Unveiling the persistent threat: recent insights into Listeria monocytogenes adaptation, biofilm formation, and pathogenicity in foodborne infections.

Listeriosis is a severe disease caused by the foodborne pathogen Listeria monocytogenes, posing a significant risk to vulnerable populations such as the elderly, pregnant women, and newborns. While relatively uncommon, it has a high global mortality rate of 20-30%. Recent research indicates that smaller outbreaks of the more severe, invasive form of the disease occur more frequently than previously thought, despite the overall stable infection rates of L. monocytogenes over the past 10 years. The ability of L. monocytogenes to form biofilm structures on various surfaces in food production environments contributes to its persistence and challenges in eradication, potentially leading to contamination of food and food production facilities. To address these concerns, this review focuses on recent developments in epidemiology, risk evaluations, and molecular mechanisms of L. monocytogenes survival in adverse conditions and environmental adaptation. Additionally, it covers new insights into strain variability, pathogenicity, mutations, and host vulnerability, emphasizing the important events framework that elucidates the biochemical pathways from ingestion to infection. Understanding the adaptation approaches of L. monocytogenes to environmental stress factors is crucial for the development of effective and affordable pathogen control techniques in the food industry, ensuring the safety of food production.

Quality Management of Probiotics: Ensuring Safety and Maximizing Health Benefits.

Consumption of probiotics, which are beneficial live microorganisms, has received a lot of attention because of their potential to improve health and wellness. Robust quality control measures are necessary to ensure the safety of probiotics and maximize their health effects. This review delves into the topic of quality management in probiotics, highlighting the significance of sticking to strict guidelines from manufacture to storage to distribution. Probiotic quality standards, Good Manufacturing Practices (GMP) implementation, quality control and testing techniques, and documentation and traceability systems are all discussed in detail. The importance of taking precautions to avoid microbial contamination, meeting all applicable regulations, and clearly marking and packaging probiotic products is also emphasized. In addition, it reviews the clinical evidence supporting the possible health advantages of probiotics and investigates the processes through which probiotics enhance health. The review continues by stressing the significance of educating and informing consumers about probiotics and their proper use in order to maximize health benefits. Probiotics' potential health benefits can be maximized and consumer faith in these helpful microbes can be bolstered by adopting thorough quality management measures to ensure their safety, efficacy, and consistency.

Efficacy of multi-strain probiotic along with dietary and lifestyle modifications on polycystic ovary syndrome: a randomised, double-blind placebo-controlled study.

PURPOSE: Effect of multi-strain probiotic along with dietary and lifestyle modifications in the management of polycystic ovary syndrome (PCOS) has rarely been reported. We thus aimed to investigate the effect of multi-strain probiotic (Lactobacillus acidophilus UBLA-34, L. rhamnosus UBLR-58, L. reuteri UBLRu-87 (each of 2 billion colony forming units (CFU)); L. plantarum UBLP-40, L. casei UBLC-42, L. fermentum UBLF-31, Bifidobacterium bifidum UBBB-55 (each of 1 billion CFU) and fructo-oligosaccharides (100 mg)) and dietary and lifestyle modifications on restoration of menstrual regularity, weight reduction, metabolic and hormonal profile in women with PCOS. METHODS: A 104 participants (age 18-40 years) were randomly allocated to receive probiotic or placebo capsules for 6 months. Baseline and end line assessment were performed for menstrual cycle regularity, ultrasonography scan for ovaries, total testosterone, dehydroepiandrosterone (DHEAS), insulin, luteinizing hormone/follicle stimulating hormone (LH/FSH) ratio, fasting blood sugar (FBS), homeostatic model assessment-insulin resistance (HOMA-IR), weight reduction, waist-/hip circumference (WC, HC), waist to hip ratio (WHR), and body mass index (BMI). Plasma lipopolysaccharide and effect of intervention on quality of life was investigated. Diet and exercise were controlled during the trial. RESULTS: Probiotic supplement along with dietary and lifestyle modifications significantly regularised menstrual cycle (p 0.023), improved levels of total testosterone (p 0.043), WC (p 0.030), WHR (p 0.027) and menstrual domain of quality of life (p 0.034) as compared to placebo. No adverse events related to study were reported. CONCLUSION: Multi-strain probiotic along with dietary and lifestyle modifications were effective in the management of PCOS. TRIAL REGISTRATION: CTRI: CTRI/2016/07/007086, dated 13 July 2016.

