Major Project #Draft 2

Reuterin production by lactobacilli isolated from pig faeces

and evaluation of probiotic traits

Rodríguez et al. (2003) conducted a study to determine the production of reuterin by lactobacilli isolated from pig faeces and to evaluate their potential as probiotic bacteria. Thirty-three samples of pig faeces were serially diluted and plated on de Man Rogosa and Sharp (MRS) agar. Approximately six colonies per sample were selected for further study. All isolates were screened for their reuterin production in the presence of glycerol by using colorimetric assay at 490 nm. Carbohydrate fermentation test kit (API 50 CHL) were used for the biochemical characterization of reuterin-producing isolates. A polymerase chain reaction (PCR) method based on amplification of 16S RNA coding sequences of L. reuteri and L. fermentum was used to identify selected isolates at species level, with primers pairs LOWLAC-REUT1 and LOWLAC-FERM1. Genomic DNA from L. reuteri CECT 925T and L. fermentum CECT 4007T were used as control in PCR reactions. Reuterin-producing isolates were exposed to HCl at pH 3 for 1 h and consecutively to 0.3% oxgall for 1 h to evaluate their survival under simulate gastric and intestinal conditions. Neutralized supernatants from cultures grown in MRS broth were used to determine the presence of bacteriocin-like antimicrobial activity by an agar diffusion assay using Enterococcus faecalis EF, L. buchneri St2A, Listeria monocytogenes Ohio, L. plantarum CECT 4645, Clostridium tyrobutyricum NZ8 and E. coli K12 as indicator strains. Antimicrobial activity was detected by the presence of inhibition zones. The results showed that 28 of 165 lactobacilli isolates produced reuterin in the presence of glycerol. Six isolates yielding high levels of reuterin (> 15 mg/mL) with respect to type strain Lactobacillus reuteri CECT 925T were identified as L. reuteri. They were able to survive at pH 3 and subsequent exposure to 0.3% oxgall and presented bacteriocin-like activities. The researchers suggested that high yields of reuterin may be obtained from selected isolates of L. reuteri. Probiotic characteristics of isolates studied could be applied in food and feed. However there are some limitations.

1) Resistance to low pH and bile salts in this study under simulate gastric and intestinal conditions in this study were not correlated with presented conditions in stomach and small intestine. According to Argyri et al. (2013), resistance to bile salts was assessed in terms of viable colony counts and enumerated after incubation culture in medium with 0.5% bile salts at 37 °C for 0, 1, 2, and 4 h, reflecting the time spent by food in the small intestine. For evaluation of resistance to low pH, the experiment was performed by incubation culture in medium with pH 2.5 at 37 °C for 0, 0.5, 1, 2, and 3 h, reflecting the time spent by food in the stomach.

2) Neutralized supernatant used for determination of bacteriocin-like antimicrobial activity were not exclude the inhibitory effect of other antimicrobial substances such as hydrogen peroxide and diacetyl which produced by lactobacilli. Therefore, inhibitory effect might not only be from bacteriocin.

3) The researchers used MRS agar without addition of CaCO₃ in the isolation of lactobacilli. Due to some microorganisms (such as yeast, mould, Bacillus, Staphylococcus) could grow in MRS medium, this isolation medium used might be difficult to select only lactobacilli. Thamacharoensuk et al. (2013) reported that lactic acid bacteria such as lactobacilli were isolated by an enrichment technique before plating medium on MRS-CaCO₃ agar. This procedure was easy to select acid-producing bacteria by occurrence of a clear zone around the colonies after incubated at room temperature for 48-72 hours.

The strength of this study is that biochemical characterization and PCR amplification of 16S RNA coding sequences used are widely accepted in many laboratories for bacterial identification in genus and species levels, respectively. Especially, 16S RNA coding sequences could differentiate bacterial species up to 99%. Another advantage of this study is that the results were clearly described with text, table, figures and conclusion which were made relation to results in this study.

References

Argyri, A. A., Zoumpopoulou, G., Karatzas, K. -A. G., Tsakalidou, E., Nychas, G. –J. E., Panagou, E. Z., & Tassou, C. C. (2013). Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests. Food Microbiology, 33: 282-291.

Rodríguez, E., Arqués, J. L., Rodríguez, R., Nuñez, M. & Medina, M. (2003). Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits. Letters in Applied Microbiology, 37: 259-263.

Thamacharoensuk, T., Thongchul, N., Taweechotipatr, M., Tolieng, V., Kodama, K., & Tanasupawat, S. (2013). Screening and characterization of lactic acid bacteria from animal faeces for probiotic properties. The Thai Journal of Veterinary Medicine, 43(4): 541-551.

