Tributyrin Agar Research Articles

Lipase and Protease Production Ability of Multi-drug Resistant Bacteria Worsens the Outcomes of Wound Infections.

Surgical site infections are one of the major clinical problems in surgical departments that cost hundreds of millions of dollars to healthcare systems around the world. The study aimed to address the pressing issue of surgical site infections, which pose significant clinical and financial burdens on healthcare systems globally. Recognizing the substantial costs incurred due to these infections, the research has focused on understanding the role of lipase and protease production by multi-drug resistant bacteria isolated from surgical wounds in the development of post-surgical wound infections. For these purposes, 153 pus specimens were collected from patients with severe post-surgical wound infections having prolonged hospital stays. The specimens were inoculated on appropriate culture media. Gram staining and biochemical tests were used for the identification of bacterial growth on suitable culture media after 24 hours of incubation. The isolated pathogens were then applied for lipase and protease, key enzymes that could contribute to wound development, on tributyrin and skimmed milk agar, respectively. Following the CSLI guidelines, the Kirby-Bauer disc diffusion method was used to assess antibiotic susceptibility patterns. The results revealed that a significant proportion of the samples (127 out of 153) showed bacterial growth of Gram-negative (n = 66) and Gram-positive (n = 61) bacteria. In total, isolated 37 subjects were declared MDR due to their resistance to three or more than three antimicrobial agents. The most prevalent bacteria were Staphylococcus aureus (29.13%), followed by S. epidermidis (18.89%), Klebsiella pneumoniae (18.89%), Escherichia coli (14.96%), Pseudomonas aeruginosa (10.23%), and Proteus mirabilis (7.87%). Moreover, a considerable number of these bacteria exhibited lipase and protease activity with 70 bacterial strains as lipase positive on tributyrin agar, whereas 74 bacteria showed protease activity on skimmed milk agar with P. aeruginosa as the highest lipase (69.23%) and protease (76.92%) producer, followed by S. aureus (lipase 62.16% and protease 70.27%). The antimicrobial resistance was evaluated among enzyme producers and non-producers and it was found that the lipase and protease-producing bacteria revealed higher resistance to selected antibiotics than non-producers. Notably, fosfomycin and carbapenem were identified as effective antibiotics against the isolated bacterial strains. However, gram-positive bacteria displayed high resistance to lincomycin and clindamycin, while gram-negative bacteria were more resistant to cefuroxime and gentamicin. In conclusion, the findings suggest that lipases and proteases produced by bacteria could contribute to drug resistance and act as virulence factors in the development of surgical site infections. Understanding the role of these enzymes may inform strategies for preventing and managing post-surgical wound infections more effectively.

Response surface optimization of lipase production by <i>Pseudomonas sp.</i> on a low-cost shea-nut cake using solid state fermentation

Shea nut cake (SNC) is the solid residue produced after the shea butter extraction process. The SNC could provide a low-cost substrate for bacterial lipase production. Greater yield are among the benefits Solid State Fermentation (SSF). However, there have been less instances of lipase production by bacteria than fungi in SSF. The current study aimed at improving bacterial lipase production on SNC in SSF using Response Surface Methodology. Lipase-producing bacteria (LPB) were isolated from the shea butter mill effluent pond and screened for lipase production on a tributyrin agar plate. The influence of four independent parameters (temperature, pH, moisture content and biosurfactant) on lipase production was studied using Box Behnken Design (BBD) of RSM. Results showed that isolate AO (Pseudomonas sp.) displayed the highest zone of hydrolysis 2 (53 mm). A quadratic regression with R = 0.931 showed that the model is best fitting, and predicted the optimum conditions of significant (P>0.05) growth parameters, including temperature at 40°C, moisture content at 50%, and biosurfactant at 2 mL, which produced 408.25 U/g of experimental lipase. The findings indicate that the model was useful for predicting optimal growth conditions for incresing lipase production in SSF and the low-cost agro- industrial SNC could be a promising substrate for industrial applications.

