Ginger Protease Research Articles

The targeted development of collagen-active peptides based on composite enzyme hydrolysis: a study on the structure-activity relationship.

Fish collagen, derived from sustainable sources, offers a valuable substrate for generating peptides with diverse biofunctionalities. In this study, alkaline, papain, and ginger protease were used to enzymatically hydrolyze fish skin collagen. The peptide molecular weight distribution and sequence were measured using HPLC and ICP-MS-MS, with papain/alkaline protease (AP) and papain/alkaline/ginger protease (APG) hydrolyzed samples compared. As the results showed, the incorporation of ginger protease was useful for increasing the degree of hydrolysis, with the content of <400 Da peptides increasing from 49.82% to 58.56%. The identified peptide sequence in the APG sample had more proline at the C-terminal. The peptides were separated into two components (different in molecular weight) using gel column chromatography. The molecular weight distribution, amino acid composition, ACE inhibitory activity, and fibroblast proliferation activity of the collected components were measured. In comparison, the contents of proline and hydroxyproline in the larger peptides decreased obviously after combined hydrolysis by ginger protease, reflecting the formation of a peptide sequence of smaller molecular weight containing glycine and hydroxyproline. The combined hydrolysis of ginger protease was beneficial for the improvement of the ACE inhibitory activity of the sample. However, the fibroblast proliferation activity of AP was higher than that of APG, indicating that further hydrolysis by ginger protease may destroy the hydroxyproline at the end of the peptide sequence. This study proposed a creative directional hydrolysis method and provided practical guidance for the production of collagen peptides with enhanced functional activity.

Identification, rapid screening, docking mechanism and in vitro digestion stability of novel DPP-4 inhibitory peptides from wheat gluten with ginger protease.

To better understand the hypoglycemic potential of wheat gluten (WG), we screened dipeptidyl peptidase IV (DPP-4) inhibitory active peptides from WG hydrolysates. WG hydrolysates prepared by ginger protease were found to have the highest DPP-4 inhibitory activity among the five enzymatic hydrolysates, from which a 1-3 kDa fraction was isolated by ultrafiltration. Further characterization of the fraction with nano-HPLC-MS/MS revealed 1133 peptides. Among them, peptides with P'2 (the second position of the N-terminal) and P2 (the second position of the C-terminal) as proline residues (Pro) accounted for 12.44% and 43.69%, respectively. The peptides including Pro-Pro-Phe-Ser (PPFS), Ala-Pro-Phe-Gly-Leu (APFGL), and Pro-Pro-Phe-Trp (PPFW) exhibited the most potent DPP-4 inhibitory activity with IC50 values of 56.63, 79.45, and 199.82 μM, respectively. The high inhibitory activity of PPFS, APFGL, and PPFW could be mainly attributed to their interaction with the S2 pocket (Glu205 and Glu206) and the catalytic triad (Ser630 and His740) of DPP-4, which adopted competitive, mixed, and mixed inhibitory modes, respectively. After comparative analysis of PPFS, PPFW, and PPF, Ser was found to be more conducive to enhancing the DPP-4 inhibitory activity. Interestingly, peptides with P2 as Pro also exhibited good DPP-4 inhibitory activity. Meanwhile, DPP-4 inhibitory peptides from WG showed excellent stability, suggesting a potential application in type 2 diabetes (T2DM) therapy or in the food industry as functional components.

Utilization of Partially Purified Milk Clotting Protease from Ginger Rhizome in the Manufacturing of Fresh Cheese

This study was carried out to utilize the partially purified ginger rhizome protease in fresh cheesemaking. The crude ginger rhizome extract was partially purified by employing 30-80% (NH4)2SO4 saturations (w/v). The maximum milk clotting activity was achieved at 50% saturation, exhibiting the purification fold of 1.64 and activity recovery of 85.76%. The milk pH and temperature for optimum time of coagulation and milk clotting activity were optimized by using response surface methodology (RSM). A numerical optimization study revealed that the optimum milk pH and temperature for producing cheese were 6.5 and 50 C respectively. The cheese produced by ginger protease had significantly (p&lt;0.05) higher moisture, ash and yield, but lower fat content than the rennet cheese. No significant difference (p&gt;0.05) was found in the calcium and protein content of both cheeses. The prepared cheese using ginger protease had significantly (p&lt;0.05) higher spreadability, but lower aftertaste than the rennet cheese. However, non-significant difference (p&gt;0.05) was observed in the flavor and overall acceptance between the two cheeses. Hence, this study demonstrated that the ginger rhizome protease has the potential to be utilized as an effective milk coagulating enzyme in the manufacturing of fresh cheese.

