Milk Agar Research Articles

Hexanic extract of Achyrocline satureioides: antimicrobial activity and in vitro inhibitory effect on mechanisms related to the pathogenicity of Paenibacillus larvae.

Paenibacillus larvae is a spore-forming bacillus, the most important bacterial pathogen of honeybee larvae and the causative agent of American foulbrood (AFB). Control measures are limited and represent a challenge for both beekeepers and researchers. For this reason, many studies focus on the search for alternative treatments based on natural products. The objective of this study was to determine the antimicrobial activity of the hexanic extract (HE) of Achyrocline satureioides on P. larvae and the inhibitory activity on some mechanisms related to pathogenicity. The Minimum Inhibitory Concentration (MIC) of the HE was determined by the broth microdilution technique and the Minimum Bactericidal Concentration (MBC) by the microdrop technique. Swimming and swarming motility was evaluated in plates with 0.3 and 0.5% agar, respectively. Biofilm formation was evaluated and quantified by the Congo red and crystal violet method. The protease activity was evaluated by the qualitative technique on skim milk agar plates. It was determined that the MIC of the HE on four strains of P. larvae ranged between 0.3 and 9.37µg/ml and the MBC between 1.17 and 150µg/ml. On the other hand, sub-inhibitory concentrations of the HE were able to decrease swimming motility, biofilm formation and the proteases production of P. larvae.

Alkaline Protease Production by Immobilized Klebsiella aerogenes Cells from Dairy Effluent Sludge

This study investigated the screening of alkaline protease microorganism from diary effluent sludge and identified by 16S ribosomal RNA nucleotide sequence as Klebsiella aerogenes with accession number MF156964.1 and maximum identity 95.45%. The cells were immobilized with coconut pod husks and optimization studies such as the effects of particle sizes, pH, temperature, agitation speed, and incubation time were determined. Out of twenty three microorganisms screened, three were potential protease producers. K. aerogenes gave the highest zone of hydrolysis (35 mm) on the skimmed milk agar plate. The particle size (0.075 mm2) of the immobilization agent gave the highest enzyme activity 176.83 U/mL. The optimum incubation time for the production of protease was 48 h with enzyme activity 143.054 U/mL which further declined. The optimum pH of the protease was pH 9.0 with activity 209.61 U/mL which made it alkaline. The agitation speed 150 rpm resulted in a protease activity 175.83 U/mL and reduced by 56.5% at 250 rpm. The optimal temperature 35 ºC was 183.78 U/mL. This study also confirmed the stability and reusability of the immobilized cells using the coconut pod husks matrix by maintaining from 100% to 76.2% up to six times recycle. Conclusively, the study established the efficiency of low cost, readily available matrix and reusability potentials of coconut pod husks for cells immobilization technology through entrapment at optimal conditions for protease production.

Assessing pH-dependent activities of virulence factors secreted by Candida albicans.

Candida albicans is an opportunistic pathogen that can thrive under adverse conditions including suboptimal pH, nutrient scarcity, and low levels of oxygen. Its pathogenicity is associated with the production of virulence factors such as extracellular hydrolytic enzymes and toxins. This study was aimed at determining the effect of external pH, substrate nature, and strain origin on protease, lipase, and hemolysin production. To achieve this objective, agar plate assays were performed at pH 5.0, 6.5, and 7.5 with substrates suitable for the detection of each family of enzymes. Moreover, the study was conducted with 20 clinical C. albicans isolates from blood, oral cavity, skin, urine, and vagina. The hydrolytic zones formed around the colonies were further measured to calculate the Ez (enzymatic zone) indexes. We found that detection of proteases in skim milk agar plates was possible for most isolates only at pH 5 (80%) and pH 6.5 (75%), whereas BSA plates could confer protease detection exclusively at pH 5 (80%). Similarly, the percentage of isolates possessing lipolytic activities was higher at pH 5 (90%) than at pH 6.5 (70%) and pH 7.5 (35%). In contrast, hemolytic activities were detected in all isolates at pH 6.5 and 7.5 but not at pH 5. Further analysis revealed that some differences in the detected activities could potentially be attributed to the anatomical origin of these isolates. Collectively, these findings suggest that the pH of the site of infection might be critical for mimicking the microenvironment employed to experimentally discover the key virulence factors.

