Some Physiological and Biological Studies on Reuterin Production from Lactobacillus reuteri

It is clearly shown that human gut bacteria consists of approximately 104 colony forming unit (cfu)/ml and about 500 to 1000 different species and live in their host in symbiosis. This bacterial population is controlled by diets [1]. From few years ago, scientists discovered beneficial bacteria in yogurt, pills and other food and called it probiotics. The Latin and a Greek word “probiotic” means “favorable to life”. According to the Food and Agricultural Organization (FAO) [2]; “they are any living microorganisms administrated in adequate amounts which have a beneficial health effect on their host. According to Fuller [3], the probiotics can be defined as “A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance.” Recently, The word ‘‘probiotic’’ means a large numbers of microorganisms, mainly bacteria (as Lactic acid bacteria (LAB)and non lactic acid bacteria) and few yeasts whom can stay a live until reach to the colon and then improve the host health by its beneficial actions on its host. For example, LAB (e.g., Lactobacillus reuteri, Lactococcus acidophilus, Enterococcus faecium, etc.) and non-lactic acid bacteria (e.g., Escherichia coli) also some yeasts (e.g., Saccharomyces cerevisiae,) are also considered as probiotics.


Introduction
It is clearly shown that human gut bacteria consists of approximately 10 4 colony forming unit (cfu)/ml and about 500 to 1000 different species and live in their host in symbiosis. This bacterial population is controlled by diets [1]. From few years ago, scientists discovered beneficial bacteria in yogurt, pills and other food and called it probiotics. The Latin and a Greek word "probiotic" means "favorable to life". According to the Food and Agricultural Organization (FAO) [2]; "they are any living microorganisms administrated in adequate amounts which have a beneficial health effect on their host. According to Fuller [3], the probiotics can be defined as "A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance." Recently, The word ''probiotic'' means a large numbers of microorganisms, mainly bacteria (as Lactic acid bacteria (LAB)and non lactic acid bacteria) and few yeasts whom can stay a live until reach to the colon and then improve the host health by its beneficial actions on its host. For example, LAB (e.g., Lactobacillus reuteri, Lactococcus acidophilus, Enterococcus faecium, etc.) and non-lactic acid bacteria (e.g., Escherichia coli) also some yeasts (e.g., Saccharomyces cerevisiae,) are also considered as probiotics.
These LAB are Gram-positive rods and usually live an-aerobically but in few cases, they can grow also on aerobic conditions [4,5]. Lindgren and Dobrogosz [6] stated that LAB can preserve dairy foods by producing some organic compounds that are antagonistic to other microorganisms. Among these compounds, the bacteriocins which are short chain proteins and have strong ability to inhibit the pathogens growth [7,8]. Many researches had focused on using bacteriocins as food preservatives and control of human bacterial pathogens and disease appearance. The probiotic activity of these bacteria is studied and recorded by Gilliand [9][10][11][12]. Bacteriocins production can be influenced by many factors, including medium composition [13] and environmental factors [14,15]. De Man Rogosa and Sharpe (MRS) medium is the standard, specific culture medium for (LAB), but it is not suitable for industrial-scale production due to its high cost. MRS had used for fermentation, while the purpose was to produce bacteriocins, lactic acid, some enzymes, or other metabolites [16,17]. The lowering of the culture media cost has been published [18][19][20], not referring to industrial fermentation. The aim of this study was to increase the production and efficiency of the studied reuterin and reduce the fermentation cost to increase the industrial production rate.

Isolation and purification of bacteriocin
Variations in the level of reuterin production were evaluated by the well diffusion assay [24] and using cork pore diameter 5 mm. Experiments were carried out in duplicate. 0.1% (v/v) of L. reuteri was inoculated at a final concentration of about 10 8 CFU/ml according to Emanuel Goldman, Lorrence H Green [25]. The antimicrobial activity of the supernatants was evaluated by the critical dilution assay of Barefoot and Klaenhammer [26] with using of Amikacin (32 µg) as antibacterial antibiotic and Fluconazole (150 µg) as antifungal antibiotic. Reuterin activity was defined as the reciprocal of the highest dilution showing definite inhibition of the indicator strains and was expressed as activity units per milliliter (AU/ml). Optimization studies: It include incubation periods, inoculum size, incubation temperature ranged from 25-45°C and initial pH ranged from 5.0 to 8.5. Effect of various carbon sources, nitrogen sources and medium salt elimination were evaluated in relation to reuterin production.