Production and Optimization of a Lipase-Producing Bacteria Enterobacter hormachei Isolated from an Oil-Contaminated Soil

Lipase enzymes possess a wide range of industrial applications. Thus, the capacity of lipase-producing bacteria to proliferate on tributyrin agar medium was used to screen for them among various sources. The lipase producing bacteria with highest zone of clearance on the screening media were preserved on the agar slants. The preserved slants were characterized by16SrRNA gene sequencing. The nucleotide sequence so obtained by the 16SrRNA gene sequence was then put through phylogenetic analysis and homology search using the NCBI’s BLAST program. The sequence of lipase producing bacteria showed maximum resemblance with Enterobacter hormaechei bacterial strain. These bacterial strains were produced by inoculating the culture in the inoculum media and allowing it to enrich over night. Subsequently, 3% of the inoculum from the inoculum media was added to the production media, which was then incubated for 48 hours in a rotary shaker. After production the media was centrifuged and supernatant was extracted and used further for optimization, Optimization of the physiochemical parameters of the bacterial strain like inoculum pH, incubation period, inoculum size was found using one factor at a time (OFAT) approach and medium parameters like different carbon source, nitrogen source, substrate, minerals, salts (11 factors) were screened using Plackett-Burman (PB) design which is a full factorial design. The Lipase activity was found by using a titrimetric method using olive oil and Arabic gum mixture as substrate mixture. The maximum lipase activity was found for inoculum pH of 5, 48 hours of incubation time, and 5% inoculum size. The results of the PB design showed the significant parameters to be glucose (carbon source), peptone (nitrogen source), KH2PO4 (salt), and NaCl (mineral). The organism of the soil sample containing bacterial strain showed maximum lipase activity of 70 U/ml and protein concentration of 4.3 μg/mL at the optimized conditions. After centrifuging the culture media that had been improved, the supernatant was collected and partially purified using dialysis and the ammonium sulfate precipitation procedure. Following precipitation, the supernatant’s activity was measured to be 74 U/mL. After being gathered, the pallet was dialyzed in a dialysis bag and added to a buffer. Both the protein content and the lipase activity were estimated. The protein concentration determined by Lowry’s technique was found to be 6.2 μg/mL, and the lipase activity was reported to be 85.22 U/mL.

Screening biosurfactant-producing actinomycetes: Identification of Streptomyces sp. RP1 as a potent species for bioremediation.

This study aimed to isolate biosurfactant-producing and hydrocarbon-degrading actinomycetes from different soils using glycerol-asparagine and starch-casein media with an antifungal agent. The glycerol-asparagine agar exhibited the highest number of actinomycetes, with a white, low-opacity medium supporting pigment production and high growth. Biosurfactant analyses, such as drop collapse, oil displacement, emulsification, tributyrin agar test, and surface tension measurement, were conducted. Out of 25 positive isolates, seven could utilize both olive oil and black oil for biosurfactant production, and only isolate RP1 could produce biosurfactant when grown in constrained conditions with black oil as the sole carbon source and inducer, demonstrating in situ bioremediation potential. Isolate RP1 from oil-spilled garden soil is Gram-staining-positive with a distinct earthy odor, melanin formation, and white filamentous colonies. It has a molecular size of ~621 bp and 100% sequence similarity to many Streptomyces spp. Morphological, biochemical, and 16 S rRNA analysis confirmed it as Streptomyces sp. RP1, showing positive results in all screenings, including high emulsification activity against kerosene (27.2%) and engine oil (95.8%), oil displacement efficiency against crude oil (7.45 cm), and a significant reduction in surface tension (56.7 dynes/cm). Streptomyces sp. RP1 can utilize citrate as a carbon source, tolerate sodium chloride, resist lysozyme, degrade petroleum hydrocarbons, and produce biosurfactant at 37°C in a 15 mL medium culture, indicating great potential for bioremediation and various downstream industrial applications with optimization.