The comparative analysis of traditional and modernized nutrient media for the cultivation of lactobacilli

Culture media for lactobacilli cultivation are used for their growth and reproduction in laboratory conditions, as well as in the production of dairy products. The composition of the developed nutrient medium includes: skimmed milk, yeast extract, lactose, lactulose, agar-agar, distilled water. This complex of components provides lactobacilli with essential nutrients, including carbohydrates, proteins, vitamins and minerals. The constituent components of the nutrient medium are affordable and cheap to purchase. The work involved the cultivation of various types of lactobacilli. (Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus bulgaricus; Lactobacillus casei) on various nutrient media (MRS, Milk agar with prebiotics). It follows from the results of the experiments that the growth rate of lactobacilli on the nutrient medium "Milk agar with prebiotics" is higher in comparison with analogues of industrial production. The greatest intensity of probiotic growth is observed in yogurt starter culture, which includes probiotics of the following species: Lactococcus lactis; Lactobacillus bulgaricus; Lactobacillus casei, grown on the nutrient medium "Milk agar with probiotics", it is 3.7 * 109 CFU/ gr. The high economic efficiency of the developed nutrient medium in comparison with foreign analogues has been confirmedA formulation of a production nutrient medium necessary for the cultivation of probiotic microorganisms has been developed. It has been established that the growth rate of various lactobacilli on this medium is higher in comparison with analogues of industrial production. The high economic efficiency of the developed nutrient medium has been confirmed in comparison with foreign analogues

Optimization of Protease Production Using Agro Wastes by Aspergillus spp. In A Solid-State Fermentation

gricultural wastes are highly nutritious and facilitate microbial growth and metabolism. Most agro-wastes are lignocellulosic in nature, composed of lignin, hemicellulose and cellulose. Agricultural residues can be used for the production of various value-added products, such as industrially important enzymes. Extracellular proteases have high commercial value and multiple applications in detergents, food, dairy, pharmaceutical, leather, diagnostics, waste management and several recovery industries. Aspergillus oryzae was isolated from rice farmland, Rigasa, Kaduna. It was screened for protease production using skimmed milk agar assay with a wider clear zone of casein hydrolysis (halo zone). The main objectives of this study were to isolate, characterized, and optimize the production conditions by Aspergillus oryzae under solid state fermentation (SSF) using locally available agricultural wastes used as substrate. The results showed that the optimum conditions for maximum protease production are 84 hours of incubation; pH 6.0; temperature of 35ᵒC; 3% inoculum size; peptone as Nitrogen source and rice husk as the best fermentation substrate. The highest protease activity of rice husk was 1350 Ug−1 of dry solids compared with an activity of corn cob 1005 Ug−1 respectively. Protease was produced from Aspergillus oryzae with broad substrate specificity, high pH and temperature stability.

ISOLATION AND SCREENING OF PROTEASE PRODUCING BACTERIA FROM SOIL

Microbial proteases account for approximately 40% of the total worldwide enzyme sales and most of the commercial proteases are of bacterial origin. Total 35 bacterial species isolated from soil and water collected from different districts of Gujarat. Isolates were screened on skim milk agar plates (pH-8.5) for their potency to secrete alkaline protease. Out of these, 14 cultures were screened based on relative enzyme activity (REA) on solid media. Cultures showing more than 3 REA were selected for further studies by using shake flask method. Bacillus spp. C6 was selected for this study and characterized by cultural, biochemical and microscopic features. All the data of the isolates that it may belong to Bacillus circulans.

