Production of alginate-based films containing the e. coli-specific bacteriophage vb ecom-p34 for use as active packaging

Abstract

The present invention relates to a method for producing sodium alginate-based films comprising the Escherichia coli-specific bacteriophage vB_EcoM-P34, intended for use as a packaging material in the food industry.

Description

[0001] PRODUCTION OF ALGINATE-BASED FILMS CONTAINING THE E. COLI- SPECIFIC BACTERIOPHAGE vB_EcoM-P34 FOR USE AS ACTIVE PACKAGING

[0002] TECHNICAL FIELD

[0003] The invention relates to a method for producing alginate-based films containing the Escherichia co / / -specific bacteriophage vB_EcoM-P34 (phage), which are intended for use as inner packaging material in the food industry.

[0004] BACKGROUND

[0005] Food safety is one of the major concerns for the food industry, governments, and consumers. Microorganisms, which are among the most significant factors causing food spoilage, adversely affect both the overall quality of products and consumer safety. Changes in retail practices — such as extended distribution periods resulting from the globalization of the food market — and shifts in consumer lifestyles associated with fast-paced living have increased expectations regarding nutrition and created a demand for easy access to high-quality food products. In response to this demand, various packaging technologies have been developed to minimize quality losses in food products and to extend their shelf life.

[0006] One of the alternative approaches to conventional packaging technologies for preserving the quality characteristics of foods and extending their shelf life is active packaging. In active packaging technology, auxiliary components are incorporated into or onto the packaging material or the headspace of the package in order to maintain product quality, ensure food safety, and prolong shelf life. Among these components, antimicrobial agents play a significant role by preserving the nutritional and sensory quality of foods while inhibiting pathogenic and spoilage microorganisms. However, there has been increasing public concern regarding the use of food additives and chemical preservatives for this purpose. Although these substances are used within the limits established by legislation, long-term accumulation in the human body may cause health problems. In addition, the widespread use of petroleum-based synthetic polymers as packaging materials has raised environmental concerns due to their non- biodegradable nature. Consequently, there is a growing interest in developing new biopolymer-based packaging materials to replace petroleum-derived synthetic polymers. Biopolymers can be used as food packaging materials due to their environmentally friendly nature, low cost, and edible film-forming properties. Edible films are considered a part of active packaging technology. Nowadays, novel technologies such as active packaging are being increasingly applied in food industry research; however, they have not yet been widely commercialized.

[0007] Although the use of active packaging in various food products around the world is still limited, it does exist; to the best of current knowledge, however, no such applications have been reported in Turkiye. According to the literature, components with antimicrobial properties used in active packaging include compounds derived from various plants and spices such as cinnamon, red pepper, clove, thyme, rosemary, onion, and garlic; bacteriocins such as nisin, natamycin, and pediocin; and substances such as sorbic acid, benzoic acid, propionic acid and their salts, ethanol, carbon dioxide, silver ions, chlorine dioxide, antibiotics, organic acids, and essential oils.

[0008] However, existing techniques suffer from the following drawbacks:

[0009] • Synthetic polymers commonly used in packaging have adverse effects on the environment and living organisms.

[0010] • Public concern has increased regarding the potential health problems associated with the long-term use of food additives and chemical preservatives in conventional preservation methods.

[0011] • Antimicrobial agents may exhibit reduced effectiveness when directly incorporated into food products.

[0012] • Direct addition of antimicrobials to the food matrix can lead to partial loss of activity due to diffusion or leakage.

[0013] • Certain antimicrobials may undergo undesirable reactions with food components such as lipids and proteins.

[0014] • Essential oils, which are frequently used in antimicrobial applications, often need to be applied at high concentrations to achieve significant antimicrobial effects; however, such concentrations may adversely affect the sensory properties of the packaged food.

[0015] • There is a need to determine a safe concentration range for essential oils intended for use in active food packaging. • Due to their volatility under ambient conditions, the antimicrobial efficacy of essential oil-based active films may decrease over time.

[0016] • Non-volatile antibacterial agents must be in direct contact with the food surface to remain active.

[0017] The following are the active antimicrobial packaging products available worldwide:

[0018] Trade name Feature of product Country of production

[0019] Wasapower™ Coated PET film and tablets Japan

[0020] Zeomic™ Films Japan

[0021] WasaOuro Plates Japan

[0022] Uvasy™ Laminated boards and pads South Africa

[0023] Microgarde™ Bags, films and packaging United States

[0024] Bactiblock Masterbatch (additive) Spain

[0025] Bioka Smail bag / pouch Finland

[0026] None of the commercial antimicrobial products listed above contain phages.

