Antimicrobial Substances Production at Refrigeration Temperatures by Lactobacillus delbrueckii MH10: A Candidate for Food Biopreservation

The objective of this study was to determine formation of antibacterial substances in supernatants of L. delbrueckii during cold storage and evaluate whether the application of this bacteria to raw ground beef would result in significant reductions of E. coli O157:H7 during refrigerated storage. Antibacterial activity of a newly isolated Lactobacillus delbrueckii MH 10 at refrigeration temperatures against food-borne pathogen Escherichia coli O157: H7 was studied. The size of inhibition zone depends on concentration of LAB cells. The cells (~10 9 CFU/ml) of L. delbrueckii produced significant amount of antibacterial substances mainly hydrogen peroxide ~ 35 μg/ml in sodium phosphate buffer (0.2 M, pH 6.5) and ~ 40 μg/ml in beef broth at 5°C during submerged cultivation without of growth. Submerged cocultivation of E. coli O157: H7 with lactobacilli in NB broth at 5°C reducing the total number of the pathogen more than 3 log for 5 days. The cell suspension intendent for treatment must contain 10 8-9 CFU/ ml of LAB. L. delbrueckii reduced initial amount 2x10 5 of E. coli O157: H7 in ground beef cocultivation up to 3 log for 3 days and become undetectable after 7 days. The application of L. delbrueckii bacteria does not cause any changes in sensory characteristics of ground beef by itself; moreover promote expanding of shelflife due to inhibition of psychrophilic spoilage microorganisms.


Introduction
For ground meat shelf life prolongation, synthetic chemicals have been traditionally used to inhibit resident pathogenic and spoilage microorganisms in refrigerated products. The increasing consumer concerns of potential health risks associated with some of synthetic preservatives has led researchers to evaluate the opportunity of using natural bio-preservatives such as Lactic Acid Bacteria (LAB) selected for their inhibitory activity towards undesirable microorganism [1,2]. For these applications lactic acid bacteria are usually chosen as they have the GRAS (Generally Recognized As Safe) status and produce a wide range of inhibitory compounds such as organic acids, hydrogen peroxide, diacetyl and bacteriocins and thus, expanding shelf life and increasing food safety [3][4][5][6][7]. The inhibitory actions of LAB toward food-borne pathogens and spoilage organisms in non-processed foods occur during entire storage period by continuous production of inhibitory compounds instead of a one-time reduction, as occurs with antimicrobial substances interventions. It has been shown that for bio-preservation the most effective are LABs able to produce hydrogen peroxide at refrigerated temperatures in absence of growth [8][9][10].
The species Lactobacillus delbrueckii subsp. lactis more of all used in food preservation, but hydrogen peroxide production is variable amongst strains [11,12]. The use of limited number of LAB strains may cause decreasing of treatment efficacy due to accumulation of deleterious mutations and/or adapting of pathogens to antibacterial substances which are produced [2]. In order to enhance biopreservation efficacy new LABs producing hydrogen peroxide should be selected and methods developed for their cultivation and application to food [13][14][15]. Because hydrogen peroxide production plays the mayor role in elimination of the pathogens at refrigerator storage, it levels should be assessed for newly selected strains.
Most raw foods are contaminated with pathogenic and spoilage microorganisms. Ground beef products are common sources of E. coli O157:H7 and its reduction is an important concern in the beef industry. Although many intervention technologies are applied to beef carcasses, ground beef processors currently do not have effective intervention steps for ground beef safe storage. There are only a few studies of LAB inhibition of E. coli O157:H7 in ground beef products and their impact on the sensory properties of these products [6,16,17].
The aim of this research was to determine formation of antibacterial substances supernatants of L. delbrueckii during cold storage and evaluate whether the application of this bacteria to raw ground beef would result in significant reductions of E. coli O157:H7 during refrigerated storage.

The Bacterial Cultures
The Lactobacillus delbrueckii MH 10 from human origin was isolated and identified in our laboratory by Hovhannisyan and Pashayan (2010) and deposited in Armenian National Microbial Depository Center (MDC) under code MDC 9617. Food born pathogen Escherichia coli O157: H7 MDC 5003 used in this study was from the MDC.

Bacteriological Analysis
Bacterial counts in liquid media were made using standard methods. For enumeration of E. coli and lactobacilli in ground beef 1 g treated meat sample was inoculated in 9 ml of sterile physiological, homogenized, made serial ten-fold dilutions and plated on Tryptose and MRS agars for determination of E. coli and LAB counts, respectively.