Protease Production by Submerged Fermentation in Shake Flasks Using Bacillus sp. Isolated from the Soil

Proteases are one of the most industrially important enzymes, which account for about 60% of total enzyme market. Protease production by submerged fermentation in shake flasks using Bacillus sp. isolated from the soil was studied. Soil samples were collected from different locations within Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra state. The soil samples were serially diluted and inoculated on sterilized skim milk agar plates. The plates were incubated at 30oC for 72 h. A clear zone around the colonies gave an indication of protease-producing bacteria isolates. The selected protease producers were subsequently used for shake flask fermentation in 50 ml sterile medium. Optimization study was conducted to determine the effect of carbon sources, nitrogen sources, trace elements, agitation rates and pH. Twenty one bacteria isolates were found to be active protease producers and isolates RS-5 and OS-9 had the highest zone of clearance of 13.5 and 12.1 mm respectively. The result of submerged production of protease by the bacteria isolates revealed that the isolates RS-5 and OS-9 accumulated maximum protease yield of 3.23 and 2.71 U/ml respectively. The isolates were Gram positive and endospore formers, and were identified as Bacillus sp. RS-5 and OS-9.The addition of Starch and maltose stimulated optimum protease production of 3.47 and 2.77 U/ml by Bacillus sp. RS-5 and OS-9 respectively. Beef extract enhanced maximum enzyme yield of 3.35 and 2.90 U/ml for Bacillus sp. RS-5 and OS-9 respectively. Maximum protease yield of 3.28 U/ml for Bacillus sp. RS-5 and 2.85 U/ml for Bacillus sp. OS-9 was obtained by the supplementation of 0.4 g/l of FeS04 respectively. The maximum protease yield was observed at agitation rate of 200 rpm for Bacillus sp. RS-5 and 170 rpm for Bacillus sp. OS-9. At pH8, protease accumulation was highest for Bacillus sp. RS-5 and OS-9. The study revealed that the soil harbours some protease-producing bacteria strains and protease production can be greatly enhanced through optimization of process parameters.

Isolation of Keratinolytic Bacteria from Common Ostrich (Struthio Camelus) and their Efficacy towards Feather Degradation of Poultry Waste

Keratinolytic microorganisms have the capacity to biosynthesize particular keratinases and determine theirprospective application in poultry waste management. Hence, the objective of this study was to utilize keratinolytic microbes for the degradation of poultry waste. For this purpose, the feathers of common ostrich (Struthio camelus) were collected, scattered along the surrounding dry soil, from a private ostrich farm in Kasur, Pakistan. Bacteria were isolated by using culture enrichment techniques and screened for their proteolytic activity on skim milk agar. The isolates were characterized clonially, morphologically, and biochemically and labeled as SNO1, SNO2, and SNO3, respectively. The effects of varied pH and temperatures were recorded on bacterial growth and feather degradation. It was noticed that the bacterial cell densities and feather degradation were high at 50˚C and pH 8.0, as compared to the rest of the culture conditions. Visual observations were made using a stereomicroscope. The maximum feather degradation in the form of white powdery mass was noticed at fourth (4 th ) week. The protein content of Struthio camelus feathers was determined as 6.5 mg/100 ml after degradation. Taken together, the authors concluded that the isolated strains were capable of feather degradation and can be used in poultry waste management.

SCREENING AND PARTIAL 16 S RRNA GENE SEQUENCING OF KERATINASE PRODUCING STENOTROPHOMONASMALTOPHILIA KARUNA5 ISOLATED FROM POULTRY WASTE

Keratinase is a protease which degrades the insoluble protein keratin that is largely found in feather waste. Fifty-seven soil samples were collected from a feather and poultry waste dumping sites. All these soil samples were subjected for enrichment in Whole feather meal medium containing 1% chicken feather as a sole source of Carbon, nitrogen and energy with pH 8.0 and incubated at 30°C on shaker condition (150 rpm) for 7 days. Out of 57 flasks only 21 showed visual feather degradation. Enriched sample from each flask was isolated on skimmed milk agar plates. Only 10 isolates demonstrated proteolytic activity by showing zone of clearance around the colony on skimmed milk agar medium. The above 10 bacterial isolates (Kar:01-12) were assayed for keratinase enzyme by growing them in Whole feather meal medium pH 8. Maximum keratinase production was demonstrated by isolate number Kar05 (41U/ml) at 30°C on shaker condition (150rpm) after 4 days of incubation. Promising isolate was identified as Stenotrophomonasmaltophila KARUNA5 by morphological, cultural, biochemical and 16S rRNA sequence analysis and was submitted to NCBI (Accession no: LC271188).

