Intestinal Barrier Protective Study of Jujube Peel Polyphenols/Zein Complexes by a Combined Caco-2 Cell and Caenorhabditis elegans Model: A Perspective of Proteomics.

Jujube peels have been recognized as a promising resource of several bioactive ingredients. The main composition of jujube peel polyphenols (JPP) has been identified as rutin, kaempferol-3-O-rutinosid, and salicylic acid. The JPP/zein complexes, whose bioavailability reached 69.73% ± 5.06% in vitro, have been formed successfully. The Caco-2 cell and Caenorhabditis elegans (C. elegans) models have been combined to detect the intestinal barrier protective effect of JPP and its complexes. Results showed that JPP/zein complexes contain better protection capability than JPP in both models. In the Caco-2 cell model, the complex relieved intestinal barrier damage by regulating the tight junction proteins. Moreover, the lysosome pathway has been activated, further regulating immune responses and lipid transportation, improving the barrier function of C. elegans after incubation with JPP/zein complexes according to the proteomics study. This work provides new insights into intestinal barrier protection with bioactive compounds.

Effect of multi-mode sweep frequency ultrasound pretreatment on properties of the zeins and ACE inhibitory peptides activity of the hydrolysates.

Effects of sweep frequency ultrasound (SFU) pretreatment of a new multifunctional ultrasonic equipment on hydrolysis characteristics of zeins and angiotensin-converting enzyme (ACE) inhibitory activity of zein hydrolysates were investigated. Degree of hydrolysis of zeins reached the highest of 25.93 % and 25.72 % at 40 kHz and 25/40 kHz, respectively. While 25/40 kHz increased solubility, surface hydrophobicity, particle size uniform of zeins and ACE inhibitory activities of the hydrolysates significantly. Endogenous fluorescence indicated that 25/40 kHz promoted unfolding of protein molecules and exposure of hydrophobic residues, thereby facilitating enzymatic hydrolysis. Circular dichroism spectrum and Fourier transform infrared spectrometer illustrated that 25/40 kHz unfolded protein molecules and decreased α-helical contents remarkably. Gel permeation chromatography showed that more small-molecule active peptides were obtained from hydrolysates at 25/40 kHz. In conclusion, SFU pretreatment at 25/40 kHz with the new equipment before proteolysis is an efficient method to improve ACE inhibitory activity of the hydrolysates.

Electrospun Mucoadhesive Zein/PVP Fibroporous Membrane for Transepithelial Delivery of Propranolol Hydrochloride.

Mucoadhesive drug delivery systems have been extensively studied to effectively reduce the limitations of conventional drug delivery systems. Zein and polyvinyl pyrrolidone (PVP) are appraised for mucoadhesive properties. This study focuses on developing a mechanically stable zein/PVP electrospun membrane for propranolol hydrochloride (PL) transport. Fourier transform infrared, Raman spectra, and swelling studies gave evidence for PVP crosslinking, whereas circular dichroism spectroscopy revealed crosslinking of zein owing to the conformational change from α-helix to ß-sheet. A 10 h thermal treatment of zein/PVP imparted 3.92 ± 0.13 MPa tensile strength to the matrix. Thermally crosslinked electrospun zein/PVP matrix showed 22.1 ± 0.1 g mm work of adhesion in porcine buccal mucosa tissue. Qualitative and quantitative evaluation of cytotoxicity in RPMI 2650 has been carried out. The in vitro drug release profile of PL from thermally crosslinked zein/PVP best fitted with the Korsmeyer-Peppas model. Immunostaining of ß-catenin adherens junctional protein confirmed the absence of paracellular transport through the junctional opening. Still, drug permeation was observed through the porcine buccal mucosa, attributed to the transcellular transport of PL owing to its lipophilicity. The ex vivo permeation of PL through porcine buccal mucosa was also evaluated.

Two γ-zeins induce the unfolded protein response.