Partial Purification, Characterization of Protease Extract from Ginger and its Effect on lipid profile and Liver Function

A ginger rhizome from the local market, Zingiber officinale var. Bentong, was used to extract protease. The activity of the protease was estimate during the extraction, the effects of pH and stabilizer were investigated. The best conditions were found to be a buffer of sodium phosphate, pH 7, 10 mM cysteine, and 5 mM EDTA as stabilizer. During the extraction process, 1% of crude ginger protease (GP) yield with a specific activity of 198 U/mg protein was generated. The protease functioned best at a pH of 7.0 and 60 °C, respectively. The effect of partially purified ginger protease on cholesterol (lipid profile) and liver function in mice was investigated.

Profile of Amino Acids and Proximate on the Extraction of Red Ginger Protease Enzymes in Commercial Feed for Aquaculture

Quality feed in aquaculture is very important to increase the growth and health of fish. Low feed efficiency can be overcome by feed supplementation using catalytic enzymes. One of the enzymes that can stimulate digestion is protease in red ginger plants. The protease enzyme in red ginger plants plays a role in breaking peptide bonds in place of carboxyl groups so that peptides are formed from complex compounds into simpler compounds. This study aims to determine the profile of amino acids and proximate in feed given red ginger enzymes. This research uses descriptive method. The feed sample used was commercial feed with 4 treatments namely Control (without red ginger enzyme), treatment A (1%), treatment B (3%), and treatment C (5%). The analyzes carried out included red ginger enzyme extraction, protease enzyme purification, proximate and amino acid analysis of each type of feed. The results showed that the results of the SDS-Page analysis on the red ginger protease sample contained at 67.1 kDa. The results of the proximate analysis showed the highest value in feed containing 3% red ginger protease (B) with a percentage of 42.69%. The resulting amino acid content consists of 8 non-essential amino acids namely, L-serine, L-glutamic acid, L-alanine, L-arginine, glycine, L-aspartic acid, L-tyrosine and L-proline. While the essential amino acids consist of 7 types of amino acids namely, L-Phenylalani, L-Isoleucine, L-Valine, L-Lysine, L-Leucine, L-Threonine and L-Histidine. The best type of feed is feed B, which is feed with the addition of 3% red ginger protease.

Identification, Screening, and Comprehensive Evaluation of Novel DPP-IV Inhibitory Peptides from the Tilapia Skin Gelatin Hydrolysate Produced Using Ginger Protease.

Inhibition of dipeptidyl peptidase-IV (DPP-IV) is an effective therapy for treating type II diabetes (T2D) that has been widely applied in clinical practice. We aimed to evaluate the DPP-IV inhibitory properties of ginger protease hydrolysate (GPH) and propose a comprehensive approach to screen and evaluate DPP-IV inhibitors. We evaluated the in vitro inhibitory properties of fish skin gelatin hydrolysates produced by five proteases, namely, neutral protease, alkaline protease, bromelain, papain, and ginger protease, toward DPP-IV. We screened the most potent DPP-IV inhibitory peptide (DIP) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with in silico analysis. Next, surface plasmon resonance (SPR) technology was innovatively introduced to explore the interactions between DPP-IV and DIP, as well as the IC50. Furthermore, we performed oral administration of DIP in rats to study its in vivo absorption. GPH displayed the highest degree of hydrolysis (20.37%) and DPP-IV inhibitory activity (65.18%). A total of 292 peptides from the GPH were identified using LC-MS/MS combined with de novo sequencing. Gly-Pro-Hyp-Gly-Pro-Pro-Gly-Pro-Gly-Pro (GPXGPPGPGP) was identified as the most potent DPP-IV inhibitory peptide after in silico screening (Peptide Ranker and molecular docking). Then, the in vitro study revealed that GPXGPPGPGP had a high inhibitory effect on DPP-IV (IC50: 1012.3 ± 23.3 μM) and exhibited fast kinetics with rapid binding and dissociation with DPP-IV. In vivo analysis indicated that GPXGPPGPGP was not absorbed intact but partially, in the form of dipeptides and tripeptides. Overall, the results suggested that GPH would be a natural functional food for treating T2D and provided new ideas for searching and evaluating potential antidiabetic compounds. The obtained GPXGPPGPGP can be structurally optimized for in-depth evaluation in animal and cellular experiments.

