A novel automated protocol for ice crystal segmentation analysis using Cellpose and Fiji.

Accurate measurement of ice crystal size is an essential step in quantitative ice recrystallization inhibition (IRI) analysis using the sucrose sandwiching assay (SSA) and splat assay (SA). Here, we introduce a novel method of measuring ice crystal size and shape using Fiji and Cellpose, an anatomical segmentation algorithm, to address the time-consuming and limited number of ice particle determination associated with the mean largest grain size measurement. This new automated approach, displaying rapid segmentation of ∼70 s per image, measures every ice crystal in an image field of view, consequently reducing bias introduced by subjectively selecting the largest crystals in an image. Consistent in determining a diverse set of crystal sizes and shapes, this method allows for the evaluation of ice crystals using Feret's diameter, a parameter that better accounts for irregular particle shape. This method provides new outputs such as standard deviation, particle size distributions of a population of ice crystals, and circularity to characterize and further provide insight into an analyte's IRI ability. Applicable to the SSA, the "shape descriptor" measurement can be used to quantify ice binding. This work presents a novel and accurate approach for ice crystal quantitative analysis.

Antioxidant, Immunostimulatory, and Anticancer Properties of Hydrolyzed Wheat Bran Mediated through Macrophages Stimulation.

Previous studies demonstrated that enzymatic hydrolysis enhances wheat bran (WB) biological properties. This study evaluated the immunostimulatory effect of a WB hydrolysate (HYD) and a mousse enriched with HYD (MH) before and after in vitro digestion on murine and human macrophages. The antiproliferative activity of the harvested macrophage supernatant on colorectal cancer (CRC) cells was also analyzed. MH showed significantly higher content than control mousse (M) in soluble poly- and oligosaccharides (OLSC), as well as total soluble phenolic compounds (TSPC). Although in vitro gastrointestinal digestion slightly reduced the TSPC bioaccessibility of MH, ferulic acid (FA) levels remained stable. HYD showed the highest antioxidant activity followed by MH, which demonstrated a greater antioxidant activity before and after digestion as compared with M. RAW264.7 and THP-1 cells released the highest amounts of pro-inflammatory cytokines after being treated with 0.5 mg/mL of digested WB samples. Treatment with digested HYD-stimulated RAW264.7 supernatant for 96 h showed the most anticancer effect, and spent medium reduced cancer cell colonies more than direct WB sample treatments. Although a lack of inner mitochondrial membrane potential alteration was found, increased Bax:Bcl-2 ratio and caspase-3 expression suggested activation of the mitochondrial apoptotic pathway when CRC cells were treated with macrophage supernatants. Intracellular reactive oxygen species (ROS) were positively correlated with the cell viability in CRC cells exposed to RAW264.7 supernatants (r = 0.78, p < 0.05) but was not correlated in CRC cells treated with THP-1 conditioned media. Supernatant from WB-stimulated THP-1 cells may be able to stimulate ROS production in HT-29 cells, leading to a decrease of viable cells in a time-dependent manner. Therefore, our present study revealed a novel anti-tumour mechanism of HYD through the stimulation of cytokine production in macrophages and the indirect inhibition of cell proliferation, colony formation, and activation of pro-apoptotic proteins expression in CRC cells.

Antioxidant and Immunomodulatory Properties of Partially purified Exopolysaccharide from Lactobacillus Casei Isolated from Chinese Northeast Sauerkraut.

BACKGROUND: Exopolysaccharides (EPS) from Lactobacillus spp. have been found to have biological activities. Our previous work demonstrated the antibiofilm activity of EPS from Lactobacillus casei NA-2 (L.casei NA-2) isolated from northeast Chinese sauerkraut (Suan Cai). The present study has focussed on the antioxidant and immunomodulatory activities of the EPS in vitro. METHODS: Antioxidant properties of the EPS were evaluated by the radical-scavenging activities in vitro. The immunomodulatory effects of EPS were assayed by measuring nitric oxide (NO), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and reactive oxygen species (ROS) in RAW 264.7 macrophages, and the mechanism was investigated through NF-κB and JNK. RESULT: EPS contains 88% total sugar, with the molecular weights (Mw) of 1.3 × 106 Da, 6.4 × 105 Da, 2.0 × 105 Da, and 1.4 × 104 Da. EPS showed antioxidant activity by scavenging hydroxyl radicals (42% at 1.2 mg/mL), superoxide radicals (76% at 100 µg/mL), and DPPH (80% at 10 mg/mL); and did not affect the proliferation of unstimulated or lipopolysaccharide (LPS)-induced RAW 264.7 cells at the concentrations ranging from 31.25 to 500 µg/mL. Results showed EPS promoted the production of ROS and TNF-α involved in NF-κB p65 and JNK signaling pathways in unstimulated RAW 264.7 cells. On the other hand, the levels of NO and iNOS were reduced after EPS treatment in LPS-induced RAW 264.7 cells. CONCLUSION: Our results showed the protective effect against oxidative damage and potential immunomodulatory and anti-inflammatory properties of EPS from Lactobacillus casei NA-2.

