Partial Purification and Characterization of Extracellular Protease from <i>Pedicoccus acidilactici</i>

Bacterial lysates have become a common ingredient for natural health care. Lactic acid bacteria (LAB) could serve as potential candidates for lysate production: the lactic acids produced by LAB have been utilized for their moisturizing, antimicrobial, and rejuvenating effects, while other substances provide topical benefits and health effects for the skin. Our study aimed to obtain lysate from a LAB S. macedonicus MBF 10-2 through an optimized fermentation using the Response Surface Methodology. Strain MBF10-2 was cultivated in a 2L fermenter tank in de Man Rogosa and Sharpe (MRS) medium and in plant-based peptone modified MRS, i.e. Soy-peptone and Vegitone. The duration and the medium composition (dextrose and soy peptone or proteose peptone) were adjusted to obtain an optimum production of cell lysate. Central Composite Design was employed for Design Expert 7.0.0 by adjusting 3 factors: dextrose (1%, 1.5%, 2%, 2.5%, 3%), soy or proteose peptone (0.5%, 0.75%, 1%, 1.25% and 1.5%), and duration of fermentation (8, 10, 12 14, and 16 h for MRS-Soy peptone and 15, 17, 19, 21, and 23 h for MRS Vegitone). Bacteriocin-Like Inhibitor Substance activity of lysate and pH were used as indicators. The optimum condition for lysate production using MRS Soy Peptone and Vegitone are as follows: dextrose concentration 2.5%, plant-based peptone 1.25%, while optimum fermentation duration were 11.18 h (MRS Soy Peptone) and 17 h (MRS Vegitone) with a starter concentration of 10% at OD600nm 0.2 ± 0.05. However, the standard MRS medium produced better quality lysate compared to MRS plant-based peptones.

The detection and isolation of lactic acid bacteria by enrichment methods from wine grapes cultivated in vineyards located in New South Wales, Australia. Enrichment cultures in de Man, Rogosa and Sharpe (MRS) broth, MRS + ethanol (5%), MRS broth supplemented with 15% (v/v) tomato juice (MRST), pH 5.5 and 3.5 and autoenrichment in grape juice homogenate were used to detect lactic acid bacteria on wine grapes. Bacteria were isolated from enrichment cultures by plating onto MRS and MRST agar and identified by 16S rDNA sequence analysis and phenotypical methods. A molecular method, PCR-denaturing gradient gel electrophoresis (DGGE) was also used to examine the bacteria that developed in enrichment cultures. Species of Lactobacillus, Enterococcus, Lactococcus and Weissella were detected in enrichments by plating and PCR-DGGE. Other bacteria (Sporolactobacillus, Asaia, Bacillus ssp.) were also found in some enrichment cultures. The principal malolactic bacterium, Oenococcus oeni, was not isolated. The incidence and populations of lactic acid bacteria on wine grapes were very low. Damaged grape berries showed a greater presence of these bacteria than undamaged berries. The diversity of bacterial species isolated from the grapes was greater than those previously reported and represented both lactic acid bacteria and nonlactic acid bacteria. Some of these bacteria (i.e. Lactobacillus lindneri, Lactobacillus kunkeei) could be detrimental to wine production. Oenococcus oeni was not found on grapes, but its recovery could be obscured by overgrowth from other species. Lactic acid bacteria are significant in wine production because they conduct the malolactic fermentation and cause stuck or sluggish alcoholic fermentation and wine spoilage. This study investigates wine grapes as a potential source of these bacteria.

