LWT - Food Science and Technology 149 (2021) 111901

Available online 11 June 20210023-6438/© 2021 Elsevier Ltd. All rights reserved.

Whey and soy proteins as wall materials for spray drying rosemary: Effects on polyphenol composition, antioxidant activity, bioaccessibility after in vitro gastrointestinal digestion and stability during storage

Mary H. Grace *, Roberta Hoskin, Jia Xiong, Mary Ann Lila Plants for Human Health Institute, Food Bioprocessing & Nutrition Sciences, North Carolina State University, North Carolina Research Campus, Kannapolis, NC, USA

A R T I C L E I N F O

Keywords: Protein-polyphenol Phenolic diterpenes Bioaccessibility Rosemary antioxidants Spray-drying

A B S T R A C T

A straightforward protocol was developed to produce rosemary particles using whey and soy protein carriers. The post-processing retention of carnosic acid (CA), carnosol (CR) and rosmarinic acid (RA), their bio-accessibility, in vitro antioxidant activity, and storage stability were investigated in protein-rosemary particles compared to rosemary extract (RME). Solids recovery was highest for whey protein or whey-inulin blend complexed with rosemary (R–WPI, R–WIN, ~90%), followed by soy protein or soy-inulin (R–SPI, R–SIN, 60% and 70%); all were higher than rosemary alone (RME, 52%). Protein or protein/inulin carriers significantly enhanced retention of CR (36.8–50.7 mg/g) and CA (17.1–19.6 mg/g) compared to RME (19.8 mg/g and 8.3 mg/ g, respectively). In vitro digestibility showed that whey protein isolate increased the bioaccessibility of CA and CR, with no effect on RA, which was highly bioaccessible in all formulations. The rosemary-protein-treatments retained high antioxidant activity measured by ROS and NO assays. CR and CA were particularly stable during 20 weeks of storage in protein-rosemary particles, and stayed at their higher concentration compared to RME. Water activity was below 0.5 and remarkable color stability was observed during storage. Overall, spray dried protein- rosemary particles constitute a creative solution to deliver preserved phytochemicals in a high-protein food format.

1. Introduction

Rosemary, a well-known perennial plant, originally native to the temperate countries of the Mediterranean region, is currently cultivated all over the world. Beyond its use as a culinary delicacy, rosemary ex-tracts are commercialized as cosmeceuticals, and as ingredients used to improve product shelf life and enhance the sensory profile of food products (Rahila et al., 2018). Moreover, rosemary is a clean label alternative to synthetic antioxidants, due to its capacity to improve the oxidative stability and antioxidant properties of food products (Soares et al., 2020).

The biological activities of rosemary are attributed to the presence of three classes of phenolic constituents: phenolic diterpenes (carnosic acid, carnosol, rosemanol, epirosemanol, methyl carnosate), flavonoids (cirsimaritin, genkwanin) and phenolic acids (rosmarinic and caffeic acids) (Grace, Qiang, Sang, & Lila, 2017). The major constituents iso-lated from rosemary extracts - carnosic acid, carnosol and rosmarinic acid - have demonstrated both in vitro and in vivo anti-obesity and

anti-diabetic properties (Sedighi, Zhao, Yerke, & Sang, 2015). However, these unstable bioactive compounds can be easily degraded during storage. In this regard, technological alternatives have been developed to slow their degradation, and improve their bioavailability in biological systems while delivering them in an easier-to-handle-and-store format (Baldim et al., 2020; Hosseini & Jafari, 2020). Spray drying microen-capsulation creates an external membrane able to entrap natural in-gredients into small particles, promoting higher stability, and minimizing losses of bioactive compounds, while preserving their functional and/or health-related properties (Ozkan, Franco, De Marco, Xiao, & Capanoglu, 2019). The drying carrier affects the overall spray drying performance and characteristics of the final product (Gonzalez et al., 2020). As a result, phytochemical-rich spray dried particles can be engineered to achieve particular properties such as improved storage stability and specific release patterns (Tarone, Cazarin & Marostica Ju-nior, 2020), and several wall materials have been tested in the literature, comprised of either individual materials or blends (Zhang, Zhang, Chen, & Quek, 2020).

* Corresponding author. E-mail address: mhgrace@ncsu.edu (M.H. Grace).