Antioxidant and angiotensin-I-converting enzyme (ACE-I) inhibitory activities of protein hydrolysates derived from water buffalo (Bubalus bubalis) liver.

In the current study, we attempted to use ginger as a novel and natural source of protease in comparison with other commercially available enzymes to extract and characterize antioxidant and antihypertensive hydrolysates from water buffalo liver, a protein rich offal. Hydrolysis of protein extracts from buffalo liver using proteinase-K, pronase-E and ginger protease significantly increased the %degree of hydrolysis (18.5-55%) and generated low-molecular weight peptides evident from SDS-PAGE. Enzyme treated hydrolysates exhibited higher (p < 0.05) DPPH radical scavenging activity (43.7-82.4%) and angiotensin-I-converting enzyme (ACE-I) inhibitory activity (46.9-50.1%) relative to control. Mass spectrometric analysis (MALDI-TOF MS) of selected gel-filtered fractions identified few important peptides derived from nuclear ribonucleoprotein, pyruvate kinase and phosphoglycerate kinase that possess strong antioxidant activity. Present findings indicate the efficacy of partially purified ginger as a novel source of protease in generating protein hydrolysates from water buffalo liver with significant antioxidant and antihypertensive activity in vitro. We successfully demonstrated the recovery of functional bioactive peptides from water buffalo liver which presents a potential opportunity for the meat industries to economically use this important byproduct.

Evaluating the Properties of Ginger Protease-Degraded Collagen Hydrolysate and Identifying the Cleavage Site of Ginger Protease by Using an Integrated Strategy and LC-MS Technology.

(1) Methods: An integrated strategy, including in vitro study (degree of hydrolysis (DH) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity) and in vivo study (absorption after oral administration in rats), was developed to evaluate the properties of the fish skin gelatin hydrolysates prepared using different proteases (pepsin, alkaline protease, bromelain, and ginger protease). Meanwhile, in order to identify the hydrolysis site of ginger protease, the peptides in the ginger protease-degraded collagen hydrolysate (GDCH) were comprehensively characterized by liquid chromatography/tandem mass spectrometry (LC-MS) method. (2) Results: The GDCH exhibited the highest DH (20.37%) and DPPH radical scavenging activity (77.73%), and in vivo experiments showed that the GDCH was more efficiently absorbed by the gastrointestinal tract. Further oral administration experiments revealed that GDCH was not entirely degraded to free amino acids and can be partially absorbed as dipeptides and tripeptides in intact forms, including Pro-Hyp, Gly-Pro-Hyp, and X-Hyp-Gly tripeptides. LC-MS results determined the unique substrate specificity of ginger protease recognizing Pro and Hyp at the P2 position based on the amino acids at the P2 position from the three types of tripeptides (Gly-Pro-Y, X-Hyp-Gly, and Z-Pro-Gly) and 136 identified peptides (>4 amino acids). Interestingly, it suggested that ginger protease can also recognize Ala in the P2 position. (3) Conclusions: This study comprehensively evaluated the properties of GDCH by combining in vitro and in vivo strategies, and is the first to identify the cleavage site of ginger protease by LC-MS technique. It provides support for the follow-up study on the commercial applications of ginger protease and bioactivities of the hydrolysate produced by ginger protease.

