Discrimination of four Cinnamomum species by proximate, antioxidant, and chemical profiling: towards quality assessment and authenticity.

Cinnamon (genus Cinnamomum) is a worldwide used spice. The highly valued, non-hepatotoxic C. verum (CV) is frequently adulterated with the cheaper hepatotoxic substitutes (C. burmannii (CB), C. cassia (CC), and C. loureiroi (CL)). Therefore, this study evaluated four major Cinnamomum species by proximate composition, antioxidant properties, and chemical analysis. The results showed that CB contained more ash and crude protein content. CC exhibited more moisture, crude fat, and nutritive value, while CV had more crude fiber and total carbohydrate content. The 80% methanol extracts of four Cinnamomum species exhibited the highest total phenolic contents (42.16 to 182.85 mg GAE/g), total flavonoid contents (0.80 to 1.07 mg QE/g), DPPH radical scavenging activities (EC50, 0.94 to 3.98 mg/mL), and ABTS radical scavenging activities (EC50, 0.09 to 0.33 mg/mL). The GC-MS based chemical profiling of CV was markedly different to those of CB, CC, and CL. Compared to the other three species, CV presented the highest eugenol content (5.77%) and the lowest coumarin content (1.90%). Principal component analysis (PCA) accounted for 94.91% of the variability, completely separating CV in quadrant I. Overall, nutritional and chemical profiles in combination with PCA could be effectively applied for monitoring Cinnamomum species, thereby ensuring food safety. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05788-y.

Authentication of olive oil in commercial products using specific, sensitive, and rapid loop-mediated isothermal amplification.

Olive oil is an important and popularly used plant oil in the daily diet or chemical industry. Due to its biological benefits on human health and higher selling prices, adulteration of olive oil for commercial fraud by other plant oils is becoming a serious issue. In this study, a specific, sensitive and rapid loop-mediated isothermal amplification (LAMP) was first developed for the detection of Olea europaea DNA for olive oil authentication. The oleosin gene was used for the primer design of the LAMP assay. After primer validation, the results showed that the LAMP primers were specific and rapid to isothermally authenticate the oleosin gene of Olea europaea within 1 h at 62 °C and had no cross-reaction with other DNA of plant oils. The sensitivity of LAMP was 1 ng of genomic DNA in olive oil, and only 1% olive oil in the sample was requisite during DNA amplification. Additionally, positive detection by LAMP in all the collected commercial olive oil products was practically performed but not in PCR assays. In conclusion, herein, the established LAMP assay with specificity could not only be capable for rapid identification but also applicable for olive oil authentication for precluding adulteration in plant oil products. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05726-y.

A Portable Continuous-Flow Polymerase Chain Reaction Chip Device Integrated with Arduino Boards for Detecting Colla corii asini.

Food security is a significant issue in modern society. Because morphological characters are not reliable enough to distinguish authentic traditional Chinese medicines, it is essential to establish an effective and applicable method to identify them to protect people's health. Due to the expensive cost of the manufacturing process and the large volume of the analytical system, the need to build a portable and cheap device is urgent. This work describes the development of a portable nucleic acid amplification device integrated with thermal control and liquid pumping connecting to Arduino boards. We present a novel microfluidic polymerase chain reaction (PCR) chip with symmetric isothermal zones. The total chip volume is small, and only one Arduino board is needed for thermal control. We assemble a miniaturized liquid pump and program an Arduino file to push the sample mixture into the chip to implement the PCR process. In the proposed operation, the Nusselt number of the sample flow is less than one, and the heat transfer is conduction only. Then we can ensure temperature uniformity in specific reaction regions. A Colla corii asini DNA segment of 200 bp is amplified to evaluate the PCR performance under the various operational parameters. The initial concentration for accomplishing the PCR process is at least 20 ng/µL at the flow rate of 0.4 µL/min in the portable continuous flow PCR (CFPCR) device. To our knowledge, our group is the first to introduce Arduino boards into the heat control and sample pumping modules for a CFPCR device.

Discrimination of Four Cinnamomum Species with Physico-Functional Properties and Chemometric Techniques: Application of PCA and MDA Models.