Bovine Milk Exosomes Affect Proliferation and Protect Macrophages against Cisplatin-Induced Cytotoxicity.

BACKGROUND: Exosomes are extracellular vesicles involved in intercellular communication. The objectives were to characterize bovine milk exosomes (BME) and determine its effect on RAW 264.7 macrophages. METHODS: BME were isolated using differential centrifugation and characterized by particle size and the presence of exosomal markers Alix, TSG101, and CD81. The effect of in vitro digestion and different pH on the stability of BME was investigated. The biological activity of BME in RAW 264.7 macrophages was conducted by assessing proliferation and cell cycle. Moreover, the protective effect of exosomes on cisplatin-induced cytotoxicity was evaluated. RESULTS: BME have an average particle size of 106.8 ± 3.4 nm and expressed Alix, TSG101, and CD81. TSG101 was detected after digestion and exposure to different pH values. Cell-cycle analysis showed that BME reduced the percentage of apoptotic cells while arresting the cells in G2/M phase accompanied by differential expression of proliferation markers p53, p21, cyclin D1, and ß-catenin. Exosomes protected macrophages against cisplatin-induced cytotoxicity. CONCLUSION: Our results showed for the first time the effect of BME on the proliferation of RAW 264.7 macrophages and its protective effect against chemotherapeutic drug-induced cytotoxicity. Potential effect of BME on immune system must be studied.

Combinatorial effect of blueberry extracts and oxaliplatin in human colon cancer cells.

The use of natural compounds to potentiate the effect of drugs and lower their adverse effects is an active area of research. The objective is to determine the effect of combined blueberry extracts (BE) and oxaliplatin (OX) in colon cancer cells. The results demonstrated that treatments of BE/OX showed inhibitory effects on HCT-116 cell and nontoxic effect on CCD-18Co normal colon cells. Flow cytometry analysis indicated that treatment with the BE, OX or in combination could induce G0/G1 cell cycle arrest, apoptosis, increase of reactive oxygen species, and induce loss of mitochondrial membrane potential in HCT-116 cells. Furthermore, after treatments, the expression of inflammatory cytokines was decreased, cyclin D1 and CDK4 were decreased; caspases-3 and 9 were activated; the Akt/Bad/Bcl-2 pathway was modulated. Moreover, the combination treatment had a considerably higher growth inhibitory effect on human colon cancer HCT-116 cells than that of BE or oxaliplatin alone. Our results showed that BE increased the anticolon cancer effect of OX making it an attractive strategy as adjuvant therapy to potentially reduce the adverse side effects associated with chemotherapeutic drugs.

Sorghum Phenolics Inhibits Inflammasomes in Lipopolysaccharide (LPS)-Primed and Adenosine Triphosphate (ATP)-Activated Macrophages.

Sorghum contains phenolic compounds with reported biological activities. The objective was to evaluate the ability of sorghum phenolic extract to inhibit inflammasomes in THP-1 human macrophages. THP-1 human macrophages was pre-treated with sorghum phenolics and the inflammasome was activated by lipopolysaccharide and adenosine triphosphate treatment. Treatment of macrophages with 50 µg sorghum extract/mL reduced IL-1ß and IL-18 secretion by 59.7 and 32.0%, respectively, associated with caspase-1 activity reduction. Moreover, the production of intracellular reactive oxygen species was reduced. Our data showed the potential role of sorghum phenolics in diseases associated with aberrant inflammasomes activation.

Epithelial-to-Mesenchymal Transition in Paclitaxel-Resistant Ovarian Cancer Cells Is Downregulated by Luteolin.