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https://doi.org/10.1016/j.lwt.2021.111901 Received 30 March 2021; Received in revised form 13 May 2021; Accepted 9 June 2021

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Currently, there is a global demand for protein-rich food ingredients and this trend is expected to continue upwards (Ismail, Hwang, & Joo, 2020). In tandem, consumer demand is increasing for natural, clean label phytochemical-rich products. In this context, we have demon-strated that the complexation of protein-rich flours with polyphenols from fruit and vegetable juices and extracts are an efficient way to produce stable dry phytochemical-rich ingredients and products (Diaz, Foegeding, & Lila, 2020). The protein-polyphenol binding creates stable aggregates with enhanced structural and health functionality, enabling their incorporation into a diverse range of product applications (Lila et al., 2017). We have shown that defatted soy protein protected an-thocyanins during transit through upper digestive tract for subsequent colonic delivery/metabolism (Ribnicky et al., 2014), and we recently reported that rice and pea proteins enhanced the stability of berry polyphenols in an in vitro gastrointestinal digestion model (Xiong et al., 2020).

In this study, we developed a straightforward protocol to produce spray dried rosemary using edible protein carriers, and evaluated the retention of the most valuable rosemary antioxidant phenolics - carnosic acid, carnosol, and rosmarinic acid. While many studies on the encap-sulation of herbs are focused primarily on their essential oils, here we present a practical approach applied to rosemary extract, a phenolic-rich substrate with important applications in the food industry. This study compared rosemary extract spray dried without carriers to groups pro-duced with selected proteins as wall materials – whey protein or soy protein isolates, with and without inulin, a prebiotic fiber with encap-sulating properties (de Abreu Figueiredo et al., 2020). The protein-polyphenol aggregate particles were tested for their ability to attain desirable water activity levels, preserved color and phenolics stability during storage. The bioaccessibility of phenolic compounds in rosemary-protein particles was evaluated before and after in vitro gastrointestinal digestion. Our hypothesis was that rosemary extract spray dried with edible protein carriers, with or without inulin, will preserve phytochemical integrity and antioxidant properties, exhibit enhanced bioaccessibility and extended stability, and therefore, might constitute a versatile clean-label ingredient for the food industry.

2. Materials and methods

2.1. Plant material

Fresh rosemary shoots (Rosmarinus officinalis, Variety, Spice Moun-tain) were obtained from John Weddington Greenhouse (Salisbury, NC, USA). The leaves were stripped off and were directly subjected to the extraction procedure.

2.2. Chemicals

Alpha-amylase from porcine pancreas Type VI-B protein, pepsin from porcine gastric mucosa 3200–4500 U/mg protein, pancreatin from porcine pancreas, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-lium bromide (MTT), 2′,7′-dichlorofluorescin diacetate (H2DCFDA), pyrrolidine dithiocarbamate (PDTC), dexamethasone (DEX), lipopoly-saccharide (LPS, from Escherichia coli 127:B8), Griess reagent, and so-dium nitrite were purchased from Sigma (St. Louis, MO, USA). Bile bovine was purchased from Chem-Impex International, Inc. (Wood Dale, IL, USA). All organic solvents were HPLC grade and obtained from VWR International (Suwanee, GA, USA). Whey protein isolate (Eidogen-Ser-tanty Inc., 90% protein), soy protein isolate (Bulk Supplements, 90% protein) and inulin fiber (Yoleez LLC, made from chicory roots, average degree of polymerization 10) were used in this study.

2.3. Extraction of rosemary leaves

Fresh rosemary leaves (moisture content, 71.3% ± 2.0%), in 200 g- batches, were extracted under vacuum with 1 L 100% ethanol using a

blender (Vita-Mix Corp, Cleveland, OH, USA) for 15 min. The mixture was sonicated at 50 ◦C for 15 min, centrifuged at 4 ◦C, and the super-natant (extract) was collected. The plant residue was re-extracted with 500 mL 80% aqueous ethanol in the same way. A third extraction was executed using 500 mL 50% aqueous ethanol. The combined superna-tant (~2.5 L) was vacuum evaporated to remove ethanol to afford a final rosemary extract (final volume of ~400 mL) containing 7.3 ± 0.2% dry matter. This extract was kept at 4 ◦C until all batches were prepared. Several batches were extracted over a period of 48 h and all were mixed together to form one single batch that was used for spray drying.

2.4. Spray drying

Five experimental groups were tested in this study: rosemary extract alone (RME, no added protein and/or inulin), rosemary extract with whey protein isolate (R–WPI), rosemary extract with soy protein isolate (R–SPI) or rosemary extract with protein-inulin blends: rosemary extract with whey protein isolate–inulin (R–WIN) and rosemary extract with soy protein isolate-inulin (R–SIN). Before each batch, the powdered protein or protein-inulin blend (4:1) was dispersed directly into the rosemary extract by vigorous mixing (PRO Scientific Bio-Gen PRO200, Oxford, CT, USA) for 2 min at 15,000 rpm until complete dissolution. The prepared feed solution was atomized using a spray dryer (B-290, Buchi Labortechnik AG, Switzerland) at 110 ◦C (outlet temperature 55–65 ◦C) following preliminary experiments (data not shown). The spray drying system operated using air in co-current flow under the following optimized conditions: 1.5 mm diameter nozzle, 10 mL/min of feed flow (controlled by peristaltic pump) kept under constant magnetic stirring during drying. For each of the four unique combinations con-taining protein or protein-inulin, the mass of protein (or protein-inulin blend) was calculated in relation to the volume of rosemary extract and kept constant as 6 g/100 mL of aq. extract. The resulting spray dried particles were collected from the collection chamber, weighed and immediately sealed in Ziploc® bags. Two independent spray dried batches were produced for each experimental group, and results repre-sent the average and standard deviation of samples.

