Impact of Sodium Alginate Packaging Film Synthesized Using Syzygium cumini Seed Extract on Multi Drug Resistant Escherichia coli Isolated from Raw Buffalo Meat

Abstract

A total of 50 Escherichia coli were isolated from buffalo meat and their antibiotic profiling was carried out. 90% E. coli isolates showed resistant to two or more classes of 21 commonly used antibiotics. Moreover, there was also variation in resistance/sensitivity behavior towards antibiotics. Highest resistance was found to be against methicillin (84%) in the isolates followed by vancomicin (70%), sulphadiazine (68%) and cefaclor (66%), whereas, resistance was less common for kanamycin (8%) and chloramphenicol (4%). ECMB1, ECMA2, ECMA8, ECMS9 and ECMA31 strains showed highest MDR pattern with presence of ^blaCTX-M, qnr S and qnr B resistant genes. ECMB1 strain was resistant to 14 antibiotics belonging to 7 different classes. Therefore, ECMB1 was selected for further studies. Sodium Alginate Film incorporated with 10, 20, and 30% ethanolic extract of Syzygium cumini (EESC) were formulated and characterized using state-of-art techniques. A dose-dependent antibacterial activity against E. coli ECMB1 was recorded by the films made from EESC (EESCF). The growth kinetics of E. coli strain ECMB1 showed 9% decrease in log CFU when it was cultured in 30% EESCF as compared to control cells after 12 h of growth. Present finding highlight the efficacy and possible use of EESCF as meat packaging film to prevent food spoilage caused by MDR bacteria.

Introduction

Microbial contamination of meat and meat products is a matter of great concern as pathogens and drug resistant bacteria may be transferred to humans via food chain and cause severe food borne illnesses, since favorable pH, high moisture content and abundance of nutrients make meat an excellent medium for growth and survival of microorganisms (yeasts and molds) in general and bacteria in particular [1–3].

An estimated 420–960 million foodborne diseases and 0.3–0.6 million deaths representing 25–46 million Disability Adjusted Life Years (DALYs) in which major causative agents were Salmonella spp., Campylobacter spp. Taenia solium, Escherichia coli, hepatitis A virus, norovirus and aflatoxin [4].

Indiscriminate use of antibiotics for non-therapeutic reasons such as growth promotion [5] and disease prevention is a common practice in intensive industrialized farm settings in the country which lead to development of antibiotic resistance in bacteria. Global usage of antimicrobials in the production of food animals was estimated at 63,151 (± 1560) tons in 2010 and is projected to increase by 67%, to 105,596 (± 3605) tons by 2030 [6]. More over 70% of the antibiotics considered medically essential to human health by the Food and Drug Administration (FDA) sold in the United States (and more than 50% in most countries around the world) are used in animals [7]. According to FDA, 93% of medically significant antibiotics were administered in US agriculture through feed or water [8]. Scientific studies also showed that 75–90% of the antibiotics tested are excreted un-metabolized form from animals and penetrate sewage systems and water bodies [9]. Animal waste therefore contains not only resistant bacteria, but also antibiotics that can contribute to resistance in bacteria outside those in the gut of an animal-including bacteria that may present a greater risk to humans [10]. Several members of Enterobacteriaceae family namely Escherichia, Salmonella and Campylobacter etc. are known to be resistant to multiple drugs and are causative agents of some major food borne illnesses. These mutated and robust bacterial strains bypass the toxic effect of antibiotics, and thus make them ineffective [11]. According to CDDS report, antibiotic resistant bacteria cause more than 2 million diseases and nearly 23,000 deaths in the USA alone in a year [12]. In India, where burden of infectious diseases and rate of antimicrobial uses are higher in the world and each year more than 58,000 babies died as a result of drug resistant bacterial infections [13, 14].

The presence of microorganisms in meat and meat products cause various undesirable physicochemical, sensory and textural changes thereby rendering them unpalatable. The main challenge for the food industry is to remove these unexpected changes in foods arising from the activity of these microorganisms and to ensure maximum food safety. Recently, different food additives such as, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nisin, sodium nitrite and sulphur dioxide (SO₂; synthetic antimicrobial) etc. are used to suppress the growth of microorganisms in various food industries [15]. Many of these food additives have safety concerns, such as BHA and PHT are reported to possess carcinogenic behavior that sometimes may cause the DNA damage [16, 17]. Therefore, to find safer alternatives that have the potential to suppress the microbial growth without having any adverse impact on the human health, is of utmost importance. Various plant based natural compounds such as spices (cloves, cinnamon, thyme, garlic, onion and sage etc.) essential oils and plant extracted compounds have been reported for their antimicrobial [18] and antioxidant [19] properties. These compounds have great applicability in the emergent area of bioactive packaging systems, wherein the natural products can be used both for packaging and antimicrobial properties simultaneously.