The rapid, massive synthesis of storage proteins that occurs during seed development stresses endoplasmic reticulum (ER) homeostasis, which activates the ER unfolded protein response (UPR). However, how different storage proteins contribute to UPR is not clear. We analyzed vegetative tissues of transgenic Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the common bean (Phaseolus vulgaris) soluble vacuolar storage protein PHASEOLIN (PHSL) or maize (Zea mays) prolamins (27-kDa γ-zein or 16-kDa γ-zein) that participate in forming insoluble protein bodies in the ER. We show that 16-kDa γ-zein significantly activates the INOSITOL REQUIRING ENZYME1/BASIC LEUCINE ZIPPER 60 (bZIP60) UPR branch-but not the bZIP28 branch or autophagy-leading to induction of major UPR-controlled genes that encode folding helpers that function inside the ER. Protein blot analysis of IMMUNOGLOBULIN-BINDING PROTEIN (BIP) 1 and 2, BIP3, GLUCOSE REGULATED PROTEIN 94 (GRP94), and ER-localized DNAJ family 3A (ERDJ3A) polypeptides confirmed their higher accumulation in the plant expressing 16-kDa γ-zein. Expression of 27-kDa γ-zein significantly induced only BIP3 and ERDJ3A transcription even though an increase in GRP94 and BIP1/2 polypeptides also occurred in this plant. These results indicate a significant but weaker effect of 27-kDa γ-zein compared to 16-kDa γ-zein, which corresponds with the higher availability of 16-kDa γ-zein for BIP binding, and indicates subtle protein-specific modulations of plant UPR. None of the analyzed genes was significantly induced by PHSL or by a mutated, soluble form of 27-kDa γ-zein that traffics along the secretory pathway. Such variability in UPR induction may have influenced the evolution of storage proteins with different tissue and subcellular localization.

Carbonized zein nanosheets with intrinsic enzyme-mimicking activities and high photothermal conversion efficiency for synergistic cancer therapy.

With the rapid development of biology and nanotechnology, designing nanomaterials with intrinsic enzyme-like activities has attracted huge attention in recent years. Herein, for the first time, we use zein as a new protein precursor to prepare N-rich carbonized zein nanosheets (C-Zein) via facile pyrolysis. Zein is an inert, biodegradable and sustainable natural biopolymer. After high-temperature carbonization, zein can be converted into highly catalytically active C-Zein, which can possess excellent peroxidase- and oxidase-like catalytic activities. Such intrinsic enzyme-like activities of C-Zein are closely related to its graphitization degree, the ratio of graphitic nitrogen and the formation of disordered graphene. Intriguingly, C-Zein also exhibits high photothermal conversion efficiency in the near-infrared (NIR) region. Coupling their unique photothermal and catalytic properties, the as-prepared C-Zein can act as a robust agent for synergistic photothermal-catalytic cancer treatment under the irradiation of NIR light. We expect that this work paves the way to use zein for designing efficient artificial enzymes and accelerate further growth in exploring its new biomedical and pharmaceutical applications.

Chondroitin sulfate-hybridized zein nanoparticles for tumor-targeted delivery of docetaxel.

Chondroitin sulfate-hybridized zein nanoparticles (zein/CS NPs) were developed for targeted delivery of docetaxel, which exhibited mean diameters of 157.8 ± 3.6 nm and docetaxel encapsulation efficiency of 64.2 ± 1.9 %. Docetaxel was released from the NPs in a sustained manner (∼72 h), following first-order kinetics. The zein/CS NPs showed improved colloidal stability, maintaining the initial size in serum for 12 h. The pre-treatment of CS reduced the uptake efficiency of the NPs by 23 % in PC-3 cells, suggesting the involvement of CD44-mediated uptake mechanism. The NPs showed 2.79-fold lower IC50 values than free docetaxel. Enhanced tumor accumulation of the NPs was confirmed in PC-3 xenograft mice by near-infrared fluorescence imaging (35.3-fold, versus free Cy5.5). The NPs exhibited improved pharmacokinetic properties (9.5-fold longer terminal half-life, versus free docetaxel) and anti-tumor efficacy comparable to Taxotere with negligible systemic toxicity, suggesting zein/CS NPs could be a promising nanoplatform for targeted cancer therapy.

In situ phase-changeable 2D MXene/zein bio-injection for shear wave elastography-guided tumor ablation in NIR-II bio-window.

