Research Journal of Biotechnology

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Vol. 10(6) June 2015

Broad-spectrum anti-yeast activity of natural lactic acid bacteria from Chinese traditional fermented food

Rao Yu and Jiang Yunlu

Lactic acid bacteria (LAB) with wide spectrum inhibitory activity against pathogenic and spoilage yeast were isolated and their inhibitory properties were analyzed. Forty-six presumptive LAB were isolated from seven Chinese traditional fermented food samples. Fourteen typical pathogenic and spoilage yeast were used as indicators. By overlay method and microtitre plate well assay, three Lactobacillus plantarum strains, two Weissella cibaria strains and one Enterococcus faecium strain with excellent anti-yeast activity were selected. Through HPLC analysis, lactic, acetic and phenyllatic acid were measured in the cell-free culture supernatants (CFSs) of anti-yeast positive LAB. By GC-MS detection, cyclic dipeptides were found in CFSs of L. plantarum AT4 and W. cibaria AT6, the latter produced cyclo(Gly-Leu), cyclo(Phe-Pro) and cyclo(Gly-Pro). The variety of cyclic dipeptides in CFS was speculated to decide the inhibitory spectrum against yeast indicators. High inhibitory rate and favorable bioactive spectrum afford the selected LAB strains greatly candidates for bio-control and bio-preservation against pathogenic and spoilage yeast.

Continuous production of inulinase by Penicillium funiculosum

Enas N. Danial and Nayera A. M. Abdelwahed

The optimization of growth conditions for the production of inulinase by Penicillium funiculosum cells was studied as well as the continuous production of the enzyme using immobilized cells. The highest amount of enzyme (163.5U/ml) was obtained when the producing cells were incubated for 96 hours at 27oC and 200 rpm in a fermentation medium containing both inulin and peptone as sole carbon and nitrogen sources respectively. However, when the cells of the tested microorganism were adsorbed on different carriers, especially linen fibers, their production ability was also successfully maintained to different extents for seven successive batches. Moreover, commercially pure inulin is very expensive in only small quantities; this fermentation medium was later substituted by a crude inulin solution obtained from Jerusalem artichoke tubers. The crude inulin juice was able to sustain inulinase production during the second batch cultivation of the Penicillium funiculosum, immobilized by their adsorption on linen fibers, in a satisfactory level of about 122U/ml. The use of the previously mentioned crude inulin preparation was also compared to the use of either complete or minimal media, composed solely of 1% pure inulin. The method, adopted in this study for inulinase production, is simple, economic, time saving, non-toxic to the microorganism and the loaded linen pads are reusable.

Breeding of new strains of Hypsizygus marmoreus by mating of monokaryotic mycelia and marker-assisted identification

Sun Shu-Jing, Zhang Hong-Mei and Hu Kai-Hui

Mating of monokaryotic mycelia by hyphal fusion is a typical method for generating new dikaryotic strains. Two monokaryons were isolated from 86 protoplast regenerated colonies of Bai yu and 13 monokaryons from 157 colonies of Hui xie. Twelve out of 26 matings were observed the formation of a clamp connection. The mated dikaryons were cultivated to investigate their morphological and physiological characteristics. Five mated strains, 13-49, 55-25, 55-4, 55-51 and 55-6 showed higher biological efficiency and a shorter cultivation period than that of the parental strains. The color of the pileus of the five mated strains was pale yellow and was intermediate between the parent strains. The strain 13-49 with long stipe has an increase of 11.8% in fruiting body production, a fruiting time shortened by 7 days and more effective for mushroom buds. The strain 13-49 is a new strain but closer to the parent strain Huixie identified by RAPD technique. The result is supported by the results of an antagonistic test. This study suggests that the mating of monokaryotic mycelia from protoplasts using molecular identification is highly utilizable approach for the development of new mushroom strains.

In Vitro Screening of Callus Cultures and Regenerants for Drought Tolerance in Upland Rice

Tripathy Swapan K.