These integrated natural compounds may enhance the durability of packaged materials by providing novel or additional functions to packaged films. The use of bioactive packaging systems having such antimicrobial compounds may be a useful tool to protect the foodstuffs against spoilage microorganisms by decreasing the risk of pathogens [20]. Syzygium cumini (L.), a common tropical Indian medicinal plant that belongs to Myrtaceae family, and seeds of S. cumini are popularly known for their anti-diabetic, anti-inflammatory [21], antibacterial [22, 23] anti-HIV [24] and antioxidant [25] properties. To the best of our knowledge no information is available on packaging films incorporated with extract of S. cumini seeds.

Present paper focused on the antibacterial properties of sodium alginate based packaging films incorporated with ethanolic extract of S. cumini seeds on MDR E. coli strains from buffalo meat. In addition to antibiotic sensitivity and resistance pattern of bacterial isolates; molecular detection of antimicrobial resistant gene (AMR) was also carried out using polymerase chain reaction (PCR). Structural and chemical characterization of EESCF using state of the art techniques HunterLab, scanning electron microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FT-IR) and determination of antibacterial properties of EESCF against selected MDR E. coli was also done.

Materials and Methods

Meat Samples Preparation

A total of 70 samples of raw buffalo minced meat were purchased from local retail shops of Aligarh City (UP), India. Raw buffalo minced meat (500 g) was collected in a sterile poly bag and transferred to the laboratory in an ice box container away from direct sunlight. 25 g of each sample were homogenized in 225 mL, buffered peptone water (0.1%) (Hi-media, Pvt., Ltd. Mumbai) and homogenate were used to make serial dilution.

Isolation, Screening and Identification of Bacteria

Levine Eosin Methylene Blue Agar (LEMB, Hi-media, Pvt., Ltd. Mumbai, Composition Pancreatic digest of gelatin 10, Dibasic potassium phosphate 2, Lactose 10, Eosin - Y 0.4, Methylene blue 0.065 and Agar 15 g/L, pH 7.1 ± 0.2) were used for selective isolation of E. coli. An aliquot of 0.1 mL of previously prepared dilution of homogenate were spread onto LEMB agar plate and incubated at 37 ºC for 24 h. The bacterial colony showing green metallic sheen were selected, purified and stored for further studies. Bacterial identity was confirmed morphologically, biochemically (IMVIC) and by MALDI-TOF-MS (Bruker Daltonics, Germany).

Antibiotic Susceptibility Testing

Antibiotic susceptibility test of bacterial isolates was carried out by Kirby-Bauer disk diffusion method according to interpretive criteria that have been recommended by Clinical and Laboratory Standards Institute (CLSI, 2018) guidelines. The following antibiotics were used in the present study: methicillin (M³⁰), vancomicin (VA³⁰), sulphadiazine (SZ³⁰⁰), cefaclor (Cj³⁰), nalidixic acid (Na³⁰), gatifloxacin (GAT⁵), gemifloxacin (GEM⁵), ertapenem (ETP¹⁰), meropenem (MRP¹⁰), imipenem (IPM¹⁰), cefepime (CPM³⁰), ceftazidime (CAZ³⁰), cefotaxime (CTX³⁰), cefotaxime/clavulanic acid (CEC^30/10), aztreonam (AT³⁰), tetracyclin (T³⁰), oxacillin (OX⁵), chloramphenicol (C³⁰), ciprofloxacin (CIP⁵), kanamycin (K³⁰) and erytromycin (E¹⁵). All the antibiotic discs were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. An aliquot of 0.1 mL of the overnight grown bacterial cultures (the turbidity of each was adjusted equal to 0.5 McFarland standard) were spread onto the Mueller-Hinton agar (MHA, HiMedia, Mumbai, India), the antibiotic discs were aseptically placed on the evenly spread plates with appropriate negative controls and incubated overnight at 37 ºC. Following incubation, the diameter of inhibition zone was measured and result were interpreted as susceptible, intermediate and resistant based on their comparison with the standards provided by CSLI. The bacterial strains harboring maximum antibiotic resistance were selected for further study. The isolates showing resistance to at least one of the key antibiotics of the three or more antimicrobial classes were designated as multidrug resistant (MDR) [26–30].