Effects of Ginger Protease on Quality of Mozzarella Cheese

Approaches in Poultry, Dairy & Veterinary Sciences Effects of Ginger Protease on Quality of Mozzarella Cheese Adhikari BR1,2*, Bhattarai RR3, Katuwal N4 and Das SKL1 1Central Department of Food Technology, Central Campus of Technology, Tribhuvan University, Nepal 2Food Management and Trading Company Limited, Provincial Office, Nepal 3School of Molecular and Life Sciences, Faculty of Science and Engineering, Curtin University, Bentley, Australia 4Food Technology and Quality Control Office, Nepal *Corresponding author: Basanta Raj Adhikari, Central Department of Food Technology, Central Campus of Technology, Tribhuvan University, Hattisar, Food Management and Trading Company Limited, Provincial Office, Biratnagar, Morang, Nepal Submission: December 07, 2021;Published: December 20, 2021 DOI: 10.31031/APDV.2021.08.000698 ISSN: 2576-9162 Volume8 Issue5

Sensory and Physical Characteristics of M. biceps femoris from Older Cows Using Ginger Powder (Zingibain) and Sous Vide Cooking.

This study aimed to evaluate the sensory and physical characteristics of zingibain-injected meat combined with sous vide cooking. M. biceps femoris (BF; n = 12) acquired from 6–7 year old Angus cows were cooked using the sous vide method at 65 °C, for 8 h or 12 h, either with ginger powder (GP) injected in a 2 g/L solution in water (treatment) or un-injected (control). The sensory attributes included flavour, juiciness, tenderness, and physicochemical characteristics were Warner-Bratzler shear (WBSF), hardness, total water content (TWC), cooking loss (CL) and collagen content. A significant improvement in tenderness with injection treatment and cooking time was observed, as evaluated through trained sensory panellists, and reduced WBSF and hardness (p < 0.05 for all). The flavour of the meat was not affected by injection treatment or cooking time (p > 0.05), but juiciness and TWC were reduced with longer cooking times (p < 0.01 for both). Soluble collagen increased with injection treatment and cooking time (both p < 0.05). Moderate to high correlations were found between sensory and physical measurements for tenderness and juiciness. The longer cooking time (12 h) with GP injection treatment caused over tenderization of the meat. The soft texture associated with over-tenderization may be suitable for some specialised consumer markets, for instance, the elderly population with chewing difficulties. Improving the eating quality of low-quality meat from old animals through sous vide cooking and the use of ginger proteases may increase the acceptability of lower value beef, potentially enhancing the commercial value of carcasses typically produced in the beef industry.

135 FH and FGSH prepared from fish skin gelatin using ginger protease alleviates DSS-induced Colitis via Nrf2 pathway

Abstract Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic inflammatory disease affecting the colon, and its incidence is rising worldwide. Hence, it is important to apply new strategies to cope with the IBD. We previously found that hydrolysate fractions (HF) and fish skin gelatin hydrolysate (FSGH), isolated from fish skin gelatin hydrolysate using ginger protease, could exert antioxidant effects in IPEC-J2 cells. Here, we aimed to evaluate the effects of FH and FGSH in a mouse model of dextran sodium sulfate (DSS)-induced colitis. Mouse were treated with 3% DSS in their drinking water for 7 days to induce acute colitis. FH (200 mg/kg) and FGSH (200 mg/kg) was administered for 7 days before and during DSS treatment via oral gavage once per day. Mouse were sacrificed at day 7 after colitis induction. The results showed that FH and FGSH significantly ameliorated the clinical symptoms of DSS-induced mice colitis, such as weight loss, disease activity index (DAI), colon shortening, spleen hypertrophy and histological scores. For MPO activity, an important indicator of neutrophil infiltration and acute inflammation in DSS-induced mouse colitis, FH and FGSH significantly reduced the level of DSS-induced hyperactivated MPO. Treatment with FH and FGSH also significantly inhibited the secretion proinflammatory cytokines TNF-α, IL-8 and IL-12 in DSS-induced mouse colitis. And FH and FGSH administration significantly increased expression of antioxidant enzymes GCLM, GCLC and GPX4 and maintained tight junction in colon tissues. Furthermore, we found that FH and FGSH significantly increased the level of Nrf2 in colon tissues, it indicated that FH and FGSH may alleviated colitis by Nrf2 pathway. Overall, these results suggested that FH and FGSH could be a potential promising candidate for the treatment of IBD.