Ovarian cancer (OVCA) is the deadliest of all gynecological cancers which is attributed to late presentation, persistence, and development of chemoresistance. The objectives were to evaluate the association between OVCA paclitaxel-resistance and epithelial-to-mesenchymal transition (EMT) and to determine the capability of luteolin to chemosensitize OVCA cells. X10 and X22 cells were 11.8-25.3-fold and 7.8-8.6-fold resistant to paclitaxel than 1AP cells. X10 and X22 cells exhibited a mesenchymal phenotype, while 1AP has an epithelial characteristics. Furthermore, the expression of the epithelial marker E-cadherin was downregulated, while mesenchymal markers Vimentin and N-cadherin were upregulated in X10 and X22 cells when compared to 1AP cells. Transcription factors Snail, Slug, and Twist1 were upregulated in X10 cells, while Twist1 was highly expressed in X22 cells. Luteolin treatment caused cytotoxicity being most potent to X10 OVCA cells. Treatment of non-cytotoxic dose of luteolin at 15.625 µM chemosensitized X10 and X22 OVCA cells to paclitaxel as evidenced by reduced ED50 values from 11.8 to 0.2 µM and 8.6 to 3.6 µM for X10 and X22 cells, respectively. Moreover, luteolin treatment led to a more epithelial phenotype of X10 and X22 cells and modification of EMT markers indicating reversal of EMT. The mechanism involved is through reduction of phosphorylation of FAK and ERK leading to reduced nuclear translocation of p65. Our results highlight the significance of EMT in OVCA resistance to paclitaxel and warrant the investigation of luteolin as a potential therapeutic agent in chemoresistant OVCA. J. Cell. Physiol. 232: 391-401, 2017. © 2016 Wiley Periodicals, Inc.

Pepsin-pancreatin protein hydrolysates from extruded amaranth inhibit markers of atherosclerosis in LPS-induced THP-1 macrophages-like human cells by reducing expression of proteins in LOX-1 signaling pathway.

BACKGROUND: Atherosclerosis is considered a progressive disease that affects arteries that bring blood to the heart, to the brain and to the lower end. It derives from endothelial dysfunction and inflammation, which play an important role in the thrombotic complications of atherosclerosis. Cardiovascular disease is the leading cause of death around the world and one factor that can contribute to its progression and prevention is diet. Our previous study found that amaranth hydrolysates inhibited LPS-induced inflammation in human and mouse macrophages by preventing activation of NF-κB signaling. Furthermore, extrusion improved the anti-inflammatory effect of amaranth protein hydrolysates in both cell lines, probably attributed to the production of bioactive peptides during processing. Therefore, the objective of this study was to compare the anti-atherosclerotic potential of pepsin-pancreatin hydrolysates from unprocessed and extruded amaranth in THP-1 lipopolysaccharide-induced human macrophages and suggest the mechanism of action. RESULTS: Unprocessed amaranth hydrolysate (UAH) and extruded amaranth hydrolysate (EAH) showed a significant reduction in the expression of interleukin-4 (IL-4) (69% and 100%, respectively), interleukin-6 (IL-6) (64% and 52%, respectively), interleukin-22 (IL-22) (55% and 70%, respectively). Likewise, UAH and EAH showed a reduction in the expression of monocyte-chemo attractant protein-1 (MCP-1) (35% and 42%, respectively), transferrin receptor-1 (TfR-1) (48% and 61%, respectively), granulocyte-macrophage colony-stimulating factor (GM-CSF) (59% and 63%, respectively), and tumor necrosis factor-α (TNF-α) (60% and 63%, respectively). Also, EAH reduced the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) (27%), intracellular adhesion molecule-1 (ICAM-1) (28%) and matrix metalloproteinase-9 (MMP-9) (19%), important molecular markers in the atherosclerosis pathway. EAH, led to a reduction of 58, 52 and 79% for LOX-1, ICAM-1 and MMP-9, respectively, by confocal microscopy. CONCLUSIONS: Extruded amaranth hydrolysate showed potential anti-atherosclerotic effect in LPS-induced THP-1 human macrophage-like cells by reducing the expression of proteins associated with LOX-1 signaling pathway.

Succinylation of zein and gelatin hydrolysates improved their ice recrystallization inhibition activity.