Potentiometric and Titratable Acidity
The pH was measured at room temperature, using a digital pH meter. Titratable acidity expressed as a percentage of lactic acid was measured by titrating 9 mL of the sample (where added 3 spots of phenolphthalein) with 0.1 N NaOH, until a pink color appeared.

Agar Disk Diffusion Method
Agar disk diffusion method was used to evaluate the antimicrobial effect of LAB suspensions. E. coli O157: H7 culture grown in NB broth for 18 h at 37°C diluted to concentration of 10 7 cells/ml and spread onto Tryptose agar. The paper discs (diameter, 5 mm) were soaked with LAB culture liquids and placed on the test culture lawn. After 2h exposition in cold the plates were incubated at 37°C for 18 h and examined for size of clear inhibitory zones.

Quantification of Antimicrobial Activity of LAB in Cold Cultivation
Lactobacilli were grown in LAPTg broth for 18 h at 37°C divided in four 10 ml aliquots, centrifuged at 12,000 × g for 10 min and each pellet resuspended in 10 ml of cold medium; sodium phosphate buffer (with or without glucose), LAPTg broth or physiological saline and incubated at 5°C. Every two days for 7 days and then each week antimicrobial activity, hydrogen peroxide amount, OD600 and pH of the cell cultures were determined.

Submerged Cocultivation of LAB Along E. Coli in Nutrient Broth (NB)
For evaluating the antagonistic activity of L. delbrueckii MH 10 against of E. coli O157:H7 overnight culture was diluted in 200 ml of fresh NB to obtain cell concentration of approximately 10 5 CFU/ml. Divided in two equal portions and supplied L. delbrueckii MH 10 in ratios 1: 100 and 1: 10. Both samples stored at 5°C and subjected to microbial analysis on days 0, 1, 3, 5, and 7.

LAB Antimicrobial Activity Determination in Ground Meat
200 g of freshly prepared commercial ground beef was obtaining from local grocery. 150 g of this ground beef was inoculated with E. coli O157 to obtain a pathogen concentration of approximately 10 5 CFU/g and divided into three equal portions. L. delbrueckii MH 10 was prepared as described previously and added individually in two of ground beef samples inoculated with E. coli O157:H7 at final concentrations of 10 7 and 10 8 CFU/g respectively. The control portions of the ground meat with and without E. coli O157:H7 were processed in the same manner. All samples were mixed, packaged in vacuum polyethylene packets, kept at 5°C and subjected for microbiological analysis on days 0, 1, 3, 5, 7 and 10.

Sensory Evaluation
Sensory evaluation of the control and the samples inoculated with the LAB strains was conducted in the open laboratory. Odor, color and appearance of slime on the external surface of ground meat were assessed.

Statistical Analysis
Statistical analysis was performed using SPSS program (Version 16). Standard deviation of mean was used to describe data. Fisher's range test was used to determine differences between tested groups. P value < 0.05 and 0.001 were considered as significant and highly significant, respectively.

Antibacterial Substances Production by Washed L. Delbrueckii Cells at 5°C
Laboratory experiments revealed that hydrogen peroxide producing ability of LAB at non permissive temperature is strongly dependent on nutrition media composition used for their prior propagation as well as media for sub cultivation at refrigeration temperatures. The largest amount of hydrogen peroxide at 5°C formed when LAB cells priory propagated in rich medium and then transferred in sodium phosphate buffer [1,18]. In this study along with phosphate buffer we used beef broth in order to ensure the lack of substances inhibitory to antimicrobials formation e. g. hydrogen peroxide.
Antimicrobial compounds production by 2 fold dilutions of washed cells L. delbrueckii MH 10 in beef broth and phosphate buffer were studied during storage at 5°C by disk diffusion method (Figures 1, 2).
The growth inhibition zones of E. coli O157:H7 around the disks, impregnated in the both media, gradually increased for 5 days period in cold storage.  Inhibitory compounds formation in beef broth by L. delbrueckii at refrigerating temperatures was shown for the first time. The inhibitory zones caused by cells inoculated in beef broth were remarkably larger than in phosphate buffer. The inhibitory zones size required high concentration of cells.