Thrombolytic potential in bacteria isolated from fermented durian tempoyak

Abstract. Priskila C, Vidian V, Sanjaya A, Sugata M, Pinontoan R. 2022. Thrombolytic potential in bacteria isolated from fermented durian tempoyak. Biodiversitas 23: 5731-5737. Cardiovascular disease is one of the leading causes of death worldwide and is primarily caused by dysregulation in the blood clotting system leading to the formation of unwanted blood clots (thrombi). Thrombolytic therapy, which aims to break down unwanted blood clots, employs various agents possessing thrombolytic activity. However, the harmful side effects of conventional thrombolytic agents necessitate the exploration of new and safer agents with high thrombolytic activities. Thrombolytic agents can be obtained from various sources, including fermented foods. Tempoyak is a condiment made from fermented durian, which is found in some regions of Indonesia, and is reported to contain beneficial microorganisms. The aim of this study was to isolate and identify thrombolytic agents from tempoyak. After several pure microbial colonies were successfully isolated from tempoyak, they were screened for proteolytic activity on skim milk agar medium. The isolated colonies with proteolytic activity were then subjected to several tests, including the whole blood clot lysis test, euglobulin clot lysis test, fibrin plate method, and zymography. Subsequently, the isolates with thrombolytic and fibrinolytic activity potential were identified in terms of their morphology and biochemistry. In this study, three bacteria with thrombolytic activity potential were successfully isolated. Fibrin zymography indicated that the molecular weights of the proteins that play roles in the fibrinolytic activity ranged from approximately 30 to 153 kDa, which were suspected to correspond to serine protease, cysteine protease, and/or metalloprotease. All three isolates were identified as belonging to the genus Corynebacterium. This is the first report discussing the thrombolytic and fibrinolytic activities of Corynebacterium sp. derived from fermented food.

Molecular Characterization, Statistical analysis of protease and its application

Proteolytic enzymes are currently attracting enormous attention because of their biotechnological potential. Currently, bacterial proteases are in great demand because of potential industrial applications that are found in soil or mud. This research was conducted to find the proteolytic bacteria isolation from PKC (Palm Kernel Cake) sample collected from Oil Palm India Limited, Kollam, Kerala and it was screened for a novel protease producing bacteria in Skim milk agar medium. Among the isolates, the S1 isolate exhibited high proteolytic activity and was selected as a novel protease-producing bacteria, which was identified by morphological and biochemical characteristics as Pseudomonas aeruginosa. The optimization study was carried out for pH, carbon, substrate, surfactants, nitrogen, metal ions, and inoculum size for determining the maximum production of protease. The results are stated thereby.

Potential use of proteolytic bacteria Paenibacillus dendritiformis (BT7) isolated from Batu tannery effluents for the detergent industry

This research was aimed at identifying a bacterium that can produce alkaline proteases. As a result, bacteria that produce proteases were isolated from Batu tannery effluents, tested for protease synthesis on skim milk agar plates, and validated with a protease assay. Microscopic and molecular phylogenetic analyses identified Paenibacillus dendritiformis (BT7) as the bacterial isolate with the highest alkaline protease production. The isolate's maximum enzyme production was obtained by 2% inoculum size, 40°C temperature, 9.0 pH, and a 48-hour incubation time with production media components such as glucose, casein, MgCl2, and 2% NaCl. The maximal enzyme activity was 270 U/mL under all optimum culture conditions. Concentrated ammonium sulfate precipitation (75%) and dialysis were employed to obtain a cell-free, partially purified protease. The specific activity of the dialysate, which accounts for 3% of the enzyme yield, was discovered to be 134 U/mL. The partially purified protease was used for application in blood stain removal. It was studied and found that the alkaline protease resistance under stringent conditions is very stable with bleach detergent. Also, this enzyme could clean blood-stained fabrics. This study shows that the alkaline protease from Paenibacillus dendritiformis - BT7 could be used in various ways in the detergent industry that are good for the environment.

