pH-promoted O-α-glucosylation of flavonoids using an engineered α-glucosidase mutant.

Retaining glycosidase mutants lacking its general acid/base catalytic residue are originally termed thioglycoligases which synthesize thio-linked disaccharides using sugar acceptor bearing a nucleophilic thiol group. A few thioglycoligases derived from retaining α-glycosidases have been classified into a new class of catalysts, O-glycoligases which transfer sugar moiety to a hydroxy group of sugar acceptors, resulting in the formation of O-linked glycosides or oligosaccharides. In this study, an efficient O-α-glucosylation of flavonoids was developed using an O-α-glycoligase derived from a thermostable α-glucosidase from Sulfolobus solfataricus (MalA-D416A). The O-glycoligase exhibited efficient transglycosylation activity with a broad substrate spectrum for all kinds of tested flavonoids including flavone, flavonol, flavanone, flavanonol, flavanol and isoflavone classes in yields of higher than 90%. The glucosylation by MalA-D416A preferred alkaline conditions, suggesting that pH-promoted deprotonation of hydroxyl groups of the flavonoids would accelerate turnover of covalent enzyme intermediate via transglucosylation. More importantly, the glucosylation of flavonoids by MalA-D416A was exclusively regioselective, resulting in the synthesis of flavonoid 7-O-α-glucosides as the sole product. Kinetic analysis and molecular dynamics simulations provided insights into the acceptor specificity and the regiospecificity of O-α-glucosylation by MalA-D416A. This pH promoted transglycosylation using O-α-glycoligases may prove to be a general synthesis route to flavonoid O-α-glycosides.

Activated STAT3 may participate in tumor progression through increasing CD133/survivin expression in early stage of colon cancer.

The activation of signal transducer and activator of transcription 3 (STAT3) by elevated interleukin (IL) levels has been reported to regulate tumorigenesis both in vitro and in vivo. However, the clinical implication of p-STAT3 expression in colon cancer is still controversial. In this study, we evaluated the effect of STAT3 inactivation on biologic behavior of primary (Caco-2) and metastatic colon cancer cells (LoVo and SNU407) and the relation of p-STAT3 expression with the invasion of colon tumor. In vitro study, the STAT3 inhibition by siRNA and stattic treatment significantly reduced colony formation and cell migration and decreased CD133 and survivin the expression compared with a control in all three cell lines. Furthermore, primary cancer cells exhibited a marked decrease in CD133 expression and increased apoptosis compared to metastatic cells after stattic treatment. The immunohistochemical assay using clinical samples of colonic tumors with various invasion depth showed that p-STAT3 expression was inversely associated with tumor invasion (p = 0.001, hazard ratio (HR) = 0.328, 95% confidence interval (95%CI): 0.170-0.632). In conclusion, p-STAT3 may participate in the progression of the early stage of colon cancer through the up-regulation of CD133, which in turn induces survivin expression. However, the regulatory mechanism of these molecules in tumor progression in vivo is need to be more verified.

Differences in clinical features and oncologic outcomes between metastatic right and left colon cancer.

Approximately 20% to 25% of patients with colorectal cancer (CRC) have distant organ metastasis at the time of initial diagnosis. The primary tumor location has been suggested as a prognostic factor for patients with metastatic CRC. In recent years, the distinction between right colon cancer (RCC) and left colon cancer (LCC) has been brought into focus due to their different outcomes, prognoses, and clinical responses to chemotherapy. In this article we aimed to review the underlying differences between metastatic RCC and LCC in terms of epidemiology, clinical features, and oncologic outcomes. The outcomes of patients with left-sided tumors were better than those of patients with right-sided tumors in terms of overall survival (OS) and objective response rate (ORR) after treatment with chemotherapy + panitumumab in the PRIME and 20050181 trials. The outcomes of patients with LCC were better than those of patients with RCC in terms of OS, progression-free survival (PFS) and ORR after treatment with FOLFIRI + cetuximab in the CRYSTAL and CALGB 80405 trials. In the FIRE-3 trial, the OS and PFS, but not the ORR, of patients with LCC were superior to those of patients with RCC. LCC and RCC exhibit distinctive clinical features and epidemiology. However, we must further investigate the impact of these distinctive features and how they influence the differential oncologic outcomes.