2.4.1. Solids recovery and polyphenol retention The solids recovery (also known as production yield) of spray dried

rosemary-protein samples was calculated as percentage (%) according to the ratio [total solids content of resulting particles (rosemary only or protein-rosemary particles)/total solids content in the feeding mixture (before spray drying)] × 100 according to our previous protocol (Hos-kin, Xiong, Esposito, & Lila, 2019). In addition, the percentage total polyphenol (TP) retention % was determined (Zhang et al., 2020) as follows:

TP retention. %=TP of SD powder, mg/gSolids in SP powder, g

÷TP of feed mixture, mg/g

Solids in feed mix, g × 100

2.4.2. Total phenolic content TP was determined spectrophotometrically according to a Folin-

Ciocalteu procedure in a microplate adapted method as previously re-ported (Xiong et al., 2020).

2.4.3. HPLC-DAD analysis for rosmarinic acid, carnosol, and carnosic acid HPLC-DAD was conducted on an Agilent 1200 series HPLC (Agilent

Technologies, Santa Clara, CA, USA) equipped with a photodiode array detector (DAD) set at 230 nm. The chromatographic separation was performed on Phenomenex Synergi 4 μm hydro-RP 80A column (250 mm × 4.6 mm × 5 μm, Torrance, CA, USA) thermostatted at 25 ◦C ac-cording to our previously described method (Grace et al., 2017). Ros-marinic acid (RA), carnosol (CR) and carnosic acid (CA) were quantified based on standard curves constructed with corresponding reference

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standards and expressed as μg/mg DW.

2.5. In vitro gastrointestinal digestion

A slightly modified static in vitro gastrointestinal digestion method (Minekus et al., 2014) was used. Simulated salivary fluid (SSF), simu-lated gastric fluid (SGF), and simulated intestinal fluid (SIF) electrolyte stock solutions were prepared with the corresponding electrolytes ac-cording to the parameters in the referenced article. In the oral phase, 100 mg RME or 200 mg rosemary-protein complexes (normalized to about 25 mg TP) suspended in 1 mL water were mixed with 0.7 mL of SSF and minced together, followed by addition of 0.1 mL of 1500 U/mL porcine pancreas α-amylase solution, 50 μL of 0.03 M CaCl2, and 150 μL of water (pH 7.0), in sequence, and thoroughly mixed for 2 min. In the gastric phase, the 2 mL oral bolus was mixed with 1.28 mL of SGF electrolyte stock solutions, 320 μL porcine pepsin stock solution of 25, 000 U/mL, 10 μL of 0.03 M CaCl2, the pH was adjusted to 3.0 with HCl, and water was added to reach 4 mL of total volume. The reaction vessel was placed in a shaking incubator at 37 ◦C for 2 h. For the intestinal phase, 4 L of gastric chyme was mixed with 2.2 mL of SIF electrolyte stock solution, 1 mL of a pancreatin solution 800 U/mL, 0.5 mL fresh bile (160 mM in fresh bile), 80 μL of 0.03 M CaCl2, the pH was adjusted to 7.0 with NaOH, water was added to reach 8 mL of total volume, and the solution was shaken for 2 h at 37 ◦C. After the intestinal digestion, samples were centrifuged to obtain the soluble fraction and the residual fraction. The digested material from individual tubes for oral, gastric, and intestinal steps of each sample was immediately frozen and freeze-dried after each step. The recovery index (RI) and bioaccessibility index (BI) for RA, CR, and CA, were calculated according to Equations (1) and (2) (Martínez-Las Heras, Pinazo):

RI (%) = (A / C) × 100 (1)

BI (%) = (B / C) × 100 (2)

Where A corresponds to the RA, CR, or CA contents (μg/mg spray dried powder) quantified in oral bolus, gastric chyme, and intestinal residue, B corresponds to the RA, CR, or CA contents (μg/mg spray dried powder) quantified in intestinal supernatant after the complete digestion process, and C is for RA, CR, or CA contents (μg/mg spray dry powder) quantified before the in vitro digestion.