Isolation of DNA and PCR Based Detection of the Antibiotic Resistant Gene

Bacterial DNA was isolated according to standard phenol-chloroform method and used as template for the presence of antimicrobial resistance (AMR) genes [31]. PCR was performed using the specific primer pair as described in Table 1. A total of 25 µL reaction mixture was used for each PCR reaction containing MgCl₂ at 2 mM, dNTPs 0.30 µL (10 mM each), Taq polymerase 1.5 U/µL. PCR amplification was carried out in Thermocycler (Bio ER XP cycler, Germany). Following PCR conditions were used; initial denaturation at 95 ºC for 2min, followed by 30 cycle of amplification at 95 ºC for 30 second, annealing temperature for 45 second, 72 ºC for qnr gene, 30 second 53.1 ºC for ^blaCTX-M gene and a final extension at 72 ºC for 5 min. An aliquot of 5 µL the amplified product was run on agarose gel (1.2%) to ascertain the size of amplicon, and images were captured using gel documentation system (Gel Doc, BioRad, USA).

Table 1.

Primers used for detection of antibiotic resistance gene, their annealing temperature and product size (bp)

Genes	Sequences (5′ to 3′)	Annealing temperature 0 C	No. cycles	Product size (bp)	References
qnrB	FW- AGCGGCACTGAATTTAT RV- GTTTGCTGCTCGCCAGTC	57	30	497	Guillard et al. [32]
qnrS	FW-GGAAACCTACAATCATACATA RV-GTCAGGATAAACAACAATACC	57	30	600	Guillard et al. [32]
blaCTX.M	Fw- ATGTGCAGYACCAGTAARGT Rv- TGGGTRAARTARGTSACCAGA	53.1	35	593	Pallecchi et al. [33]

Y, (C + T); R, (A + G); S, (C + G)

Preparation of S. cumini Seed Extract

50 g of S. cumini seed powder (supplied by the Himalaya Drug Company, Bengaluru, India) was mixed in 500 mL ethanol (50%) and stirred in shaking incubator at 40 ºC for 24 h. Then the extract was filtered through Whatman No.1 filter paper using Buchner funnel. The mixed ethanolic filtrate was then evaporated at 40 ºC in a vacuum evaporator to dryness. The dried crude extract was stored at 4 ºC for further study.

Preparation of Antibacterial Sodium Alginate Film (SAF)

The formulation of antibacterial SAF was done with the method described previously [34, 35] with some modification. Ethanolic extract of S. cumini (EESC), sodium alginate (SA) and glycerol (BIO BASIC CANADA INC) as a plasticizer were used for preparation of film. One gram of SA (supplied by Loba Chemie Pvt. Ltd. India) were dissolved in 100 mL distilled water at 90 ºC under constant stirring for 2 h using hot plate magnetic stirrer. Glycerol (1.5 g^− 1 of SA) was added to the film solution. The different formulation of films is shown in Table 2.

Table 2.

Formulation of films and their constituents

Sample	1% SA + 0.5% Gly (mL)	EESC (mL)
SAF	100	00
10% EESCF	90	10
20% EESCF	80	20
30% EESCF	70	30

SA (Sodium Alginate), Gly (Glycerol) and EESC (Ethanolic extract of S. cumini)

The formulated films were designated as SAF (Sodium Alginate Film), 10% EESCF, 20% EESCF and 30% EESCF containing 10%, 20% and 30% ethanolic extract of S. cumini seeds, respectively. The film forming solution (100 mL) were poured into 13 × 12 cm gel casting tray and oven dried at 40 ºC. Upon drying films were dipped in 50 mL of 2% Calcium chloride (CaCl₂) for 30 seconds and re-dried at the same temperature. The films were removed from tray and stored in a desiccator for characterization.

Characterization of Formulated Films

Films Thickness

Film thickness was measured with a digital Vernier Caliper (Mitutoyo 150 mm Mitutoyo corporation, Japan). The mean film thickness was measured from 6 random points of each film with corresponding to standard deviation.

Moisture Content and Water Solubility

To determine the moisture content, 0.2 g of film samples were weighted in a crucible and oven dried at 105 ºC for 24 h. Moisture content was measured in terms of percentage of the initial films weight lost during drying and reported on a wet basis.