Utilization of shea-nut cake for lipase production by thermophilic Bacillus valezensis EAC 9 isolated from hot compost and optimization of nutritional parameters

The use of oily waste as a cost-effective substrate for lipase production is gaining more importance. However, shea-nut cake (SNC) remains under-explored in this regard. Lipases of thermophilic origin such as hot compost bacteria, are of significant biotechnological and industrial importance due to favorable robust properties. This paper describes the isolation of potential lipase producing thermophilic bacteria from hot compost and improvement of its lipase through optimization of nutritional parameters. The bacteria were isolated on tributyrin agar plate and applied for the production of lipase on Olive oil, Shea-nut cake (SNC) and the mixture of olive oil and SNC. Using Plackett-Burman Design (PBD) for screening and Central Composite Design (CCD) of Response Surface Methodology (RSM) for optimization studies, factors influencing lipase production on SNC substrate were identified. One (EAC 9) out of the 4 most potent isolates was identified as Bacillus velezensis EAC9 using 16S rRNA, and produced the highest lipase activity on mixture of olive oil and SNC (103.66 U/ml) than olive oil (65.22) and SNC (41.72 U/ml) alone. Optimization model validity was verified and revealed an optimum medium containing olive oil and Tween 80 at 1.0 % v/v, sucrose of 1.0 % w/v and (NH4)2SO4 of 0.1 % w/v led to maximum lipase production at 200 U/ml which was 4.79-fold increase from the unoptimized. The results indicate that SNC is a better prospective substrate for improving lipase production by thermophilic Bacillus valezensis EAC9, and that statistical methods may be used to deduce optimal parameters for effective lipase production.

Isolation, Identification and Screening of Potential Marine Actinomycete for Lipase Production under Submerged Fermentation by using Tributyrin substrate

Ninety-six isolates were collected at various depths and sea shores of the Bay of Bengal near Visakhapatnam Coastal of Andhra Pradesh. These isolates were screened for the isolation of lipase activity by tributyrin agar clearing method and cultured under submerged fermentation and assayed for extra cellular lipase production by using tributyrin as substrate. Cultural characteristics of the selected strain was done by incorporating different media. Various parameters namely morphological, biochemical, physiological and molecular were used for the characterization and identification of actinomycetes isolates. Results indicated that all the isolates showed lipolytic activity after primary screening. The marine isolate T9 was the most active one thus the same was selected for production of lipase. The 16S rDNA amplification for phylogenetic study revealed that the isolate was highly related to Nocardiopsis dassonvillei. In conclusion, these results increased the scope of finding industrially important marine actinomycetes from Visakhapatnam Coastal of Andhra Pradesh.

Screening For Lipolytic Bacteria from Bonoloyo Cemetery, Surakarta

A cemetery is a burial place managed by the government. Inside the TPU, the body is decomposed by microorganisms, one of which is lipolytic bacteria, because the human body is composed of about 12.5–13.60% lipids. Research on lipolytic bacteria from TPU in Indonesia has not been found, even though it has the potential to obtain lipolytic bodies. Therefore, this research was conducted to select lipolytic bacteria from TPU and provide simple assistance. A total of 45 bacterial isolates from TPU Bonoloyo Surakarta were selected for their lipolytic activity using tributyrin agar media. Lipolytic activity was determined using the lipolytic index (LI). Bacterial isolates with lipolytic potential were identified based on colony morphology and Gram staining. The results showed that 30 isolates (67%) showed lipolytic activity, with the highest LI value of 5.43 (BLB 9) after 2x24 hours of incubation. Bacterial isolates that have lipolytic activity are white colonies with circular shapes. The results of Gram staining showed that the bacterial isolates belonged to a group of Gram-negative bacteria in the form of cocci. The conclusion put forward is that public cemeteries (TPU) have the potential to store isolates of lipolytic bacteria dominated by Gram-positive bacteria by as much as 57%

Isolation and Growth Optimization of Lipase-Producing Staphylococci for Industrial Applications