Proliferated calli from mature caryopsis culture of six upland rice cultivars (cv. Khadagiri, Mandakini, Annada, Sahbhagidhan , N22 Selection and Vandana) were cultured on selection medium supplemented with polyethylene glycol (PEG6000:0.0, 0.5, 1.0, 1.5 and 2.0M) / mannitol / sorbitol (0.0, 2.5, 5.0, 7.5 and 10M) used as selective agents for osmotolerance. The drought tolerant calli were selected and also cultured for two cycles, each of two weeks on regeneration medium supplemented with PEG6000/ mannitol /sorbitol. Increased concentration of PEG resulted in a progressive reduction in callus fresh weight whereas it was more than double in no stress or control after four weeks of sub-culture in the callus induction medium. Significant reduction in callus fresh weight was observed in response to PEG at 1.5-2.0M concentration. Increased concentration of PEG/mannitol/sorbitol in the medium resulted in decreased plant regeneration frequency. However, the effect of drought stress was more prevalent in PEG at even lower concentration(s) than mannitol and sorbitol. Therefore, calli of rice genotypes could be tested for drought tolerance at 1.5-2.0M PEG. N22 selection was tested as highly drought tolerant while Khandagiri and Annada were highly sensitive to PEG based on callusing ability and plant regeneration among the present set of six upland genotypes. A number of putative drought tolerant plants were obtained from N 22 sel., Sahbhagidhan and Mandakini and those were forwarded for field evaluation following a sequence of hardening process. Besides, the method adopted for in vitro screening of plant regenerants may pave the way for improvement of drought tolerance.

Influence of conjugated linoleic acid (CLA) on growth performance, body composition and cholesterol metabolism of large yellow croaker (Pseudosciaena crocea R.) ‏

Wang Jinbo, Han Fei and Qi Lili

The present study was conducted to determine the effects of conjugated linoleic acid (CLA) on growth performance and fat metabolism of large yellow croaker. We found that the dietary CLA had no effect on feed conversion ratio (FCR), specific growth rate (SGR) and condition factor (CF) of fish. Dietary CLA supplementation increased the contents of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) and decreased monounsaturated fatty acid (MUFA) content in muscle (P<0.05). The deposition of the cis-9, trans-11 and trans-10, cis-12 CLA isomers in muscle was significantly increased by CLA (P<0.05). A increasing of C18:2 n-6, C20:5 n-3 (eicosapentaenoic acid, EPA) and C22:6 n-3 (docosahexaenoic acid, DHA) was observed in muscle of fish fed 2-4% CLA (P<0.05). Fish fed CLA exhibited lower concentrations of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) compared with the control (P<0.05). Free cholesterol (FC) and high-density lipoprotein cholesterol (HDL-C) contents were unaffected by CLA. The results indicate that large yellow croakers are capable of absorbing and depositing CLA and long-chain n-3 PUFA in muscle, dietary CLA could possess anti-hypercholesterolemia activity in this species. Taken together, large yellow croaker fed with CLA has beneficial effects on human health as a food source.

Research on the meat quality produced by Polyodon Spathula sturgeons species related to human nutritional requirements

Simeanu D., Creangă Ş. and Simeanu Cristina

The objective of the present study was to evaluate the main physico-chemical and histological traits of the paddlefish meat (Polyodon spathula) from an aquaculture pond located in the North-East of Romania. A total of 40 fish samples from 40 individuals with different ages (each ten individuals from 1st to 4th summer), were randomly harvested. The samples were examined by macro and microscopic and some physico-chemical analysis such as meat pH determination, water, protein, lipid and mineral contents. The results revealed that Polyodon spathula sturgeons belong to the protean fish group with low fat content. Paddlefish meat is also rich in monounsaturated and polyunsaturated fatty acids (linoleic, linolenic, arachidonic, eicosapentaenoic, docosapentaenoic and docosahexaenoic acids), as well as low cholesterol level. Therefore, this sturgeon could be considered as a source of “healthy fats”. The ratio between connective and muscular tissues was favorable for the muscular one.

Emetic and enterotoxin profiles of food-borne Bacillus cereus strains from Turkey

Karagoz Kenan, Adiguzel Gulsah and Dikbas Neslihan

To determine the role of Bacillus cereus as a potential pathogen in food poisoning, the production of an emetic toxin (cereulide) and enterotoxigenic by B. cereus was isolated in various food sources. Ninety- two strains of B. cereus isolated from milk, cereal, chicken and meat products have been used to study profiles in detail and by polymerase chain reaction (PCR) for presence of toxin genes. B. cereus can cause diarrheal and emetic type of food poisoning but little study has been done on the main toxins of food poisoning caused by B. cereus in Turkey. The objective of this study is to characterize the toxin gene profiles and toxin-producing ability of 92 B. cereus isolates from food samples in Turkey. The detection rate of nheA, hblC, cytK and EM1 genes among all B. cereus strains was 42, 55,4, 14 and 6,5% respectively. The EM1 gene encoding emetic toxin was not detected in strains from chicken and cereal. B. cereus strains carried at least 1 of the 3 enterotoxin genes classified into 9 groups according to the presence or absence of 4 virulence genes. The 2 major patterns, nheA, hblC, gene accounted for 48,7 % of all strains (92 B. cereus isolates). Our finding detected that NHE and HBL enterotoxins encoded by nhe and hbl genes were the major toxins among B. cereus tested in this study and enterotoxic type of B. cereus was predominant in Turkey. Thus, emetic toxin and enterotoxin genes should be frequently screened to provide insight into B. cereus food poisoning.