Water solubility at 25 ºC was determined according to [36] and percentage water solubility was calculated using the following formula:

$$\text{WS}(\%)=\left[\frac{\text{W}0-\text{Wf}}{\text{W}0}\right]\times 100$$

where W0 and Wf represents initial and insolubilized dry matter, respectively.

Instrumental Measurement of Films Color

The color of film samples was determined with the help of HunterLab ColorFlex EZ 45/0º color spectrophotometer serial No. CFEZ0352 (HunterLab, Reston, VA, USA), with D65, 10º observer according to ASTM E308. The measurement was conducted on CIE lab scale. Prior to measurements, the instrument was calibrated with a black and white tile (L^* 93.47, a^* 0.83 and b^* 1.33). Results are presented with following parameters:

L^* (luminosity),

a^* (red/green) and.

b^* (yellow/blue).

The overall color difference (ΔE) was calculated using the formula:

$$\text{E}=\sqrt{{{(\text{L}}^{\ast }-{\text{L}}^{\prime })}^2{{{+(\text{a}}^{\ast }-{\text{a}}^{\prime })}^2{+(\text{b}}^{\ast }-{\text{b}}^{\prime })}^2}$$

where L^*, a^* and b^* represent the color parameter values of the samples and L′, a′ and b′ represent the values of standard white plate used as the films background (L′= 66.39, a′= -0.74 and b′= 1.25). The study was carried out in triplicate with three repetitions. The mean value was calculated for the top and bottom sides.

Scanning Electron Microscopy (SEM)

The surface morphology of SAF were analyzed using scanning electron microscope (JEOL JSM-6510 LV, Japan). A small piece of each SAF samples were coated with gold (8 nm) before visualization. The images were captured at 1000 X magnification.

Fourier Transform Infrared Spectroscopy (FT-IR)

FT-IR spectra of SAF was recorded over the range of 4000 − 400 cm^− 1 on a FT-IR spectrophotometer (PerkinElmer In., spectrum2, USA) using the KBr pellet.

Antibacterial Activity of Films

Antibacterial activity of prepared films was carried out using both qualitative and quantitative approaches. Qualitative approach involves agar diffusion assay, same as performed for antibiotic sensitivity testing with some modifications. The overnight grown culture of E. coli was spread onto MHA plates, after that 10-mm diameter disks of each EESC films including control SAF were placed and incubated at 37 ºC for 24 h, the results were recorded and the diameter of inhibition zone around disks was measured with a Caliper.

The quantitative approach for antibacterial activity of the prepared films uses broth medium as described [37] with some modifications, 100 mg of each EESC films including control SAF was added in 10 mL of MHB. After that 100 µl of the overnight grown culture (standardised to 0.5 McFarland turbidity) inoculated into the respective broth medium and incubated at 37 ºC for 12 h in a shaker incubator. Colony forming unit (CFU) was counted by withdrawing aliquots after every 2 h by serial dilution. CFU was preferred to the OD, because the amended compound diffuses into the broth and imparts it dark color making measurement of OD at 620 nm quite difficult.

Statistical Analysis

All experiments were carried out in many independent replicates as stated individually in each case and data is shown as mean ± standard deviation. Differences between the mean values of the films ‘properties were measured using by the Duncan’s multiple range tests (DMRT).

Result and Discussion

Characterization and Antibiotic Susceptibility of E. coli Isolates

A total of 50 bacterial isolates from 70 buffalo meat samples were identified as E. coli by morphological, cultural (sheen on LEMB) and biochemical characteristics. Furthermore, five isolates harbouring highest MDR pattern with AMR gene was also ascertained by MALDI-TOF-MS. The antimicrobial susceptability test of E. coli isolates showed that 90% of the isolates were resistant to multiple antibiotics. Resistance was most frequently observed against methicillin (84%), vancomicin (70%), sulphadiazine (68%), cefaclor (66%), oxacillin (52%), nalidixic acid (40%), tetracyclin (36%), erytromycin (24%), gemifloxacin (18%), aztreonam (16%) cefotaxime (14%), imipenem (12%), cefepime (12%), ciprofloxacin (8%), kanamycin (8%), chloramphenicol (4%) and gatifloxacin (2%) (Fig. 1a). while 100% of the isolates were susceptible to ertapenem, meropenem, ceftazidime and combined disc cefotaxime/clavulanic acid, and 96% for chloramphenicol 92% for gatifloxacin, ciprofloxacin and kanamycin, (32%) for sulphadiazine, (30%) vancomicin and least susceptibility (16%) was recorded against methicillin. A study on the antimicrobial susceptibility and incidence of E. coli isolates from beef meet in Ghana found that 44.44% resistance was recorded against tetracycline and chloramphenicol, and 68.89% to erythromycin, while 95.56% of the isolates were susceptible to ciprofloxacin [38]. Aslam and Service [39] reported chloramphenicol (2.45%) and tetracycline (38%) resistance in E. coli originated from beef sources. The E. coli isolated from Egyptian beef carcasses were resistant to chloramphenicol (23.8%), ciprofloxacin (42.8%), gentamicin/kanamycin (19%) and sulfamethazole (80.9%) [40]. Another study found antimicrobial resistance in E. coli isolates (from beef sample) demonstrated that 85.71% isolates were resistant to erythromycin, 71.43% to oxy-tetracycline while 100% of these isolates showed sensitivity to ciprofloxacin and gentamicin [41]. Resistance to third generation antibiotics were also found in E. coli strains from food sources with resistance pattern as cefotaxime (5.3%) and amoxyclave (4.6%) [42]. E. coli strains isolated from seafood items has also been found resistant to ceftazidime (14.29%), cefotaxime (10.71%) and ceftriazone (10.71%) [43].