Discrimination of highly valued and non-hepatotoxic Cinnamomum species (C. verum) from hepatotoxic (C. burmannii, C. loureiroi, and C. cassia) is essential for preventing food adulteration and safety problems. In this study, we developed a new method for the discrimination of four Cinnamomum species using physico-functional properties and chemometric techniques. The data were analyzed through principal component analysis (PCA) and multiclass discriminant analysis (MDA). The results showed that the cumulative variability of the first three principal components was 81.70%. The PCA score plot indicated a clear separation of the different Cinnamomum species. The training set was used to build the discriminant MDA model. The testing set was verified by this model. The prediction rate of 100% proved that the model was valid and reliable. Therefore, physico-functional properties coupled with chemometric techniques constitute a practical approach for discrimination of Cinnamomum species to prevent food fraud.

Specific, sensitive and rapid Curcuma longa turmeric powder authentication in commercial food using loop-mediated isothermal nucleic acid amplification.

Turmeric (Curcuma longa) is a rhizomatous plant of the ginger family Zingiberaceae that is usually dried and ground into powder for use as a seasoning. Because turmeric has become increasingly popular in the functional food market, adulteration of C. longa by other turmeric species is becoming an increasingly significant problem. In this study, loop-mediated isothermal amplification (LAMP) was developed for the detection of C. longa DNA for turmeric authentication. ITS2-26S rDNA was used for the LAMP primer designation. The results demonstrated that the specific primers exhibited high specificity, authenticated C. longa DNA within 30 min at 65 °C isothermally and had no cross-reaction with other adulterants. LAMP was sensitive to 0.1 ng of turmeric C. longa DNA, and only 0.01% of C. longa turmeric powder in the sample was required for DNA amplification. The sensitivity of LAMP was 10-fold higher than that of PCR (0.1%) from a previous report. Moreover, all the collected commercial turmeric products were positively detected by LAMP and RtF-LAMP (real-time fluorescence LAMP). The developed LAMP assay not only had higher specificity and rapidity than that of other methods but could also be applied to authenticate turmeric to prevent adulteration in food products.

Specific, sensitive and rapid authentication of donkey-hide gelatine (Colla corii asini) in processed food using an isothermal nucleic acid amplification assay.

Donkey-hide gelatine (DHG) is a well-known, animal-derived traditional Chinese medicine material called Colla corii asini (known in Chinese as "E'jiao"). Because DHG is claimed to have properties that are beneficial to health, its consumption has increased, but its production has decreased. Thus, the incidence of DHG adulteration has become increasingly serious. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the authentication of DHG. Identification of donkey DNA from DHG was performed specifically and rapidly within one hour by LAMP primers. Moreover, the sensitivity of LAMP in authenticating DHG was 10-3 ng, which revealed a 105-fold higher sensitivity than that of conventional PCR. The relative detection limit was 0.1% DHG in the adulterants, including gelatines of horse, cow, pork, goat, sheep or chicken origins. When genomic DNAs extracted from heat-treated DHG samples, including boiling or autoclaving for 40 min, were used as templates, DHG detection by LAMP was unchanged and reproducible. In conclusion, the LAMP assay established herein could potentially be applied for the authentication of DHG and DHG-related products in herbal or food markets.

Development of a specific isothermal nucleic acid amplification for the rapid and sensitive detection of shrimp allergens in processed food.

Food allergens that cause anaphylactic reactions have become an important health problem worldwide. Among them, shrimp is a popular seafood in many cuisines. The best way to avoid allergic reactions is to mitigate the intake of food allergens. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of shrimp DNA. Using LAMP primers, the identification of shrimp DNA by the LAMP assay was specific and rapid (within 30 min). It exhibited no cross-reaction with the DNA of other Crustacea, including crabs and lobster, and at least 0.01% shrimp DNA existed in the test sample. Additionally, the sensitivity of LAMP for detecting shrimp DNA was 100-fold greater than that of conventional PCR. LAMP for the detection of shrimp DNA was reproducible regardless of whether the genomic DNA was extracted from boiled, steamed or roasted shrimp samples. In summary, the LAMP assay established herein not only could be potentially used for diagnosing shrimp DNA but could also be applicable for identifying shrimp allergens in commercial food products in marketplaces.