Genetic and Phenotypic Characteristics of a Multi-strain Probiotic for Broilers.

Bacteria isolated from different segments of the gastro-intestinal tract (GIT) of healthy free-range broilers were screened for probiotic properties. Six strains were selected and identified as Lactobacillus gallinarum, Lactobacillus johnsonii, Lactobacillus salivarius, Lactobacillus crispatus, Enterococcus faecalis and Bacillus amyloliquefaciens based on 16S rRNA, gyrB and recA gene sequence analyses. All six strains produced exopolysaccharides (EPS) and formed biofilms under conditions simulating the broiler GIT. Lactobacillus johnsonii DPN184 and L. salivarius DPN181 produced hydrogen peroxide, and L. crispatus DPN167 and E. faecalis DPN94 produced bile salt hydrolase (BSH) and phytase. Bacillus amyloliquefaciens DPN123 produced phytase, amylase, surfactin and iturin A1. No abnormalities were observed when broilers were fed the multi-strain combination, suggesting that it could be used as a probiotic.

Correction to: Genetic and Phenotypic Characteristics of a Multi-strain Probiotic for Broilers.

The authors would like to correct the errors in the publication of the original article.

Understanding the antimicrobial activity behind thin- and thick-rolled copper plates.

The aim of this study was to compare the antibacterial properties of the surfaces of copper plates that were rolled to a thickness of 25 and 100 µm. Differences in topology of 25- and 100-µm-thick copper plates were studied using scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray diffraction (XRD). Antibacterial activity of the copper surfaces was tested against strains of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Streptococcus sp. BY1, Enterococcus sp. BY2, and Bacillus cereus BY3. Changes in viable cell numbers were determined by plating onto optimal growth media and staining with LIVE/DEAD BacLight™. Changes in metabolic activity were recorded by expression of the luciferase (lux) gene. Cell morphology was studied using SEM. Accumulation and diffusion of copper from cells were recorded using inductively coupled plasma mass spectroscopy (ICP-MS). Lipid and protein oxidation were recorded spectrophotometrically. Surfaces of 25-µm-thick copper plates were rough compared to that of 100-µm-thick copper plates. For most species, a five-log reduction in cell numbers, cell membrane instability, and a decline in metabolic activity were recorded after 15 min of exposure to 25-µm-thick copper plates. Copper accumulated in the cells, and lipids and proteins were oxidized. The rough surface of thinner copper plates (25 µm thick) released more copper and was more antimicrobial compared to thicker (100 µm) copper plates. Cell death was attributed to destabilization of the cell membrane, lipid peroxidation, and protein oxidation.

Antimicrobial Hyaluronic Acid-Cefoxitin Sodium Thin Films Produced by Electrospraying.

The healing properties of hyaluronic acid (HA) in the recovery of wounds are well known. Cefoxitin (Cef), a cephalosporin antibiotic, is generally used to prevent and treat postoperative infections. In this study, we describe the incorporation of Cef in HA thin films (Cef-HAF) by using electrospraying. Scanning electron microscopy images showed that HA-containing thin films (HAF) were composed of numerous nanoparticles (255 ± 177 nm in diameter) with irregular surfaces, connected to each other with nanofibers of 50 ± 11 nm in diameter. Cef-HAF contained fewer, but larger, particles (551 ± 293 nm) with smooth surfaces and were interconnected with nanofibers of 61 ± 13 nm in diameter. Differences in surface morphology between HAF and Cef-HAF were confirmed by atomic force microscopy. Fourier transform infrared and X-ray diffraction analyses revealed that Cef was not modified when incorporated into Cef-HAF and remained active against Klebsiella pneumoniae Xen 39, Staphylococcus aureus Xen 36 and Listeria monocytogenes EDGe. Nanofiber scaffolds of HA-containing Cef may be used in dressings to control postoperative infections.

Co-spinning of Silver Nanoparticles with Nisin Increases the Antimicrobial Spectrum of PDLLA: PEO Nanofibers.