Data analysis:
The recorded data and results were summarized and analyzed in the tables according to Coulombier et al. [28]. Table 1 clearly shown that the reuterin production by L. reuteri being affected by incubation time, it reached a maximum at 24 and 48 hr. which was 19.0 mm inhibition zone for E. coli and 22.0 mm for both Staph. aureus or Candida albicans comparing to positive control (Amikacin 32 µg/ml as antibacterial agent and Fluconazole 150 µg/ml as antifungal agent) in addition to sterile distilled water as negative control. So 24 hr. is the best incubation time for reuterin production, there for we select 24 hr. incubation period for reuterin production.

Effect of using different initial pH values
According to the nature of the studied organism which is acidophilic, the reuterin production by L. reuteri was highly affected by the initial pH values of the media. Eight pH values were selected to study their effects on reuterin production ranged from 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5. All environmental and nutritional factors were fixed and incubated for 24 hr. Table 2 clearly illustrate that the pH 6.5 and 7.0 were the best pH values for maximum reuterin production than that of others under the same experimental conditions. The maximum production of reuterin is after 24 hr. and pH 6.

Effect of using different incubation temperatures
At this experiment, the inoculated test tubes will incubated at 25, 30, 37 and 45°C for 24 hr. and pH 6.5 with fixation of other environmental and nutritional factors and the cell biomass, final pH and reutrin productivity were recorded and listed in Table 3.
The recorded data show that the reuterin productivity was clearly affected by the temperature of incubation. The reuterin productivity was maximized at 30 -37°C while at elevated temperature (45°C) or low temperature (25°C) the activity decreased. At 25°C the activity decreased to 19 mm inhibition zone diameter while at high temperature (45°C) there was no activity.

Effect of using different inoculum sizes
The Table 4 clearly illustrate the effect of using different inoculum sizes (3,6,9,12,15,18,21,24 or 27 × 10 8 CFU/ml) on reuterin production with the fixation of all other environmental or nutritional factors according to the best final condition. According to the Table 4, the best inoculum size gives the highest reuterin production was 21 × 10 8 .

Effect of using different carbon sources
Carbon source is the most important nutritional factor for both growth and reuterin production of L. reuteri strain. This study discusses the effect of using of different carbon sources instead of 2% glucose of the reuterin production medium. Carbon sources include; fructose, lactose, sucrose or cane sugar molasses in a concentrations raged from 1-4%. Table 5 show that the best carbon sources were glucose (2%) and lactose (2%) followed by sucrose while fructose gave weak results on other hand, cane sugar molasses was not suitable for reuterin production (negative results).

Effect of using different nitrogen sources
six nitrogenous compounds were used instead of nitrogenous component of the reuterin production medium, at fixed optimum conditions (i.e., at pH 6.5, temperature 37°C, inoculum size 21 × 10 8 with 2% glucose). These nitrogen sources were; peptone, yeast extract, beef extract, casein hydrolysate, SBE and CSL (according to the equivalent N-basis). Table 6 showed that the most favourable nitrogenous compound was yeast extract which gave the highest activity expressed by inhibition zone diameter (The maximum production of reuterin is 22.2 mm inhibition zone for E. coli and 22.5 mm for Staph. aureus and 22.8 mm for Candida albicans comparing to positive controls) followed by corn steep liqour which gave almost the near same reuterin productivity. Results of Table 6 revealed that there is inversely proportionality between reuterin productivity and the cell biomass i.e., the reuterin production will decrease when the cell biomass increased (and vice versa).

Effect of salt elimination
The results illustrated in Table 7 showed that elimination of one salt resulted in decrease of reuterin productivity, so the reuterin productivity resulted from original MRS medium is highly affected by the culture medium component and elimination of one salt will decrease the reuterin productivity. So, all the medium salts are important for reuterin production with concentrations as in MRS medium.