Characterization of divergent pseudo-sucrose isomerase from Azotobacter vinelandii: Deciphering the absence of sucrose isomerase activity.

Among members of the glycoside hydrolase (GH) family, sucrose isomerase (SIase) and oligo-1,6-glucosidase (O16G) are evolutionarily closely related even though their activities show different specificities. A gene (Avin_08330) encoding a putative SIase (AZOG: Azotobacterglucocosidase) from the nitrogen-fixing bacterium Azotobacter vinelandii is a type of pseudo-SIase harboring the "RLDRD" motif, a SIase-specific region in 329-333. However, neither sucrose isomerization nor hydrolysis activities were observed in recombinant AZOG (rAZOG). The rAZOG showed similar substrate specificity to Bacillus O16G as it catalyzes the hydrolysis of isomaltulose and isomaltose, which contain α-1,6-glycosidic linkages. Interestingly, rAZOG could generate isomaltose from the small substrate methyl-α-glucoside (MαG) via intermolecular transglycosylation. In addition, sucrose isomers isomaltulose and trehalulose were produced when 250 mM fructose was added to the MαG reaction mixture. The conserved regions I and II of AZOG are shared with many O16Gs, while regions III and IV are very similar to those of SIases. Strikingly, a shuffled AZOG, in which the N-terminal region of SIase containing conserved regions I and II was exchanged with the original enzyme, exhibited a production of sucrose isomers. This study demonstrates an evolutionary relationship between SIase and O16G and suggests some of the main regions that determine the specificity of SIase and O16G.

The short-term and oncologic outcomes of laparoscopic versus open surgery for T4 colon cancer.

PURPOSE: To compare R0 resection rates and short-term and oncologic outcomes between laparoscopy and open surgery for T4 colon cancer. METHODS: Patients with non-metastatic T4 colon cancer (n = 117) underwent treatment either through laparoscopy (n = 51) or open surgery (n = 66). Conversion to open surgery occurred in seven cases (13.7%). RESULTS: History of abdominal surgery (2.0 vs. 12.1%) and emergency operation (2.1 vs. 24.2%) were less frequent in the laparoscopy group. Conversion to open surgery occurred in seven cases (13.7%). Resection of adjacent organs was less frequently performed in the laparoscopy group (27.5 vs. 53.0%, p = .005). The mean operative time (189 vs. 210 min) and rate of 30-day postoperative complications (12 vs. 24%) were similar between the two groups. Shorter time to soft diet (7 vs. 9 days, p = .018) and hospital stay (14 vs. 18 days, p = .044) were observed in the laparoscopy group. T4b tumor was also less frequent in the laparoscopy group (3.9 vs. 18.2%, p = .018), while R0 resection rates were similar between the laparoscopy (96.1%) and open surgery group (95.5%). The mean number of lymph nodes was 22 in the laparoscopy group and 27 in the open surgery group (p = .021). No differences in 3-year overall survival rate (82.5 vs. 75.7%), recurrence-free survival rate (61.9 vs. 63.5%), and local recurrence-free survival rate (89.8 vs. 88.5%) were observed between the groups. Operation time, blood loss, 30-day complication rate, time to diet, duration of hospital stay, R0 resection rate, 3-year overall and local recurrence-free survival rates showed no difference between the converted and open surgery groups. CONCLUSIONS: Our results indicate that laparoscopy is a surgically safe and oncologically acceptable approach and thus could be considered for well-selected patients with T4 colon cancer in order to allow faster short-term recovery.

Construction of an antimyoglobin single-chain variable fragment with rapid reaction kinetics.

