Assessing safety of Lactobacillus plantarum MTCC 5690 and Lactobacillus fermentum MTCC 5689 using in vitro approaches and an in vivo murine model.

Lactobacilli have a long history of safe use in human nutrition, however, inclusion of any new strain, despite its safe usage evidence, warrants proper analysis of its safety and toxicity under the purview of existing regulations. In the present investigation, Lactobacillus plantarum MTCC 5690 and Lactobacillus fermentum MTCC 5689 were evaluated for their safety and toxicity using both in vitro and in vivo approaches. The in vitro assays included mucin degradation, hemolytic activity, biogenic amine production and platelet aggregation assay. The safety was also assessed using acute, subacute and subchronic assays, bacterial translocation studies, intravenous and intravenous administration and genotoxicity assay in murine model. The outcome of this toxicological safety assessment indicated that both the test strains lacked any harmful metabolic activity or any genotoxic effects. Furthermore, the results of oral toxicity studies in mice revealed that short term administration of high cell mass concentration of 1012 cfu/animal as well as long term feeding of the probiotic strains did not alter any hematological, general health parameters or cause any organ specific disorder. Based upon these scientific assessments and supported by long history of safe use, both MTCC 5690 and MTCC 5689 may be considered safe for human consumption.

Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).

We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), ß-actin (ACTB), ß-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, ß-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

Chymosin and other milk coagulants: sources and biotechnological interventions.

Calf rennet, which consists of over 90% chymosin, is commonly used in cheese industries for the curdling of milk. Various animal, plant and microbial sources have been exploited as possible alternatives to calf rennet. The coagulating properties of the enzymatic preparations (coagulants) from these sources differ in terms of their physicochemical factors. The cheese industry has always sought out novel and stable enzyme sources, and recombinant chymosin has been found to be an effective alternative since it possesses several advantages over plant and microbial milk-clotting enzymes. This paper reviews the use of various milk coagulants, especially animal coagulants, for cheese making. Advancements in genetic and protein engineering to produce recombinant chymosin are discussed in addition to evaluating its identity to the rennet available from natural sources.

Characterisation of pSMA23, a 3.5 kbp plasmid of Lactobacillus casei, and application for heterologous expression in Lactobacillus.

The complete nucleotide sequence of plasmid pSMA23 isolated from Lactobacillus casei A23 was determined. Plasmid pSMA23 is a 3497bp circular molecule with a G+C content of 38.18%. Four putative open reading frames were identified. Based on homology, two orfs were identified as genes encoding replication initiation (Rep) and mobilisation (Mob) protein, respectively. Various regulatory regions like promoters, ribosome binding site (RBS), transcriptional terminators were deduced from the sequences of rep and mob. The origin of replication (dso) was predicted. Shuttle vectors pL142 and pL157 were constructed for Escherichia coli and Lactobacillus using rep gene and ori of pSMA23 for replication in Lactobacillus, the ori of the commercial vector pBluescript SkII+ for replication in E. coli, and the erythromycin and chloramphenicol resistance genes of pE194 and pC194, respectively, as selection markers. Transformants of E. coli and Lactobacillus were obtained on media containing erythromycin and chloramphenicol, respectively, suggesting expression of the ermC and cat194 genes in both organisms. The shsp gene of plasmid pSt04 of Streptococcus thermophilus encoding a small heat shock protein and the Lactobacillus plantarum cbh gene encoding conjugated bile salts hydrolase were cloned and successfully expressed in the heterologous host Lb. casei LK1 with the aid of pSMA23-derived vectors.

NEED FOR PRECISE PROTOCOL IN MAGNETIC RESONANCE IMAGING OF THE POST OPERATIVE SPINE.

Gadolinium enhanced Magnetic Resonance Imaging (MRI) for the evaluation of the post operative lumbo-sacral spine is a sensitive and specific imaging technique. A need for establishing a specific protocol for Failed Back Surgery Syndrome (FBSS) for use in the service hospitals is highlighted for convenience of patient management and preservation of active manpower. The MR scan of 50 patients performed over a span of six months, who complained of persistent low backache even after surgery, were retrospectively analysed. The specificity of this series using MRI in indicating the exact cause of FBSS was clocked at only 30%. The conditions diagnosed were rectifiable. The balance of the patients who could not be offered any specific diagnosis towards the cause, were being managed conservatively / placed in low medical category for a considerable period. It was noted with concern that there was non prevalence of sequence like the fast spin echo with gadolinium enhancement as a routine.

Purification and characterization of a bacteriocin-like compound (Lichenin) produced anaerobically by Bacillus licheniformis isolated from water buffalo.

