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.

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.

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.

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.

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.

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.

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.

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.

Enterocin typing of enterococci isolated from dried infant foods.

One hundred and fifty enterococcal isolates recovered from 16 market samples of infant foods and 35 from other sources were characterized and subjected to enterocin typing with 18 indicator strains. Among 150 enterococcal isolates, 114 (76%) were able to be typed by the indicator strains. Although 24 enterocin patterns were observed with these enterococci, the most prevalent types were X-9, 224, and 65-603. Occurrence of pattern X-9 either singly or in combination with many other types was most frequent. Many of the enterocin patterns in enterococcal isolates were recovered from samples of dairy water supply and hand washings of personnel working in a dairy plant that manufactured infant food; this suggests the possibility of these as sources of contamination. Enterocin typing of enterococci could prove useful in epidemiological studies.

Changes in enterococcal population during storage of reconstituted infant food.

A six-fold increase in the enterococcal population was observed in reconstituted infant food samples after storage for 2 h at 37 degrees C. The increase in enterococcal counts at 40 degrees C and 45 degrees C was approximately five-fold during the same period. However, the corresponding total viable counts increased by twelve fold at these temperatures after 2 h. After 12 h, the enterococcal and total viable counts increased to 39 x 10(4) and 36 X 10(7) colony forming units per ml, at 37 degrees C respectively. A similar pattern in enterococcal and total bacterial count was observed at 40 degrees and 45 degrees C. TNase was detected in reconstituted infant food samples held at 37 degrees, 40 degrees and 45 degrees C, after 12 h, while pH values declined to 5.0, 5.1 and 5.2, respectively at the above temperatures. From TNase positive samples, an isolate S. faecium IF-100 capable to produce TNase was recovered. Storage of reconstituted infant food samples in the refrigerator (5 degrees C) resulted in a gradual increase in enterococcal population which reached 39 X 10(3) c.f.u. per ml after 12 days. However, TNase was not detected in any of these samples.

Variation in the behaviour of enterotoxigenic Staphylococcus aureus after heat stress in milk.

The survival of several strains of Staphylococcus aureus after heat stress in different menstrua was not logarithmic and F-values were determined to express their resistance to heat. Of the strains tested, Staph. aureus 234 (enterotoxin B) was the most heat resistant and Staph. aureus 790 (enterotoxin E) was the most heat sensitive. Buffalo milk gave the best protection to all the strains of Staph. aureus against heat, followed by cow's milk; phosphate-buffered saline gave the least protection. Soyabean casein digest agar gave maximum recovery of survivors followed by brain heart infusion and Baird-Parker medium. At 50 degrees C there was no marked variation in coagulase production by the surviving strains but at 55 and 62.5 degrees C there was complete loss of coagulase activity. There was a decreased deoxyribonuclease (DNase) production by all the strains of Staph. aureus after heat stress. Heat-treatment at 55 and 62.5 degrees C resulted in loss of enterotoxin production by all the survivors except S6 and 234, the surviving cells of which still produced enterotoxin B after heat treatment at 55 degrees C. Most of the survivors regained lost characteristics such as coagulase, DNase and enterotoxin production after four to five passages through BHI which suggests that subculture of Staph. aureus recovered from heat-processed milk is necessary to avoid false results.