The rapid and sensitive detection of edible bird's nest (Aerodramus fuciphagus) in processed food by a loop-mediated isothermal amplification (LAMP) assay.

Edible bird's nest (EBN) is a well-known and precious traditional Chinese herbal material (CHM). Because of this, preventing the adulteration of EBN efficiently and precisely is crucial to protect consumers' interests and health. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of EBN using specifically designed LAMP primers. The results demonstrated that the identification of EBN by LAMP assay was specific and rapid (within 1 h). It had no cross-reaction with EBN adulterants, including white fungus, egg white and pig skin, in different ratios. The relative detection limit was 0.01% EBN in the adulterants. Moreover, the sensitivity of LAMP in authenticating EBN was 10-8 µg, it showed higher sensitivity than that of conventional PCR with 105 fold. When genomic DNAs extracted from boiled or steamed EBN samples were used as templates, LAMP for EBN detection was not affected and was reproducible after heat processing. In conclusion, the LAMP assay established herein could be applicable for authenticating EBN and for identifying commercial EBN products in herbal markets.

Development of loop-mediated isothermal amplification (LAMP) assays for the rapid detection of allergic peanut in processed food.

Peanut is a widely and common used in many cuisines around the world. However, peanut is also one of the most important food allergen for causing anaphylactic reaction. To prevent allergic reaction, the best way is to avoid the food allergen or food containing allergic ingredient such as peanut before food consuming. Thus, to efficient and precisely detect the allergic ingredient, peanut or related product, is essential and required for maintain consumer's health or their interest. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of allergic peanut using specifically designed primer sets. Two sets of the specific LAMP primers respectively targeted the internal transcribed sequence 1 (ITS1) of nuclear ribosomal DNA sequence regions and the ara h1 gene sequence of Arachia hypogeae (peanut) were used to address the application of LAMP for detecting peanut in processed food or diet. The results demonstrated that the identification of peanut using the newly designed primers for ITS 1 sequence is more sensitive rather than primers for sequence of Ara h1 gene when performing LAMP assay. Besides, the sensitivity of LAMP for detecting peanut is also higher than the traditional PCR method. These LAMP primers sets showed high specificity for the identification of the peanut and had no cross-reaction to other species of nut including walnut, hazelnut, almonds, cashew and macadamia nut. Moreover, when minimal 0.1% peanuts were mixed with other nuts ingredients at different ratios, no any cross-reactivity was evident during performing LAMP. Finally, genomic DNAs extracted from boiled and steamed peanut were used as templates; the detection of peanut by LAMP was not affected and reproducible. As to this established LAMP herein, not only can peanut ingredients be detected but commercial foods containing peanut can also be identified. This assay will be useful and potential for the rapid detection of peanut in practical food markets.

The development of loop-mediated isothermal amplification (LAMP) assays for the rapid authentication of five forbidden vegetables in strict vegetarian diets.

Plant-based food ingredients such as garlic, Chinese leek, Chinese onion, green onion and onion are widely used in many cuisines around the world. However, these ingredients known as the "five forbidden vegetables" (FFVs) are not allowed in some vegetarian diets. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of FFVs using five respective LAMP primer sets. The specific primers targeted the ITS1-5.8S-ITS2 nuclear ribosomal DNA sequence regions among the five vegetables. The results demonstrated that the identification of FFVs using the newly developed LAMP assay is more sensitive than the traditional PCR method. Using pepper, basil, parsley, chili and ginger as references, established LAMP primer sets showed high specificity for the identification of the FFV species. Moreover, when FFVs were mixed with other plant ingredients at different ratios (100:0, 50:50, 20:80, 10:90, 5:95, 2:98, and 1:99), no cross-reactivity was evident using LAMP. Finally, genomic DNAs extracted from boiled and steamed FFVs in processed foods were used as templates; the performance of the LAMP reaction was not influenced using validated LAMP primers. Not only can FFV ingredients be identified but commercial foods containing FFVs can also be authenticated. This LAMP method will be useful for the authentication of FFVs in practical food markets in the future.