AIMS: To characterize a bacteriocin-like factor from Bacillus licheniformis 26 L-10/3RA isolated from buffalo rumen. METHODS AND RESULTS: The culture supernatant exhibited the antibacterial activity against a number of indicator organisms in a cut-well agar assay under anaerobic conditions. The inhibitory component was purified by following ammonium sulphate precipitation, gel filtration and ion exchange chromatography and confirmed to be a single peptide. A single band on tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity and having an estimated molecular mass of approximately 1400 dalton. Complete amino acid sequence of the peptide yielded 12 amino acids from the N-terminal end (ISLEICXIFHDN). No homology with previously reported bacteriocins was observed and has been designated as Lichenin. Lichenin was found to be hydrophobic, sensitive to atmospheric oxygen, retained biological activity even after boiling for 10 min and was active over a pH range of 4.0-9.0. CONCLUSIONS: The Lichenin represents the first anaerobiosis specific expression of bacteriocin-like compound isolated from Bacillus licheniformis 26 L-10/3RA of buffalo rumen origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Lichenin could be a potential candidate for manipulating the rumen function at molecular level intended for improving the productivity of the ruminant.

IMPROVED PRESERVATION OF FACIAL NERVE IN TRANSTEMPORAL APPROACHES TO THE SKULL BASE.

Surgery of the skull base has evolved over the past 100 years. The importance of combined otorhinological and neurosurgical approach in the management of lesions of the skull base has been realised in recent years. Through this strategy, lesions which were once thought unresectable are being removed completely with reduction in mortality. However, the morbidity due to facial palsy in lateral skull base surgery is common. To avoid this, intraoperative facial nerve monitoring was introduced in the late 70's and has been refined to such a point that it is now possible to safely predict the recovery of the temporary facial paralysis after lateral skull base surgery. The present study which was carried out in the Armed Forces for the first time has evaluated this by comparing 5 patients who underwent this surgery without intraoperative facial nerve monitoring with 5 patients who were operated with intraoperative facial nerve monitoring. The results have proved the efficacy of this tool in reducing the severity and duration of facial palsy after this surgery.

SPHINCTER INVOLVEMENT IN LUMBAR DISC HERNIATION.

A retrospective analysis of 30 operated cases of prolapsed lumbar disc with cauda equina syndrome was carried out. 27 were male and 3 were female. Age varied from 27 to 60 years. 10 were acute presentation and 20 had previous history of backache-sciatica syndrome. The most common sphincter dysfunction was urinary retention and other neurological findings, pointing to sphincter involvement were impairment of sensation in the perineum, lax anal sphincter and absent superficial anal reflex. The other motor or sensory deficit depended on level of disc herniation. The majority of our patients reached us between 7-14 days after onset of sphincter dysfunction. Emergency myelogram was the most common investigation done and L 4/5 was most common disc herniation. Emergency surgery was done in 24 cases and semi emergency in 6 cases. Patients were followed up and at 1 year follow-up 12 had recovered fully, 7 partially and in rest 11 patients there was no significant improvement in sphincter functions.

Bovine chymosin: production by rDNA technology and application in cheese manufacture.

Bovine chymosin, an aspartyl protease extracted from abomasum of suckling calves, is synthesized in vivo as preprochymosin and secreted as prochymosin which is autocatalytically activated to chymosin. Chymosin is bilobular, with Asp 32 and Asp 215 acting as the catalytic residues. Chymosin A and chymosin B have pH optima of 4.2 and 3.8, respectively, and act to initiate milk clotting by cleaving kappa-casein between Phe 105 and Met 106. The gene encoding chymosin has been cloned and expressed in suitable bacteria and yeast hosts under the control of lac, trp, trp-beta, gly A genes, and serine hydroxymethyl-transferase promoters. Protein engineering of chymosin has also been attempted. A number of companies are now producing recombinant chymosin for commercial use in cheese manufacture.

Vestibular Schwanomma with normal hearing.

Schwanomma of the vestibular nerves usually present with sensorineural hearing loss of varying degrees. We report here a large (5.5cm x 3.5cm) vestibular schwanomma with normal hearing. Other unusual features of this case were the young age (20 yrs) of the patient and absence of any otological symptoms.

FACIAL PALSY - TREATMENT OPTIONS.

Facial palsy poses a diagnostic and therapeutic challenge to the doctor. Definite treatment modalities, medical, surgical and physical have evolved and can be used either singly or in combination to treat this condition successfully. 25 cases of facial palsy of varied aetiology managed over 2 year period in the Neurotology clinic of Armed Forces Medical College are presented. 10 cases underwent medical management only. 15 cases underwent surgical management, consisting of facial nerve decompression (10), nerve approximation (2), nerve grafting (1) and hypoglossal facial anastamosis [2]. All patients underwent physiotherapy to the paralysed face. Patients with Bell's palsy had 83.5% recovery (5 out of 6 cases), CSOM cases after surgical decompression of facial nerve had a 100% recovery (3 out of 3 cases), iatrogenic facial palsy 80% (8 out of 10 cases) and patients after tumour excision 68% (4 out of 6 cases) recovery. The diagnostic approach, method of evaluation of degree of facial palsy based on clinical, electrodiagnostic tests and the various treatment modalities are discussed.