Silver nanoparticles (AgNPs), synthesized using N,N-dimethylformamide (DMF), were electrospun with nisin in a 50:50 blend of 24 % (w/v) poly(D,L-lactide) (PDLLA) and poly(ethylene oxide) (PEO). Addition of AgNPs decreased the average diameter of the nanofibers [silver nanofibers (SF)] from 588 ± 191 to 281 ± 64 nm, or to 288 ± 63 nm when nisin was co-spun with AgNPs. Nanofibers containing AgNO3 (SF) had a beads-on-string structure, whereas nanofibers with AgNPs and nisin [silver plus nisin nanofibers (SNF)], nanofibers with only nisin [nisin nanofibers (NF)], and nanofibers without AgNPs and nisin [control nanofibers] had a uniform structure. The irregular topography was confirmed by atomic force microscopy. No interactions occurred between silver, nisin, PDLLA, and PEO, as confirmed with Fourier transform infrared spectroscopy. Most of the AgNPs (18 ± 2.8 ppm) and nisin (78.1 ± 1.2 µg/ml) were released within the first 2 h. SF and SNF inhibited the growth of gram-positive and gram-negative bacteria, whereas NF failed to inhibit gram-negative bacteria. A wound dressing with broad-spectrum antimicrobial activity may be developed by the incorporation of nanofibers containing a combination of AgNPs and nisin.

2,3-dihydroxybenzoic acid-containing nanofiber wound dressings inhibit biofilm formation by Pseudomonas aeruginosa.

Pseudomonas aeruginosa forms biofilms in wounds, which often leads to chronic infections that are difficult to treat with antibiotics. Free iron enhances biofilm formation, delays wound healing, and may even be responsible for persistent inflammation, increased connective tissue destruction, and lipid peroxidation. Exposure of P. aeruginosa Xen 5 to the iron chelator 2,3-dihydroxybenzoic acid (DHBA), electrospun into a nanofiber blend of poly(d,l-lactide) (PDLLA) and poly(ethylene oxide) (PEO), referred to as DF, for 8 h decreased biofilm formation by approximately 75%. This was shown by a drastic decline in cell numbers, from 7.1 log10 CFU/ml to 4.8 log10 CFU/ml when biofilms were exposed to DF in the presence of 2.0 mM FeCl3 6H2O. A similar decline in cell numbers was recorded in the presence of 3.0 mM FeCl3 6H2O and DF. The cells were more mobile in the presence of DHBA, supporting the observation of less biofilm formation at lower iron concentrations. DHBA at MIC levels (1.5 mg/ml) inhibited the growth of strain Xen 5 for at least 24 h. Our findings indicate that DHBA electrospun into nanofibers inhibits cell growth for at least 4 h, which is equivalent to the time required for all DHBA to diffuse from DF. This is the first indication that DF can be developed into a wound dressing to treat topical infections caused by P. aeruginosa.

2,3-Dihydroxybenzoic acid electrospun into poly(D,L-lactide) (PDLLA)/poly(ethylene oxide) (PEO) nanofibers inhibited the growth of Gram-positive and Gram-negative bacteria.

Widespread emergence of antibiotic-resistant pathogens in recent years has restricted the treatment options for various infectious diseases. Investigation of alternative antimicrobial agents and therapies is thus of utmost importance. Electrospinning of 50 mg/ml 2,3-dihydroxybenzoic acid (DHBA) into 24 % (w/v) poly(D,L-lactide) (PDLLA) and poly(ethylene oxide) (PEO) (1:1) produced nanofibers with an average diameter of 401 ± 122 nm. DHBA released from the nanofibers (315 ± 0.04 µg/ml within 2 h) inhibited the growth of Pseudomonas aeruginosa Xen 5, Klebsiella pneumoniae Xen 39, Escherichia coli Xen 14, Salmonella typhimurium Xen 26, and Staphylococcus aureus strains Xen 30, Xen 31, and Xen 36. The reason for the rapid diffusion of DHBA from PEO:PDLLA may be due to formation of hydrogen bonds between the hydroxyl groups of DHBA and the C=O groups of the PDLLA. DHBA formed a strong interaction with PDLLA and increased the thermal stability of the nanofiber mesh. The DHBA-containing nanofibers were non-hemolytic, suggesting that they may be incorporated in the development of a wound dressing.

In Vitro Human Gastrointestinal Tract Simulation Systems: A Panoramic Review.