Fig. 1.

a. Resistance and susceptibility profile of E. coli isolates against tested antibiotics b Multidrug Resistant Pattern of E. coli isolates from raw buffalo meat. Key antibiotics: Methicillin (M³⁰), Vancomicin (VA³⁰), Sulphadiazine (SZ³⁰⁰), Cefaclor (Cj³⁰), Oxacillin (OX⁵), Nalidixic acid (Na³⁰), Tetracyclin (T³⁰), Erytromycin (E¹⁵), Gemifloxacin (GEM⁵), Aztreonam (AT³⁰), Cefotaxime (CTX³⁰), Cefepime (CPM³⁰), Imipenem (IPM¹⁰), Ciprofloxacin (CIP⁵), Kanamycin (K³⁰) Chloramphenicol (C³⁰), Gatifloxacin(GAT⁵), Ertapenem (ETP¹⁰), Meropenem (MRP¹⁰), Ceftazidime (CAZ³⁰), Cefotaxime/Clavulanic (CEC^30/10) 

Present study revealed that the emergence of resistance against 3rd and 4th generation quinolones/Fluor quinolones (gatifloxacin and gemifloxacin) along with 1st and 2nd generation (nalidixic acid and ciprofloxacin) in meat originated E. coli is a matter of great concern to public health threat. The significant findings of our study is there was resistance against fourth generation cephalosporin such as, cefepime (12%), broad spectrum imipenem (12%) and wide range newly developed monobactums such as aztreonam (16%).

In the present study the multi drug resistant characteristics of each isolates were phenotypically identified by observing the resistance pattern of the isolates against tested antibiotics/drugs. All the fifty isolates exhibited 44 different antibiotic resistance patterns, which revealed the strain diversity among the E. coli isolates (Fig. 1b). The worrying aspect of the current study is that 45 (90%) of the isolates were found to be multidrug-resistant. Two of the isolates ECMB1 and ECMA2 showed highest MDR pattern against 14 antibiotics of 7 different classes such as, β-lactam, quinolones, sulfonamides, Tetracycline, glycopeptide, macrolides and aminoglycosides. Saud et al. (2019) reported multidrug resistance in bacteria isolated from buffalo meat and found 69.81% E. coli isolates were MDR [44].

The Multiple Antibiotic Resistance Index (MARI) was derived using a mathematical formula as described earlier and expressed as MAR _Index = a/b, where ‘a’ defined the number of antibiotics to which the isolates were resistant and ‘b’ the total number of antibiotics to which an individual isolate was tested [45]. The MARI analysis revealed that 25 (50%) isolates of MDR E. coli strains had very high MARI value (> 0.2) which is an indication of high-risk source of contamination augmented in food chain through imprudent use and greater exposure of antimicrobials in humans, veterinary, poultry and livestock. The MARI value of E. coli isolated from beef ranged from 0.11 to 0.78 [46]. The high prevalence of MDR E. coli in buffalo meat reveals the unhygienic production and processing of meat which may cause serious public, health threats. The finding of this study indicates for a strict monitoring and implementation of effective biosafety and hygienic measures in the whole food chain in meat as well as prudent use of antibiotics.