2.6. Cell culture assays

2.6.1. Cell viability assay Mouse macrophage RAW 264.7 (ATCC TIB-71) cell line was

routinely maintained according to our previous report (Hoskin et al., 2019). For the cell viability assay, cells were treated with different treatment concentrations (50–200 μg/mL) for 24 h in sterile 96-well plates. Cell viability was measured in triplicate after 2 h of exposure to MTT and quantified spectrophotometrically at 550 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

2.6.2. In vitro reactive oxygen species (ROS) assay The ROS assay was conducted according to a modified method (Choi,

Hwang, Ko, Park, & Kim, 2007). Briefly, RAW 264.7 macrophage cells were seeded into sterile 24-well plates overnight, and exposed to 50 μM H2DCFDA solution for 30 min. After washing, cells were treated with 100 μg/mL of samples and positive control 40 μM of PDTC for 4 h and induced by 10 ng/mL of LPS for 18 h. The fluorescence of 2′, 7′-dichlorofluorescein (DCF) was measured at 485 nm (excitation) and 520 nm (emission) on the microplate reader. Results were expressed as ROS production (%) relative to LPS induction.

2.6.3. In vitro nitric oxide (NO) assay The colorimetric assay protocol consisted of measuring the accu-

mulation of NO in culture media using the Griess reagent. RAW 264.7

cells in 24-well plates were treated with rosemary samples at 100 μg/mL for 4 h, except for LPS control. All treated and untreated cells were then induced by 10 ng/mL with LPS for 18 h 100 μL of cell-free supernatant was mixed with 100 μL of Griess reagent. The mixture was incubated for 10 min in the dark at room temperature and the absorbance was read at 520 nm. Dexamethasone (DEX) at 40 μM was used as positive control. The nitric oxide concentrations were calculated based on a sodium ni-trite calibration curve (1–40 μM).

2.7. Storage stability

Samples (2–3 g) of spray dried rosemary alone or rosemary-protein particles were transferred to 20 mL transparent screw-cap vials and kept in the dark at room temperature (21 ◦C). At defined intervals (4, 8, 12, 16, and 20 weeks), duplicate sets of samples were collected and evaluated for concentrations of RA, CR, and CA (μg/mg) as described in section 2.4.3. In addition, a reflectance spectrophotometer (CR-400, Konica, Minolta, Japan) previously calibrated with white and black standards was used to determine the instrumental color of samples during storage. Results lightness (L*), greenness (− a*) or redness (+a*), and blueness (− b*) or yellowness (+b*) were used to calculate the total color difference between samples collected at each time point (2, 4, 8, 12, 16, and 20 weeks) and time zero (immediately after spray drying) according to a previous protocol (Khalifa, Li, Mamet, & Li, 2019). Finally, the water activity of all samples were measured using an Aqualab water activity meter (Decagon, Pullman, WA, USA).

2.8. Statistical analysis

Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used to perform the two-way ANOVA analysis and Tukey multiple comparison tests, or Dunnett’s multiple comparison, with statistical significance determined at p < 0.05. Results are presented as the mean ± SD.

3. Results and discussion

3.1. Solids recovery and polyphenol retention

Solids recovery is a useful index to determine the production yield and feasibility of a spray drying process. In this study, the final spray dried rosemary products were collected from the collection vessel only, and all the other particles that adhered to the drier walls and/or pipes were discarded. Our results show a clear solids recovery increase (p <0.05) when protein carriers (alone or blended with inulin) were used to spray dry the aqueous rosemary extract (Fig. 1). Drying carriers are used to increase process yield by decreasing particle losses to the dryer chamber caused by heat-induced physical changes that lead to unde-sirable stickiness. In fact, some fruits and vegetables cannot be feasibly spray dried without the incorporation of drying carriers, due to large solids deposits on the equipment walls that jeopardize the production (Khalifa et al., 2019). In particular, the addition of whey protein had a beneficial effect on the solids recovery in this study, since both groups (R–WPI and R–WIN) presented higher recovery (~90%) when compared to soy protein isolate samples (R–SPI and R–SIN, 60% and 70% respectively). Similar improvement based on drying carriers was observed elsewhere (Bilusic et al., 2020; Tontul, Topuz, Ozkan, & Kar-acan, 2016). It has been suggested that the efficiency of solids recovery can be modulated by differences in protein content of the carrier (Tontul et al., 2016) but in this study, both protein isolates had similar protein content (80%) and therefore, this factor should not play a significant role on the observed results. However, whey protein is more soluble than soy protein, and this higher solubility in water might have contributed to a better mixture of the drying feed, resulting in higher solids recovery (Jayasundera et al., 2009). Moreover, the presence of inulin favored the recovery of solid particles only when soy protein was used, as R–SIN showed better recovery (p < 0.05) than R–SPI group, but