Simulated human gastrointestinal (GI) tract systems are important for their applications in the fields of probiotics, nutrition and health. To date, various in vitro gut systems have been available to study GI tract dynamics and its association with health. In contrast to in vivo investigations, which are constrained by ethical considerations, in vitro models have several benefits despite the challenges involved in mimicking the GI environment. These in vitro models can be used for a range of research, from simple to dynamic, with one compartment to several compartments. In this review, we present a panoramic development of in vitro GI models for the first time through an evolutionary timeline. We tried to provide insight on designing an in vitro gut model, especially for novices. Latest developments and scope for improvement based on the limitations of the existing models were highlighted. In conclusion, designing an in vitro GI model suitable for a particular application is a multifaceted task. The bio-mimicking of the GI tract specific to geometrical, anatomical and mechanical features remains a challenge for the development of effective in vitro GI models. Advances in computer technology, artificial intelligence and nanotechnology are going to be revolutionary for further development. Besides this, in silico high-throughput technologies and miniaturisation are key players in the success of making in vitro modelling cost-effective and reducing the burden of in vivo studies.

Complete Genome Sequence and Probiotic Characterization of Lactobacillus delbrueckii subsp. Indicus DC-3 Isolated from Traditional Indigenous Fermented Milk.

In this study, Lactobacillus delbrueckii subsp. indicus DC-3 was isolated from Indian traditional indigenous fermented milk Dahi and identified using whole genome sequencing. The safety of the strain was evaluated using genetic and phenotypic analyses, such as the presence of virulence factors, mobile and insertion elements, plasmids, antibiotic resistance, etc. Besides this, the strain was comprehensively investigated for in vitro probiotic traits, biofilm formation, antibacterials, and exopolysaccharide (EPS) production. In results, the strain showed a single circular chromosome (3,145,837 bp) with a GC content of 56.73%, a higher number of accessory and unique genes, an open pan-genome, and the absence of mobile and insertion elements, plasmids, virulence, and transmissible antibiotic resistance genes. The strain was capable of surviving in gastric juice (83% viability at 3 h) and intestinal juice (71% viability at 6 h) and showed 42.5% autoaggregation, adhesion to mucin, 8.7% adhesion to xylene, and 8.3% adhesion to Caco-2 cells. The γ-hemolytic nature, usual antibiotic susceptibility profile, and negative results for mucin and gelatin degradation ensure the safety of the strain. The strain produced 10.5 g/L of D-lactic acid and hydrogen peroxide, capable of inhibiting and co-aggregating Escherichia coli MTCC 1687, Proteus mirabilis MTCC 425, and Candida albicans ATCC 14,053. In addition, the strain showed 90 mg/L EPS (48 h) and biofilm formation. In conclusion, this study demonstrates that L. delbrueckii subsp. indicus DC-3 is unique and different than previously reported L. delbrueckii subsp. indicus strains and is a safe potential probiotic candidate.

Antioxidative potential of folate producing probiotic Lactobacillus helveticus CD6.

Folate producing Lactobacillus sp. CD6 isolated from fermented milk showed 98% similarity with Lactobacillus helveticus based on 16S rRNA gene sequence analysis. It was found to produce a folic acid derivative 5-methyl tetrahydrofolate (5-MeTHF). The intracellular cell-free extract of strain demonstrated antioxidative activity with the inhibition rate of ascorbate autoxidation in the range of 27.5% ± 3.7%. It showed highest metal ion chelation ability for Fe(2+) (0.26 ± 0.06 ppm) as compared to Cu(2+). The DPPH (α,α-Diphenyl-ß-Picrylhydrazyl) radical scavenging activity for intact cells were found to be 24.7% ± 10.9% proved its antioxidative potential. Furthermore, it demonstrated 14.89% inhibition of epinephrine autoxidation, 20.9 ± 1.8 µg cysteine equivalent reducing activity and 20.8% ± 0.9% hydroxyl radical scavenging effect. The strain was evaluated for probiotic properties as per WHO and FAO guidelines. It showed 90.61% survival at highly acidic condition (pH 2.0), 90.66% viability in presence of synthetic gastric juice and 68% survivability at 0.5% bile concentration for 24 h. It was susceptible to many antibiotics which reduces the prospect to offer resistance determinants to other organisms if administered in the form of probiotic preparations. It showed in vitro mucus binding and antimicrobial activity against enteric pathogens like Salmonella typhimurium (NCIM 2501), Streptococcus pyogenes (NCIM 2608), and Staphylococcus aureus (NCIM 5021) and moreover it showed non- hemolytic activity on sheep blood agar.