The Application of Crude Ginger Protease as an Inhibitive Assay for Heavy Metals

In this work, a novel source of protease for the bioassay of heavy metals has been developed using proteases extracted from ginger. The result shows that the optimum protease activity was reached at 1 mg/mL protein concentration of the ginger protease. The optimum casein concentration toward crude ginger protease activity was 1.75 mg/mL. The most suitable pH for protease from crude ginger protease was within the range from pH 5.0 to 7.0. The proteases exhibited high protease activity in a broad range of temperature from 20 to 60 oC. The optimum incubation time for the enzyme occurred at minute 30. Among the six heavy metals tested, only three heavy metals inhibited proteolytic activity of ginger crude with an inhibition more than 30% at 1 mg/L. The calculated LC50 for mercury, copper and silver were, 0.182, (95%, confidence interval (C.I.) 0.134 to 0.283), 0.071, (95% C.I. of 0.056 to 0.096), 0.054, (95% C.I. of 0.039 to 0.085), respectively. Data on the sensitivity of various proteases to heavy metals shows that the crude ginger protease is comparable in sensitivity for mercury to the achromopeptidase, bromelain, papain assays. The crude ginger protease assay for copper is comparable in sensitivity to the papain assay and appears to be more sensitive than the rest of the assays. For silver, the ginger protease was the most sensitive while other assay methods are either unable or be able to detect higher concentration of silver. The crude proteases extracted from ginger showed a good potential for the development of a rapid, sensitive, and economic inhibitive assay for the biomonitoring of mercury, copper, and silver in the environment.

Antioxidative peptides of hydrolysate prepared from fish skin gelatin using ginger protease activate antioxidant response element-mediated gene transcription in IPEC-J2 cells

Excess production of reactive oxygen species (ROS) causes oxidative stress, which is associated with oxidative damage and a number of human chronic diseases. Here, novel antioxidant hydrolysates were obtained from fish skin, pig skin, pig bone and bovine skin gelatins using ginger protease, and were compared with those produced using pepsin-pancreatin. Ginger protease could hydrolyze the gelatin from fish skin to peptides with low average molecular weights (<690 Da) more efficiently than that from pig skin, pig bone and bovine skin. All gelatin hydrolysates showed higher antioxidative activities than non-hydrolysed gelatins. Especially, fish skin gelatin hydrolysate (FSGH) obtained using ginger protease exhibited the highest degree of hydrolysis (13.08%) and antioxidant activity towards DPPH radical (97.21%) and lipid peroxidation (48.46%). The FSGH was then separated into three fractions by ultrafiltration. The fraction with a molecular weight below 1000 Da demonstrated the strongest scavenging capacity for DPPH and hydroxyl radicals and inhibitory effect on lipid peroxidation. Further HPLC-ESI-QqQ-MS results revealed that this fraction mainly included two types of peptides: Gly-Pro-Y-type and X-Hyp-Gly-type tripeptides. The ARE-luciferase activity of a selection of the identified oligopeptides was examined using the IPEC-J2 cell model. The most active peptide was Gly-Pro-Ala, which showed an approximately 2.5-fold increase in ARE-luciferase activity. Additionally, Gly-Pro-Ala (0–2.5 mg/mL) activated the expression of ARE-driven antioxidant enzyme genes in a dose dependent manner, and subsequently suppressed the H2O2-induced intracellular ROS production. Overall, our results demonstrate that FSGH prepared using ginger protease could serve as a source of peptides with high antioxidative activities.

Fish Skin Gelatin Hydrolysate Production by Ginger Powder Induces Glutathione Synthesis To Prevent Hydrogen Peroxide Induced Intestinal Oxidative Stress via the Pept1-p62-Nrf2 Cascade.