Lipases are an extremely significant group in the biotechnologically related enzymes. Lipases have massive applications in various industries that include detergent, dairy, food, and pharmaceutical industries. Lipase producing bacteria were isolated from different oil contaminated sites using tributyrin agar as the media. All the isolates belonged to the genera Staphylococci. The isolates with highest lipolytic activity were selected by using titrimetric assay. The lipolytic activity of 2S, 10S and 12S was more noticeable than other isolates. The effects of different parameters like temperature, pH and incubation time on lipolytic activity were assessed. The isolates showed maximum activity at 30°C 2S and 12S displayed least activity at 45°C and 10S displayed least activity at the lowest provided temperature that was 25°C. Incubation time of 20-30 minutes was effective in increasing the enzyme activity considerably. 25 minutes was shown to decrease the lipolytic potential of 10S and 45 minutes of isolate 2S and 12S. Thin layer chromatography was also employed for the visualization of fatty acids bands. The isolates were identified as Staphylococcus species through 16S rRNA gene sequencing. Such isolates can be of industrial importance due to their ability to produce industrially important enzymes.

Isolation, Identification and Characterization of Thermoduric Psychrotrophic Bacteria from Market Samples of Pasteurized Milk Collected from Mannuthy, Kerala State, India

Aims: To isolate, identify and characterize thermoduric psychrotrophic bacteria from locally available market samples of pasteurized milk.
 Study Design: Market samples of pasteurized milk were collected, subjected to laboratory pasteurization and thermoduric psychrotrophic bacteria were enumerated after 10 days of storage at 7ºC.The isolates obtained were identified by 16S rRNA sequencing and subsequent blasting. The isolates were characterized in terms of haemolytic, lipolytic and proteolytic activities. Selected isolates were also assessed for their decimal reduction time at 63ºC. 
 Place and Duration of Study: Verghese Kurien Institute of Dairy and Food Technology, (Formerly College of Dairy Science and Technology), Mannuthy, Thrissur, Kerala between September, 2019 to March, 2021.
 Methodology: A total of 42 market samples of pasteurized milk belonging to seven different brands were assessed for their thermoduric psychrotrophic bacterial population. The thermoduric psychrotrophic bacterial isolates were identified by 16S rRNA sequencing and the sequences obtained were searched with the NCBI BLAST program (http://www.ncbi.nlm.nih.gov) for their closest relatives/reference strains with a homology of over or equal to 99 per cent. Haemolytic, proteolytic and lipolytic activities of the isolates were determined by streaking them on blood agar, skim milk agar (SMA) and tributyrin agar respectively and grading them based on the presence or absence of zone of clearance around the colonies developed. Decimal reduction time at 63ºC of two selected isolates was also determined.
 Results: Six thermoduric psychrotrophic bacteria were isolated from the 42 market samples of pasteurized milk i.e., only 14.3 % of tested samples had thermoduric psychrotrophic bacterial population. 16S rRNA sequencing and subsequent blasting identified the isolates as Aeromonas caviae, Moraxella osloensis, Delftia tsuruhatensis, Staphylococcus arlettae and two strains of Carnobacterium maltaromaticum. On assessing the haemolytic activity of the isolates, A.caviae DMV01 exhibited α haemolytic activity whereas Staphylococcus arlettae DMV02, M. osloensis DMV03, C. maltaromaticum DMV05 and C. maltaromaticum DMV06 exhibited ϒ haemolytic activities. A. caviae DMV01 was found to be lipolytic and the two Carnobacterium strains exhibited proteolytic activities. Staphylococcus arlettae DMV02 was found to be both proteolytic and lipolytic. Aeromonas caviae DMV01 exhibited D63 value of 4 minutes 38 seconds whereas for Moraxella osloensis DMV03 it was 25 minutes 18 seconds.
 Conclusion: Exhibition of heat resistance by microorganisms capable of low temperature growth presents quite a challenging situation in terms of quality assurance and shelf life extension of pasteurized milk. So it is high time that dairy industry takes up initiatives to remain updated with the changes happening in pasteurized milk microflora in order to address the challenges such microorganisms may pose in future.