Major Project #Draft 1

Reuterin production by lactobacilli isolated from pig faeces

and evaluation of probiotic traits

Rodríguez et al. (2003) conducted a study to determine the production of reuterin by lactobacilli isolates from pig faeces and to evaluate their potential as probiotic bacteria. Thirty-three samples of pig faeces were used to isolate lactobacilli in this study. Samples were serially dilutions and plated on MRS agar. All isolates were screened for their reuterin production in the presence of glycerol by using colorimetric assay at 490 nm. API 50 CHL strips were used for the biochemical characterization of reuterin-producing isolates. A polymerase chain reaction (PCR) method based on amplification of 16S RNA coding sequences of L. reuteri and L. fermentum was used to identify selected isolates at species level, with primers pairs LOWLAC-REUT1 and LOWLAC-FERM1. Genomic DNA from L. reuteri CECT 925T and L. fermentum CECT 4007T were used as control in PCR reactions. Reuterin-producing isolates were exposed to HCl at pH 3 for 1 h and consecutively to 0.3% oxgall for 1 h to evaluate their survival under simulate gastric and intestinal conditions. Neutralized supernatants from cultures grown in MRS broth were used to determine the presence of bacteriocin-like antimicrobial activity by an agar diffusion assay using Enterococcus faecalis EF, L. buchneri St2A, Listeria monocytogenes Ohio, L. plantarum CECT 4645, Clostridium tyrobutyricum NZ8 and E. coli K12 as indicator strains. Antimicrobial activity was detected by the presence of inhibition zones. The results showed that 28 of 165 lactobacilli isolates produced reuterin in the presence of glycerol. Six isolates yielding high levels of reuterin (> 15 mg/mL) with respect to type strain Lactobacillus reuteri CECT 925T were identified as L. reuteri. They were able to survive at pH 3 and subsequent exposure to 0.3% oxgall and presented bacteriocin-like activities. The researchers suggested that high yields of reuterin may be obtained from selected isolates of L. reuteri. Probiotic characteristics of isolates studied could be applied in food and feed. However there are some limitations.

1) Resistance to low pH and bile salts in this study under simulate gastric and intestinal conditions in this study were not correlated with presented conditions in stomach and small intestine. According to Argyri et al. (2013), resistance to bile salts was assessed in terms of viable colony counts and enumerated after incubation culture in medium with 0.5% bile salts at 37 °C for 0, 1, 2, and 4 h, reflecting the time spent by food in the small intestine. For evaluation of resistance to low pH, the experiment was performed by incubation culture in medium with pH 2.5 at 37 °C for 0, 0.5, 1, 2, and 3 h, reflecting the time spent by food in the stomach.

2) Neutralized supernatant used for determination of bacteriocin-like antimicrobial activity could not exclude the inhibitory effect of other antimicrobial substances such as hydrogen peroxide and diacetyl which produced by lactobacilli. Therefore, inhibitory effect might not be from bacteriocin.

The strength of this study is that biochemical characterization and PCR amplification of 16S RNA coding sequences used are widely accepted in many laboratories for bacterial identification in genus and species levels, respectively. Especially, 16S RNA coding sequences could differentiate bacterial species up to 99%. Another advantage of this study is that the results were clearly described with text, table, figures and conclusion which were made relation to results in this study.

References

Rodríguez, E., Arqués, J.L., Rodríguez, R., Nuñez, M. and Medina, M. (2003). Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits. Letters in Applied Microbiology, 37: 259-263

Argyri, A. A., Zoumpopoulou, G., Karatzas, K. -A. G., Tsakalidou, E., Nychas, G. –J. E., Panagou, E. Z., and Tassou, C. C. (2013). Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests. Food Microbiology, 33: 282-291.

Minor project

My research question is the factors affecting preservative activity of reuterin can provide possibilities to apply reuterin in many kinds of food. Reuterin is an antimicrobial compound produced by the food grade lactic acid bacterium Lactobacillus reuteri. This substance has high potential as a broad spectrum food preservative. The issue is contested with some researcher saying reuterin can be apply as preservative in food and others saying it can be easily converted to inactive form which depends on environmental condition of food.

Researchers who have looked at this subject are El-Ziney et al. (1999) and Lüthi-Peng et al. (2002). The former encourages the use of reuterin produced from Lactobacillus reuteri as preservative for microbial decontamination in meat products and the latter found that reuterin can be easily transformed to other compounds that cannot exhibit antimicrobial activity.

El-Ziney et al. (1999) reported that Escherichia coli O157:H7 count on the surface of cooked pork meat with 100 AU/g of reuterin decrease by 5.0 log cfu/g after 1 day of storage at 7 °C. Reuterin at a concentration of 250 AU/g reduce 3.0 log cfu/g of Listeria monocytogenes after 1 week of storage at 7 °C. They suggest that reuterin could be used as food preservative, especially in cold stored food.

Lüthi-Peng et al. (2002) state that reuterin is less stable in milk and de Man, Rogosa and Sharpe (MRS) medium than in water. It can react with other components in the food or dehydrate to acrolein which is inactive form at higher temperatures above 20 °C.

Debate centers on the basic issue of environmental conditions in food that affect on stability and preservative activity of reuterin have to be investigated because there is no study has been done on this point.

My work will be closer to Lüthi-Peng et al. (2002) because I will elucidate a number of parameters, such as pH, salt, concentration, and temperature that influence on the stability and preservative activity of reuterin. Due to the applicability of reuterin in foodstuffs, an understanding of the factors that influence the stability and preservative activity will be determined.