This study aimed to determine the survival and growth of Escherichia coli O157:H7 and Salmonella enterica subsp. enterica in a medium supporting the growth of a Lactic Acid Bacteria (LAB) food antimicrobial culture. Foodborne pathogens and LAB were cultured individually in tryptic soy broth (TSB), tryptic soy broth supplemented with one g l(-1) Tween 80(®) (TSB-T80), and de Man, Rogosa and Sharpe (MRS) broth. Growth of E. coli O157:H7 and Salmonella was similar in TSB and TSB-T80 but was significantly less in MRS. Conversely, LAB growth was similar in MRS and TSB-T80 but was significantly less in TSB. Supplementation of TSB with Tween 80(®) allows growth of LAB to levels similar to that observed with MRS but does not inhibit the growth of E. coli O157:H7 and Salmonella. We present the formulation of a medium useful in studies useful for evaluating competitive inhibition of foodborne pathogens by LAB in vitro. This study reports the utility of TSB-T80 for the completion of in vitro competitive inhibition assays incorporating a Lactic Acid Bacteria food safety culture.

Tuberculosis (TB) is one of the infectious diseases that have become a major problem in Indonesia. This disease is caused by the Mycobacterium tuberculosis bacteria. The bacteria are commonly treated with antibiotics. However, the use of irrelevant antibiotics is the most influential factor of antibiotic resistance. Therefore, natural ingredients are required as novel antibiotic agents, some of which are sourced from lactic acid bacteria. This study aims at investigating the antituberculosis activities of Pediococcus acidilactici and Lactobacillus plantarum isolated from breast milk against M. tuberculosis. Pediococcus acidilactici and Lactobacillus plantarum breast milk isolates were rejuvenated with MRS (De Man, Rogosa and Sharpe) broth media, and the supernatant was then neutralized to pH 7.0 using a pH meter by adding 1N NaOH solution. Antituberculosis activity test was performed with Lowensten Jensen media to investigate the growth of M. tuberculosis. The results of this study showed that Pediococcus acidilactici had antituberculosis activity at 48 hours at concentrations of 80% and 90%, while the secondary metabolites of L. plantarum had antituberculosis activity at 24 hours and 48 hours at concentrations of 60%, 70%, 80%, 90%. Therefore, this study concludes that P. acidilactici and L. plantarum bacteria have the potential to be developed as antituberculosis agents.

ABSTRACT: Milk and its products contain large number of Lactic acid bacteria (LAB) that are difficult to culture under laboratory conditions because of their complexity and diversity under dynamic ecosystem. MRS (de Man, Rogosa and Sharpe) medium was used to culture of LAB from milk and its products under aerobic and anaerobic conditions separately. The same samples were used to culture LAB on new Soybean culture medium (per litre) containing extract of 20 g soya nutri nuggets, 5 g yeast powder extract, 20 mL coconut milk and agar 20 g. The samples on both media were incubated at 37±1℃ for 48-72 h and examined for growth of different LAB. The cost of MRS medium available in the market ranges from U$ 50 to 100 per 500 g of which 50-60 g is used to prepare 1 litre of culture medium depending upon the manufacturer. The minimum cost of 1 litre MRS comes to U$ 5 while the cost of ingredients used in Soyanutri Jaggery Agar (SJA) medium is only 50 cents i.e. ten times cheaper than MRS and the number of colonies isolated from milk and its products were 8-10x higher than MRS medium. Jaggery (concentrated raw sugarcane juice) is available at the 5 Kg for 1U$ and easily available. 500 g of Soyanutri cost only U$ 1. Further researches are being conducted in our laboratories and it is suggested that Soyanutri Jaggery Agar medium may be used for routine culture of LAB in laboratories for the demonstration and practice for the students and also for screening of samples on large scale.