Purification and characterization of a robust thermostable protease isolated from Bacillus subtilis strain HR02 as an extremozyme.

Since the hot water of Genow, a village in Isin rural district in the central district of Bandar Abbas, Hormozgan Province, Iran, has a rich source of thermophilic bacteria, the current study aimed to find a new thermophilic protease enzyme with suitable properties to be used in different industries. Water and sediment samples were collected from the hot water of Genow, and finally, 20 colonies were isolated. Among these isolated colonies, two bacterial strains grew on the skim milk agar medium, and a clear halo was formed around the colony, which was accurately identified by 16S rRNA gene sequence analysis. The comparison of 16S rRNA gene sequence analyses of isolated strains HR01 and HR02 with registered sequences of 16S rRNA genes in NCBI showed that the two isolates had the most similarity to Bacillus sonorensis and Bacillus subtilis, respectively. Among the two bacterial strains, the highest enzymatic activity was observed in B. subtilis strain HR02, from which the protease purification process was performed. A putative native B. subtilis strain HR02 protease (BSHR02PR) was purified by the UNO Q-6 ionic exchange chromatography method. Biochemical analyses revealed a monomeric enzyme, BsHR02Pro, with a molecular weight of 25 kDa, showing the maximum activity at 70°C and pH8.0. Moreover, the purified enzyme was stable up to 80 °C and in a pH range of 6.0-12.0. The steady-state kinetic analysis for colloidal casein showed that the Km , Vmax and kcat values of the purified enzyme were 25.7μM, 93.2μM min-1 and 2.18 s-1 , respectively. The hot water of Genow is a rich source of protease-producing bacteria. Sediments are a better source for the isolation of these types of bacteria than spring water. Overall, our results demonstrated a potential bacterial enzyme BsHR02Pro as a suitable catalyst to be used in the various industries.

Evaluate the effect of the formula containing partial purified of protease produced from Bacillus spp

In this study was used Bacillus spp. isolate after collection and identification and screening as the best isolate for protease production by using skim milk agar as screening media then the isolate that give the highest proteolytic activity was chosen and the protease was partially purified by the ammonium sulfate and sephadex G-50 to give activity 320 and 640 U/ml subsequently , then the activity was examined in vitro with formula by using skim milk agar as a screening media to detect of protease activity within the formula the result proved increasing of protease activity by increasing of clearing zone around well of well diffusion assay of skim milk and can used this formula for medical application.

The Potential of Phylogenetically Diverse Culturable Actinobacteria from Litopenaeus vannamei Pond Sediment as Extracellular Proteolytic and Lipolytic Enzyme Producers.

Enzymes are catalysts that can increase the reaction time of a biochemical process. Hydrolytic enzymes have a pivotal role in degrading organic waste in both terrestrial and aquatic environments. The aims of this study were (1) to investigate the ability of actinobacteria isolated from Litopenaeus vannamei pond sediment to produce proteolytic and lipolytic enzymes, (2) to identify promising candidates using 16S rRNA gene amplification, and (3) to construct a phylogenetic tree based on the 16S rRNA genes. A skim milk agar medium was used in the preliminary experiment of the proteolytic assay, and a Tween 20/80 medium was used in the lipolytic assay. Fifteen and 20 (out of 40) actinobacterial isolates showed great potential for proteolytic and lipolytic activities, respectively. Furthermore, four actinobacteria isolates produced both enzyme types with proteolytic and lipolytic index scores of 1-6.5. The most promising candidates were SA 2.2 (IM8), SC 2.1 (IM6), SD 1.5 (IM6) and SE 1.1 (IM8). BLAST homology results showed a high similarity between the actinobacteria isolates and Streptomyces verucosisporus, S. mangrovicola, S. barkulensis and Nocardiopsis lucentensis, respectively. Therefore, actinobacteria from Litopenaeus vannamei pond sediment are high-potential proteolytic and lipolytic enzyme producers.