Antibodies with rapid reaction kinetics (high association and dissociation rates), named reversible antibodies, are used to perform continuous monitoring of sensitive disease biomarkers. In cases of acute myocardial infarction (AMI), continuous monitoring and early diagnosis are important. Human myoglobin (Myo) is a useful biomarker for AMI during the early stage after the onset of symptoms. In this study, a single-chain variable fragment (scFv) specific to Myo was derived from an IgG antibody that has rapid reaction kinetics. Enzyme-linked immunosorbent assay revealed that recombinant scFv exhibited 3.8-fold reduced affinity compared with the parent IgG antibody based on the antibody concentration necessary for 50% of the maximum signal. The scFv retained the rapid reaction kinetic mode with average kon and koff of 2.63 × 10(5) M(-1) Sec(-1) and 3.25 × 10(-3) Sec(-1) , respectively, which were reduced to 10- and 2.3-fold compared with those of the parent antibody. The equilibrium constant for the association of the scFv (KA = 8.09 × 10(7) M(-1) ) was 4.6-fold lower than that of its parent IgG antibody. This scFv may be a starting point for further mutagenesis/kinetic and structural analyses providing valuable insight into the mechanism of reversible antibodies.

The safety and prognostic factors for mortality in extremely elderly patients undergoing an emergency operation.

PURPOSE: As the number of elderly people has increased, the number of elderly patients who need emergency operations has also increased. Although there are many models to evaluate the risk of surgery in elderly patients, they all are associated with limitations. We herein evaluated the prognostic factors for surgical mortality in elderly patients more than 80 years old who needed emergency operations. METHODS: A total of 171 patients more than 80 years old underwent emergency operations from January 2001 to December 2012. Among them, 79 patients with acute cholecystitis, panperitonitis and intestinal obstruction with strangulation, which included mortality cases, were included. We retrospectively reviewed the medical records of the patients and analyzed the prognostic factors for surgical mortality. RESULTS: Forty-eight patients had a co-morbidity. Thirty-one patients initially had systemic inflammatory response syndrome. There were 27 surgical mortality cases. A univariate analysis revealed that panperitonitis, a positive blood culture and the level of albumin were significant prognostic factors predicting a worse prognosis. However, a multivariate analysis revealed that a serum albumin level more than 3.5 g/dL was the only significant prognostic factor (p = 0.037). CONCLUSION: Surgeons cannot fully evaluate the risk of emergency operation cases. However, our data indicate that if patients do not show hypoalbuminemia, the surgeon may be able to perform an emergency operation without a high risk of surgical mortality.

Structural and biochemical characterization of the broad substrate specificity of Bacteroides thetaiotaomicron commensal sialidase.

Sialidases release the terminal sialic acid residue from a wide range of sialic acid-containing polysaccharides. Bacteroides thetaiotaomicron, a symbiotic commensal microbe, resides in and dominates the human intestinal tract. We characterized the recombinant sialidase from B. thetaiotaomicron (BTSA) and demonstrated that it has broad substrate specificity with a relative activity of 97, 100 and 64 for 2,3-, 2,6- and 2,8-linked sialic substrates, respectively. The hydrolysis activity of BTSA was inhibited by a transition state analogue, 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid, by competitive inhibition with a Ki value of 35µM. The structure of BSTA was determined at a resolution of 2.3Å. This structure exhibited a unique carbohydrate-binding domain (CBM) at its N-terminus (a.a. 23-190) that is adjacent to the catalytic domain (a.a. 191-535). The catalytic domain has a conserved arginine triad with a wide-open entrance for the substrate that exposes the catalytic residue to the surface. Unlike other pathogenic sialidases, the polysaccharide-binding site in the CBM is near the active site and possibly holds and positions the polysaccharide substrate directly at the active site. The structural feature of a wide substrate-binding groove and closer proximity of the polysaccharide-binding site to the active site could be a unique signature of the commensal sialidase BTSA and provide a molecular basis for its pharmaceutical application.

Characterization of a galactosynthase derived from Bacillus circulans ß-galactosidase: facile synthesis of D-lacto- and D-galacto-N-bioside.

Glycosynthases-retaining glycosidases mutated at their catalytic nucleophile-catalyze the formation of glycosidic bonds from glycosyl fluorides as donor sugars and various glycosides as acceptor sugars. Here the first glycosynthase derived from a family 35 ß-galactosidase is described. The Glu→Gly mutant of BgaC from Bacillus circulans (BgaC-E233G) catalyzed regioselective galactosylation at the 3-position of the sugar acceptors with α-galactosyl fluoride as the donor. Transfer to 4-nitophenyl α-D-N-acetyl-glucosaminide and α-D-N-acetylgalactosaminide yielded 4-nitophenyl α-lacto-N-biose and α-galacto-N-biose, respectively, in high yields (up to 98%). Kinetic analysis revealed that the high affinity of the acceptors contributed mostly to the BgaC-E233G-catalyzed transglycosylation. BgaC-E233G showed no activity with ß-(1,3)-linked disaccharides as acceptors, thus suggesting that this enzyme can be used in "one-pot synthesis" of LNB- or GNB-containing glycans.