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similar behavior was not observed for R–WPI or R–WIN counterparts. Once again, we believe that the increase in solubility caused by the presence of inulin in the R–SIN group might explain the observed dif-ferences. It has been demonstrated that proteins are able to form a glassy film on the surface of the phytomolecules, and prevent excessive adherence to the walls of the spray drier chamber, resulting in higher solids recovery and more prolific production (Fang & Bhandari, 2012). In addition, protein’s surface activity leads to an effective migration to the air/droplet interface and helps for a rapid formation of the afore-mentioned protein layer (Shi, Fang, & Bhandari, 2013).

Previous reports suggested that it is possible to spray dry rosemary extracts without carriers, but some of them failed to disclose the actual solids recovery of the process or did not clearly demonstrate the re-covery difference when spray drying with or without drying aids (Bilusic et al., 2020; Couto et al., 2012). Moreover, some studies on spray drying rosemary using drying carriers did not make any reference to the solids recovery (de BarrosFernandes, R.V., Borges, S.V., Botrel, D. A., 2014), or did not show the difference in performance between groups with different carriers (Bilusic et al., 2020; Choi et al., 2007), which makes it difficult to assess their importance to the production yield. Spray drying of rosemary extracts without carriers resulted in production yields be-tween 17.1 and 74.96%, in a previous report (Chaul, Conceiçao, Bara, Paula, & Couto, 2017), in contrast to the 87.1–92.7 recoveries reported here for R–WPI and R–WIN, respectively. Similar solids recovery (75%) was reported when aqueous rosemary extracts were freeze dried and used to prepare extracts that were subsequently spray dried with whey protein isolate (Bilusic et al., 2020). In another study, spray dried pro-liposomes prepared with freeze dried rosemary extracts were obtained with recoveries of only 20.1–45.8%, substantially lower than our results (Bankole, Osungunna, Souza, Salvador, & Oliveira, 2020). Our design allows the preparation of aqueous rosemary extracts without an addi-tional freeze drying step, which makes for a simpler, cost effective and more straightforward production protocol.

When formulating functional, phytochemical-rich food products, the major impacts of manufacturing operations on the concentration and bioaccessibility of natural phytoactive compounds have to be taken into account. In fact, despite all the benefits resulting from processing – for example, improved texture, taste, and extended shelf life - thermal processing can induce nutritional and/or phytochemical modifications in food (Rawson et al., 2011). Therefore, in this study we evaluated the retention of polyphenols after spray drying, and compared the results among groups in order to determine the differences between spray dried rosemary with and without protein/inulin carriers. The addition of

protein or protein/inulin carriers led to significantly higher polyphenol retention (p < 0.05) when compared to spray dried rosemary extract (RME) alone (Fig. 1). The phenolic compounds present in the protein-rosemary particles come primarily from the rosemary extract, since the TPC of protein sources was negligible (<2 mg/g). In fact, all treatment groups (R–WPI, R–WIN, R–SPI, R–SIN), independently of the type of protein or presence of inulin, showed enhanced ability to retain the polyphenol content of the rosemary extracts after spray drying. Our hypothesis is that soy and whey protein isolates – with or without inulin - surround the ephemeral rosemary polyphenol molecules, creating a physical barrier that protects them from thermal degradation during spray drying due to their excellent film forming properties (Fang & Bhandari, 2012; Tontul & Topuz, 2017). Spray drying pomegranate extract with soy protein isolate gave higher polyphenol recovery than spraying with maltodextrin due to a better protein-bioactive interaction (Robert et al., 2010). In our study, inulin was added in a fixed amount and accounts for only 1/5 of the added drying aid, thus, we hypothesize that the positive experimental effects result mainly from the protein portion of the carrier.

On the molecular level, the retention of the three major antioxidants RA, CR, and CA was examined in the four spray dried protein-rosemary formulations compared to RME. Taking into account the dilution due to the addition of the protein carriers, concentrations were normalized to rosemary extract’s dry weight content (100% for RME, 55% for all other protein-rosemary complexes). Spray drying with protein or protein/ inulin carriers resulted in a significant retention enhancement of CR concentrations (36.8–50.7 mg/g) compared to 19.8 mg/g for RME (Fig. 2). CA similarly showed significantly enhanced retention (17.1–19.6 mg/g) for rosemary-protein formulations compared to 8.3 ±0.6 mg/g in RME, indicating that whey or soy protein isolates or their blends with inulin were able to protect less polar compounds (CR and CA) during spray drying (thermal) process. As illustrated in Fig. 2, the concentrations of the more polar phenolic dicarboxylic acid RA (24.4 ±1.4 mg/g in the RME) remained nearly the same in all rosemary-protein treatments. The interaction between RA and bovine milk whey protein was previously studied, where authors noted a loss of antioxidant ac-tivity due to non-covalent interactions between RA and whey protein, decreasing the amount of free RA (Ferraro, Madureira, Sarmento, Gomes, & Pintado, 2015). RA has demonstrated moderately strong binding affinity to human serum albumin (HSA) through hydrophobic interactions, and the binding site in HSA was assigned. This reference further suggested that the protein carriers were not able to protect RA from degradation during the spray drying processes, unless a lower