Hydrogen Peroxide Production by L. delbrueckii During Storage at 5°C
The dominant inhibitory factor produced by lactobacilli at refrigerating temperatures was identified as hydrogen peroxide [11,[19][20][21]. In this study production of hydrogen peroxide by L. delbrueckii at 5°C storage were obtained. The hydrogen peroxide accumulation by lactobacilli in both media phosphate buffer and beef broth gradually increased and riche maximum after five days of cold storage. Cell suspensions in beef broth showed higher accumulation of H 2 O 2 in comparison to phosphate buffer. The overall H 2 O 2 amount in phosphate buffer and beef broth were approximately 35 and 40µg/ml, respectively (Fig. 3, 4). Thus, the strain MH 10 by hydrogen peroxide production was not inferior to known strains L. delbrueckii [20,22].  It was revealed that the character of hydrogen peroxide accumulation was similar to antibacterial activity growth in LAB suspension (see figures 1, 2) which indicates on main role of hydrogen peroxide in inhibitory action lactobacilli against E. coli O157: H7 at refrigerator storage.

Cells Viability and Acidity Changes During Cold Storage
During the entire period of storage at 5°C live cells count and culture pH and titrable acidity were monitored (Table 1). No significant differences were found in the population levels of LAB cultures during entire storage period at 5°C indicating that LAB reproduction was not necessary for the inhibition of pathogens. These findings come in accordance with the observations of other authors who also suggested that the production of inhibitory metabolites can occur by LAB during storage in the absence of growth [1,18]. Any significant changes of pH and titrable acidity in all of media weren't detected which indicated that the organic acids are not formed.

Antagonistic Action of L. delbrueckii on E. coli O157:H7 in NB at 5°C
L. delbrueckii was added to NB along with E. coli O157:H7 in order to determine its antagonistic activity against the pathogen at 5°C. Two ratios of E. coli O157:H7 to L. delbrueckii 1: 100 and 1: 10 were tested. The total number of E. coli O157:H7 cells in both treatments were determined on days 0, 1, 3, 5 and 7 by plating appropriate dilutions on Tryptose agar and incubation at 37°C for 24 hours.

LAB Inhibitory Effect on E. coli O157:H7 in Ground Meet at 5°C
Cell suspension of L. delbrueckii was tested in packaged ground meat stored for their ability to reduce the viability of Escherichia coli O157:H7 during storage temperature at 5°C. Fresh ground meat was inoculated with 10 5 CFU/g of E. coli. The trial samples were treated with L. delbrueckii, at a level of 10 7 and 10 8 CFU/g and stored at 5°C for 7 days in plastic vacuum bags. Samples were analyzed for E. coli O157:H7 survivors and lactic acid bacteria on days 1 to 7. Towards the end of ground meat vacuum storage E. coli O157:H7 quantity, depending of L. delbrueckii ratio, was 2-3 log lower than those in the control. But the LAB count in treated and control samples after refrigeration storage didn't significantly change for 10 days. Growth of LAB in a fresh meat held at refrigeration temperature is not desirable because it would lead to premature spoilage of the product. Furthermore, the viable L. delbrueckii cells in ground beef, continuous protection by preventing secondary contamination during entire storage period.
It was revealed that the application of L. delbrueckii bacteria doesn't causes any changes in sensory characteristics of ground beef entire 10 days storage, moreover promote expanding of shelf-life due to inhibition of psychrophilic spoilage microorganisms which is in agreement with other authors [6,[24][25][26][27]. Thus, the LAB treated samples keep good quality for ten days whilst in the control sample at 7 day of storage appeared undesirable odor, greening and smooth on surface. Therefor the selected strain L. delbrueckii MH 10 is able to synthesize antimicrobial compounds in amounts sufficient to inhibit the growth of pathogens and spoilages.
The results of the experiments suggest that L. delbrueckii MH 10 has a potential to be used as a candidate culture in biopreservation method to improve the safety and extend the shelf life of meat products in cold storage.

Conclusion
The cells of L. delbrueckii MDC 9617 produced significant amount of antibacterial compounds mainly hydrogen peroxide in beef broth and sodium phosphate buffer at 5°C in absence of growth. This strain exerts inhibitory action against E. coli O157:H7 and dramatically (more than 95%) reduced number of pathogen bacteria in both submerged and solid-stat cocultivation trials at refrigeration temperatures. Thus, L. delbrueckii can be recommended as candidate bio-preservative for use in commercial applications.