Neutrophil-lymphocyte ratio predicts pathologic tumor response and survival after preoperative chemoradiation for rectal cancer.

BACKGROUND: Neutrophil-lymphocyte ratio (NLR) reflects the balance between pro- and anti-tumor immune activities. We evaluated whether NLR is associated with pathologic tumor response and prognosis in rectal cancer patients that underwent preoperative chemoradiaton therapy (CRT) with surgery. METHODS: One hundred two patients with rectal cancer that were treated by preoperative CRT followed by surgery were enrolled. A total of 50.4 GY of radiation and 5-FU-based chemotherapy were delivered. An NLR ≥ 3 was considered to be elevated. Pathologic tumor response based on ypTNM stage was categorized into two groups, good response (n = 35, pathologic complete response and ypTNM I) and poor response groups (n = 67, ypTNM II, III, and IV). RESULTS: Twenty-five patients (24.5%) had elevated NLR. Multivariate analysis showed that an elevated CEA level (p = 0.001), larger tumor (p = 0.03), and elevated NLR (p = 0.04) were significant predictors for a poor response. Poor pathological tumor response and elevated NLR were risk factors for cancer-specific and recurrence-free survivals. CONCLUSION: An elevated NLR before CRT can be used as predictors for poor tumor response and unfavorable prognostic factors. Dominant pro-tumor activities of neutrophils or reduced anti-tumor immune response by lymphocytes, as determined by NLR, may have a impact on poor tumor response and unfavorable prognosis.

Refolded scFv antibody fragment against myoglobin shows rapid reaction kinetics.

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the V(H)-V(L) sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10⁻4 M⁻¹·s⁻¹ and 6.29 × 10⁻³ s⁻¹, respectively, with an affinity value exceeding 107 M⁻¹ (k(on)/k(off)), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor.

Visual detection of biogenic monoamines based on amine oxidase-peroxidase coupling reaction using ascorbic acid as H2O2 scavenger.

This study represents a visual detection for total biogenic monoamines with naked eye as a simple and rapid semi-quantitative method for biogenic amine monitoring. The equivalent reaction of H2O2 with ascorbic acid resulted in color development by an amine oxidase-peroxidase coupling reaction in the samples containing the biogenic monoamines higher than the subjected ascorbic acid by 10 µM. Upon employing the commercial doenjang extracts as a model food, an additional heating step was requested, and the expected ranges for the biogenic monoamines from 360 to 480 µM covered the real contents of the samples (360.2-407.3 µM). Therefore, this visual detection method makes it possible to decide with naked eye whether the sample contains the biogenic monoamines higher than the ascorbic acid supplemented as much as a control level on manufacturing sites without instrumental analysis.

Overproduction of a thermostable 4-α-glucanotransferase by codon optimization at N-terminus region.

BACKGROUND: 4-α-Glucanotransferases are useful enzymes to modify starch owing to their transglycosylation activity. In this study, codon optimizations were conducted to overproduce a thermostable 4-α-glucanotransferase from Thermus thermophilus (TTαGT). RESULTS: Two variants, termed TTαGT-P4CCG and TTαGT-mut6, were constructed, which have the optimized codon at the first rare codon and optimized codons at all six chosen rare codons at the N-terminus of TTαGT, respectively. In the Escherichia coli system, the expression of both optimized genes was enhanced by about 100-fold relative to that of the original gene, whereas all six mutated codons contributed to the overall enhancement of TTαGT production in Bacillus subtilis. On the basis of the αGTase activity of the crude cell extracts, relative activities of 1:2.9:5.8 were determined for TTαGT, TTαGT-P4CCG and TTαGT-mut6, respectively, in B. subtilis. In addition, the activity of TTαGT-mut6 from B. subtilis grown without antibiotics was as much as that with the antibiotics. Finally, after heat treatment, the specific activity of TTαGT-mut6 from B. subtilis was 1.5-fold greater than that from E. coli. CONCLUSION: The codon-optimized TTαGT that was produced in a GRAS microorganism, B. subtilis, without the selection antibiotics is potentially useful in the food industry as a food-grade enzyme.