The goal of this research was to enhance the ice recrystallization inhibition (IRI) activity of zein and gelatin hydrolysates (ZH and GH, respectively) by succinylation modification. ZH was prepared by Alcalase treatment for 3 h and then modified by succinic anhydride (SA); whereas GH was made by Alcalase hydrolysis for 0.25 h and succinylated by n-octylsuccinic anhydride (OSA). After 0.5 h of annealing at -8 °C at 40 mg/mL, modified hydrolysates decreased the average Feret's diameter of ice crystal from 50.2 µm (polyethylene glycol, negative control) to 28.8 µm (SA modified ZH) and 29.5 µm (OSA modified GH) in comparison to the unmodified hydrolysates, which had the crystal size of 47.2 µm (ZH) and 45.4 µm (GH). Also, the two succinylated samples had altered surface hydrophobicity, which potentially contributed to their enhanced IRI activity. Our results indicate that succinylation of food-derived protein hydrolysates can improve their IRI activity.

Bioactive compounds, antioxidant capacity and anti-inflammatory activity of native fruits from Brazil.

The purpose of this study was to extract, identify, and quantify the phenolic compounds in grumixama (Eugenia brasilienses Lam.) and guabiju (Myrcianthes pungens), native fruits from southern region of Brazil, and to explore their antioxidant and anti-inflammatory properties. The phenolic compounds were extracted with acidified water and acidified methanol and evaluated for their bioactive constituents, antioxidant capacity, and anti-inflammatory properties. Spectrophotometric quantification shows tannins to be the most prevalent at 2.3 to 5.8 g/100g fresh fruit with acidified methanol containing higher concentrations of different phenolics than acidified water. HPLC analysis indicates that gallic acid, catechin, vanillic acid, and ellagic acid are the most prevalent phenolics in the two fruits extracts. Scavenging of DPPH and NO radicals showed inhibition by as much as 95% and 80%, respectively, at 2.5 gallic acid equivalent (GAE)/mL of the extract. At 50 µg GAE/mL, the release of pro-inflammatory molecules NO and IL-6 was significantly reduced with acidified methanol extract having higher inhibitory activity. Our results revealed that these native fruits, grown in the south of Brazil, are rich sources of phenolic compounds and have great antioxidant and anti-inflammatory activity.

Ice Recrystallization Inhibition Activity of Soy Protein Hydrolysates.

Identifying and developing ice recrystallization inhibitors from sustainable food proteins such as soy protein isolate (SPI) can lead to practical applications in both pharmaceutical and food industries. The objective of this study was to investigate the ice recrystallization inhibition (IRI) activity of SPI hydrolysates, and this was achieved by using an IRI activity-guided fractionation approach and relating IRI activity to interfacial molecular activity measured by vibrational sum frequency generation (VSFG). In addition, the impact of molecular weight (MW) and enzyme specificity was analyzed using three different proteases (Alcalase, trypsin, and pancreatin) and varying hydrolysis times. Using preparative chromatography, hydrolysates from each enzyme treatment were fractionated into five different MW fractions (F1-F5), which were then characterized by high-performance liquid chromatography (HPLC). All SPI hydrolysates had IRI activity, resulting in a 57-29% ice crystal diameter reduction when compared to native SPI. The F1 fraction (of 4-14 kDa) was most effective among all tested hydrolysates, while the lower MW peptide fractions lacked activity. One sample (SPI-ALC 20-F1) had a 52% reduction of ice crystal size at a lower concentration of 2% compared to the typical 4% used. SFG showed a difference in H-bonding and hydrophobic interactions of the molecules on the water/air interface, which may be linked to IRI activity. This study demonstrates for the first time the ability of SPI hydrolysates to inhibit ice crystal growth and the potential application of SFG to study molecular interaction at the interface that may help illustrate the mechanism of action.

Lunasin reduces the susceptibility of IL-10 deficient mice to inflammatory bowel disease and modulates the activation of the NLRP3 inflammasome.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that can cause severe damage to the gastrointestinal tract leading to lower quality of life and productivity. Our goal was to investigate the protective effect of the soy peptide lunasin in an in vivo model of susceptibility to IBD and to identify the potential mechanism of action in vitro. In IL-10 deficient mice, oral administration of lunasin reduced the number and frequency of mice exhibiting macroscopic signs of susceptibility to inflammation and significantly decreased levels of the proinflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-18 by up to 95%, 90%, 90%, and 47%, respectively, in different sections of the small and large intestines. Dose-dependent decrease of caspase-1, IL-1ß, and IL-18 in LPS-primed and ATP-activated THP-1 human macrophages demonstrated the ability of lunasin to modulate the NLRP3 inflammasome. We demonstrated that lunasin can decrease susceptibility to IBD in genetically susceptible mice by exerting anti-inflammatory properties.