Gelatin hydrolysate was reported to contain large amounts of biologically active peptides with excellent antioxidant properties. However, its inducement of antioxidant response within cells and the underlying molecular mechanism are far from clear. Here, gelatin from Nile tilapia skin was hydrolyzed by ginger protease to produce antioxidant hydrolysate, and three fish skin gelatin hydrolysate fractions (FSGHFs) were obtained by ultrafiltration. Fractionation of the hydrolysate increased the free radical scavenging capacity of the FSGHFs, particularly FSGHF3, which showed the lowest molecular weight (below 1000 Da). Furthermore, FSGHF3 treatment prior to H2O2 exposure increased cell viability and membrane integrity in IPEC-J2 cells. H2O2-induced ROS production and epithelial barrier damage were suppressed by FSGHF3 pretreatment. FSGHF3 stimulated the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), along with increases in the mRNA and protein expression of catalytic and modulatory subunits of γ-glutamylcysteine ligase as well as in the level of glutathione. Silencing of Nrf2 or p62 (an upstream regulator of Nrf2) suppressed FSGHF3-induced Nrf2 activation and its protection against H2O2-induced oxidative stress. Moreover, oligopeptides in FSGHF3 may mediate the cytoprotective effect against oxidative stress, which was confirmed by the result that FSGHF3 failed to inhibit the ROS production in H2O2-exposed cells with the knockdown of Pept1 (an oligopeptide transporter). Therefore, FSGHF3 can induce glutathione synthesis and prevent oxidative stress through the Pept1-p62-Nrf2 cascade and thus may be a functional food for gastrointestinal dysfunction.

Collagen-derived X-Hyp-Gly-type tripeptides promote differentiation of MC3T3-E1 pre-osteoblasts

Oral ingestion of collagen hydrolysate leads to significantly high blood levels of various hydroxyproline (Hyp)-containing oligopeptides, such as Pro-Hyp that has been reported to promote osteoblast differentiation. Using ginger protease, we recently developed a novel type of collagen hydrolysate enriched with X-Hyp-Gly-type tripeptides, which were efficiently absorbed into the blood after oral administration of the ginger-degraded collagen hydrolysate. In the present study, we demonstrated that X-Hyp-Gly promoted the differentiation of MC3T3-E1 pre-osteoblasts more effectively than Pro-Hyp, as evidenced by increases in alkaline phosphatase activity and mineralization. In addition, collagen secretion was enhanced by Ala-Hyp-Gly and Leu-Hyp-Gly, particularly at an early stage of differentiation. We detected the uptake of Ala-Hyp-Gly and Leu-Hyp-Gly as well as Pro-Hyp into MC3T3-E1 cells. These results suggest that the collagen hydrolysate prepared using ginger protease can be used as a functional food for the prevention and treatment of bone diseases, such as osteoporosis.

Producing armyworm (spodoptera sp.) Bioinsecticide based on cysteine protease of red ginger (zingiber officinale var. Rubrum)

Armyworm (Spodoptera sp.) is highly polyphagous defoliator on various horticulture and grain plants. Various chemical insecticides have been created to control it. There is a need to create an eco-friendly and specific insecticide which only affect armyworm’s nervous system. This research investigates cysteine-protease’s enzyme activity of red ginger (Zingiber officinale var. Rubrum) which is called zingibain. Its catalytic site matches with residue site in armyworm’s body so it can be used as bioinsecticide raw material which meets the criterias above. Fresh red ginger rhizomes were washed and extracted. The juice was then deposited in low temperature and centrifuged to get rid of its starch content. It was filtrated to remove large contaminants and poured into Potassium Phospate buffer. The liquid was then centrifuged again for 30 minutes before collecting the supernatant. Fresh leaves were then dipped into crude ginger protease extract and fed to fourth instar-armyworms. Leaves dipped into non-diluted extract were barely eaten by armyworm while the 50% and 25% dilution was half eaten and most eaten. The crude red ginger extract was not strong enough to kill them although the research showed its enzymatic activity reaches up to 169 PU. It still needs improvement to be produced as commercial bioinsecticide.