PCR based detection of AMR gene

All the MDR E. coli were screened for the AMR gene by PCR. Gel electrophoresis of PCR product showed the presence of 593 bp band of ^blaCTX-M gene (Fig. 2; Table 3) that is responsible for production of β-lactamase. The ^blaCTX-M gene in multidrug-resistant E. coli isolated from minced meat has been reported in scientific literature [47]. Moreover, qnr S1 gene were also present along with ^blaCTX-M-15 in E. coli isolated from Asian seafood (prawns) imported to Denmark [48]. Resistance to third generation antibiotics cephalosporin was mediated by extended spectrum β-lactamase (ESBL) producing gene such as ^blaCTX-M which is generally described in bacteria from human and animal origins [49–51]. This ESBLs were also sporadically identified in isolates of Danish production animals and Danish retail meat and they are one of the most frequently detected ESBLs in humans.

Fig. 2.

Agarose gel electrophoresis showing PCR based amplification of a ^blaCTX-M, b qnr S, c qnr B gene in MDR E. coli isolates from raw meat samples. lane L: DNA molecular weight marker (Gene Ruler 100 bp DNA Ladder; Fermentas) used as reference marker; lane PC: positive control (E. coli RP4 for ^blaCTX-M, Klebsiella K8-5 qnrB and E. coli qnr S1); lane NC: negative control; lane 1–5: ECMB1, ECMA2, ECMA8, ECMS9 and ECMA31 E. coli isolates

Table 3.

Phenotypic and genotypic characteristics of selected E. coli isolates

E. coili isolates		Phenotypic antibiotic resistant pattern	Genotype
1	ECMB1	M Cj Na SZ GAT GEM VA CTX AT OX T CIP K E	qnr S, qnr B, blaCTX-M
2	ECMA2	M Cj Na SZ GEM VA CPM CTX AT OX T CIP K E	qnr S, qnr B, blaCTX-M
3	ECMA8	M Cj Na SZ GEM VA CPM CTX OX T CIP K	qnr S, qnr B, blaCTX-M
4	ECMS9	M Cj Na SZ GEM VA CPM CTX AT OX	qnr S, blaCTX-M
5	ECMA31	Cj Na SZ V CPM CTX AT OX E	qnr B, blaCTX-M

A 600 and 497 bp band showed the presence of quinolone antibiotic resistant qnr S and qnr B gene (Fig. 2; Table 3). Quinolone is a broad spectrum antibiotic and widely used in treatment of disease specially in urinary tract infections with low side effect [52, 53]. These antibiotics tend to bind tightly to the bacterial enzymes (DNA gyrase or topoisomerase IV), and inhibit the bacterial growth, has been reported worldwide is the incidence of plasmid-mediated resistance genes [54]. The presence of plasmid mediated quinolone resistance (PMQR) genes qnr S and qnr B in E. coli facilitates the resistance to quinolone antibiotics such as, nalidixic acid, gemifloxacin, ciprofloxacin and gatifloxacin as phenotypically detected in E. coli isolates (Table 3). These are highly significant antimicrobials of highest importance in the immuno suppressed patient. This phenotype is unusual in food, and imports can be a source of PMQR genes for human [55]. Most of the AMR genes detected in these isolate was previously identified on mobile genetic elements and could be transferred to the pathogens.

.

Film Thickness, Moisture Content and Solubility in Water

Film thickness is a crucial parameter of a packaging films in terms of evaluating the mechanical and barrier properties. Film thickness including the control (SAF) ranged from 0.11 mm to 0.18 mm (Table 4). The result insight a slight difference in thickness of EESC films in comparison to control film (SAF). The thickness of EESCF was found to be slightly increased with the increasing concentration of EESC seed due to high amount of solid contents. Similar results were demonstrated earlier when ethanolic extract of Guava leaf was added to sodium alginate films [56]. There was an in increase in thickness of packaging films prepared from sodium alginate incorporated with anti-oxidant extracted from agro-industrial sub-products [57].

Table 4.