Isolation and optimization of thermophilic lipase producing bacteria from soil contaminated with used engines oil

Thirty two of thermophilic bacterial isolates producing lipase were selectively isolated from soil samples contaminated with used engines oil using Tributyrin agar medium. The qualitative assay of lipase activity was done on Tributyrin agar plates by measuring diameter of the clear zone. The isolates 6T, 14T and 29T of the isolated strains exhibited a greater zone of clearance than the others, indicating higher lipase activity. Secondary screening for lipase production was done using liquid basal medium with 1% (w/v) olive oil and pH 7. Results from quantitative assay showed that 6T, 14T and 29T isolates appeared highest ability to produce lipase enzyme 4 U/ml, 10 U/ml and 7 U/mlrespectively. Therefore, the 14T isolate was selected and identified based on their morphological and physiological characteristics which was subsequently identified and designated as Bacillus licheniformis. The effect of incubation time, temperature and pH influencing lipase production from Bacillus licheniformis14T isolate was determine. Results showed that the maximal lipase production was 11 U\ml after 3 day of incubation at medium pH7 and temperature 50 0C.

Identification and Optimization Study of Lipase Producing Bacteria Isolated from Municipal Waste and Bio-deteriorated Waste

Bio-deteriorated waste is the leftover organic matter of unwanted raw food which if not handled properly or left for natural degradation can cause health issues. Microorganisms have the ability to biodegrade waste by secreting enzymes. The aim of the work was to isolate and identify lipase-producing bacteria from waste polluted (Bio-deteriorated waste and Municipal Solid Waste) dumping sites of Gandhinagar, Gujarat, India. Lipase-producing bacteria were isolated using tributyrin agar as a selective medium. Out of 7 bacterial isolates, 1 isolate (HAL-2) gave the highest lipolytic activity. HAL-2 was identified as Bacillus pumilus by 16S rRNA sequencing. The bacterial isolate gave maximum lipase activity (0.68 U/mL) at 37°C and pH 7.0 Culture medium parameters such as carbon source, nitrogen source, pH, and inoculum size were varied for the purpose of optimization. The maximum lipase production was observed at pH 7.0, 37°C temperature. Inoculum size had an effect of direct proportionality on lipase activity. Glycerol tributyrate was found to be the best substrate (0.68 U/mL). Sucrose and Tryptone in the medium increased enzyme production when compared with other carbon and nitrogen sources.

Characterization of lipase from lipase-producing bacteria and its application in the reduction of triglyceride content in milk sample

Lipases hydrolyse esters of long-chain fatty acids. They have several biotechnological applications such as in the field of food technology, leather, cosmetics, detergents, textiles, oleochemicals, pharmaceuticals and industrial waste. Lipases occur widely in nature in animals, plants and microorganisms; however, in terms of commercial significance, microbial lipases are the most beneficial owing to the ease of production. This study aimed to characterise lipase isolated from lipase-producing bacteria, which were screened on Tributyrin agar to confirm lipase production. Growth parameters of the isolates including their temperature and pH tolerance were determined so that they can be applied for potential industrial uses. The enzymatic activity, pH optimum and temperature optimum of the lipase were determined by the p-nitro phenyl palmitate assay. Finally, the lipase was used to reduce triglyceride content in full-fat buffalo milk.

Pseudomonas fluorescens and Pseudomonas putida from refrigerated raw milk: genetic diversity and lipoproteolytic activity.