A study was conducted to investigate the effect of particle size and micro-organism on fermentation of sorghum and maize for poultry feed. Sorghum ( Sorghum bicolour L. Moench) and maize were milled in a hammer mill and separated into coarse, medium, fine and very fine particles sizes with a stack of sieves of apertures 2.5 mm, 850 μm and 500 μm, from the first to the last sieve and ending in a pan with the very fine particles. Samples were weighed into 100 g sachets and irradiated using 60 Co at 25 kGy γ-radiation. Grains were fermented with sterile distilled water for 24 h at a ratio of 1 feed:1.4 water and inoculated with 0.01 ml of an overnight culture of De Man, Rogosa and Sharpe (MRS) broth containing Pediococcus acidilactici (PA1) or Lactobacillus plantarum (SLP) (ca 10 9 cfu/ml). The medium was incubated at 30°C simultaneously with a control treatment without lactic acid bacteria (LAB). Subsamples were collected aseptically at the beginning of the fermentation (0 h) and at 4, 8, 24 h after fermentation for pH, sugar and organic acids analysis. Significant reductions in the pH of maize and sorghum for LAB treatments (PA1 and SLP) were evident after 8 hours of fermentation. Twenty four hour lactic acid concentrations from coarse particle size fermentations were not significantly different from concentrations in the medium and fine particle size fermentations. The choice of LAB did not affect the concentration of lactic acid for any particle size. However, acetic acid production from fermentation with PA1 was significantly higher (P<0.01) than the concentration obtained with SLP. Results suggest that moderate grain processing may be enough to permit production of biosafe levels of lactic acid in fermented feed for poultry birds. Keywords : Fermentation, lactic acid bacteria, maize, particle size, sorghum African Journal of Biotechnology Vol. 12(26), pp. 4147-4157

Orthogonal array deciphering MRS medium requirements for isolated Lactobacillus rhamnosus ZY with cell properties characterization

Lactobacillus delbrueckii subsp. delbrueckii KCTC 1047, grown in de Man, Rogosa and Sharpe (MRS) or soymilk media, completely hydrolyzed the isoflavone glucosides, genistin and daidzin at 50 μg ml−1, into their respective aglycones, genistein and daidzein within 30 min. Other lactic acid bacteria did not produce β-glucosidase, the enzyme responsible for the hydrolysis of isoflavone glucosides, when cultured in MRS medium. Glucoside-hydrolyzing activity was induced in some lactic acid bacteria when cultured in soymilk medium. These strains hydrolyzed 70–80% of genistin into genistein and 25–40% of daidzin into daidzein.

One of the functional foods in Bali, especially in Gianyar Regency, is teh wong (mushroom), which has a sweet-sour taste. The purpose of the study was to determine the characteristics of probiotic candidates isolated from the fermented tea wong drink and to determine the LAB of Teh Wong which could potentially be probiotic candidates. The samples used were 20 lactic acid bacteria (LAB) isolates from wong tea. The process of isolating the LAB candidate used the spread plate method from the fermented liquid of Teh Wong for 7 days. Isolation was carried out after the plates were incubated for 24 hours at 37oC. Colonies suspected of being LAB were then streaked on new De Man, Rogosa and Sharpe (MRS) Agar media to obtain a single colony. The total LAB produced from LAB isolates ranged from 3.0 x 105 cfu/ml to 7.0 x 108 cfu/ml. Of the 20 isolates from gram staining, it was found that the LAB Teh Wong isolate was dominated by rods (bacilli) and only 4 isolates were spherical (coccus).

Chapter 10 Media for the detection and enumeration of bifidobacteria in food products

In recent years, the development of probiotic-based fermented products with halal status has been a concern. The use of growth medium has relied on de Man Rogosa and Sharpe (MRS) as a relatively expensive standard medium, and its halal status is still uncertain. Extensive research has been carried out to investigate the development of low-cost halal alternative media for the cultivation of probiotic lactic acid bacteria (LAB). This study aimed to develop a probiotic halal and low-cost culture medium using a tofu whey-based medium. This study used three tofu whey-based media – A (tofu whey 100%), B (tofu whey 94.5%, molasses 3%, skim milk 2.5%), C (tofu whey 92.5%, molasses 3%, cheese whey 2.5%, tomato extract 2%), and MRS broth as a standard medium. Bacterial populations, total sugars utilized, total lactic acids produced, low pH (2.0) tolerance, and high bile salt concentration (oxgall 1.5%) were assayed. The highest bacterial population after 48 h of incubation was shown by medium B compared to medium MRS (12.34 ± 0.87 and 11.48 ± 0.3 log CFU/mL). Total sugars utilized by 48.28 ± 2.89, 38.89 ± 6.94, 39.14 ± 4.24, and 76.00 ± 1.41 %; on the other hand, total lactic acids produced by 0.16 ± 0.12, 0.03 ± 0.04, 0.31 ± 0.03, and 2.25 ± 1.48 % in A, B, C, and MRS, respectively. Probiotic tolerance at low pH and the presence of bile salts of Lactiplantibacillus plantarum D4 consistently showed a high survival rate in medium B compared to MRS. Based on these results, the components and proportions used in medium B were suitable for the growth of L. plantarum D4 as a halal probiotic starter candidate.