High molecular weight of polysaccharides from Hericium erinaceus against amyloid beta-induced neurotoxicity.

BACKGROUND: Hericium erinaceus (HE) is a well-known mushroom in traditional Chinese food and medicine. HE extracts from the fruiting body and mycelia not only exhibit immunomodulatory, antimutagenic and antitumor activity but also have neuroprotective properties. Here, we purified HE polysaccharides (HEPS), composed of two high molecular weight polysaccharides (1.7 × 10(5) Da and 1.1 × 10(5) Da), and evaluated their protective effects on amyloid beta (Aß)-induced neurotoxicity in rat pheochromocytoma PC12 cells. METHODS: HEPS were prepared and purified using a 95 % ethanol extraction method. The components of HEPS were analyzed and the molecular weights of the polysaccharides were determined using high-pressure liquid chromatography (HPLC). The neuroprotective effects of the polysaccharides were evaluated through a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and an MTT assay and by quantifying reactive oxygen species (ROS) and mitochondrial membrane potentials (MMP) of Aß-induced neurotoxicity in cells. RESULT: Our results showed that 250 µg/ml HEPS was harmless and promoted cell viability with 1.2 µM Aß treatment. We observed that the free radical scavenging rate exceeded 90 % when the concentration of HEPS was higher than 1 mg/mL in cells. The HEPS decreased the production of ROS from 80 to 58 % in a dose-dependent manner. Cell pretreatment with 250 µg/mL HEPS significantly reduced Aß-induced high MMPs from 74 to 51 % and 94 to 62 % at 24 and 48 h, respectively. Finally, 250 µg/mL of HEPS prevented Aß-induced cell shrinkage and nuclear degradation of PC12 cells. CONCLUSION: Our results demonstrate that HEPS exhibit antioxidant and neuroprotective effects on Aß-induced neurotoxicity in neurons.

Development of qualitative and quantitative PCR analysis for meat adulteration from RNA samples.

Total RNA samples were used to establish qualitative and quantitative PCR-based methods for assessing meat adulteration. The primers were designed based on the mRNA sequences of troponin I (TnI), mitochondrial ribosomal protein (MRP) and tropomodulin genes to distinguish chicken, pork, goat, beef and ostrich. There was no cross reaction between the primers, and the detection limit of the cDNA template was 0.01 and 20 ng in simplex PCR and multiplex PCR, respectively. In the low temperature storage test, the detection limits of cDNA template with 10 and 1 ng were determined at 4 °C and -80 °C. In quantitative assay, the precision of real-time PCR analysis expressed as a coefficient of variation (CV) ranged from 0.25% to 5.24% and the trueness, expressed as an error, ranged from 0.28% to 6.98% for adulteration. Thus, herein, we provided alternative tools for the assessment of meat adulteration using mRNA-based PCR methods.

Estrogen degradation and sorption onto colloids in a constructed wetland with different hydraulic retention times.

Endocrine disrupting compounds are a global concern, owing to their interference with the endocrine system of wildlife. In particular, natural estrogens at concentrations as low as ng/L level can interrupt the endocrine system of many organisms. A constructed wetland is an effective means of removing the residual levels of estrogen. This study investigates the estrogen degradation and sorption on colloids in a constructed wetland at hydraulic retention times (HRTs) of 27.5, 45.9, and 137.5h. Three natural estrogens (i.e. estrone (E1), 17ß-estradiol (E2), and estriol (E3)) are analyzed with liquid chromatography/tandem mass spectrometry. At HRT=27.5h, no degradation occurs; at HRT=45.9h, the degradation rates are 0-46.2%; and at HRT=137.5h, the degradation rates are 40-84.3%. Additionally, estrogen sorption coefficients (logKCOC values) range from 3.37 to 4.89. Average logKCOC values are 4.08±0.33, 4.04±0.34, and 4.11±0.28 for E1, E2, and E3, respectively. At different HRTs, values of logKCOC increase with an increasing HRT. Analytical results indicate that constructed wetlands can remove residual natural estrogens. With an increasing HRT, the estrogen degradation rate increases as well as the estrogen sorption on colloids.