In this research communication the genetic diversity of Pseudomonas fluorescens (n = 67) and Pseudomonas putida (n = 44) isolated from refrigerated raw milk from bulk tank trucks were verified. The relationship between the genetic profile of the isolates and their lipoproteolytic potential was evaluated using skim milk agar and tributyrin agar (21°C/72 h). The lipoproteolytic potential (low or high), evaluated by the diameter of the halos (cm), was correlated with the number of milk producing properties that contributed to each sample (one sample = one bulk tank truck; 8-80 producers/sample) and the distance between the dairy properties and the processing plant (21-370 km). P. fluorescens was confirmed in all samples, while P. putida in 60% samples. For both species, two clusters (I and II) were observed, and the first one showed lower genotypic diversity and the presence of isolates with 100% similarity. P. fluorescens isolates presenting at least 70% similarity were 83.9% in Cluster I (n = 31) and 44.4% in Cluster II. In both clusters (I and II) observed in the P. fluorescens dendrogram, the occurrence of high proteolytic and lipolytic potential were equivalent. The higher the number of farms per milk sample, the greater the lipoproteolytic intensity of P. fluorescens isolates. In relation to P. putida isolates, 74% presented at least 50% similarity in Cluster I (n = 27) and only 35% in Cluster II (n = 17). The occurrence of high proteolysis linked to P. putida was proportional between both Clusters, but the occurrence of high lipolysis was greater in Cluster II. No significant association was detected between P. putida isolates and the variables studied. The results indicate the circulation of P. putida and P. fluorescens with 100% similarity in different milk producing regions. The level of genetic diversity was related only to the lipolytic capacity of P. putida.

Cloning, Expression, and Purification of a GDSL-like Lipase/Acylhydrolase from a Native Lipase-Producing Bacterium, Lactobacillus fermentum

Background: Lipase enzymes are of great importance in various industries. Currently, extensive efforts have been focused on exploring new lipase producer microorganism as well as genetic and protein engineering of available lipases to improve their functional features. Methods: For screening lipase-producing lactobacilli, isolated strains were inoculated onto tributyrin agar plates. Molecular identification of lipase-producing Lactobacilli was performed by sequencing the 16Sr DNA gene, and a phylogenetic tree was constructed. The LAF_RS05195 gene, encoding lipase protein in L. fermentum isolates, was identified using specific primers, amplified by PCR (918 bp) and cloned into the pET28a (+) vector. The recombinant proteins were expressed 2, 4, 6, and 12 hours after induction with IPTG and assessed using the SDS-PAGE. Enzymatic activity of the purified recombinant protein was measured at 410 nm in the presence of ρ-NPA and ρ-NPP. Results:Among five identified native lipase-producing isolates, one isolate showed 98% similarity with Enterococcus species. The other four isolates indicated 98% similarity to L. fermentum. After purification steps with Ni-NTA column, a single protein band of about 34 kDa was detected on SDS- PAGE gel. The enzymatic activity of purified recombinant protein alongside ρ-NPA and ρ-NPP was measured to be 0.6 U/ml and 0.2 U/ml, respectively. Conclusion:In the present research, a novel lipase/esterase from L. fermentum was cloned and expressed. The novel lipase/esterase has the merit to be further studied due to its substrate specificity.

Identification of lipase producing bacteria from palm oil sewage sludge processing plant at Malimping, Banten, Indonesia

Abstract. Layli IR, Meryandini A, Helianti I, Astuti RI. 2021. Identification of lipase-producing bacteria from palm oil sewage sludge processing plant at Malimping, Banten, Indonesia. Biodiversitas 22: 4512-4524. Lipase (triacylglycerol hydrolase E.C.3.1.1.3) is an enzyme that catalyzes the hydrolysis reaction of triacylglycerol into free fatty acids and glycerols. Lipase has broad application spectrum and unique catalytic mechanism, as one of important enzymes for industries, and almost all enzymes used for industries are originated from microbes. Identification of lipase-producing bacteria from palm oil processing sewage sludge was carried out to determine the potential of isolates and the application of lipase enzymes for industry. This study identified lipase-producing bacteria from the screening and isolation results from palm oil sewage sludge in Malimping-Banten. Two isolates were obtained from screening and isolation results. The morphological and observation and Gram staining method resulted in mlp-1 colony isolate was Gram-negative with basil cell shape, while mlp-2 colony isolate was Gram-positive with basil cell shape. Qualitative test on two isolates in tributyrin agar showed that two isolates produced clear zone or lipase enzyme. Molecular identification using 16SrRNA sequence showed that mlp-1 colony isolate was Chryseobacterium gleum, while mlp-2 colony was Bacillus velezensis. Literature study stated that Chryseobacterium gleum are pathogenic bacteria, resulting in uncontinued identification process, while Bacillus velezensis are non-pathogenic bacteria that have potential for industrial application. Identification of Bacillus velezensis was continued by performing biochemical test and inhibitory test. The continued identification results supported the molecular identification of lipase-producing enzyme for Bacillus velezensis.