The aim of this study is to purify and characterize an acid protease from Aspergillus carbonarius. The protease was purified to apparent homogeneity by 4 M sucrose concentration, ion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on Phenyl Sepharose CL-4B and gel filtration chromatography through Sephadex G-100. A 10-fold purification with specific activity of 485.47 Umg-1 protein was achieved. SDS-PAGE and zymogram analysis of the protease indicated an estimated molecular mass of 72 kDa. The optimum temperature and pH for the proteolytic activity were 40°C and pH 3.0, respectively. The enzyme was active over a wide range of temperature from 40 to 80°C. The enzyme retained about 70% of its original activity (40°C) at 90 and 100°C. The enzyme was most stable at pH of 3.0 and temperature of 50°C. The enzyme has a relatively broad pH range of 4.0 to 10.0 after 30 min. The enzyme was slightly significantly (P < 0.001) stimulated by K+, Ba2+ while Ca2+ and Zn2+ moderately enhanced it. Mn2+, Fe2+ and Cu2+ strongly significantly (p<0.001) enhanced the enzyme activity while it was significantly (p<0.001) inhibited by Hg2+. The enzyme was not affected by Sr2+ and Mg2+ but Co2+ and Na+ elicited slight repression of the enzyme activity. The protease was strongly significantly (p < 0.001) inhibited by E-64, IAA and DIMSO while EDTA and 2-ME appreciably enhanced the activity of the protease. The enzyme is a cysteine protease as indicated by its inhibition studies. The protease may find potential applications in food and brewing industries as well as in meat tenderization. Key words: Aspergillus carbonarius, protease, purification, characterization, kinetic properties.

Simple SummaryThis study focused on isolation and identification of xylose utilizing lactic acid bacteria from the midgut of Eri silkworm to understand their characteristics such as tannin tolerance, production of cellulolytic enzymes, and antimicrobial activity against insect pathogenic bacteria. The Enterococcus was found as the dominant genus among xylose utilizing lactic acid bacteria. Within this genus, Enterococcus hirae SX2 showed the potential to be used as a probiotic in Eri silkworm culture due to its tannin tolerance and antimicrobial activity against insect pathogenic bacteria. The trial experiment for applying live E. hirae SX2 supplemented to castor leaves in Eri silkworm rearing showed a positive effect for improving larval weight and survival. These findings led to the development of a new probiotic for Eri culture and also could be the experimental model for screening of the potential probiotic from mulberry silkworm (Bombyx mori).A total of 51 pentose utilizing lactic acid bacteria (LAB) were isolated from acid-forming bacteria in the midgut of healthy mature Eri silkworm using de Man, Rogosa and Sharpe (MRS) agar containing 10 g/L xylose (MRS-xylose) as the carbon source supplemented with 0.04% (w/v) bromocresol purple. Further analysis of 16S rRNA gene sequences revealed the highest prevalence of up to 35 enterococci isolates, which included 20 isolates of Enterococcus mundtii, followed by Entercoccus faecalis (eight isolates), Weissella cibaria (four isolates), Enterococcus hirae (two isolates), Enterococcus lactis (one isolate), and Enterococcus faecium (one isolate). All 51 LAB isolates showed positive growth on MRS containing a range of polysaccharides as the sole carbon source. All isolates were able to grow and form clear zones on MRS supplemented with 1 g/L xylose, while E. faecalis SC1, E. faecalis SCT2, and E. hirae SX2 showed tannin tolerance ability up to 5 g/L. Moreover, five isolates showed antimicrobial activity against Eri silkworm pathogens, including Bacillus cereus, Staphylococcus aureus, and Proteus vulgaris, with E. hirae SX2 having the highest inhibitory effect. Supplementation of live E. hirae SX2 on castor leaves significantly improved the weight and reduced the silkworm mortality when compared with the control group (p < 0.05). This cocci LAB can be considered as the new probiotic for Eri culture. Additionally, this finding presented the perspective of non-mulberry silkworm that could also be used as the model for further applying to new trends of the sericulture industry.

