Efficacy of Purified Bacteriocin of “Brevibacillus laterosporus TK3” against Listeria monocytogenes and Staphylococcus aureus in Chicken

The microbiological spoilage of raw chicken is due to the biochemical activity of microorganisms causing changes in its appearance, odour, texture or taste. Several bacterial pathogens including Salmonella, Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Clostridium botulinum are found associated with many food borne illnesses which are serious public health concern worldwide. So to maintain the quality and safety of foods various measures are generally adopted in food industry i.e. good manufacturing practices, good hygienic practices etc. but preservation of food by a suitable means is the key of food quality and safety. There are number of preservation techniques started from low temperature preservation like refrigeration, freezing etc. and thermal preservation techniques like pasteurization, sterilization and preservation using certain chemicals (Singh, 2018).
       
Generally, food industry depends on chemicals for the preservation of foodstuff and to increase the shelf life of food. Chemical preservatives and other conventional preservation strategies fail to deliver the requisite health benefits and cause serious disorder thus necessitates seeking alternatives (Sarika et al., 2019). Hence, according to an increased negative perception towards chemical preservatives and a trend towards natural food additives so called “clean- labeling” has driven exploring of effective natural antimicrobial compounds as an alternative to synthetic food additives (Castillano et al., 2008). The use of bacteriocins is a promising ongoing development in food preservation as bacteriocins have strong antagonism against most of the food borne pathogens. In the food industry, bacteriocins have been widely utilized for the biopreservation of various foods, either alone, or in combination with other methods of preservation known as hurdle technology (Galvez et al., 2007; Barathiraja et al., 2015). Incorporation of bacteriocins into the food packaging film or surfaces has been explored as well (Zendo, 2013). Bacteriocins are ribosomally synthesized extracellularly released bioactive peptides or peptide complexes that vary in spectrum of  activity, mode of action, molecular weight, genetic organization and considered to be safe biopreservatives  since they can be digested by proteases thus having no or little influence on the gut microbiota (Padmaja et al., 2011) The lantibiotic nisin, which contains unusual amino acids such as lanthionine and β-methyllanthionine, is the most studied bacteriocin to date and is the only bacteriocin currently used as a food additive (Zheng et al., 1999), however, the use of nisin is limited due to its very low activity at neutral or alkaline pH. Therefore, the search for novel bacteriocins with improved biochemical properties, including stability over a wide pH range, thermostability and a broad antimicrobial spectrum, is of great interest for applications in foods.  As different types of  bacteria produce  different  kind of  bacteriocins, therefore, there is a pressing need to explore the nascent field of  natural food biopreservation by isolating different bacteria from new sources capable of producing novel bacteriocins and to characterize  them  to  be  added to food. Therefore, in the present investigation an attempt has been made to use purified bacteriocin of Brevibacillus laterosporous TK3 to preserve raw chicken.

The  experiments were conducted  at Department  of  Basic  Sciences (Microbiology  Section),  Dr Y. S. Parmar University  of  Horticulture  and  Forestry,  Nauni,  Solan,  H.P. India in  the year 2018-19. Bacteriocin producing Brevibacillus laterosporus TK3, isolated from “Tatwakhar”- a flour prepared from seeds of Indian Horse Chestnut (Aesculus indica). The bacteriocin was purified by single step gel exclusion chromatography. This thermostable bacteriocin with molecular weight 6 kDa, specific activity 34,482.0 AU/mg and with strong antagonistic potential against L. monocytogenes and S. aureus was applied as a biopreservative to enhance shelf life of chicken. Application of purified bacteriocin as a biopreservative was studied for fresh raw chicken. Two variants of raw chicken i.e. chicken cubes and minced chicken were used. Pathogenic bacteria viz. L. monocytogenes and S. aureus were used as test inoculums to study the comparative effect of purified bacteriocin and chemical preservatives i.e. sodium nitrite in the food samples. Controls without any preservative were run in parallel.
 
Preservation study in fresh raw chicken cubes
 
Fresh raw chicken (1 kg) was procured from local market. Chicken cubes of size 1 x1 x 1cm using sterile measuring scale were cut by sterilized surgical blade. These were surface sterilized by instant dipping in 60% ethanol and were further dried in aseptic conditions for two hours under laminar flow to evaporate excess residual ethanol. A treatment of 20 chicken cubes each was used for every treatment. These chicken cubes were given uniform dipping in purified bacteriocin (2000 AU/ml), test indicators viz. L. monocytogenes and S. aureus (10⁸CFU/ml) and sodium nitrite (200 mg/l) in a sterilized beaker for two min. In total 18 Treatments were prepared as given below:
 