Hopefully my contribution will provide a systematic investigation of the antimicrobial effect of reuterin under different conditions of concentration, temperature, salt and pH relevant to food products, and describe the interactions between these environmental factors and reuterin.

Reference list

El-Ziney, M.G., van den Tempel, T., Debevere, J.M., Jakobsen, M. (1999). Application of reuterin produced by Lactobacillus reuteri 12002 for meat decontamination and preservation. Journal of Food Protection, 62, 257-261.

Lüthi-Peng, Q., Schärer, S. and Puhan, Z. (2002). Production and stability of 3-hydroxypropionaldehyde in Lactobacillus reuteri. Applied Microbiology and Biotechnology, 60, 73-80

Assignment 2 : Introduction

Helicobacter pylori eradication by lactic acid bacteria

Stage1

Helicobacter pylori is a curve rod-shaped, Gram-negative, microaerophilic bacterium usually presented in the human stomach. It is a major cause of chronic gastritis and peptic ulcer disease. H. pylori is believed to associate with an increased risk of gastric carcinoma (Heavey and Rowland, 2004). It is classified to Class I (definite) biological carcinogen in humans by the International Agency for Research into Cancer (IARC) and the World Health Organization (WHO) (Park et al., 2004). The prevalence of H. pylori infection is high as 80% in developing countries and up to 40% in developed countries (Cover and Blaser, 1995; Dubois, 1995). At present, a combination therapies consisting of acid suppression drugs (proton pump inhibitors) and antibiotics (amoxicillin, clarithromycin, or nitroimidazoles) are used as a treatment for H. pylori eradication (Hamilton-Miller, 2003). However, antibiotic-based therapy would not be cost effective, cause side effect and would lead to widespread of antibiotic resistance (Michetti, 2001). For these reasons, this regimen is not recommended and the alternative strategies are developed to decrease H. pylori colonization.

Stage 2

Many studies reported that some strains of lactic acid bacteria showed antagonistic effect against H. pylori. Tsai et al. (2004) found that Enterococcus faecium TM39 isolated from infant faeces was able to inhibit the growth of H. pylori. Colonization of H. pylori could be reduced by ingestion of probiotic product containing Lactobacillus johnsonii La1 (Cruchet et al., 2003). In addition, chitosan microsphere containing the precipitant produced by L. casei ATCC 393 displayed growth inhibition and anti-adhesive activity on H. pylori (Ko et al., 2011).

Stage 3

However, the potential uses of probiotics especially, lactic acid bacteria to inhibit H. pylori infections, characterization of antimicrobial metabolites produced and their responsible mechanisms, are less documented.

Stage 4 & 5

Therefore, the objectives of this study was to select the most effective lactic acid bacteria for H. pylori eradication as well as to characterize the predominant antimicrobial metabolites secreted by selected strain. The final purpose was to evaluate the potential use of selected strain for H. pylori eradication in food products. Lactic acid bacteria may represent an approach to establish a harmless relationship with H. pylori. Approaches based on simple dietary changes are likely to meet public acceptance and may prove safer and cheaper than complex drug regimens.

I already comment on friend's blog: 1) http://chawanphatnn.blogspot.com/2015/02/assignment-2-writing-introduction_4.html#comment-form and 2) http://woratouch.blogspot.com/2015/02/assignment-2-writing-introduction.html#comment-form

Assignment 1 : Citation

In situ reuterin production by Lactobacillus reuteri in dairy products

Abstract

In situ reuterin production during the manufacture and storage of two dairy model systems elaborated from milk supplemented with an optimized concentration of glycerol (50 mM) and inoculated with a commercial starter and with different reuterin-producing Lactobacillus reuteri strains was investigated. L. reuteri was able to survive and to produce reuterin in cheese and yogurt models. The highest reuterin production was achieved by L. reuteri INIA P572 and INIA P579, which displayed reuterin concentrations up to 5.5 mM in cheese and up to 1.5 mM in yogurt. The addition of reuterin-producing L. reuteri and glycerol to milk reduced the viable counts of the cheese starter from day 10 onwards, while did not influence the counts of the yogurt starter, compared to control dairy models without L. reuteri. Strains L. reuteri INIA P572 and INIA P579 could be promising candidates in the development of bioprotective cultures to control pathogenic microorganisms in dairy products due to the potentially inhibitory concentrations of reuterin achieved in situ.

Reference

Langa, S., Landete, J. M., Martín-Cabrejas, I., Rodríguez, E., Arqués J. L., and Medina, M. (2013). In situ reuterin production by Lactobcillus reuteri in dairy products. Food Control 33, 200-206.

Results/Findings

Since the ability to produce the highest content of reuterin, selected Lactobacillus reuteri isolates (INIA P572 and INIA P579) could be used as commercial starter cultures in dairy products.

APA Citation format

Langa et al. (2013) found that Lactobacillus reuteri INIA P572 and INIA P579 displayed the maximum production of reuterin, as a consequence, these strains might be applied as starter cultures in dairy products

Strains of Lactobacillus reuteri INIA P572 and INIA P579 have the potential to use as starter cultures in dairy products due to their highest production of reuterin (Langa et al. 2013).