Optimization Studies on Extracellular Protease Production by Aspergillus niger and Aspergillus terreus Using Skim Milk Casein as Substrate

Aim: Proteases are proteolytic enzymes that have a wide range of applications in the industrial sectors. This study investigated the production and optimization of protease by protease-producing fungi, (Aspergillus niger and A. terreus) using skim milk casein as substrate.
 Materials and Methods: Aspergillus terreus and A. niger obtained from the Department of Plant Science and Biotechnology, University of Jos, Nigeria were screened for extracellular protease on skim milk agar medium. Optimization studies across different parameters: temperatures (30-80oC), pH (3-10), substrate Concentration (0.25-2 %), and incubation period (6 days) using submerged fermentation were done for maximum protease activity.
 Results: The maximum temperature of protease activity was 50oC (116.5 IU/ml) for Aspergillus niger and 40oC (121.86 IU/ml) for Aspergillus terreus. The Optimum pH for protease activity was pH 9 (106.85 IU/ml) for Aspergillus niger and pH 8 (107.90 IU/ml) for Aspergillus terreus. Optimum protease activity (29.36 IU/ml) at an incubation period of 48 hours was recorded for Aspergillus niger and 72 hours (182.2 IU/ml) for Aspergillus terreus. At 2% substrate concentration a maximum activity of 87.21 IU/ml and 76.5 IU/ml was recorded for Aspergillus niger and A. terreus, respectively.
 Conclusion: The test fungi Aspergillus niger and Aspergillus terreus exhibited maximum hydrolytic potential for protease production which has a vast application in different industries.

Isolation of Keratinolytic Bacteria from Common Ostrich (Struthio Camelus) and their Efficacy towards Feather Degradation of Poultry Waste

Keratinolytic microorganisms have the capacity to biosynthesize particular keratinases and determine their prospective application in poultry waste management. Hence, the objective of this study was to utilize keratinolytic microbes for the degradation of poultry waste. For this purpose, the feathers of common ostrich (Struthio camelus) were collected, scattered along the surrounding dry soil, from a private ostrich farm in Kasur, Pakistan. Bacteria were isolated by using culture enrichment techniques and screened for their proteolytic activity on skim milk agar. The isolates were characterized clonially, morphologically, and biochemically and labeled as SNO1, SNO2, and SNO3, respectively. The effects of varied pH and temperatures were recorded on bacterial growth and feather degradation. It was noticed that the bacterial cell densities and feather degradation were high at 50˚C and pH 8.0, as compared to the rest of the culture conditions. Visual observations were made using a stereomicroscope. The maximum feather degradation in the form of white powdery mass was noticed at fourth (4th) week. The protein content of Struthio camelus feathers was determined as 6.5 mg/100 ml after degradation. Taken together, the authors concluded that the isolated strains were capable of feather degradation and can be used in poultry waste management.

CHICKEN FEATHER DEGRADATION BY KERATINOLYTIC ASPERGILLUS SPECIES ISOLATED FROM FEATHER WASTE DUMPING SOIL

Slaughterhouses and poultry processing units worldwide dispose of huge amount of chicken feathers that accumulate as solid waste in the environment and cause serious consequences. Keratinases are a group of enzymes, produced by microorganisms have the ability to degrade tough recalcitrant keratin that is abundantly present in chicken feathers. Soil sample was collected from a feather dumping site at Virudhunagar (9° 35' 13.9524'’ N Latitude and 77° 57' 5.1516'’ E Longitude), Tamil Nadu, India. Soil sample was serially diluted, inoculated on to feather meal agar plates and incubated at 37 °C for 7 days to isolate keratinolytic fungal strains. The isolated strains were screened for proteolytic activity on skim milk agar for 8 days and three better strains were identified based on morphology, mycelial growth, and structure of hyphae using lactophenol cotton blue staining and confirmed by ITS primer sequencing method as Aspergillus niger, Aspergillus terreus and Aspergillus flavus respectively. Different range of pH (7 to 11), temperature (25 to 45 °C), substrate (bovine serum albumin, feather, peptone and gelatin) and feather concentration (0.5g, 1g, 1.5g and 2g) were tested to find out the optimum conditions for the growth and keratin degradation of the chosen fungal strains. The temperature and feather concentration for the optimum growth of A. niger, A. terreus and A. flavus were 25 °C and 1.5g, 45 °C and 1g and 30 °C and 1.5g respectively. All three Aspergillus species exerted better growth at pH 9 and with bovine serum albumin as the substrate.