Bovine milk exosomes affected proliferation of macrophages under hypoxia.

Exosomes are extracellular vesicles implicated in cell-to-cell communication. The objective was to investigate the effect of exosomes in macrophages under hypoxia. Exosomes were isolated from skim milk using differential centrifugation and was characterized by particle size and exosomal markers TSG101, CD81, and ALIX. The effect of exosomes on macrophages under hypoxia was investigated by assessing proliferation, cytokine and reactive oxygen species (ROS) production, and cell cycle. Exosomes treatment increased the cell viability under hypoxia while ROS production was significantly reduced. The production of TNF-α was not affected by hypoxia alone but increased in a dose-dependent manner in cells treated with exosomes under hypoxic condition. Hypoxia arrested cells in the G0/G1 phase whereas exosome treatment reduced the cell in this phase. Our study found that bovine milk exosomes affect the proliferation of macrophages under hypoxia and are able to reverse the adverse effects of hypoxia on cell viability.

Nanoencapsulation of apigenin with whey protein isolate: physicochemical properties, in vitro activity against colorectal cancer cells, and bioavailability.

Incorporating lipophilic phytochemicals with anti-cancer activities in functional beverages requires an appropriate nanoencapsulation technology. The present objective was to encapsulate apigenin with whey protein isolate (WPI) utilizing a pH-cycle method and subsequently characterize physicochemical properties, the in vitro anticancer activities against human colorectal HCT-116 and HT-29 cancer cells, and the in vivo bioavailability. Up to 2.0 mg/mL of apigenin was nanoencapsulated with 1.0 mg/mL WPI, with an encapsulation efficiency of up to 98.15% and loading capacity of up to 196.21 mg/g-WPI. Nanodispersions were stable during storage, and apigenin became amorphous after encapsulation. Nanoencapsulation and in vitro digestion did not reduce the anti-proliferative activity of apigenin. Nanoencapsulation of apigenin enhanced the cellular uptake, the pro-apoptotic effects, and the bioavailability in the mice's blood and colon mucosa when comparing to the unencapsulated apigenin. Therefore, the present work may be significant to incorporate lipophilic phytochemicals in functional beverages for disease prevention.

The role of nutraceutical proteins and peptides in apoptosis, angiogenesis, and metastasis of cancer cells.

The process of carcinogenesis is complex and not easy to eliminate. It includes the initial occurrence of genetic alterations which can lead to the inactivation of tumor-suppressor genes and further accumulation of genetic alterations during tumor progression. Looking for food and food components with biological properties, collectively called nutraceuticals, that can hinder such alterations and prevent the inactivation of tumor-suppressor genes is a very promising area for cancer prevention. Proteins and peptides are one group of nutraceuticals that show potential results in preventing the different stages of cancer including initiation, promotion, and progression. In this review, we summarized current knowledge on the use of nutraceutical proteins and peptides in cancer prevention and treatment. We focused on the role of plant protease inhibitors, lactoferrin and lactoferricin, shark cartilage, plant lectins, and lunasin in the apoptosis, angiogenesis, and metastasis of cancer cells. Also included are studies on bioavailability and clinical trials conducted on these promising proteins and peptides.

Common bean (Phaseolus vulgaris L.) hydrolysates inhibit inflammation in LPS-induced macrophages through suppression of NF-κB pathways.

The objectives of this study were to evaluate the antioxidant capacity of protein hydrolysates of the common bean (Phaseolus vulgaris L.) varieties Negro 8025 and Pinto Durango and determine their effect on the markers of inflammation in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Cell viability was determined and the percentage of viable cells was calculated and concentrations that allowed >80% cell viability were used to determine the markers of inflammation. Alcalase hydrolysates and pepsin-pancreatin hydrolysates showed the highest antioxidant capacity after 80 and 120min of hydrolysis, respectively. Alcalase hydrolysates of the common bean Pinto Durango at 120min inhibited inflammation, with IC50 values of 34.9±0.3, 13.9±0.3, 5.0±0.1 and 3.7±0.2µM, while var. Negro needed 43.6±0.2, 61.3±0.3, 14.2±0.3 and 48.2±0.1µM for the inhibition of cyclooxygenase-2 expression, prostaglandin E2 production, inducible nitric oxide synthase expression and nitric oxide production, respectively. Also, hydrolysates significantly inhibited the transactivation of NF-κB and the nuclear translocation of the NF-κB p65 subunit. In conclusion, hydrolysates from the common bean can be used to combat inflammatory and oxidative-associated diseases.

