https://doi.org/10.17113/ftb.57.01.19.5879

     

In Vitro Assessment of the Antioxidant Properties of Aqueous Byproduct Extracts of Vitis vinifera


Roberto Puglisi^1*, Alex Severgnini¹, Aldo Tava² and Marina Montedoro<sup>1


1</sup>Istituto Sperimentale Italiano Lazzaro Spallanzani, Loc. La Quercia, 26027 Rivolta d’Adda (CR), Italy
²CREA-ZA Centro di Ricerca Zootecnia e Acquacoltura, Viale Piacenza 29, 26900 Lodi, Italy



Article history:
Received: 1 June 2018
Accepted: 31 January 2019




Key words:
non-cytotoxic effect, extraction at low temperature, human dermal fibroblasts, oxidative status, mitochondrial membrane

Summary:
Aqueous extracts were obtained at low temperature with the Naviglio technology from grapevine stalks (Merlot), marc (Merlot and Cabernet Sauvignon) and leaves (Merlot) as typical byproducts of winemaking industry, and their properties were evaluated cytofluorometrically on human dermal fibroblasts. Leaf extracts had the greatest total phenolic ((47.6±3.5) mg/g) and proanthocyanidin ((24.2±0.1) mg/g) contents compared to the others. The preliminary colorimetric MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay individuated two consecutive non-toxic volume fractions of each extract (from 0.8 to 12.8 %) that were adopted for three cytofluorometric tests. The first cell membrane test did not evidence any harmful effects against plasma membranes at the two non-toxic volume fractions. The second mitochondrial membrane test showed a decreased (p<0.01) percentage of cells ((15.7±8.3) vs (32.5±1.3) %) with active polarized mitochondrial membranes at the higher non-cytotoxic volume fractions of extracts from Cabernet Sauvignon marc in response to 4.5 mM H₂O₂, and from Merlot stalks (p<0.05) at 1.5 mM H₂O₂ ((49.3±6.1) vs (64.6±2.4) %) and without H₂O₂ ((89.7±2.4) vs (96.9±1.8) %), compared to the controls submitted to the same H₂O₂ concentration. Conversely, mitochondrial activity of leaf extracts significantly (p<0.05) increased ((96.3±1.8) and (96.4±1.4) %) after treatment with 0.5 mM H₂O₂ at both non-cytotoxic volume fractions compared to control ((88.2±1.1) %). Finally, as evidenced by the third oxidative status test, stalk extracts did not evidence relevant effects on the cellular oxidative state, while the extracts of marc and leaves demonstrated significantly medium (p<0.05) to highly (p<0.001) positive effects following exposure to H₂O₂ ranging from 0.5 to 4.5 mM, compared to controls.

*Corresponding author:    +39036378883
                                             +390363371021
                                             roberto.puglisi@istitutospallanzani.it

https://doi.org/10.17113/ftb.57.01.19.5865

       

Development of a Transgenic Flammulina velutipes Oral Vaccine for Hepatitis B


Li-Hsin Huang¹, Hao-Yeh Lin², Ying-Tzu Lyu¹, Chiau-Ling Gung¹ and Ching-Tsan Huang<sup>2*


1</sup>MycoMagic Biotechnology Co. Ltd., 8F-1, 12, Lane 270, Sec. 3, Beishen Road, New Taipei City, Taiwan
²Department of Biochemical Science and Technology, National Taiwan University, 1, Sec. 4, Roosevelt Road, Taipei, Taiwan



Article history:
Received: 24 May 2018
Accepted: 31 January 2019




Key words:
Agrobacterium-mediated transformation, Flammulina velutipes, hepatitis B, mating, oral vaccine

Summary:
Orally administered fungal vaccines show promise for the prevention of infectious diseases. Edible mushrooms are deemed appropriate hosts to produce oral vaccines due to their low production cost and low risk of gene contamination. However, their low expression level of antigens has limited the potential development of oral vaccines using mushrooms. The low expression level might result from impurity of the transgenic mycelia since dikaryotic mycelia are commonly used as transformation materials. In this study, stable transgenic hepatitis B virus surface antigen (HBsAg) in Flammulina velutipes transformants was obtained by Agrobacterium-mediated transformation, followed by fruiting and basidiospore mating. The formation of HBsAg was detected by western blot analysis. The expression levels of HBsAg in transgenic F. velutipes fruiting bodies were (129.3±15.1), (110.9±1.7) and (161.1±8.5) ng/g total soluble protein. However, the values may be underestimated due to incomplete protein extraction. Two of the four pigs in the experimental group produced positive anti-HBsAg-specific IgG after being fed the HBsAg transgenic F. velutipes fruiting bodies for 20 weeks, while no anti-HBsAg antibody was detected in the control group. One of the positive pigs had HBsAg titres of 5.36 and 14.9 mIU/mL in weeks 10 and 14, respectively, but expression faded thereafter. The other positive pig displayed HBsAg titres of 9.75, 17.86 and 39.87 mIU/mL in weeks 14, 18 and 20, respectively. The successful immunogenicity in pigs fed transgenic F. velutipes fruiting bodies demonstrated the potential of using the fungus as an oral vaccine.