THE EFFECT OF Bacillus sp. ADDITION ON THE VIRULENCE OF Vibrio parahemolyticus TOWARD AXENIC CULTURE OF Artemia franciscana

Research on the benefits of Bacillus sp. in aquaculture has been done a lot. Currently, the aquaculture industry, especially shrimp farming, is facing AHPND disease, which is known to be caused by Vibrio parahaemolyticus. This study aims to see the effect of Bacillus sp. against virulence of V. parahaemolyticus in vitro using caseinase production as an indicator and survival of Artemia franciscana cultured axenically when challenged with V. parahaemolyticus. In in vitro tests, cultures of V. parahaemolyticus were grown with and without the addition of Bacillus sp. on Luria Bertani (LB) liquid media, and then the caseinase test was carried out using skim milk (SM) agar as a growth medium. In the challenge test, 10 axenic cultured Artemia sp. were distributed in falcon tubes containing 10 ml of sterile seawater. Challenge test treatments consisted of treatment A as control of Artemia culture without the addition of bacteria, treatment B Artemia culture adding by 106 CFU/ml V. parahaemolyticus, treatment C Artemia culture with the addition of 106 CFU/ml Bacillus sp., and treatment D Artemia culture with the addition of 106 CFU/ml V. parahaemolyticus and 106 CFU/ml Bacillus sp. All treatments were done in triplicates. The results showed that the addition of Bacillus sp. was able to reduce the caseinase production of V. parahemolyticus up to 29% indicated by the decrease of the clearing zone diameter formed on SM agar. Furthermore, the addition of Bacillus sp. in treatment D was able to significantly increasing (P<0.05) of Artemia's survival when challenged with V. parahamolyticus. This indicates that Bacillus sp. has a potential as a probiotic candidate to prevent disease caused by V. parahaemolyticus.

Evaluation of probiotic Lactobacillus plantarum CS fermented culture and its crude enzymes as chicken growth enhancers

Studies have shown that supplementation of important enzymes in poultry feed can improve the growth of birds. However, there were little or no available information on the utilization of such enzymes or the probiotic fermented culture in local feed production in Nigeria. Therefore, the aim of the study was focused on the evaluation of probiotic Lactobacillus plantarum fermented culture and its crude enzymes as chicken growth enhancers. The organism was first screened for amylase and protease producing potential using starch agar and skim milk agar respectively. Thereafter, confirmation of the produced amylase and protease enzymes via shake-flask fermentation at 30oC for 3 days was by 3,5-dinitro salicylic acid and Folin ciocalteu reagents respectively. The produced fermented culture and its recovered crude enzymes (2 %v/v) were respectively supplemented in the locally produced chicken feed for 10weeks. The chicken fed without supplement served as control and all their growths were determined every week. The screening results revealed that L. plantarum CS produced more amylase(29 mm) than protease(19 mm) with a total enzyme yield of 22.50 U/ml and 20.92 U/ml respectively, The chicken fed with supplements containing either the enzymes or the fermented culture grew faster and significantly higher (5 kg /10 wk) than those without the supplement (3.0 kg/10 wk)at p>0.05. The growth enhanced by the fermented culture ((5 kg/10 wk) was more than that stimulated by either the amylase (3.52 kg/10 wk) or the protease (3.86 kg/10 wk), although not significant. Supplementation with protease enzyme resulted in significant robust and faster growth (3.57 kg/ 7 wk) than that with amylase enzyme (3.37 kg/7 wk). These results indicated the great potentials of both L. plantarum fermented culture and its crude enzymes especially protease as chicken growth stimulants.