Fig. 1. Solids recovery and total polyphenol retention of spray dried rosemary-protein particles: RME: rosemary extract only; R–WPI: rosemary-whey protein isolate; R–WIN: rosemary-whey protein isolate/ inulin blend; R–SPI: rosemary-soy protein isolate; R–SIN: rosemary-soy protein isolate/ inulin. Bars indicate standard deviation. Samples marked with asterisk are signifi-cantly different compared to spray dried rosemary extract alone (RME, no protein or protein/inulin): **p < 0.01; ****p < 0.0001.

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amount of protein (0.05 wt % WPI) was used (Pang, Yusoff, & Gimbun, 2014). In addition, their results indicated that RA degrades quickly during spray drying, and they attributed this to presence of many hy-droxyl groups in its molecular structure. In contrast, as indicated in this study, soy and whey protein isolates surrounded the rosemary poly-phenol molecules, creating a physical barrier that protected them from thermal degradation during spray drying (Fang & Bhandari, 2012).

3.2. In vitro gastrointestinal digestion, recovery and bioaccessibility

In vitro digestion models are the most widely used approach to mimic in vivo digestion. They are relatively inexpensive, simple and rapid with no ethical restrictions, compared to in vivo protocols. Separation of the soluble compounds by centrifugation, one of the most common and efficient techniques for simulating the bioaccessible fraction of food and extract, was carried out in this study. In order to determine the per-centage of compounds that reach different digestion phases, the recov-ery index (RI%) and intestinal bioaccessibility index (BI%) (percentage of compounds in the bioaccessible fraction that potentially have been taken up by enterocytes) were analyzed. The results presented in Fig. 3 compared the RI% of RA, CR and CA of the spray dried rosemary par-ticles, with or without carriers, through oral, gastric, and intestinal digestion phases, and in the bioaccessible fraction. For RA, all four protein-rosemary complexes had levels comparable to RME after the oral phase, then the concentration in the rosemary-protein treatments slightly decreased after the gastric phase. After intestinal digestion, the RA recovery (in the solid pellet) diminished, while larger amounts appeared in the bioaccessible fractions (average 46%–63%). R–WIN showed highest recovery of RA of in the bioaccessible fraction. Our re-sults agree with the reported BI% for RA (30%–45%) using filtration, centrifugation, and dialysis membrane methods after intestinal diges-tion (Gayoso et al., 2016). For CR digestibility in RME, there was no significant change when comparing RI% in the three digestion phases. However, the RI% of the four rosemary-protein complexes showed a gradual decrease oral > gastric > intestinal. As far as BI, results varied from 9 to 46% and were similar for all treatments (p > 0.05). For CA, a sharp decrease was observed in the intestinal digestion phase, and its bioaccessibility (8%–30%) was the lowest among the three compounds. R–WPI formulation showed the highest significant BI% for CA. When considering the total sum of the three compounds RA, CA and CR, all samples had similar recovery (27%–43% in intestinal digestion phase) and bioaccessibility (23%–41%).

We can attribute the differences between the bioaccessibility of RA compared to the less polar CR and CA to the lower water solubility of these latter compounds. In previous work, the bioaccessibility of CR and CA was significantly increased when rosemary extract was supple-mented with soy lecithin and sunflower oil (Soler-Rivas et al., 2010). The bioaccessibility of CA lecithin-based nanoemulsion versus CA sus-pension in a TNO’s gastrointestinal model demonstrated that CA bio-accessibility in the nanoemulsion increased 5-fold compared to the CA suspension (Zheng et al., 2021). Complexation and encapsulation of such compounds with protein-based formulation is an effective approach to improve their solubility, bioaccessibility and bioavail-ability. Therefore we conclude that the complexation with WPI or SPI protects CR and CA compounds and improves their in vitro bio-accessibility when compared to RME.