Isolation and Optimization of Lipase Producing Bacteria from Oil-Contaminated Soils

This research aimed to isolate lipase-producing bacteria from soil samples contaminated with vegetable oil or petrol, and then optimize the conditions for lipase production. The bacteria that could be grown on tributyrin agar supplemented with 1% palm oil and produced a wide clear zone were selected. These selected bacterial isolates were tested for lipase activity, among them, the isolate PKRU-9 had the highest lipase activity. Then, the optimum conditions for lipase production of the PKRU-9 were optimized. The maximum lipase production was obtained when PKRU-9 was cultured in a basal medium containing 0.5% yeast extract as a nitrogen source and 0.5% soybean oil as a carbon source at 200 rpm. After cultivation for 54h, the maximum lipase production was reached. Treatment of wastewater contaminated with lipid of PKRU-9 was studied by culturing the bacterium in synthetic wastewater medium containing 1% palm oil, and in wastewater contaminated with fat. PKRU-9 was able to decrease the value of BOD5 by 74% and 90%, respectively comparing to the starting BOD5. The highest percent reduction of BOD5 was found on day 3 of cultivation. These results suggested that the PKRU-9 has the potential to be used for treating municipal wastewater contaminated with fat.

Inhibitory Effect of Cinnamon and Thyme Essential Oils Against Acinetobacter baumannii strains Isolated from Raw Milk and Some Milk Products

Abstract A total of 250 random samples including raw milk samples collected from street vendors, dairy shops and dairy houses in addition to some milk products including Kariesh cheese, Domiati cheese, cream, and milk powder, were collected from different areas in Luxor city, Egypt. for detection of Acinetobacter species by conventional method and PCR for the presence of blaox51 like gene. results revealed that the 10% of Acinetobacter spp. were recovered from whole raw milk samples, species were A. baumannii, A. calcoaceticus and A. haemolyticus with percentages of 5.3, 2.7 and 2%, respectively. street vendors raw milk samples showed the highest prevalence (14%) of Acinetobacter spp. Regarding milk products samples, 8% of Acinetobacter spp. were isolated from kariesh cheese. A higher percentage of 25% were recorded from cream samples. All Acinetobacter baumanni strains harbored blaox-51 like gene, showing strong lipolytic activity on tributyrin agar, the antibacterial effect of cinnamon oil and thyme oil against 16 Acinetobacter baumanni isolates showed high antibacterial activity. Keywords. Acinetobacter species., Dairy products, PCR, blaox51 like gene, Essential oils

ISOLATION OF THERMOPHILIC LIPASE PRODUCING BACTERIUM FROM HOT SPRINGS AT THE EAST COAST OF PENINSULAR MALAYSIA

Hot spring is a natural habitat for thermophilic bacteria and the primary source of thermostable enzymes useful in industrial applications. In Malaysia, the search for thermophilic organisms has been focused on hot springs, especially on the peninsular West coast. In this work, lipase or esterase producing thermophilic microorganisms were isolated from East coast hot springs in Pahang and Terengganu's states. Morphological and biochemical analysis were carried out on Isolates LH1, LH2, LH3, LH4, LH5, B2B2 and S1B4, which showed that they are gram positive, aerobic, spore forming, and motile organisms. All of the seven isolates showed the ability to grow at 45°C and formed hydrolysis zones on tributyrin agar plates. However, only isolate B2B2 and S1B4 were able to thrive at higher temperatures of up to 65°C. The genotypic characterisation was carried out using 16S rRNA sequencing. Bacillus and Geobacillus species were found to be the dominant bacteria isolated from these hot springs. From La hot spring, isolate LH1 (MT 645486), Isolates LH2 (MT645483), LH3 (MT645484), LH4 (MT 645485) and LH5 (MT 645487) were all closely related to Bacillus sp. (at 97.3-97.9%). Meanwhile, from Bentong and Sungai Lembing hot springs, isolates B2B2 (MT668631) and S1B4 (MT668632) were near related to either Geobacillus kaustophilus or Geobacillus thermoleovorans; each at 98.5% and 97.9% similarity, respectively. These strains from Geobacillus sp. were able to thrive at higher temperature and their thermostable esterases or lipases have properties useful for biotechnological applications.