Localized tumor photothermal cancer ablation is a minimally invasive therapeutic modality for combating cancer, but it often suffers from low therapeutic efficacy and poor precision due to the poor accumulation and non-uniform distribution of used photothermal-conversion agents in tumor tissue via the typical intravenous administration. To address this, an injectable and phase-changeable composite bio-injection consisting of biocompatible two-dimensional (2D) niobium carbide (Nb2C) MXene and the plant-originating protein, zein, has been engineered for near infrared (NIR)-II-triggered tumor photothermal ablation. Zein can respond to aqueous microenvironments and also external photo-triggers from the NIR-II bio-window (1064 nm), and transforms into a solid bio-implant after solvent exchange between ethanol and water. Which, thus, traps Nb2C MXene and heat, improving ablation efficiency and enabling the precise and complete eradication of 4T1 breast tumor cells without additional safety concerns. More significantly, shear wave elastography (SWE) as a deep-penetration imaging mode that can reflect the ablated outcomes via monitoring tissue density variation, has been employed to guide the photo-thermal ablation process to further improve the ablation precision. Thus, this compatible and phase-changeable bio-injection capable of improving photo-thermal ablation efficiency holds great potential in clinical applications.

Characterization of the protein and peptide of excipient zein by the multi-enzyme digestion coupled with nano-LC-MS/MS.

Zein, a class of prolamine proteins extracted from maize, is extensively used in the food and pharmaceutical industries. Characterization of its components is essential for quality control and safety evaluation. We performed in silico digestion of zein proteins using tandem combinations of different proteinases, to improve protein sequence coverage and subsequent identification by nano-LC-MS/MS analysis. Trypsin/chymotrypsin yielded the highest protein sequence coverage of up to 79.5% and increased the number of proteins from 11 to 35 compared to trypsin/Lys-C. Besides, SDS-PAGE analysis revealed 37 proteins in the zein extract, as well as the possibility of protein polymers. Also, 420 peptides originating from 71 proteins were identified, of which 116 were predicted as bioactive by in silico approach. In conclusion, in silico prediction coupled with multi-enzyme digestion can significantly improve the coverage of complex zein protein proteome, and the potential function of zein proteins and peptides need be further investigated.

Plant-derived protein bodies as delivery vehicles for recombinant proteins into mammalian cells.

The encapsulation of biopharmaceuticals into micro- or nanoparticles is a strategy frequently used to prevent degradation or to achieve the slow release of therapeutics and vaccines. Protein bodies (PBs), which occur naturally as storage organelles in seeds, can be used as such carrier vehicles. The fusion of the N-terminal sequence of the maize storage protein, γ-zein, to other proteins is sufficient to induce the formation of PBs, which can be used to bioencapsulate recombinant proteins directly in the plant production host. In addition, the immunostimulatory effects of zein have been reported, which are advantageous for vaccine delivery. However, little is known about the interaction between zein PBs and mammalian cells. To better understand this interaction, fluorescent PBs, resulting from the fusion of the N-terminal portion of zein to a green fluorescent protein, was produced in Nicotiana benthamiana leaves, recovered by a filtration-based downstream procedure, and used to investigate their internalization efficiency into mammalian cells. We show that fluorescent PBs were efficiently internalized into intestinal epithelial cells and antigen-presenting cells (APCs) at a higher rate than polystyrene beads of comparable size. Furthermore, we observed that PBs stimulated cytokine secretion by epithelial cells, a characteristic that may confer vaccine adjuvant activities through the recruitment of APCs. Taken together, these results support the use of zein fusion proteins in developing novel approaches for drug delivery based on controlled protein packaging into plant PBs.

Biodegradable zein film composites reinforced with chitosan nanoparticles and cinnamon essential oil: Physical, mechanical, structural and antimicrobial attributes.

Natural bio-based zein films were prepared by incorporating cinnamon essential oil (CEO) and chitosan nanoparticles (CNPs) at 2% and 4% (w/w) amounts, respectively, in order to provide mechanical and antimicrobial functionalities. The physical, mechanical, structural and antibacterial properties of the enriched zein films were also scrutinized. The results showed that the combination of CEO-CNPs significantly improves the tensile strength and decreases the elongation of zein film composite. According to X-ray diffraction (XRD) results, zein film experienced more crystallinity in the presence of CNPs and also combination of CNPs-CEO. Nano-scale size of CNPs and their uniform distribution within the zein film were monitored by scanning electron microscopy (SEM) and proved by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The antimicrobial properties were investigated against Escherichia coli and Staphylococcus aureus, observing that their growth was considerably inhibited by addition of CEO alone and in combination with CNPs in zein films, while CNPs-loaded zein film had no significant effect on the growth of microorganisms. Thus, it can be concluded that the reinforced zein based composites could be suggested as potential degradable film-forming materials for food packaging applications.