Determination of glucose concentration in various optical phantoms by indigenously developed ultrasound collaborated infrared technology

Srivastava Anuj, Chowdhury Md. Koushik, Sharma Shiru and Sharma Neeraj

The advent of a successful noninvasive blood glucose determining technology would revolutionize the diabetes management protocols. Here indigenously developed techniques based on amplitude modulated ultrasound with infrared technique have been utilized to detect particular glucose concentrations in various phantoms. This research paper explains the impact of the glucose concentration determinations in different types of optical tissue property resembling phantoms. Various samples utilized here were distilled water, commercialized milk, chicken breast tissue and human whole blood. Concentrations of the glucose molecules were varied in phantom samples and its effects were observed through indigenously developed modulated ultrasound and infrared technique based unit. Actually, in the indigenously developed unit, the ultrasound in modulating wave characteristics generates molecular vibrations throughout the phantom sample. The infrared light captures the glucose based vibrational patterns in solitary mode to yield the amount of glucose concentration present in various phantom samples. The result signifies the good functioning effectiveness of our indigenously developed MUS-IR (Modulated Ultrasound-Infrared) unit for measuring Dextrose (glucose) concentrations in particular optical phantoms. In near future, this new hybridized technology of ultrasound and infrared units could be helpful for designing and developing noninvasive blood glucose monitoring devices.

Oscillatory Patterns of leaf growth in Epiphytic Plants: The role of Arthropods and Litter

Castaño-Meneses Gabriela, Romo-Herrera José-Manuel, José Garcia and Castaño Victor M.

The effect of arthropod presence and litter accumulation on the vegetative growth of the vascular epiphytes, on the leaves growth pattern in Tillandsia violacea was conducted in an Abies-Quercus temperate forest in Central Mexico. The leave production was recorded every month for one year in 40 relatively small plants of T. violacea (< 20 cm at the base of the rosette) revealing that in plants with litter and arthropods accumulation, there is an oscillatory period of 9.6787 month in the leaves production whereas when arthropod presence and litter accumulation are restricted, the oscillatory period is lost showing an irregular growth pattern. These results suggest the importance of arthropods and litter accumulation on the development of the epiphytic plants.

Development and Validation of a Residual Host DNA Detection Method for Biopharmaceutical Products

Chen Renhai and Liu Quanhai

A commercial kit, consisting of magnetic particle-based DNA extraction, followed by a quantitative PCR analysis, was commonly used to detect the residual host DNA of biopharmaceuticals made in CHO cells. However, the recovery of the target DNA was just no more than 30% which is much lower than that the regulatory guideline expected between 80% to 120% range, especially when the kit was applied to the residual CHO DNA detection in monoclonal antibody samples. In this study, we describe our improvements in the recovery on extracting residual DNA from monoclonal antibody samples that often contains high protein concentration. Enzyme/protein ratio, incubate conditions and elution conditions were developed and the optimized method resulted in the increasing of DNA recovery from 30% to 80% and above. Besides, high speed centrifugation turned out to help lowering the particle loss risk and shortening the time of the magnetic particles attracted by the magnetic stand. The optimized method was validated formally to identify its accuracy, precision and robustness. At last, the validated method was applied to samples analysis from different purification processes as well as the final drug product release successfully.

Improved Agrobacterium-mediated transformation of Vinca minor via combination of dynamic transfection and vacuum infiltration

Wang G. R. and Tang Ning

An improved Agrobacterium-mediated transformation method was developed by combining dynamic transfection with vacuum infiltration and challenged for Vinca minor hairy roots induction. The transfection was conducted in a simple device including a funnel-shaped bottle, a vacuum pump and a liquid pump. During the process of transformation, the bacterial solution continuously circulated in the funnel-shaped bottle and the combination of dynamic transfection and vacuum infiltration was achieved by using the liquid pump and vacuum pump simultaneously. The results indicated that the transformation efficiency was affected by filter size, solution circulation velocity as well as vacuum level and the maximum transformation rate of 82.5% was obtained under the appropriate conditions which was higher than that 25.3% in control.