Immunomodulating activity of Nymphaea rubra Roxb. extracts: activation of rat dendritic cells and improvement of the T(H)1 immune response.

Polysaccharides play a key role in enhancing immune function and facilitating cellular communication. Here, we purified Nymphaea rubra Roxb. polysaccharides (NR-PS) by treating them with pullulanase. They were then cultured with immature dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs). After treatment with bioactive NR-PS with a degree of polymerization (DP) value of 359.8, we found that the DCs underwent morphological changes indicative of activation. CD80/86 (87.16% ± 8.49%) and MHC class II (52.01% ± 10.11%) expression levels were significantly up-regulated by this treatment compared to the controls (65.45% ± 0.97% and 34.87% ± 1.96%). In parallel, endocytosis was also reduced (167.94% ± 60.59%) after treatment with 25 µg/mL of NR-PS as measured by the medium fluorescence intensity compared to the control (261.67% ± 47.26%). Furthermore, the DCs after treatment with 25 µg/mL NR-PS showed increased IL-12 (102.09 ± 10.16 to 258.78 ± 25.26 pg/mL) and IFN-γ (11.76 ± 0.11 to 15.51 ± 1.66 pg/mL) secretion together with reduced IL-10 secretion (30.75 ± 3.35 to 15.37 ± 2.35 pg/mL), which indicates a T(H)1 immune response. In conclusion, NR-PS exhibits stimulatory effects on rat DCs and promotes the secretion of T(H)1 cytokines. Taken together, our studies are the first to show that NR-PS is an immunomodulator affecting the maturation and functioning of DCs.

Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus.

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-ß-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.

Identification of the NLS and NES motifs of VP2 from chicken anemia virus and the interaction of VP2 with mini-chromosome maintenance protein 3.

BACKGROUND: VP2 of chicken anemia virus (CAV) is a dual-specificity phosphatase required for virus infection, assembly and replication. The functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP2 in the cell, however, are poorly understood. Our study identified the presence of a NLS in VP2 and showed that the protein interacted significantly with mini-chromosome maintenance protein 3 (MCM3) in the cell. RESULTS: An arginine-lysine rich NLS could be predicted by software and spanned from amino acids 133 to 138 of VP2. The critical amino acids residues between positions 136 and 138, and either residue 133 or 134 are important for nuclear import in mammalian cells based on systematic mutagenesis. A NES is also predicted in VP2; however the results suggest that no functional NES is present and that this protein is CRM1 independent. It was also shown that VP2 is a chromatin binding protein and, notably, using a co-immunoprecipitation assay, it was found that VP2 association with MCM3 and that this interaction does not require DSP activity. CONCLUSIONS: VP2 contains a NLS that span from amino acids 133 to 138. VP2 is a CRM1 independent protein during nuclear export and associates with MCM3 in cells.

Composition analysis and immuno-modulatory effect of okra (Abelmoschus esculentus L.) extract.

The aim of this study was to analyse the composition of okra (Abelmoschus esculentus L.) extract and investigate the effect of A. esculentus L. polysaccharides (AE-PS) on the maturation and function of dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs) in vitro. BMHC-derived immature DCs (BMHC-imDCs) were extracted from rats and treated with AE-PS. The hydrolysed okra extract contained 0.6% ß-1, 3-D-glucan. AE-PS induced the presence of polymorphic nuclei and elongated protrusion in the BHMC-imDCs, indicating DC activation. Treatment with 100 µg/mL of AE-PS increased the MHC class II and CD80/86 expression levels by 41% and 42%, respectively. Treated cells had reduced endocytosis activity. The secretion of IL-12 and IFN-γ increased significantly by 120% and 75%, respectively, when treated with 100 µg/mL of AE-PS. Moreover, IL-10 production was reduced by 66%. In conclusion, AE-PS exhibits stimulatory effects on rat dendritic cells and promotes the secretion of T(H)1 cytokines.