Cellular Uptake and Intracellular Antioxidant Activity of Zein/Chitosan Nanoparticles Incorporated with Quercetin.

In this work, zein/chitosan nanoparticles (ZCPs-Q) were developed for encapsulating quercetin to overcome its lower water solubility and instability, and to concomitantly enhance its cellular uptake and intracellular antioxidant activity. This strategy enhanced quercetin solubility 753.6 and 9.95 times in water and PBS (7.4), respectively, and quercetin encapsulated in ZCPs remained stable after UV irradiation and heat treatment. ZCPs-Q could significantly attenuate AAPH induced erythrocyte hemolysis through the inhibition of ROS generation. It restored intracellular antioxidant enzyme (SOD and GSH-Px) activities to normal levels and inhibited intracellular malondialdehyde (MDA) formation. Simultaneously, ZCPs-Q showed a strong antioxidant activity in HepG2 cells with an EC50 value of 31.18 µg/mL, which was lower than free quercetin's 41.02 µg/mL. ZCPs enhanced the uptake efficiency of quercetin in Caco-2 cells, which contributed to the improvement of cellular antioxidant activities (CAA) evaluated with the CAA assay and AAPH-induced erythrocyte hemolysis assay. The designed route is particularly suitable for the encapsulation of water-insoluble nutraceuticals and for enhancing cell uptake and CAA.

Uniparental and transgressive expression of α-zeins in maize endosperm of o2 hybrid lines.

The α-zein gene family encodes the most abundant storage proteins of maize (Zea mays) endosperm. Members of this family are expressed in a parent-of-origin manner. To characterize this phenomenon further, we investigated the expression of a subset of α-zein polypeptides in reciprocal crosses between o2 lines that were characterized by a simplified α-zein pattern. Maize lines that suppressed the expression of α-zeins when used as female parents were identified. The suppression was cross-specific, occurring only when specific genetic backgrounds were combined. Four α-zein sequences that were sensitive to uniparental expression were isolated. Molecular characterization of these α-zeins confirmed that their expression or suppression depended on the genetic proprieties of the endosperm tissue instead of their parental origin. DNA methylation analysis of both maternally and paternally expressed α-zeins revealed no clear correlation between this epigenetic marker and parent-of-origin allelic expression, suggesting that an additional factor(s) is involved in this process. Genetic analyses revealed that the ability of certain lines to suppress α-zein expression was unstable after one round of heterozygosity with non-suppressing lines. Interestingly, α-zeins also showed a transgressive expression pattern because unexpressed isoforms were reactivated in both F2 and backcross plants. Collectively, our results suggest that parent-of-origin expression of specific α-zein alleles depends on a complex interaction between genotypes in a manner that is reminiscent of paramutation-like phenomena.

The ZmbZIP22 Transcription Factor Regulates 27-kD γ-Zein Gene Transcription during Maize Endosperm Development.

Zeins are the most abundant storage proteins in maize (Zea mays) kernels, thereby affecting the nutritional quality and texture of this crop. 27-kD γ-zein is highly expressed and plays a crucial role in protein body formation. Several transcription factors (TFs) (O2, PBF1, OHP1, and OHP2) regulate the expression of the 27-kD γ-zein gene, but the complexity of its transcriptional regulation is not fully understood. Here, using probe affinity purification and mass spectrometry analysis, we identified ZmbZIP22, a TF that binds to the 27-kD γ-zein promoter. ZmbZIP22 is a bZIP-type TF that is specifically expressed in endosperm. ZmbZIP22 bound directly to the ACAGCTCA box in the 27-kD γ-zein promoter and activated its expression in wild tobacco (Nicotiana benthamiana) cells. 27-kD γ-zein gene expression was significantly reduced in CRISPR/Cas9-generated zmbzip22 mutants. ChIP-seq (chromatin immunoprecipitation coupled to high-throughput sequencing) confirmed that ZmbZIP22 binds to the 27-kD γ-zein promoter in vivo and identified additional direct targets of ZmbZIP22. ZmbZIP22 can interact with PBF1, OHP1, and OHP2, but not O2. Transactivation assays using various combinations of these TFs revealed multiple interaction modes for the transcriptional activity of the 27-kD γ-zein promoter. Therefore, ZmbZIP22 regulates 27-kD γ-zein gene expression together with other known TFs.