Characterization of amine oxidases from Arthrobacter aurescens and application for determination of biogenic amines.

Biogenic amines (BAs) that are produced through naturally occurring decarboxylation of amino acids have toxicological effects on humans. Bacterial amine oxidases are useful tools for the rapid quantification of BAs in foods. To develop amine oxidases for the rapid detection of BAs, the genes for amine oxidases from Arthobacter aurescens TC-1, designated AMAO1, AMAO2, and AMAO3, respectively, were cloned and expressed in Escherichia coli. AMAO1 was catalytically inactive to BAs, and AMAO3 showed a narrow substrate spectrum. In contrast, AMAO2 exhibited activity with relative k cat/K M values of 100:49.6:7.6 for 2-phenylethylamine, tyramine, and histamine, respectively. AMAO2 also utilized putrescine and spermidine as substrates, with four or five orders of magnitude lower k cat/K M values than that of 2-phenylethylamine. AMAO2 and AMAO3 were seriously affected by substrate inhibition. Using BA mixtures (consisting of 2-phenylethylamine, tyramine, and histamine) as samples, the detection range of the enzymatic analysis of BA using AMAO2 was determined to be 2.5-120 µM, and its detection limit was 2.3 µM. Analysis of five commercial cheese products revealed that the BA contents determined by the enzymatic methods showed a good agreement with the sum of three monoamines and histamine by HPLC. Therefore, the enzymatic assay using AMAO2 can be used in quality control of food products through rapid, sensitive, and preliminary estimation of major BAs including the most important TyrN and HisN in foods.

Fermented Soybean Paste Attenuates Biogenic Amine-Induced Liver Damage in Obese Mice.

Biogenic amines are cellular components produced by the decarboxylation of amino acids; however, excessive biogenic amine production causes adverse health problems. The relationship between hepatic damage and biogenic amine levels in nonalcoholic fatty liver disease (NAFLD) remains unclear. In this study, mice were fed a high-fat diet (HFD) for 10 weeks to induce obesity, presenting early-stage of NAFLD. We administered histamine (20 mg/kg) + tyramine (100 mg/kg) via oral gavage for 6 days to mice with HFD-induced early-stage NAFLD. The results showed that combined histamine and tyramine administration increased cleaved PARP-1 and IL-1ß in the liver, as well as MAO-A, total MAO, CRP, and AST/ALT levels. In contrast, the survival rate decreased in HFD-induced NAFLD mice. Treatment with manufactured or traditional fermented soybean paste decreased biogenically elevated hepatic cleaved PARP-1 and IL-1ß expression and blood plasma MAO-A, CRP, and AST/ALT levels in HFD-induced NAFLD mice. Additionally, the biogenic amine-induced reduction in survival rate was alleviated by fermented soybean paste in HFD-induced NAFLD mice. These results show that biogenic amine-induced liver damage can be exacerbated by obesity and may adversely affect life conservation. However, fermented soybean paste can reduce biogenic amine-induced liver damage in NAFLD mice. These results suggest a beneficial effect of fermented soybean paste on biogenic amine-induced liver damage and provide a new research perspective on the relationship between biogenic amines and obesity.

Site-Directed Mutagenic Engineering of a Bifidobacterium Amylosucrase toward Greater Efficiency of Turanose Synthesis.