Treatment A₁ - Control (chicken cubes without any treatment)
Treatment A₂ - Control (Minced chicken without any treatment)
Treatment B₁ - Chicken cubes + S. aureus
Treatment B₂- Minced chicken +S.aureus
Treatment C₁ - Chicken cubes + L. monocytogenes
Treatment C₂ - Minced chicken + L. monocytogenes
Treatment D₁ - Chicken cubes + purified bacteriocin + S. aureus
Treatment D₂ - Minced chicken + purified bacteriocin + S. aureus
Treatment E₁ - Chicken cubes + purified bacteriocin + L. monocytogenes
Treatment E₂ - Minced Chicken + purified bacteriocin + L. monocytogenes
Treatment F₁ - Chicken cubes + purified bacteriocin
Treatment F₂-   Minced Chicken+ purified bacteriocin
Treatment G₁ - Chicken cubes + chemical preservative (NaNO₂)
Treatment G₂- Minced Chicken + chemical preservative (NaNO₂)
Treatment H₁ - Chicken cubes + NaNO₂+ S. aureus
Treatment H₂ - Minced Chicken + NaNO₂+ S. aureus
Treatment I₁ -  Chicken cubes + NaNO₂+ L. monocytogenes
Treatment I₂- Minced chicken_+ NaNO₂+ L. monocytogenes
 
These samples were stored in sterilized zip-lock bags and were kept at 20°C+2 for 30 days for storage studies.
 
Preservation study of fresh raw minced chicken
 
Minced chicken was prepared by grinding 1.0 kg boneless chicken in mixer- grinder. It was  surface sterilized by instant dipping in 60% ethanol and was further dried in aseptic conditions for two hours under laminar flow to evaporate excess residual ethanol. 20 gm of minced chicken was used for every treatment. Uniform dipping was given in purified bacteriocin (2000 AU/ml), test indicators viz. L. monocytogenes and S. aureus (108CFU/ml) and sodium nitrite (200 mg/l) in a sterilized beaker for two min. Treatments were prepared as described previously.
 
Microbiological evaluation of fresh raw chicken cubes and minced chicken
 
Microbiological evaluation of the samples was done periodically on 0, 3, 5, 7, 10, 15, 21 and 30 days. CFU/ml for each sample was calculated on nutrient agar and the total count was evaluated in terms of log CFU/ml (Gautam and Sharma, 2014).

Thermostable bacteriocin of Brevibacillus laterosporus TK3-NCBI accession no KP861913.1 is used in preservation of  chicken. The consumption of poultry products has been steadily increasing worldwide, not only because of their relatively low cost but for the high nutritional value that it contains. Poultry meat support the growth of pathogens e.g. L. monocytogenes, S. aureus and Salmonella, are the most commonly reported pathogens implicated in food borne outbreaks (Jofre et al., 2008).  L. monocytogenes is known as the causative agent of listeriosis, a disease chiefly dangerous to certain risk groups, such as immune-compromised patients, pregnant women, new born, the elderly and (Shamloo et al., 2019). Staphylcoccal food poisoning is a gastrointestinal illness caused by food contaminated with toxins produced by S. aureus (Landgraf and Destro, 2013). Hence, chicken was used in the present study to compare the preservative effect of purified bacteriocin and chemical preservative against pathogens associated with chicken.
 
Comparison of preservative potential of bacteriocin with chemical preservative in raw chicken cubes against L. monocytogenes
 
The biopreservative effect of purified bacteriocin of Brevibacillus laterosporus TK3 against L. monocytogenes in chicken cubes is represented in Table 1. Surface sterilized chicken cubes were taken to determine the effect of biopreservation during storage at room temperature and compared with chemical preservative i.e. sodium nitrite. Bacteriocin and chemical preservative were applied in chicken cube within permissible limit. Results of preservation studies performed with chicken cubes were quite interesting when performed on 0, 3^rd, 5^th, 7^th, 10^th, 15^th, 21^st and 30^th days. The total bacterial count (TBC) in control (Treatment A₁) started developing 6.30 log CFU/ml on the third day which rose to log 7.41 CFU, on fifth day, log 8.01 on seventh day, 8.14 on tenth day, 8.30 on fifteenth day, 8.48 on 21^st day and became countless afterwards. Chicken cubes inoculated with purified bacteriocin (Treatment F₁) and chicken cubes with chemical preservative (Treatment G₁) were found able to prevent the growth of bacteria up to 5^th day and meager growth was observed from 7^th day onwards. On 21^st day and  30^th day slight increase in the bacterial  growth was  observed (Table 1). In case of chicken cubes inoculated with L. monocytogenes (Treatment C₁), growth started developing from 3^rd day onwards with rapid rise in growth of L. monocytogenes on 5^th, 7^th and 10^th day. On 15^th day and afterwards growth became countless. In chicken cubes with purified bacteriocin plus L. monocytogenes (Treatment E₁) and chicken cubes inoculated with chemical preservative plus L. monocytogenes (Treatment I₁), purified bacteriocin as well as chemical preservative were able to contain the growth of L. monocytogenes notably up to 7^th day of storage. Growth of L. monocytogenes started developing from 10^th day onwards with log CFU 7.99 in chicken cubes with bacteriocin and log 7.76 in chicken cubes with chemical preservative with considerable rise in counts on day 15^th, 21^st and on 30^th day respectively. From the results of this experiment it was observed that the purified bacteriocin of Brevibacillus laterosporus TK3 was effective in controlling the total bacterial count in chicken cubes up to 7th day of storage with very slow increase in bacterial count up to 30^th day of storage almost at par with chemical preservative as compared to control in which bacterial count appeared on 3^rd day and reached to infinity from fifteenth day onwards. Similarly, the purified bacteriocin was successful in controlling the growth of food borne pathogen  L. monocytogenes up to 7^th day as compared to control inoculated with L. monocytogenes which is almost at par with the results achieved with chemical preservative. An analogous study by (Sarika et al., 2012) has been demonstrated in literature. They reported that bacteriocin PSY2 could provide a promising alternative to harmful chemical preservatives in stored fish as it was effective against L. monocytogenes when compared with chemical preservative.
 