Pseudomonas cremoris sp. nov., a novel proteolytic species isolated from cream.

During a study investigating the microbiota of raw milk and its semi-finished products, strains WS 5106T and WS 5096 were isolated from cream and skimmed milk concentrate. They could be assigned to the genus Pseudomonas by their 16S rRNA sequences, but not to any validly named species. In this work, a polyphasic approach was used to characterize the novel strains and to investigate their taxonomic status. Examinations based on the topology of core genome phylogenomy as well as average nucleotide identity (ANIm) comparisons suggested a novel Pseudomonas species within the Pseudomonas fluorescens subgroup. With pairwise ANIm values of 90.1 and 89.8 %, WS 5106T was most closely related to Pseudomonas nabeulensis CECT 9765T and Pseudomonas kairouanensis CECT 9766T. The G+C content of strain WS 5106T was 60.1 mol%. Morphologic analyses revealed Gram-stain-negative, aerobic, catalase and oxidase positive, rod-shaped and motile cells. Proteolysis on skimmed milk agar as well as lipolysis on tributyrin agar occurred at both 28 and 6 °C. Tolerated growth conditions were temperatures between 4 and 34 °C, pH values between 6.0 and 8.0, and salt concentrations of up to 5 %. Fatty acid profiles showed a pattern typical for Pseudomonas, with C16 : 0 as the dominant component. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol and the dominating quinone was Q-9. Based on these results, it is proposed to classify the strains as a novel species, Pseudomonas cremoris sp. nov., with WS 5106T (=DSM 111143T=LMG 31863T) as type strain and WS 5096 (=DSM 111129=LMG 31864) as an additional strain.

Isolation, screening and identification of Lipase producing fungi from cotton seed soapstock

Background/Objectives: The present study was focused to exploit the indigenous strains of fungi isolated from cotton seed soapstock for the production of the extracellular lipase through submerged fermentation technique. Methods/ Statistical analysis: Cotton seed soapstock samples used in the study contains gelatinous oil richer chemical constituents. In addition, their enrichment and diluted materials were used for the isolation of lipase producing microorganisms on tributyrin agar plates. All isolates were lipase positive confirmed by a qualitative plate assay. Quantitative estimation of Lipase production activity was measured spectrophotometrically using pnitrophenyl palmitate (p-NPP) as a substrate. In order to exploit the isolated fungal strain for industrial usage, other cellulase and protease enzymes were tested by plate assay. Morphological and molecular characteristics of selected isolates were studied. Findings: From enriched soapstock samples, a total of 49 cultures were isolated among them 19 fungal isolates were screened for lipase, cellulase, and protease activity qualitatively by plate assay. Out of 19, six fungi were selected based on their lipase activity. Highly potent Fusarium solani 7F had the ability to produce 5.95 U/mL/min. crude lipase whereas Aspergillus niger 13F has 4.2 U/mL/min. after 4days incubation at 30oC. Potent fungi culture was identified by morphological, cultural, and molecular characteristics (18s rRNA gene sequence and phylogenetic analysis) revealed them as Penicillium griseofulvum 5F, Aspergillus flavus 6F, Fusarium solani 7F, Aspergillus niger 12F, Aspergillus niger 13F, and Aspergillus terreus 17F. Novelty: Fungi was the first time reported and isolated from cotton seed soapstock materials. In future studies, this enzyme will be used in the degradation of soapstock and also in the production of biosurfactant from soapstock. Keywords: Cotton seed soapstock; Fusarium solani 7F; p-nitrophenyl palmitate; Tributyrin agar plates; degradation