Measurement of thickness, solubility and moisture content of the prepared packaging films

Samples	Thickness (mm)	Solubility (%) in water at 25 ºC	Moisture content % (Dry basis)
Control SAF	0.11 ± 0.02 c	22.5 ± 3.53d	21 ± 1a
10% EESCF	0.12 ± 0.01 b	26.78 ± 2.52c	12 ± 1b
20% EESCF	0.12 ± 0.01 b	33.25 ± 6.6b	11 ± 1c
30% EESCF	0.18 ± 0.02 a	39.38 ± 1.6a	08 ± 1d

Data is shown as mean value ± SD (for thickness n = 6, for water solubility and moisture content n = 2). Letters (a-d) indicates significant difference (p < 0.05)

The moisture content of the amended films decreased in contrast to control SAF, and ranged from 21 to 8% (Table 4). The plenty of hydrophilic groups such as hydroxyl (-OH) and carboxylic (-COOH) in sodium alginate molecules provide much higher moisture content to the control SAF. On addition to EESC seeds in SAF, the -OH groups in the extracts may form intermolecular hydrogen bonds with hydrophilic groups in SA, which minimized the interaction between the water and the composite matrix. Similar findings in tendency of moisture contents were also reported when ethanolic extract of Guava leaf was added to sodium alginate films [56]. Water solubility is a reflection of films and coating resistance to water. The control SAF showed least solubility in water (22.5%). On increasing the concentration of EESC seeds the water solubility of EESCF increases (Table 4). The increased water solubility has been attributed to hydrophilic polyphenol groups in EESC which can easily bind to water. A similar finding in chitosan film incorporated with thinned young apple polyphenols has been reported previously [58].

Color Appearance of Films

Color of packaging films is an important attribute which affect not only the consumers’ acceptance but also the products appearance, therefore it is of prime importance to preserved its transparency or it shows a color as close as possible to the natural pigment of foods to which the film is applied. The control film (SAF) appeared clear and transparent, on addition of EESC seed it looks yellowish and on increasing EESC concentration it becomes brown (Fig. 3). Our findings are in agreement with those of [59]. The color parameters obtained by HunterLab for EESCFs including control (SAF) is given in Table 5. The ^‘L^’ value of EESC incorporated films decrease in comparison to the control film (SAF) which indicated that the color of films tends to darken. The higher value of EESC concentration showed lower ^‘L^’ value, while ^‘b^’ value gradually decreases and ^‘a^’ value increases (except in 10% EESCF), indicated that tendency toward redness, and statistically significant color variations (P < 0.05) (shown by parameter ΔE) have been observed. In fact, the EESCF was more coloured in comparison to control as observed visually.

Fig. 3.

Color variation of EESCFs with respect to control SAF on increasing concentration of EESC

Table 5.

HunterLab values and color parameter of EESCFs

Samples	L*	a*	b*	ΔE
Control SAF	11.5d ± 0.74	-0.13c ± 0.11	-0.3d ± 0.6	-
10% EESCF	22.68a ± 0.12	-1.09d ± 0.14	2.68a ± 0.75	12.71a ± 0.1
20% EESCF	20.18b ± 0.70	0.32b ± 0.24	2.47b ± 0.85	11.43a ± 0.35
30% EESCF	16.13c ± 0.55	0.79a ± 0.17	1.77c ± 0.32	07.3b ± 0.03

Data is given as the mean ± SD (n = 3). Letters (a-d) represent the significant difference (p < 0.05)

Scanning Electron Microscopy (SEM)

There is involvement of hydrogen bonds, electrostatic interactions, covalent bonds, or other charge-shift interactions among the constituents for the formation of sodium alginate packaging films. Film characteristics such as surface structure, morphology, and roughness depend on the type of materials used and their interactions [60]. SEM surface images of sodium alginate packaging films (with different concentration of EESC) and control SAF is shown in Fig. 4. The microscopic images of the control film (without treatment) shows that there was a smooth and homogeneous matrix without pores (Fig. 4a). The treatment of films with different concentration of ethanolic extract of S. cumini (seed) resulted in disruption of the homogeneous film surface started as observed form the surface morphology (Fig. 4b). Similar findings were also observed earlier for chitosan film containing Eucalyptus globulus essential oil [61]. The findings concluded that an increasing concentration of extract in the film led to deposition on the film’s surface which is essential for antimicrobial activity. The irregularity imparted by the extract on the film’s surface induces the antimicrobial activity when it comes in contact with food material.

Fig. 4.

SEM surface micrograph of sodium alginate based packaging films a control SAF, b 10% EESCF, c 20% EESCF, d 30% EESCF (incorporated with ethanolic extract of Syzygium cumini seed powder)

FT-IR Spectra

To obtain a detailed insight of the chemical nature and functional groups in the synthesized films, FT-IR analysis was performed (Fig. 5). SAF and EESCF exhibited very similar absorption peaks with minor differences. The % transmittance maxima near 3400 cm^− 1 in both the samples (i.e. SAF and EESCF) represents the O-H stretching vibration and presence of water molecules. The transmittance bands near 2930 and 1620 cm^− 1 is assigned to C-H stretching and C = O vibration respectively. The absorption band ranging from 1260 to 1000 cm^− 1 is due to the stretching vibrations of the C-OC and C-OH groups. An obvious increase in most of the bands were recorded except for the peak at 3400 cm^− 1. The absorption peaks in SAF located near 3400, 2935, and 1622 cm^− 1 slightly shifted in EESCF, indicating an interaction between sodium alginate and S. cumini extract.