Optimization of liquid fermentation conditions for biotransformation zein by Cordyceps militaris 202 and characterization of physicochemical and functional properties of fermentative hydrolysates

Abstract Cordyceps militaris 202 is a potential fungus for biotransformation zein, due to its various proteases, high tolerance and viability in nature. In this article, single factor experiment and response surface methodology were applied to optimize the liquid fermentation conditions and improve the ability of biotransformation zein. The optimized fermentation conditions were as follows: inoculum concentration of 19%, volume of liquor of 130 mL/500 mL and pH of 4.7. Under this condition, the degree of hydrolysis (DH) was 27.31%. The zein hydrolysates from fungi fermentation maintained a high thermal stability. Compared to the original zein, the zein hydrolysates were found to have high solubility, which most likely results in improved foaming and emulsifying properties. Overall, this research demonstrates that hydrolysis of zein by C. militaris 202 is a potential method for improving the functional properties of zein, and the zein hydrolysates can be used as functional ingredients with an increased antioxidant effect in both food and non-food applications.

Zein Microneedles for Localized Delivery of Chemotherapeutic Agents to Treat Breast Cancer: Drug Loading, Release Behavior, and Skin Permeation Studies.

Localized delivery of chemotherapeutic agents to treat breast cancer could limit their adverse drug reactions. The aim of this study was to investigate the influence of physico-chemical properties of chemotherapeutic agents in their loading, release behavior, and skin permeation using microneedles. Zein microneedles were fabricated using the micromolding technique containing 36 microneedles in a 1-cm2 area. These microneedles were loaded with two anti-breast cancer drugs, tamoxifen and gemcitabine, having different water solubilities. Entrapment or surface coating of chemotherapeutic agents in zein microneedles was optimized to achieve greater loading efficiency. The greatest loading achieved was 607 ± 21 and 1459 ± 74 µg for tamoxifen and gemcitabine using the entrapment approach, respectively. Skin permeation studies in excised porcine skin showed that the coating on microneedles approach results in greater skin deposition for tamoxifen; while the poke-and-patch approach would provide greater skin permeation for gemcitabine. Taken together, it can be concluded that different loading strategies and skin penetration approaches have to be studied for delivery of small molecules using polymeric microneedles.

Identification of New Anti-inflammatory Peptides from Zein Hydrolysate after Simulated Gastrointestinal Digestion and Transport in Caco-2 Cells.

Chronic inflammation is an underlying contributor to various chronic diseases. The objectives of this study were to investigate the anti-inflammatory activity of zein hydrolysate after simulated gastrointestinal digestion and Caco-2 cell absorption and to identify novel anti-inflammatory peptides after transport across Caco-2 cells. Three zein hydrolysates were prepared and further digested using gastrointestinal proteases; their transports were studied in Caco-2 cells. Anti-inflammatory activity was studied in endothelial EA.hy926 cells. Three zein hydrolysates and their digests significantly decreased the expression of tumor necrosis factor-α (TNF-α) induced pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) by 37.3-66.0%. Eleven novel peptides with 5-9 amino acid residues were sequenced; three peptides showed strong anti-inflammatory activity by inhibiting the VCAM-1 by 54-38.9% and intercellular cell adhesion molecule-1 (ICAM-1) by 36.5-28.6% at 0.2 mM. A new approach to identify novel anti-inflammatory peptides that could survive gastrointestinal digestion and absorption was developed.

Engineering sulfur storage in maize seed proteins without apparent yield loss.

Sulfur assimilation may limit the pool of methionine and cysteine available for incorporation into zeins, the major seed storage proteins in maize. This hypothesis was tested by producing transgenic maize with deregulated sulfate reduction capacity achieved through leaf-specific expression of the Escherichia coli enzyme 3'-phosphoadenosine-5'-phosphosulfate reductase (EcPAPR) that resulted in higher methionine accumulation in seeds. The transgenic kernels have higher expression of the methionine-rich 10-kDa δ-zein and total protein sulfur without reduction of other zeins. This overall increase in the expression of the S-rich zeins describes a facet of regulation of these proteins under enhanced sulfur assimilation. Transgenic line PE5 accumulates 57.6% more kernel methionine than the high-methionine inbred line B101. In feeding trials with chicks, PE5 maize promotes significant weight gain compared with nontransgenic kernels. Therefore, increased source strength can improve the nutritional value of maize without apparent yield loss and may significantly reduce the cost of feed supplementation.