 
Comparison of preservative potential of bacteriocin with chemical preservative in raw chicken cubes against S. aureus
 
Fig1 represents the comparative biopreservative effect of purified bacteriocin of Brevibacillus laterosporus TK3 against S. aureus in chicken cubes during 30 days of storage at room temperature. Control (Treatment A₁) started developing total bacterial count i.e. 6.30 log CFU/ml on the third day which rose to log 7.41 CFU on fifth day, log 8.01 on seventh day, 8.14 on tenth day, 8.30 on fifteenth day, 8.48 on 21^st day and became countless afterwards. Chicken cubes inoculated with purified bacteriocin (Treatment F₁) and with chemical preservative (Treatment G₁) were found able to prevent the growth of bacteria up to 5^th day and meager growth of total bacteria was observed from 7^th day onwards i.e. log 6.48 and 6.30, log 7.20 and log 7.08 on day 10^th, log 7.46 and log 7.36 on fifteenth day, log 7.66 and log 7.59 on 21^st day and again with slight increase on 30^th day with log 7.71and log 7.67 respectively. In case of chicken cubes inoculated with S aureus (Treatment B₁), heavy growth of S. aureus developed from 3^rd day onwards with log 8.40 CFU/ml with rapid rise reaching to log 8.48 on day five and uncountable afterwards. Chicken cubes inoculated with purified bacteriocin plus S. aureus (Treatment D₁) and chicken cubes with chemical preservative plus inoculated with S. aureus (Treatment H₁₎, bacteriocin as well as chemical preservative were able to contain the growth of S. aureus up to 5^th day of storage. Growth of S. aureus started developing from 7^th day onwards with log 7.88 in chicken cubes with bacteriocin and log 7.80 in chicken cubes with chemical preservative with considerable rise in counts on 10th day i.e. log 8.11 and log 8.10 log 8.26 and log 8.22 on 15^th day, log 8.42 and log 8.40 and log 8.48 and log 8.46 on 30^th day respectively. From the results of this experiment it was observed that the purified bacteriocin was successful in prevent the growth of food spoilage S. aureus up to 7^th day almost at par with results achieved with chemical preservative as compared to control inoculated with S. aureus in which heavy growth of S. aureus appeared on 3^rd day only and reached to uncountable preposition from fifth day onwards.
 

 
Comparison of preservative potential of bacteriocin with chemical preservative in minced chicken L. monocytogenes
 
The biopreservative effect of purified bacteriocin of Brevibacillus laterosporus TK3 against L. monocytogenes in minced chicken is shown in the Table 2. Biopreservation was compared with commercial chemical preservatives i.e. sodium nitrite to evaluate its effect in minced chicken. Preservatives, bacteriocin and chemical preservative were applied in minced chicken within permissible limits. Results of preservation studies performed with minced chicken were quite encouraging when performed on 0, 3^rd, 5^th, 7^th, 10^th, 15^th, 21^st and 30^th days. The total bacterial count (TBC) in control (Treatment A₂) were recorded 7.95 log CFU/ml on the third day which rose to log 8.20 CFU on fifth day, log 8.37 on seventh day, 8.43 on tenth day, 8.48 on fifteenth day and became countless afterwards. Minced chicken inoculated with purified bacteriocin (Treatment F2) and minced chicken with chemical preservative (Treatment G₂) were found able to prevent the growth of bacteria up to 3^th day and meager growth was observed from 5^th day to  21^st day and again with slight increase on 30^th was  observed.
 