PCR-based rapid detection of Mycobacterium tuberculosis in blood from immunocompetent patients with pulmonary tuberculosis.

A PCR test based on insertion sequence IS1081 was developed to detect Mycobacterium tuberculosis complex organisms in the peripheral blood. The method was applied to blood samples from immunocompetent individuals with localized pulmonary tuberculosis. Seven of 16 (43.75%) blood samples were found to be positive for the circulating DNA copies of M. tuberculosis complex.

Cerebello-pontine angle choroid plexus papilloma.

A 23 years male presented with clinical and rardiological features of right CP angle papillary tumour. Histopathology confirmed it to he choroid plexus papilloma, which is a rare CP angle tumour.

Spinal epidural tuberculoma simulating prolapsed intervertebral lumber disc.

50 year old man presented with right sciatica syndrome of 10 months duration. Clinically and radiologically he seemed to have prolapse of intervertebral disc at L5 - S1. At surgery a well demarcated greyish yellow mas sticking to S1 root and adjacemt dura was found. Histology confirmed it to be tuberculoma. Patient was put on anti-tubercular chemotherapy and made good recovery.

Antifungal attributes of lactic acid bacteria--a review.

Molds constitute a very important contaminating flora of dairy products. Contamination with undesirable molds has been a serious and frequently disturbing problem in the dairy industry that results in huge losses due to spoilage of cheese and other fermented foods incriminated by a variety of mycoflora such as Aspergillus, Penicillium, Fusarium, Rhizopus, and Mucor. The considerable drop in pH caused by the growth of lactic acid bacteria (LAB) in fermented milk makes such foods a breeding ground for the highly opportunistic fungi to proliferate and thrive, spoiling the products and effecting cost and its commensurate accessories. The major antimicrobial substances isolated from the LAB are found effective against bacteria only and their inhibition toward the growth of contaminating bacteria has been explored in detail. However, studies on the fungistatic properties of LAB are relatively rare. This article reviews the investigative studies on the antifungal aspects of different lactic acid bacteria and the prospects of this exceptional trait as a potential food biopreservative.

Production of antifungal substance by Lactococcus lactis subsp. lactis CHD-28.3.

Six of the 2100 colonies of lactic acid bacteria isolated from 4 month old Cheddar cheese and raw buffalo milk showed antifungal activity against Aspergillus flavus IARI when tested by the well agar diffusion assay on Potato Dextrose Agar containing 0.1% Triton X-100. Out of these, the most promising isolate having a broad spectrum of antifungal activity including Aspergillus flavus IARI, A. flavus NCIM 555, A. parasiticus NCIM 898 and Fusarium spp. was identified as Lactococcus lactis subsp. lactis CHD-28.3. Among the mold cultures used as indicator strains, the most sensitive towards antifungal substance produced by the test culture was A. flavus IARI. The cell-free supernatant of the test culture in Elliker's broth adjusted to pH 6.8 produced an inhibition zone of 15-19 mm against A. flavus IARI, A. flavus NCIM555 and A. parasiticus NCIM898. The isolate when grown at 30 degrees C for 48 h in Elliker's broth showed optimum antifungal activity. When the supernatant was neutralized to pH 7.0 or 7.5, there was little reduction in activity. However, after enzymatic treatment of supernatant with chymotrypsin, trypsin and pronase E, the antifungal activity disappeared which indicated the proteinaceous nature of the antifungal substance.

Production and properties of crude enterotoxin of Pseudomonas aeruginosa.

Pseudomonas aeruginosa CTM-3 was found to be the most potentially enterotoxigenic strain out of the 12 isolates recovered from milk, as a high fluid length ratio, i.e. F/L (1.1) in rabbit gut and a strong permeability response in rabbit skin (38.5 mm2 necrotic zone) was obtained with this culture. No clear-cut relationship between the two tests was observed. Six of the ethidium bromide (300 micrograms/ml) cured variants of this culture completely lost their ability to produce enterotoxin indicating the possible involvement of a plasmid in enterotoxin synthesis. The crude enterotoxin from P. aeruginosa CTM-3 was completely inactivated in 15 s at 72 degrees C. However, it was fairly stable at pH values in the range 4.5-7.5. Both pepsin and trypsin inactivated the enterotoxin activity at a concentration of 40 micrograms/ml. Organic acids, formalin and hydrogen peroxide had no significant effect on the enterotoxin activity. The need for further investigations with purified preparations is emphasized.