3.3. Cell viability and antioxidant activity

The cytotoxicity of spray dried rosemary samples on the viability of RAW 264.7 cells was evaluated after 24 h of exposure using the MTT assay. Extracts from samples corresponding to 50–200 μg/mL were tested. Percentages of cell viability above 80% were considered as non- cytotoxic. No cytotoxicity was observed up to the highest sample con-centrations, therefore, a concentration of 100 μg/mL was selected for evaluation of the in vitro antioxidant activity.

Many environmental stimulants prompt cells to produce ROS, which might induce a number of damaging events in cell (Sanchez-Marzo et al., 2020). NO has important biological functions, but over production of NO provokes diverse autoimmune disorders (Lo, Liang, Lin-Shiau, Ho, & Lin, 2002). Results indicated that the cell-based antioxidant activity of RME and rosemary-protein samples were comparable to the positive controls (PDTC or DEX for ROS and NO, respectively) (Fig. 4). RME contained 100% rosemary extract, while the rosemary-protein

Fig. 2. Concentrations of rosemary marker antioxidant compounds, rosmarinic acid (RA), carnosol (CR), and carnosic acid (CA), in spray dried rosemary- protein particles. RME: rosemary extract only; R–WPI: rosemary-whey protein isolate; R–WIN: rosemary-whey protein isolate/inulin blend; R–SPI: rosemary- soy protein isolate; R–SIN: rosemary-soy protein isolate/inulin. Concentra-tions were calculated as μg/mg based on the percentage of rosemary extract in each formulation; 100% in RME, and 55% in rosemary-protein formulations. Different letters denote statistical differences among mean values according to Turkey’s test, p < 0.005.

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formulations contained rosemary extract diluted with protein at a 55:45 ratio. However, rosemary-protein particles showed comparable antiox-idant capacity to RME. These results correlate well with the higher content of antioxidant compounds RA, CR and CA found in the spray dried rosemary-protein samples (Fig. 2).

3.4. Storage stability

3.4.1. Storage effect on rosmarinic acid, carnosol and carnosic acid concentrations

Interestingly, we could not find reports describing the retention of the three major phytoactive rosemary components over storage.

Therefore, we monitored the concentrations of RA, CR, and CA in all samples for 20 weeks of storage (Fig. 5). Both CA and CR were very stable along the storage time with no significant differences between the start and 20-week time points (p > 0.05). The RA concentration of all groups presented higher fluctuations along the studied storage period when compared to CA and CR. RA showed significant decrease in its concentration for all samples by the end of the storage time. On the other hand, CR and CA were very stable until the end of storage time. These findings demonstrate that CA and CR were more stable compared to RA at room temperature storage conditions.

Fig. 3. Recovery index (RI) and bioaccessibility index (BI) for rosmarinic acid (RA), carnosol (CR), and carnosic acid (CA) for spray dried rosemary-protein particles within the three stage in vitro gastrointestinal digestion (oral, gastric and intestinal). RME: rosemary extract only; R–WPI: rosemary-whey protein isolate; R–WIN: rosemary-whey protein isolate/inulin blend; R–SPI: rosemary-soy protein isolate; R–SIN: rosemary-soy protein isolate/inulin. Different letters denote statistical differences among mean values according to Turkey’s test, p < 0.05.

Fig. 4. Effect of spray dried rosemary- protein particles on reactive oxygen species (ROS) and nitric oxide (NO) production in LPS-induced macrophages RAW 264.7 at 100 μm/mL 40 μM pyrrolidine dithiocarba-mate (PDTC) or Dexamethasone (DEX) was used as positive controls. RME: rosemary extract only; R–WPI: rosemary-whey protein isolate; R–WIN: rosemary-whey protein isolate/inulin; R–SPI: rosemary-soy protein isolate; R–SIN: rosemary-soy protein isolate/ inulin. Post hoc analyses were made using the Dnnett’s multiple comparison tests. Samples marked with asterisks are signifi-cantly different compared to lipopolysac-charide (LPS) treated group. ****p <

0.0001.

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3.4.2. Storage effect on water activity and color Water activity is a key parameter that governs microbiological

growth and degradation reactions, such as lipid oxidation, non- enzymatic browning and enzyme activity (Zhang et al., 2020). All spray dried rosemary particles presented initial Aw levels around 0.2, which is typical of spray dried powders. Interestingly, immediately after production (time zero) RME samples presented lower Aw when compared to all other groups (p < 0.05; Fig. 6 A). This behavior likely occurred due to the high water affinity of whey and soy protein isolates caused by the presence of hydrophilic groups that promote increased water binding (Abaee, Mohammadian, & Jafari, 2017). However, after only four weeks of storage, this initial difference could not be observed anymore and all groups presented similar water activity levels that was kept until the end of the 20 weeks at room temperature.