Common Kilka Hydrolysis: Investigation of Functional and In Vitro Antioxidant Properties of Hydrolysates Obtained by Kiwifruit and Ginger Proteases

ABSTRACTThe functional and in vitro antioxidant properties of common kilka fish protein hydrolysates with different degree of hydrolysis (DH) obtained by kiwifruit protease (KP) and ginger protease (GP) were evaluated. The electrophoretic patterns of hydrolysates showed the presence of one major band in different DHs with a molecular weight of less than 25 kDa. Protein solubility was positively correlated with DH, and the solubility of KP and GP hydrolysates (HKP and HGP, respectively) at 15% DH was higher than other DHs (p ≤ 0.05). Higher emulsifying and foaming properties were observed in HKP over a pH range of 2–10 (p ≤ 0.05), and in vitro antioxidant activity was higher in HKP at 15% DH as seen from 2,2-diphenyl-1-picrylhydrazyl radical scavenging and ferric-reducing power. Thus, the results reveal that DH and enzyme type affects functional properties and antioxidant activity of the hydrolysates.

Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing Cyclic Dipeptides with High Oral Bioavailability: Efficient Formation of Cyclo(X-Hyp) from X-Hyp-Gly-Type Tripeptides by Heating.

Cyclic dipeptides (2,5-diketopiperazines) are present in a variety of foods and are reported to demonstrate antioxidant, antidepressant, and other beneficial effects. We recently developed a novel collagen hydrolysate characterized by a high content of X-hydroxyproline (Hyp)-Gly-type tripeptides using ginger protease. In the present study, we found that, through heating, X-Hyp-Gly can be easily converted into Hyp-containing cyclic dipeptides. After heating for 3 h at 85 °C and pH 4.8, Ala-Hyp-Gly was almost completely cyclized to cyclo(Ala-Hyp), in contrast to a slight cyclization of Ala-Hyp. The contents of cyclo(Ala-Hyp) and cyclo(Leu-Hyp) reached 0.5-1% (w/w) each in the ginger-degraded collagen hydrolysate under the heating conditions. Oral administration experiments using mice revealed that cyclo(Ala-Hyp) and cyclo(Leu-Hyp) were absorbed into the blood at markedly higher efficiencies compared to collagenous oligopeptides, including Pro-Hyp. The high productivity and oral bioavailability of the collagen-specific cyclic dipeptides suggest significant health benefits of the heat-treated ginger-degraded collagen hydrolysate.

Production of a novel wheat gluten hydrolysate containing dipeptidyl peptidase-IV inhibitory tripeptides using ginger protease.

Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P2 position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of<600Da. In addition, the gluten hydrolysate contained substantial amounts of tripeptides, including Gln-Pro-Gln, Gln-Pro-Gly, Gln-Pro-Phe, Leu-Pro-Gln, and Ser-Pro-Gln (e.g. 40.7mg/g at pH 5.2). These gluten-derived tripeptides showed high inhibitory activity on dipeptidyl peptidase-IV with IC50 values of 79.8, 70.9, 71.7, 56.7, and 78.9μM, respectively, suggesting that the novel gluten hydrolysate prepared using ginger protease can be used as a functional food for patients with type 2 diabetes.

Milk clotting activity of protease, extracted from rhizome of Taffin giwa ginger ( Zingiber officinale ) cultivar, from northwestern Nigeria

The increasing prices of calf rennets, their accessibility and ethical concerns associated with the production of such enzymes for general cheese making have led to systematic investigations on the possibility and suitability of their substitution by other enzymes of plant origin. In this study, ammonium sulphate ((NH 4 ) 2 SO 4 ) fractionation, characterization and milk clotting activity (MCA) of protease extracted from Taffin Giwa ginger rhizome cultivar of the family Zingiberaceae from northwestern Nigeria were carried out. The protease extracted showed optimum activity at temperatures near 60 °C and pH value of 6.5 with a relative activity in a broad pH range of 5.0 to 8.0 accordingly. The enzyme was completely denatured at higher temperature of 100 °C and higher pH range of 12.0. The milk clotting property of the protease indicated 3.1 and 2.2 folds of MCA and MSCA respectively in relation to the commercial calf rennet with MCA/PA ratio of 2.52. The properties of Taffin Giwa protease shown in this study, especially its milk clotting activity, make it a potential candidate for substituting calf rennet in the food industries, particularly in cheese making processes. Keywords: Ginger Protease, Milk Clotting Activity, Calf rennet, Characterization, Extraction