Synergistic anti-inflammatory activity of apigenin and curcumin co-encapsulated in caseins assessed with lipopolysaccharide-stimulated RAW 264.7 macrophages.

Dietary polyphenols are potential anti-inflammatory agents, and their combinations with enhanced biological activities may lower toxicity and side effects. The objective of this work was to investigate the potential synergistic anti-inflammatory activities of apigenin and curcumin co-nanoencapsulated in sodium caseinate, with comparison to unencapsulated polyphenol combinations. Non-toxic concentrations of apigenin, curcumin, and their combinations in the free and co-encapsulated forms were studied in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. Combinations of free polyphenols produced stronger inhibition of nitric oxide (NO) production, more significant at a higher proportion of curcumin, which was further enhanced after co-encapsulation. The enhanced reduction of NO was concomitant with the decreased expression of iNOS, the enhanced inhibition of pro-inflammatory cytokines of IL-6 and TNF-α, and the reduced production of intracellular reactive oxygen species. The potential multi-target effects and the enhanced solubility, proximity, and bioavailability of AP and CUR after co-encapsulation contributed to the synergistic activities. These results demonstrated that co-nanoencapsulation of apigenin and curcumin may enable the practical application utilizing the synergistic anti-inflammation effects to improve health.

Physicochemical, functional and bioactive properties of hempseed (Cannabis sativa L.) meal, a co-product of hempseed oil and protein production, as affected by drying process.

Hempseed meal after protein isolation (HM-PI) is a co-product obtained from hempseed. The objectives were to characterize and determine the effect of drying on HM-PI. HM-PI was produced using three drying methods: freeze (FD), vacuum oven (VOD), and oven drying (OD). HM-PI contained over 70% protein and had similar or higher level of essential amino acids than recommended values for human adults. Osborne fractionation indicated that glutelin was the most dominant fraction in HM-PI. FD HMPI has a significant lower surface hydrophobicity and higher in vitro protein digestibility than OD and VOD HM-PI. FD HM-PI demonstrated better functional properties than OD and VOD HM-PI. Pepsin-pancreatin digestion of VOD, FD and OD resulted in comparable and considerable antioxidant and anti-inflammatory properties. This is the first report on the characterization of HM-PI, a co-product of hempseed processing. HM-PI could serve as a novel food protein ingredient resulting in increase utilization of hempseed.

Utilization of tofu processing wastewater as a source of the bioactive peptide lunasin.

The goal of our study was to design a simple and feasible method to obtain lunasin, a naturally-occurring bioactive peptide, from tofu whey wastewater. A combination of alcoholic precipitation of high-molecular weight proteins from the whey, isoelectric precipitation of lunasin enriched material, and purification via gel filtration chromatography was selected as the best approach using tofu whey prepared at the laboratory scale. This process was applied to tofu whey produced by a local tofu factory and 773 mg of 80% purity lunasin was obtained per kg of dry tofu whey. Significant reduction of nitric oxide, and pro-inflammatory cytokines TNF-α and IL-6 over lipopolysaccharide activated murine macrophages demonstrate its biological activity. Our three-step process is not only simpler and faster than the previously reported methods to obtain lunasin but provides a sustainable approach for the valorization of a waste product, promoting the better utilization of soybean nutrients and active compounds.

Effect of heating on the digestibility of isolated hempseed (Cannabis sativa L.) protein and bioactivity of its pepsin-pancreatin digests.

The objective was to investigate the effects of heat pretreatment and simulated gastrointestinal digestion on potential antioxidant, anticancer and anti-inflammatory activities of hempseed (Cannabis sativa L.) proteins. Unheated isolated hempseed protein (IHP) and its heated counterparts (100 °C, 15 min and 30 min, termed as HP15D and HP30D) were hydrolyzed sequentially with pepsin and pancreatin and analyzed for digestibility and bioactivity (antioxidant, anti-proliferative and anti-inflammatory properties). Heat pretreatment led to an increase of low molecular weight proteins and degree of hydrolysis, and decrease of concentration of soluble protein, which means heat pretreated can significantly improve the digestibility of IHP. Pepsin-pancreatin digests released from heat pretreated IHP possessed less antioxidant, antiproliferative and anti-inflammatory properties than digests from unheated IHP. In conclusion, heat pre-treatment improved the digestibility of IHP but the resulting digests from heated IHP had lower bioactivity.