 
In case of minced chicken inoculated with L. monocytogenes (Treatment C₂), growth started developing from 3^rd day onwards with rapid rise in growth of L. monocytogenes i.e. on day five, 7^th day, 10^th day and uncountable afterwards as shown in  Table  2.  In minced chicken with purified bacteriocin and inoculated with L. monocytogenes (Treatment E₂) and minced chicken with chemical preservative plus inoculated with L. monocytogenes (Treatment I₂), purified bacteriocin as well as chemical preservative were able to contain the growth of  L. monocytogenes notably up to 5^th day of storage. Growth of L. monocytogenes started developing from 7^th day onwards with log 7.89 in minced chicken with bacteriocin and log 7.84 in minced chicken with chemical preservative with considerable rise in counts on day ten i.e. log 8.00 and log 7.88 log 8.14 and log 8.09 on 15^th day, log 8.32 and log 8.28 on 21^st day, log 8.47 and log 8.40 on 30^th day respectively.
       
From the results of this experiment it was observed that the purified bacteriocin of Brevibacillus laterosporus TK3 was effective in controlling the total bacterial count in minced chicken up to 3^rd day of storage with slow increase in bacterial count up to 30^th day of storage, which was even better than the chemical preservative when compared to control in which bacterial count appeared on 3^rd day and reached to infinity from fifteenth day onwards. Similarly, the purified bacteriocin was successful in containing the growth  of L. monocytogenes up to 7^th day as compared to control inoculated with L. monocytogenes which is even better than with the results achieved with chemical preservative. These findings are in agreement with the few results currently available in the literature, including the work of (Chakchouk-Mtibaa et al., 2017), where the effect of the semi purified bacteriocin BacFL31 was investigated on the shelf life of refrigerated raw ground turkey meat. The findings indicated that BacFL31 treatments were effective against Listeria monocytogenes and Salmonella Typhimurium.
 
Comparison of preservative potential of bacteriocin with chemical preservative in minced chicken against S. aureus
 
Fig 2 represents the comparative biopreservative effect of purified bacteriocin of  Brevibacillus laterosporus TK3 against S aureus in minced chicken during 30 days of storage at  room  temperature. On the initial 0 day log CFU/ml for minced chicken were not detected. The total bacterial count (TBC) in control (Treatment A_­2) were recorded 7.95 log CFU/ml on the third day which rose to log 8.20 CFU on fifth day, log 8.37 on seventh day, 8.43 on tenth day, 8.48 on fifteenth day and became countless afterwards. In case of minced chicken inoculated with S aureus (Treatment B₂), heavy growth of S. aureus developed and reached to uncountable level on 3^rd day only with log 8.48 CFU/ml. In minced chicken with purified bacteriocin inoculated with S. aureus (treatment D₂) purified bacteriocin was able to contain the growth of S. aureus upto 5th day whereas in case minced chicken with chemical preservative and inoculated with S. aureus (Treatment H₂), growth of log 6.85 of S. aureus appeared on 5^th day itself. On 7^th day log 7.98 of S. aureus was observed in minced chicken with bacteriocin and log 7.99 was noted in minced chicken with chemical preservative which rose to log 8.12 and log 8.14 on 10^th day, log 8.21and log 8.25 on 15^th day, log 8.45 and log 8.46 on 30^th day, respectively.
 

       
From the results of this experiment it was observed that the purified bacteriocin was successful in containing the growth of S. aureus up to 5^th day whereas chemical preservative was able to contain the growth of S. aureus up to 3^rd day only as compared to control inoculated with S. aureus. The results found in these experiments for application of bacteriocin as biopreservative in chicken as an alternative to chemical preservative are quite encouraging and satisfactory. However, these findings could be refined and used meticulously after combining the use of bacteriocin along with other novel approaches like, anti-microbial film packaging, hurdle technologies etc. But, definitely this study has indicated the strong possibility of success of using bacteriocin at commercial level for biopreservation of chicken after the trials at larger scale. There are similar reports in the literature regarding bio preservative potential of becteriocins for enhancing the shelf life of meat products. According to one such report the application of bacteriocin plantaricin IIA-1A5 of Lactobacillus plantarum IIA-1A5 was compared with 0.3% nitrite to extended the shelf life of beef meatball. Interestingly, 0.3% plantaricin IIA-1A5 displayed the ability to inhibit a population of S. aureus as strong as 0.3% nitrite during the storage period demonstrating promising potential use of plantaricin as a nitrite replacer. (Sarika et al., 2015).

In the present investigation efforts were made to use purified bacteriocin of Brevibacillus laterosporous TK3 as biopreservative to enhance the shelf life of chicken products. Purified bacteriocin was found almost as effective as chemical preservative in comparative studies. Encouraging results of the study suggests that the bacteriocin of Brevibacillus laterosporus TK3 has a strong potential as an alternative to replace synthetic food additives- sodium nitrite for safer preservation of food products in food industry.