Zein-based Nanocarriers as Potential Natural Alternatives for Drug and Gene Delivery: Focus on Cancer Therapy.

Protein nanocarriers possess unique merits including minimal cytotoxicity, numerous renewable sources, and high drug-binding capability. In opposition to delivery carriers utilizing hydrophilic animal proteins, hydrophobic plant proteins (e.g, zein) have great tendency in fabricating controlled-release particulate carriers without additional chemical modification to stiffen them, which in turn evades the use of toxic chemical crosslinkers. Moreover, zein is related to a class of alcohol-soluble prolamins and generally recognized as safe (GRAS) carrier for drug delivery. Various techniques have been adopted to fabricate zein-based nanoparticulate systems including phase separation coacervation, spray-drying, supercritical anti-solvent approach, electrospinning and self-assembly. This manuscript reviews the recent advances in the zein-based colloidal nano-carrier systems such as nanospheres, nanocapsules, micelles and nanofibers with a special focus on their physicochemical characteristics and drug delivery applications.

Green self-assembly of zein-conjugated ZnO/Cd(OH)Cl hierarchical nanocomposites with high cytotoxicity and immune organs targeting.

Inorganic nanomedicines in the fight against cancer have progressed rapidly during recent years, with the synergistic advantages of multifunctional nanosystems compared to single component. Herein, a drug-combination opinion was introduced into "nanomedicine" based on the understanding of Trojan horse-anti-tumor mechanism of inorganic nano-medicines. Moreover, we reported the green and facile synthesis route of mono-dispersed and rod-like zein-conjugated ZnO/Cd(OH)Cl hierarchical nanocomposites. We found that the nanocomposites exhibited high-efficiency killing ability to tumor cells through lipid peroxidation mediated-membrane disintegration route. The safety studies in BALB/c mice didn't detect injection anaphylaxis, hemolysis and cytotoxicity. More interestingly, the nano-composites could specially accumulate in liver and kidney, which will be helpful for targeting cure to these regional cancers.

Dietary actinidin from kiwifruit (Actinidia deliciosa cv. Hayward) increases gastric digestion and the gastric emptying rate of several dietary proteins in growing rats.

Dietary actinidin influences the extent to which some dietary proteins are digested in the stomach, and it is hypothesized that the latter modulation will in turn affect their gastric emptying rate (GE). In this study, the effect of dietary actinidin on GE and gastric digestion of 6 dietary protein sources was determined in growing rats. Each dietary protein source [beef muscle, gelatin, gluten, soy protein isolate (SPI), whey protein isolate, and zein] was included in 2 semisynthetic diets as the sole nitrogen source. For each protein source, 1 of the 2 diets contained actinidin [76.5 U/g dry matter (DM)] in the form of ground freeze-dried green kiwifruit (Actinidia deliciosa cv. Hayward), whereas the other diet contained freeze-dried gold kiwifruit (Actinidia chinensis cv. Hort16A), which is devoid of actinidin (3.4 U/g DM). For both diets, dietary kiwifruit represented 20% of the diet on a DM basis. The real-time GE was determined in rats gavaged with a single dose of the diets using magnetic resonance spectroscopy over 150 min (n = 8 per diet). Gastric protein digestion was determined based on the free amino groups in the stomach chyme collected from rats fed the diets (n = 8 per diet) that were later killed. GE differed across the protein sources [e.g., the half gastric emptying time (T(½)) ranged from 157 min for gluten to 266 min for zein] (P < 0.05). Dietary actinidin increased the gastric digestion of beef muscle (0.6-fold), gluten (3.2-fold), and SPI (0.6-fold) and increased the GE of the diets containing beef muscle (43% T(½)) and zein (23% T(½); P < 0.05). There was an inverse correlation between gastric protein digestion and DM retained in the stomach (r = -0.67; P < 0.05). In conclusion, dietary actinidin increased gastric protein digestion and accelerated the GE for several dietary protein sources. GE may be influenced by gastric protein digestion, and dietary actinidin can be used to modulate GE and protein digestion in the stomach of some dietary protein sources but not others.