulated gastric juice particularly for control sample, which the reduction was more than CBWFY samples [34]. This can be attributed that the enzymatic activity, that breakdown control protein easily during GIS digestion. These results were similar from those who obtained by [24], in which  shown in Figure 3A.  Figure 3B Based on ABTS .+ scavenge radical capacity result, again CBWFY showed higher scavenging ability as compare to control, regardless for sample black bean whey which low value was obtained, suggesting that CBWFY had better antioxidant ABTS .+ radical scavenging ability comparing to control. In terms of four CBWFY, kidney bean whey demonstrated signifi cantly (p < 0.05) higher scavenging activity followed by cranberry, white and black samples. The peak antioxidant activity displaying by the CBWFY after P3-120 phase of duodenal digestion, addressed by the this assay is accredited to the connecting capability of hydrophobic proteins with ABTS .+ hydrophilic radicals [24]. This result is similar with fi nding [24], that up to two hours black bean protein hydrolysates showed 73% inhibition of ABTS .+ scavenging activity. Obtained result, suggests that enzymatic activity during GIS digestion capable to generate high amount of antioxidant fractions from CBWFY, probably due to its higher proteolytic activity on this food matrix. Hydroxyl radical scavenging ability: As shown in Figure   3D, CBWFY samples demonstrated strong hydroxyl radicals scavenging activity compared to the control sample. In terms of four CBWFY, CM fortifi ed with kidney bean whey revealed signifi cantly (p < 0.05) higher hydroxyl radical scavenging capability. Higher values of hydroxyl radicals scavenging attributed to the CBWFY particularly for sample kidney bean might be due to their greater phenolics contents. It seems that kidney bean whey might contain more hydroxyl radical scavenging ingredients which are created during fermentation.
Plenty investigations have revealed that phenolics and isofl avones demonstrate hydroxyl radical scavenging activity  [36]. Earlier insights have reported that the presence of phenolic compounds plays an imperative role in sample as a ferrous ion chelating agents [37].
To conclude, CBWFY exhibited higher antioxidant activity compared to the control through different methods and mechanisms of antioxidant activity measurement. However, in terms of four CBWFY, CM fortifi ed with kidney bean whey demonstrated higher antioxidant activity which was signifi cantly (p < 0.05) higher than three other and control.
Higher antioxidant activity in CBWFY might be attributed to their phenolic content of CBW. In addition, [38] reported that colored beans have greater antioxidant and antiradical attributes than less colored beans. Thus, this fi nding further support our observation which CM fortifi ed with white bean whey exhibited low value of antioxidant activity as compare to reaming three varieties that measured via different methods.
[39] also reported that kidney bean extract showed higher antioxidant activity than those from white bean extract .

ACE inhibitory properties
For prediction the destiny of the ingested ACE inhibitory peptides, in vitro GIS digestion is an advantageous scheme [40]. ACE inhibitory activity of CBWFY and control after P3-120 phase of duodenal digestion was shown in Figure 4. Based on the result, all samples of CBWFY showed (p < 0.05) superior ACE inhibitory activity comparing to control, indicating that CBW supplementation enhanced the ACE inhibitory activity of fi nal product. This is might due to hydrolyse of cow milk and common bean whey proteins, mainly casein and 7S vicilin into peptides during fermentation and further hydrolyzed by GIS digestion into smaller peptides. Among all four CBWFY, the ACE inhibitory activity of kidney bean whey was higher followed by black, cranberry and white samples. This result is in accordance with the peptides content in current study. It seems that the most bioactive peptides were produced at duodenal digestion, especially for samples CBWFY. Therefore, CM fortifi ed with CBW increased the production of bioactive peptides [41].
Reported that in fermented dairy product, generation of special proline endopeptidase enzymes such as (PepP, PepR, PepX) during fermentation, will enhances the amount of proline containing C-terminal peptides which directly linked with the ACE-inhibitory activity. A study investigated the effect of enzymatic hydrolysis on goat and cow milk and found that as the hydrolysis extended, the enzyme activity continuously breakdown the peptides, and consequently a number of amino acids at the C-or N-terminal produced that usually have strong affiliation with the active site of ACE [42]. These results demonstrated that after the treatment of CBWFY and control samples with the gastrointestinal enzymes, novel ACE inhibitory peptides were produced, which proposed that this product might have health promoting effect when reached to the digestive system.

Sensory evaluation of CBWFY
The results of sensory evaluation are shown in Figure 5.  [43].  The values related to taste including sweet, milk taste and aftertaste that represents the taste of novel product. Compared to CM, the taste score for CM supplemented with CBW was higher, except low score was obtained for CM supplemented with cranberry bean whey due to low score for milk taste and aftertaste parameters. This could be probably due to incomplete fermentation. Our result demonstrated that CM supplemented with CBW stimulate fermentation process as well. Among all varieties of CBW, the taste score was higher for CM supplemented with white bean whey followed by black, kidney and cranberry. Our result exhibited that the CM supplemented with white bean whey was more acceptable due to high score for sweet and aftertaste, whereas the taste result for CM supplemented with cranberry bean whey had less acceptability which resulted in a low score for sweet and aftertaste and might be due to increased acidity. According to [43,44], they have reported that, fermented milks fortifi ed to 18% total solids showed higher taste than control sample.
Taste problems in common bean whey yogurts attributed to fermented dairy aroma and common bean whey fl avor which was also due to sharp lactic acid production. Another study showed that, reduction in lactose concentrations is associated with decreases in sensory sweetness insights.
Finally, all indicative parameters related to fl avor including refreshing degree, viscosity, delicate degree and sour/sweet ratio which indicate fl avor of fi nal product. The fl avor score was higher for CM supplemented with CBW in comparison to CM, except low score was obtained for CM supplemented with kidney bean whey, due to low score for refreshing degree and delicate degree. This implies that supplementation with CBW improved the product fl avor. Regarding all varieties of CBW, the fl avor score was higher for CM supplemented with white bean whey followed by black, cranberry and kidney. This result showed that CM supplemented with white bean whey was more acceptable due to high score for viscosity, delicate degree and sour/sweet ratio which also related to the taste result, while the result for CM supplemented with kidney was less acceptable regarding low score for refreshing degree, viscosity, delicate degree and sour/sweet ratio.

Conclusion
In the current investigation, we studied the protein digestibility, antioxidant activity and ACE inhibitory properties of yogurt fortifi ed with four varieties of CBW (namely white, black, kidney and cranberry) and control were assessed by using an "standardized static in vitro gastrointestinal digestion" procedure. Results showed that, compared to the control, common beans whey fortifi ed yogurt (CBWFY) had lower protein digestibility at gastric phase particularly for CM fortifi ed with black bean which was confi rmed with peptide and electrophoresis results. The lower digestibility indicated that protein aggregation between CM and CBW proteins were occurred. All samples of CBWFY exhibited higher antioxidant activity, particularly for CM fortifi ed with kidney bean whey demonstrated higher antioxidant activity which was signifi cantly (p < 0.05) higher than three other and control.
However, CBWFY, showed high ACE inhibitory activities and higher (p < 0.05) value was obtained for CM fortifi ed with kidney bean whey, which is in accordance with the peptide content results. In summary, our study could be a good contribution for better utilization of CBW as effi cient and valuable components for development of novel foods. Therefore, in vitro analysis of anti-nutritional factors of CBWFY could be addressed hereafter.