The aim of this study was to establish one of the most efficient biocatalytic processes for turanose production by applying a robust Bifidobacterium thermophilum (BtAS) mutant developed through site-directed mutagenesis. A gene encoding the amylosucrase of B. thermophilum (BtAS) was cloned and used as a mutagenesis template. Among the BtAS variants generated by the site-directed point mutation, four different single-point mutants (P200R, V202I, Y265F, and Y414F) were selected to create double-point mutants, among which BtASY414F/P200R displayed the greatest turanose productivity without losing the thermostability of native BtAS. The turanose yield of BtASY414F/P200R reached 89.3% at 50 °C after 6 h with 1.0 M sucrose + 1.0 M fructose. BtASY414F/P200R produced significantly more turanose than BtAS-wild type (WT) by 2 times and completed the reaction faster by another 2 times. Thus, turanose productivity (82.0 g/(L h)) by BtASY414F/P200R was highly improved from 28.1 g/(L h) of BtAS-WT with 2.0 M sucrose + 0.75 M fructose.

Evaluating the efficacy of slow-releasing carbon source tablets for in situ biological heterotrophic denitrification of groundwater.

Slow-releasing precipitating tablets (SRPTs) and slow-releasing floating tablets (SRFTs) were formulated to release fumarate as a carbon source (CS) and/or electron donor (ED) in an in situ biological heterotrophic denitrification system. These tablets were prepared using pharmaceutical manufacturing. Soil column tests were conducted to evaluate nitrate denitrification efficacy, microbial population changes, and mass balance of fumarate and potential electron acceptors. Significant and simultaneous consumption of both fumarate and nitrate, and the production and consumption of nitrite were observed in both SRPT-treated and SRFT-treated soil columns. These results suggest that SRPT and SRFT releasing fumarate, induce heterotrophic biological denitrification. In the SRPT- and SRFT-treated columns, 65% and 73% of fumarate were associated with heterotrophic denitrification, respectively. Particularly, surplus citric acid, originally designed to serve as a floating agent, was utilized for 36% and 28% for SRFT flotation and denitrification, respectively. The results of 16s RNA analyses revealed that a bacterium that shared 99% 16s rRNA sequence similarity with those of Azoarcus sp. AN9, and Pseudogulbenkiania sp. NH8B, a facultative heterotrophic denitrifier, was detected in the column effluent. This study confirms that SRPT and SRFT can effectively operate long-term in situ biological denitrification processes, because it is possible to supply detailed CS and/or ED uniformly by applying both SRPT and SRFT in the well.

Double primary malignancies associated with colon cancer in patients with situs inversus totalis: two case reports.

Situs inversus totalis (SIT) is not itself a premalignant condition, however, rare synchronous or metachronous multiple primary malignancies have been reported. Herein we present a case of synchronous transverse and sigmoid colon cancers and a case of metachronous rectosigmoid colon and gastric cancers in patients with SIT.A 66-year-old male with SIT was referred for a two-month history of hematochezia. Synchronous colonic tumors were found on the proximal transverse and sigmoid colon. The patient underwent open total colectomy and was discharged without incident. A 71-year-old female with rectosigmoid colon cancer and SIT underwent laparoscopy-assisted low anterior resection. Fourteen months after the surgery, the patient developed a single hepatic metastasis and underwent hepatic segmentectomy (S6). Forty-six months after laparoscopy-assisted low anterior resection, the patient developed metachronous early gastric cancer on the antrum and underwent radical subtotal gastrectomy with gastroduodenostomy. The patient is doing well without recurrence for 28 months.

Solid Fat Replacement with Canola Oil-Carnauba Wax Oleogels for Dairy-Free Imitation Cheese Low in Saturated Fat.

Canola oil was structured into oleogels with different amounts of carnauba wax, and their processing performances were assessed as an alternative to solid fat for imitation cheese low in saturated fat. The contents of solid fat in the oleogels were less vulnerable to the change in temperature than the palm oil. The replacement of palm oil with oleogels produced cheese samples with harder and more cohesive/chewy textures. Dynamic and transient viscoelastic measurements demonstrated that the use of oleogels was effective in increasing the elastic nature of the cheeses. Two distinct components with different proton mobilities were observed in the imitation cheeses, and longer T2 relaxation times were detected in the oleogel samples. The meltability of the cheese with palm oil was not significantly different from those with 3% and 6% oleogels. The saturated fat level of the oleogel cheese was significantly reduced from 45.70 to 5.20%. The application of canola oil-carnauba wax oleogels could successfully produce imitation cheese high in unsaturated fat and low in saturated fat. This study thus demonstrated that the health-functional properties of imitation cheese could be enhanced by using oleogels.