Fig. 5.

FT-IR spectra of EESCF and Control SAF

The FT-IR spectrum of sodium alginate packaging films functionalized by Guava leaf extracts exhibited a similar pattern as in our case [59]. SAF exhibited characteristic bands at 3400, 2935, 1622, 1420, and 1020 cm^− 1. Li et al. (2015) also recorded 2930 and 1620 cm^− 1 band which represent C-H stretching in a study conducted on antioxidant and immunomodulatory activities of polysaccharides from Prunella vulgaris [62]. The absorption band near 1620 cm^− 1 in FT-IR spectrum is representation of C = O vibration [63]. The changes in peak intensity and position of FT-IR spectra is induced by the hydrogen bonds between various phytochemicals and sodium alginate [64]. A strong intermolecular bonding may improve the physico-chemical properties of the packaging film.

Antibacterial Activity of EESCFs

The packaging films containing active compounds such as natural antioxidants or antimicrobials will guarantee food products quality and safety. The agar diffusion assay (qualitative approach) for antibacterial property of EESCF against selected MDR E. coli exhibited a marginal increase in zone size as the concentrations of EESCF increases (Figs. 6 and 7). At the 10% and 20% EESCF 20 ± 1 mm zone of inhibition was recorded, whereas, at 30% EESCF, an increase of 5% (21 ± 2 mm) in zone size was observed, while no zone of inhibition in control SAF.

Fig. 6.

Zone of inhibition (mm) demonstrated by EESCFs against tested E. coli, bar diagram represents the mean ± S.D. values of three independent replicates (n = 3)

Fig. 7.

Antimicrobial activity of packaging film by agar diffusion method [1: a 10% EESCF, b 20% EESCF, c 30% EESCF; 2: Control SAF] against selected MDR E. coli

After assessing the qualitative antibacterial assays, the quantitative measurement of antibacterial activity of EESCF was evaluated by growing the cells of E. coli in MHB medium supplemented with varying concentrations of EESCF (10, 20 and 30%) along with control SAF. In all the treatment, a gradual increase in cell number was noticed with incubation period (Fig. 8). In case of untreated control, bacterial cells were increased from 2.15 to 9.21 log CFU/mL after 12 h of incubation. In contrast, a marginal decline was observed in EESCF treated E. coli cells, as seen 9% log CFU/mL reduction in 30% treated EESCF in contrast to control cells after 12 h of growth. In accordance with our findings, Norajit and Ryu (2011) have been reported the antibacterial activity of packaging film containing ginseng extract against E. coli. [36]. Similarly, the antimicrobial potential of sodium alginate-based green packaging films incorporated with guava leaf extract has recently been reported against S. aureus and E. coli. [56]. The present finding suggested that EESCF was inhibitory against the MDR in general and E. coli in particular.

Fig. 8.

Growth of E. coli strain ECMB1 under the influence of different concentrations of EESCF along with control SAF demonstrating the reduction in Cell count (log CFU/mL)

Conclusions

Our study provides an insight into the occurrence of multidrug resistance among E. coli strains in raw buffalo meat. E. coli isolates were highly resistant to multiple antibiotics but susceptible to ertapenem, meropenem, ceftazidime and cefotaxime/clavulanic acid. The resistance to quinolone/fluoroquinolone and β-lactam antibiotics is due to the presence of AMR genes (qnr S, qnr B and ^blaCTX-M) in E. coli isolates. High MARI value (> 0.2) indicated high-risk source of contamination augmented in food chain through indiscriminate use and greater exposure of antimicrobials in livestock. Therefore, it is of great public health concern that meat infected with such MDR bacteria may be a severe cause of foodborne infections when ingested. The study further highlights the importance of formulated sodium alginate packaging film with S. cumini seed extract having good antimicrobial property against the isolated MDR bacteria. Therefore, it may be a safer packaging alternative for meat industries to prevent the spoilage and minimize the contamination of the antibiotic resistant bacteria.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest associated with this manuscript.