Evaluation of Hyacinth bean (Lablab purpureus) genotypes for salinity-tolerance

Kokila S. and Varadahally R. Devaraj

Soil salinity is one of the most severe factors limiting growth and productivity of crops. Changes in antioxidants and antioxidant enzymes in response to salinity stress of four Dolichos lablab (Lablab purpureus L. sweet) genotypes were investigated. Stress indicators like H2O2, lipid peroxidation, antioxidants such as GSH, proline, Asc and antioxidant enzymes such as GPOX, CAT and GR, metabolic enzymes like AP and AMY were analyzed in ten days old seedlings under salt stress. Salt-induced oxidative stress was observed in all four genotypes with greater lipid peroxidation in HA4. Inter-varietal differences were observed in extent of elevation in GSH, Asc and Proline. The genotypes also differed in terms of antioxidant and metabolic enzyme pattern. While three varieties seem to rely on antioxidants for salt stress response, HA4 appeared to rely on both antioxidants and antioxidant enzyme.

An improved double-staining fluorescence assay for early apoptosis detection by flow cytometry

Li Gang, Zhong Yanping, Shen Qingrong and He Min

Apoptosis is a fundamental feature of many biological processes. The exposure of Phosphatidylserine (PS) at cell surface during the early stages of apoptosis is a characteristic cellular change which can be detected by Flow cytometry. The apoptosis of Jurkat cells was induced by different doses of EGCG[(-)-Epigallocatechin Gallate]. The apoptotic cells were collected and detected with AnnexinV-FITC/PI staining and Anti PS- AF488/7-AAD staining respectively. The percentage of apoptotic cells was analyzed and quantified by flow cytometry. In comparison of AntiPS-AF488/7-AAD staining with AnnexinV-FITC/PI staining, we found that the two methods presented the similar tendency in detecting the proportion of viable cells, early apoptotic cells and late apoptotic cells. Both methods showed that the percentage of late apoptotic Jukat cells was remarkably increased and that of the viable cells was decreased. However, when the cells were treated by EGCG with a concentration of 20 mg/L and 80 mg/L, the percentage of early apoptotic cells measured by AntiPS-AF488/7-AAD staining was obviously higher than that of the cells measured by AnnexinV-FITC/PI staining (p < 0.05), but no difference between these two methods was detected in measurement of late apoptosis (p > 0.05). AntiPS-AF488/7-AAD staining method could increase the sensitivity in detection of early apoptotic cells by flow cytometry and it might be a new staining method for early detection of apoptosis.

Impact of transgenic events on Helicoverpa armigera (Hubner) and Spodoptera litura (Fabricius) on cotton in India

Chinna Babu Naik V., Subbanna A.R.N.S., Suneetha P. and Krishna M.S.R.

Four Bt cotton hybrids viz, RCH2 Bt, JK Durga Bt, Nath baba Bt and RCH BG II representing Mon 531, JK cry1Ac, GFM and Mon 15985 events with their corresponding non Bt genotypes were evaluated under field conditions at RARS, Lam, India. The comparative incidence and damage of Helicoverpa armigera (Hubner) and Spodoptera litura (Fabricius) over two years revealed the superiority of Bt cotton hybrids in managing H. armigera. However, the incidence of S. litura is comparable in both Bt and non Bt hybrids except RCH BG II. This second generation Bt cotton hybrid was found superior over the other tested hybrids with no signs of incidence and damage of both target pests. Despite the low levels of damage on all Bt hybrids, little progression was observed after 130 DAS corresponding to decreased levels of toxin expression. The present investigation can be helpful for IPM in Bt cotton.

Identification of heat stress-induced differentially expressed genes of Siberian wildrye grass (Elymus sibiricus L.) leaves

Lee Sang-Hoon, Lee Dong-Gi and Lee Ki-Won

The present study investigated the responses of differentially expressed genes (DEGs) in Siberian wildrye grass leaf to heat stress. Siberian wildrye grass seedlings were subjected to heat treatment at 42°C for 6 h. To identify heat-responsible DEGs in Siberian wildrye grass leaves, we performed an annealing control primer-based differential display reverse transcriptase PCR (ACP-based DDRT-PCR) strategy. Using 120 arbitrary ACP sets, a total of 3 up-regulated and 3 newly synthesized DGEs were visualized via agarose gel stain following sequencing. To gain functional information about these genes and investigate their tentative homologies, a BlastX search was applied using the highest homology. These DEGs were identified as serine carboxypeptidase-like protein, ribosomal protein S19, RNA polymerase beta subunit, heat shock protein (HSP)16.9C, HSP 83 and 17.9 kDa class I HSP-like. The present study showed that 3 HSPs, including 2 small HSPs, were newly synthesized by heat stress. The identification of heat-responsible genes may offer a better understanding of the heat stress response in crop plants. Furthermore, newly induced genes may play pivotal role for protection of cellular damage caused by heat stress.