*Corresponding author:   +886233664454
                                            +886223634796
                                             cthuang@ntu.edu.tw

https://doi.org/10.17113/ftb.57.01.19.5909

     

Proteinaceous Pancreatic Lipase Inhibitor from the Seed of Litchi chinensis


Sveeta V. Mhatre^1,2, Amita A. Bhagit^1,2 and Raman P. Yadav<sup>2*


1</sup>Department of Medical Biotechnology, MGM School of Biomedical Sciences, MGM Institute of Health Sciences, Sector 1, Kamothe 410209, Navi Mumbai, Maharashtra, India
²MGMIHS OMICS Research Center, MGM Central Research Laboratory, MGM Medical College and Hospital, MGM Institute of Health Sciences, Sector 1, Kamothe 410209, Navi Mumbai, Maharashtra, India



Article history:
Received: 22 June 2018
Accepted: 11 December 2018




Key words:
fruit seed, Litchi chinensis, proteinaceous pancreatic lipase inhibitor, obesity treatment

Summary:
A study of the pancreatic lipase inhibitory activity of a protein from the seed of Litchi chinensis was carried out. Protein was isolated by 70 % ammonium sulphate precipitation followed by dialysis. Lipase inhibitory activity of the protein was evaluated using both synthetic (p-nitrophenyl palmitate) and natural (olive oil) substrates. Protein at the final concentration of 100 μg/mL was able to inhibit 68.2 % pancreatic lipase on synthetic substrate and 60.0 % on natural substrate. Proteinaceous nature of the inhibitor was determined using trypsinization assay. Pancreatic lipase inhibitory protein was sensitive to 0.05 % trypsin treatment with the loss of 61.9 % activity. IC₅₀ of this proteinaceous pancreatic lipase inhibitor was 73.1 μg/mL using synthetic substrate. This inhibitory protein was sensitive to pH, with the highest inhibitory activity at pH=8.0 and the lowest at pH=3.0. Protein was further analyzed using 10 % non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and, interestingly, it showed the presence of a single band of (61±2) kDa when stained with Coomassie brilliant blue. The isolated protein was finally crystallized to see its homogeneity by batch crystallization method. Crystals were well formed with distinct edges. The isolated protein showed good pancreatic lipase inhibitory activity.

*Corresponding author:  +912227432890
                                                +912227437693
                                           +912227431094
                                           ramanpyadav@gmail.com

https://doi.org/10.17113/ftb.57.01.19.5983

       

Multiplex Real-Time PCR with HRM for Detection of Lactobacillus sakei and Lactobacillus curvatus in Food Samples


Konstantin A. Kurbakov^1*, Evgenii A. Konorov^1,2,3, Mikhail Y. Minaev¹ and Oksana A. Kuznetsova<sup>1


1</sup>V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences, Talalikhina 26, 109316, Moscow, Russian Federation
²Vavilov Institute of General Genetics, Gubkina 3, 119333, Moscow, Russian Federation
³Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov University, Malaya Pirogovskaya 20-1, 119435 Moscow, Russian Federation



Article history:
Received: 9 August 2018
Accepted: 17 January 2019




Key words:
real-time PCR, HRM, starter cultures, fermented sausages, Lactobacillus sakei, Lactobacillus curvatus

Summary:
Optimization of fermentation processes requires monitoring the species composition of starter cultures and their growth during fermentation. Most starter cultures contain closely related species. Nowadays, high-resolution melting (HRM) analysis is extensively used for multiplex identification of closely related species. In the present paper, we applied real-time polymerase chain reaction (PCR) with HRM analysis for the detection and differentiation of Lactobacillus sakei and L. curvatus. A primer pair was selected for the site of the rpoA gene of Lactobacillus spp. Eleven starter cultures and fifteen fermented sausages with a known bacterial composition were successfully tested using real-time PCR with HRM analysis with the developed primer pair.

*Corresponding author:  +79629529223
                                           homo_ludens@vniimp.ru