Fermentation of Lactic Acid Bacteria (LAB) Pediococcus pentosaceus can improve the quality of food and its shelf life. Using commercial LAB specific media, de Man Rogosa and Sharpe (MRS) for growth on industrial scale application is not efficient. Tapioca wastewater (TW) still contains some of the nutrients needed for the growth of P. pentosaceus , but needs the enrichment of carbon sources (5% of glucose) and nitrogen sources (ammonium sulfate). This study aimed to use tapioca industrial wastewater with the addition of glucose and ammonium sulfate as an alternative growth media for lactic acid bacteria P. pentosaceus E.1222. The results showed that glucose and nitrogen had no significant effect on the number of bacterial colonies. The highest substrate efficiency was tapioca wastewater (86.81%), MRS broth (53.73%), and TW with 5% of glucose and 1% of ammonium sulfate (43.53%) respectively. Maximum growth rate (μmaks) was found in TW with 5% of glucose and 1% of ammonium sulfate (0,52 hours -1 ). Increasing the starter volume until 1000 mL in TW with 5% of glucose and 1% of ammonium sulfate showed a slight decrease in the log number of bacteria from 8,836 (50 mL), 8,401 (500 mL), to 8,063 (1000 mL).

To isolate and characterize lactic acid bacteria (LAB) and determine whether they could potentially be used as heavy metal (cadmium and lead) absorbing probiotics. The study used 53 environmental (mud and sludge) samples to isolate cadmium- and lead-resistant LAB, by following spared plate technique. A total of 255 cadmium- and lead-resistant LAB were isolated from these samples. The survival of 26 of the LAB was found after passing through sequential probiotic characterizations. These 26 probiotic LAB exhibited remarkable variations in their metal-resistant and metal-removal abilities. Of 26, seven (Cd54-2, Cd61-7, Cd69-12, Cd70-13, Pb82-8, Pb96-19 and Cd109-16) and four (Pb71-1, Pb73-2, Pb85-9 and Pb96-19) strains displayed relatively elevated cadmium- and lead-removal efficiencies from water, respectively, compare with that of the remaining strains. Strains Cd70-13 and Pb71-1 showed the highest cadmium (25%) and lead (59%) removal capacity from MRS (De Man, Rogosa and Sharpe) culture medium, respectively, amongst the selected strains and showed a good adhesive ability on fish mucus. A phylogenetic analysis of their 16S rDNA sequences revealed that the strains Cd70-13 and Pb71-1 belong to Lactobacillus reuteri. Excellent probiotic, metal sorption and adhesive characteristics of newly identified Lact. reuteri strains Cd70-13 and Pb71-1 were isolated, which indicated their high potential abilities to survive in the intestinal milieu and to uptake the tested metals from the environment. To our knowledge, this is the first study that has aimed to isolate, characterize and identify metal-resistant LAB strains that have potential to be a probiotic candidate for food and in vivo challenge studies in the intestinal milieu of fish for the uptake and control of heavy metal bioaccumulation.