Besides the aroma, one of the most desired attributes of rosemary is the bright natural green color of its leaves. The apparent color of the spray dried rosemary particles is a result of the combination of the carrier and rosemary extract, the effect of the thermal drying and modifications that occurred to the natural pigments during storage. Interestingly, we could not find reports assessing the color of spray dried

rosemary in the literature. Considering that the color of herbal products influences their market value and is a major quality factor for con-sumers, we evaluated the experimental color stability of samples based on total color difference ΔE, a numerical index that indicates the color variation between the spray dried rosemary samples at each time point along storage and the color of freshly prepared samples.

For the duration of the storage period, the spray dried ingredients showed a clear pattern, as seen in Fig. 6B. While R–SPI exhibited min-imal color changes (groups R–SPI and R–SIN), R–WPI samples presented the more pronounced color changes among samples. Spray dried rose-mary without protein (RME) exhibited intermediate color changes, positioned between SPI and WPI groups (Fig. 6B). Overall, the color changes observed during 20 weeks of storage at room temperature ranged between 13.46 > ΔE > 3.88 for all experimental groups. Ac-cording to Obon (Obon et al., 2009) AE < 1.5 represents imperceptible changes to human eyes, while AE between 1.5 and 5 are detectable, but still considered discrete color modifications. For AE > 5, the color changes become more evident, possibly as a result of degradation and modifications of rosemary natural pigments (Çalıskan Koç and Nur Dirim, 2017). All spray dried groups presented good color stability, since the initial color change observed after four weeks of storage was maintained until the end of the 20-week storage (p > 0.05), with little fluctuation (Fig. 6B). The modifications observed for all spray dried rosemary groups were gentle when compared, for example, to the hot air-rotary drum drying of green peas that caused AE between 41.81 and 64.95 to the final product (Kaveh, Abbaspour-Gilandeh, & Chen, 2020).

Fig. 5. Concentrations of rosmarinic acid, carnosol, and carnosic acid in spray dried rosemary-protein particles along storage at room temperature. RME: rosemary extract alone; R–WPI: rosemary-whey protein isolate; R–WIN: rosemary-whey protein isolate/inulin; R–SPI: rosemary-soy protein isolate; SIN: rosemary-soy protein isolate/inulin. Concentrations were calculated based on the percentage of rosemary extract in each formulation, 100% in RME, and 55% in rosemary-protein formulations.

Fig. 6. Water activity (A) and total color difference (B) of spray dried rosemary-protein particles during storage at room temperature. RME: rosemary extract only; R–WPI: rosemary-whey protein isolate; R–WIN: rosemary-whey protein isolate/inulin blend; R–SPI: rosemary-soy protein isolate; R–SIN: rosemary-soy protein isolate/inulin. Bars indicate standard deviation.

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4. Conclusion

Our findings show improved solids recovery when protein carriers were used as wall material for spray drying of rosemary extract. Whey protein presented higher recovery when compared to soy protein sam-ples, and both were significantly higher than spray dried rosemary. Whey and soy proteins, with or without inulin, protected the integrity of the high valued antioxidants carnosic acid and carnosol from thermal degradation during spray drying, yielding higher concentrations in the final products when compared to spray dried rosemary extract alone. We also demonstrated that whey protein increased the bioaccessibility of carnosol and carnosic acid. The in vitro cell reactive oxygen species (ROS) and nitric oxide (NO) assays indicated that the rosemary-protein formulations at 55% rosemary extract were comparable to the 100% rosemary extract without protein, meaning that the protein protected the antioxidant compounds from thermal degradation during spray drying and preserved their antioxidant activities. Stability study for 20 weeks showed that the concentration of carnosic acid and carnosol remained stable, and retained higher concentration levels compared to spray dried rosemary alone. All spray dried groups presented good color stability during 20 weeks of storage at room temperature. Overall, we demonstrated that spray dried rosemary extracts with whey or soy protein isolates constitute a creative solution to deliver preserved phy-tochemicals in a high-protein food format. All findings in this study favor the use of whey and soy protein isolates, not only as production enhancers, but also as valuable assets to protect the most valuable rosemary antioxidants during digestion and storage. The simplicity of the preparation method of these protein-polyphenol particles and rela-tively low cost of spray drying encourage the use of these particle in the food industry.

Funding

This work was supported by USDA NIFA Hatch Project 02689.

CRediT authorship contribution statement

Mary H. Grace: Conceptualization, Methodology, Investigation, Writing – original draft, Formal analysis. Roberta Hoskin: Conceptu-alization, Methodology, Investigation, Writing, Formal analysis. Jia Xiong: Methodology, Formal analysis, Writing, Visualization. Mary Ann Lila: Supervision, Writing – review & editing, Funding acquisition.

Declaration of competing interest

The authors have declared no conflicts to declare.

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