Understanding Osteoporosis: Human Bone Density, Genetic Mechanisms, Gut Microbiota, and Future Prospects.

Osteoporosis is a systemic condition of the skeleton that leads to diminished bone mass, a breakdown in the bone tissue's microscopic architecture, and an elevated risk of breaking a bone. The elderly and women particularly after menopause are disproportionately affected, and the condition generally stays undiagnosed until a broken bone causes severe pain and immobility. Causes of osteoporosis include low bone mass, more than normal bone loss, changes in hormone levels (decreased estrogen or testosterone), certain diseases and therapies, and lifestyle factors like smoking and inactivity. The spine, hip, and forearm are particularly vulnerable to osteoporosis-related fractures. The purpose of this article is to present a thorough understanding of osteoporosis, including the disease's connection to bone density in humans, and the major part played by genetic pathways and gut flora. The causes of osteoporosis, the effects of aging on bone density, and why some groups experience a higher incidence of the disease than others are investigated. The paper also includes animal and human experiments investigating the link between gut flora and osteoporosis. Finally, it looks to the future and speculates on possible developments in osteoporosis prevention and therapy.

Genetic and phenotypic assessments for the safety of probiotic Bacillus clausii 088AE.

In this study, we report on whole genome sequence analysis of clinically documented, commercial probiotic Bacillus clausii 088AE and genome features contributing to probiotic properties. The whole genome sequence of B. clausii 088AE generated a single scaffold of 4,598,457 bp with 44.74 mol% G + C. This assembled genome sequence annotated by the RAST resulted in 4371 coding genes, 75 tRNAs, and 22 rRNAs. Gene ontology classification indicated 39.5% proteins with molecular function, 44.24% cellular component, and 16.25% proteins involved in biological processes. In taxonomic analysis, B. clausii 088AE shared 99% identity with B. clausii DSM 8716. The gene sequences related to safety and genome stability such as antibiotic resistance (840), virulence factors (706), biogenic amines (1), enterotoxin (0), emetic toxin (0), lanthipeptides (4), prophage (4) and clustered regularly interspaced short palindromic repeats (CRISPR) sequences (11), were identified and evaluated for safety and functions. The absence of functional prophage sequences and the presence of CRISPR indicated an advantage in genome stability. Moreover, the presence of genome features contributing to probiotic characteristics such as acid, and bile salt tolerance, adhesion to the gut mucosa, and environmental resistance ensure the strains survivability when consumed as a probiotic. In conclusion, the absence of risks associated with sequences/genes in the B. clausii 088AE genome and the presence of essential probiotic traits confirm the strain to be safe for use as a probiotic.

Identification and characterisation of antimicrobial compound produced by probiotic Alkalihalobacillus clausii 088AE.

In the present study, Alkalihalobacillus clausii 088AE was evaluated for the production of antimicrobial compounds, their characterisation, and identification. The results showed that, 48-h-old A. clausii supernatant is able to inhibit the growth of indicator bacteria Micrococcus luteus MTCC 106T (7.0 ± 0.51 mm). The cultivation of 088AE on solid media along with XAD16N beads for 5 days, and isopropanol extraction of antimicrobial compound thereof showed enhanced antimicrobial activity (19.67 ± 0.58 mm) against indicator strain. Further purification with a reversed-phase C18 cartridge yielded a powder with 389.12 ± 10.08 µg of protein per mg. The UV spectra of the sample revealed the characteristic aromatic amino acid peaks at 262 and 276 nm. The results of triple-quadrupole mass spectrometry showed a peak of 1055 m/z, confirming the existence of 10542+, i.e., 2108 Da, suggestive of class I lantibiotic clausin. Furthermore, in silico analysis confirmed the presence of genes responsible for the production of lanthipeptide clausin. The clausin is stable in the presence of proteases (trypsin and pepsin), high temperature (100 °C), pH up to 11 and showed antimicrobial activity against Listeria monocytogenes ATCC 19115, M. luteus, and Staphylococcus aureus ATCC 6538P. Therefore, it could be useful to control gut infections caused by Gram-positive pathogens.