The minimum inhibitory concentration (MIC) test
The results of Table 8 show that, the best volume for culture filtrate (free of cells) containing reuterin is 0.05 ml, below which negative effect of reuterin against studied bacterial and yeast strains was recorded and then it can be considered as the MIC. On the other hand, the volume 0.15 ml as well as 0.2 ml and 0.25 ml of culture filtrate were the best concentrations gives the highest reuterin production and antimicrobial activity against all studied pathogenic strains.

Discussion
This study focused on the effect of two growing factors; environmental and nutritional factors. The effect of environmental factors on reuterin production, including incubation periods, pH values, incubation temperatures and inoculum size were studied by many researchers [29]. On other hand, the nutritional factors (medium composition) play an important role in bacteriocin production [30].   Furthermore, reuterin production is strongly affected by medium composition [31]. Our study showed that, the pH values ranged from 6.5 to 7.0 were the optimum pH values gave the highest reuterin productivity with moderate cell biomass of the studied L. reuteri. Above or bellow this pH value will lead to decrease in reuterin productivity. These results agree with Leroy and De Vuyst [32], they proved that the bacteriocin activity of B. bifidum was lower at pH ranging from 7.5 to 8.0 and from 5.5 to 6.5.
Generally, reuterin production by L. reuteri was considered as a temperature sensitive process. Furthermore, the optimal growth temperature definitely not as like as the optimal bacteriocin production temperature [33] and it consistent with our results which exhibited a temperatures ranged from (30-37°C) were optimum for reuterin production while temperature 37°C only is optimum for cell growth. A higher production of bacteriocin in the range of temperature for optimal strain development has been observed in many LAB strains, such as E. faecium RZS C5 [32], Lactobacillus sakei Lb 706 [24]. However, no significant differences were reported for bacteriocin production at 30 and 37°C, which may prove that growth temperature in that range does not play a defining role in bacteriocin activity and it coincide with Todorov and Dicks [34] with regards to L. plantarum bacteriocins.
The studying of using different nutritional factors on reuterin production aimed to attain higher reuterin yields with very low cost media as food industry wastes (e.g., sugar cane molasses and CSL) or low cost proteins as SBE. With regards to studied carbohydrates, low reuterin production was produced when using sucrose and fructose while sugar cane molasses does not initiate reuterin production for the studied strain, it may be due to that the studied organism did not have the enzymatic system responsible for the digestion of sugar cane molasses. Since fructose and molasses reduced cell biomass and reuterin productivity, we deduced that the studied probiotic could not utilize these saccharides as a sole carbon source, on the other hand, the sole carbon sources which were readily to be utilized and gave high reuterin productivity were glucose, lactose followed by sucrose. Bing et al. observed that the glucose concentration over 2.0% could reduce the reuterin yield [35]. On the other hand, the nitrogenous compound SBE reduces markedly the reuterin production but the cell growth was too high. It explain that the studied probiotic used the SBE to produce cells only not for reuterin production while CSL gave high reutern production (more than the control medium) with moderately high cell growth, this agree with Helal et al. [36], although several researchers observed that higher bacteriocin production were recorded when increasing the concentrations of nitrogen content of the medium [37,38]. Salt elimination test explain clearly that all medium salts needed for high reuterin production in a different degrees and the less effective one was MnSO 4 4H 2 O which gave slight effect.
Finally, our study tested the effect of reuterin concentrations in the culture filtrate on the studied pathogens, it was found that the MIC for activity is 0.05 ml of the reuterin below which there is no antimicrobial activity and concentration 0.15 ml of the culture filtrate is the optimal concentration gave the best antimicrobial activity, this results agree with Helal et al. [36].

Conclusion
This study revealed that the reuterin productivity and cell biomass of the studied Lactobacillus reuteri is highly sensitive to environmental and nutritional factors which affecting on the studied probiotic. The relation between the cell biomass and reuterin productivity is not in linear relationship i.e., reuterin productivity is optimized with    -ve: negative (no reuterin productivity) Table 8: The effect of using different culture filtrate concentrations on reuterin production as inhibition zone diameter (mm).