Microencapsulation of riboflavin-producing Lactiplantibacillus Plantarum MTCC 25,432 and Evaluation of its Survival in Simulated Gastric and Intestinal Fluid.

Microencapsulation is an optimistic method for the delivery of live microbial cells through different food products. In this study, riboflavin-producing probiotic strain Lactiplantibacillus plantarum MTCC 25,432 was encapsulated using a spray drying technique with different wall materials including Inulin, maltodextrin (MD), and MD + Inulin (1:1). The obtained spray dried powder was investigated for probiotic viability, encapsulation efficiency, particle size, water activity, moisture content, hygroscopicity, bulk and tapped densities, storage stabilities, Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). Besides this, the viability of the free and encapsulated probiotic cells was tested under simulated gastric and intestinal fluid conditions. In the results, microcapsules produced with the combination of MD + Inulin showed higher dry powder yield (36.5%) and viability of L. plantarum MTCC 25,432 (7.4 log CFU / g) as compared with individual coating materials. Further characterization revealed that MD + Inulin microcapsules are spherical (3.50 ± 1.61 µm in diameter) in shape with concavities, showed the highest encapsulation efficiency (82%), low water activity (0.307), moisture content (3.67%) and good survival ability at low pH (pH 2.0 and 3.0), high bile salt concentrations (1.0% and 2.0%), and long storage conditions. No differences in FTIR spectra were observed among the tested samples. However, TGA showed enhanced thermal stability of probiotic-loaded microcapsules when MD + Inulin was used together. In conclusion, MD + Inulin could be a potential encapsulation material for riboflavin-producing probiotic bacteria L. plantarum MTCC 25,432.

Predictive Modeling of Riboflavin Production in Lactiplantibacillus plantarum MTCC 25432 Using Fuzzy Inference System.

Riboflavin (Vitamin B2) is an essential vitamin and a microbial metabolite produced by some lactic acid bacteria (LAB). This investigation aims to study the overproduction of riboflavin in selected Lactiplantibacillus plantarum strain by using the one factor at a time (OFAT) tool coupled with the Fuzzy Inference System (FIS) and its validation through fermentative production in semi-defined media. Out of three Lactiplantibacillus strains used in this study, the maximum riboflavin producing strain was selected based on its ability to grow and produce higher levels of riboflavin. In results, Lactiplantibacillus plantarum strain MTCC 25432 was able to produce 346 µg/L riboflavin in riboflavin deficient assay medium and was investigated further. By using the OFAT-fuzzy FIS system, casamino acid in the range of 5-20 g/L, GTP 0.01-0.04 g/L, sodium acetate 5-15 g/L, and glycine 5-15 g/L were used to predict their effect on riboflavin production. The conditions optimized with modeling showed a 24% increment in riboflavin production (429 µg/L) by Lactiplantibacillus plantarum MTCC 25432 vis-a-vis the unoptimized counterpart (346 µg/L). In conclusion, an FIS-based predictive model was effectively implemented to estimate the riboflavin within an acceptable limit of 3.4%. Riboflavin production enhancing effects observed with various levels of sodium acetate, casamino acid, and GTP could be useful to re-design matrices for riboflavin production.

Complete genome sequence of Lactiplantibacillus plantarum BBC32B isolated from human feces sample.

We report complete genome sequence of Lactiplantibacillus plantarum BBC32B, which was isolated from human feces sample and submitted to Microbial-Type Culture Collection (MTCC), India with deposition number MTCC 25432. The bacteria from Lactobacillaceae family contained 3,411,152 bp; 3,425 protein coding genes, sharing 69.67% average nucleotide identity with closest species of Lactobacillus brevis ATCC367.

Cholesterol assimilation and biotransformation by Lactobacillus helveticus.

Lactobacillus helveticus, grown at 37°C in MRS medium supplemented with 3 mM cholesterol, assimilated all the cholesterol in 42 h having 68 U mg(-1) of intracellular cholesterol oxidase activity. The strain transformed 1 g cholesterol to 0.05 g of androsta-1, 4-diene-3, 17-dione and 0.04 g of androst-4-ene-3, 17 dione within 48 h at 37°C with extracellular cholesterol oxidase activity at 12 U mg(-1) and intracellular oxidase at 0.5 U mg(-1).