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention relates to the production of an alginate film incorporating the bacteriophage vB_EcoM-P34 for use in packaging, particularly aimed at eliminating E. coli in foods that pose a risk of contamination by this pathogenic bacterium. Preferably, the invention is intended for use in packaging Ka§ar cheese, a commonly consumed product.

[0029] Due to the adverse environmental and biological effects of synthetic polymers, there has been an increasing need to develop new packaging materials and a corresponding shift toward naturally occurring, biodegradable polymers such as alginate, chitosan, pectin, starch, and proteins. Alginate is a natural polysaccharide obtained from brown seaweed and certain bacteria. It is one of the most suitable biopolymers for the production of edible films for use in foods because it is natural, inexpensive, biocompatible, biodegradable, generally recognized as safe (GRAS), mechanically stable, and highly tolerant to salts and chelating agents.

[0030] The film is a thin-layered material obtained by casting a film-forming solution into solid and flexible layers, followed by drying. Films used to wrap food may be consumed as part of the food or removed prior to consumption. Plasticizers are employed to enhance the physical and mechanical properties of alginate-based films.

[0031] Films produced through the combination of biopolymers and various antimicrobial agents, which have potential use as active packaging, aim to extend the shelf life of food and ensure the delivery of safe products to consumers by slowing the rate of spoilage reactions and inhibiting microbial growth.

[0032] The incorporation of bacteriophages into biodegradable polymer films enables the development of antibacterial properties. Although bacteriophages were first discovered in 1915-1917 (D’Herelle, 1917; Twort, 1915), they have recently gained prominence due to the increasing resistance of pathogenic bacteria to antibiotics and their environmentally friendly characteristics.

[0033] Lytic bacteriophages are viruses specific to bacteria that infect bacterial cells, lyse them, and are subsequently released from the lysed cells to infect neighboring host cells. Due to their high specificity, bacteriophages inhibit only the target host bacteria without affecting the natural flora, unlike antibiotics. Furthermore, they replicate specifically in the locations where host bacteria are present. In the absence of a suitable host, bacteriophages are eliminated by the host organism’s immune system. This characteristic of bacteriophages is referred to as automatic dosing. Additional advantages of bacteriophages include their effectiveness at low doses, the ability to eliminate certain biofilm populations, and their relatively simple and cost- effective production technologies.

[0034] As natural antibacterial agents, bacteriophages are recommended for incorporation into films, as they do not alter the nutritional value, taste, or aroma of food when compared to other antimicrobial agents. In addition, the United States Food and Drug Administration (FDA) has approved the use of various phage-based preparations designed to combat pathogens such as Salmonella spp., Escherichia coli, Shigella spp., and Listeria monocytogenes in food products.

[0035] In the present invention, the E. coli O157:H7-specific bacteriophage vB_EcoM- P34, a phage with high lytic activity, has been employed. The vB_EcoM-P34 phage, isolated from slaughterhouse wastewater, belongs to the Myoviridae family. Its genome does not contain the virulent genes stx1 or stx2. In addition to E. coli O157:H7 strains, vB_EcoM-P34 is also effective against certain Salmonella enterica serovars. This phage, characterized by high lytic activity, exhibits a burst size and latent period of 102 PFU / cell and 20 minutes, respectively. Moreover, it retains its activity under high temperature conditions (70°C for 30 minutes) and across a wide pH range (pH 2-11 ).

[0036] The technical effects of the present invention are summarized as follows:

[0037] • The use of edible films as part of active packaging, in place of synthetic packaging, contributes to solving environmental pollution. • While antimicrobial agents directly added to food formulations may negatively affect quality attributes such as taste, color, or viscosity, the use of edible films in active packaging prevents these adverse effects.

[0038] • There is no need for additional processes such as spraying, mixing, or dipping to apply the antimicrobial agent to the food.

[0039] • Upon release of the bacteriophage from the films, protection continues as long as the target pathogenic bacteria are present in the environment.

[0040] • Phages are natural components and do not harm living organisms other than their specific hosts.

[0041] • Since bacteriophages are host-specific, they do not inhibit all bacteria as antibiotics or other antibacterial agents do; thus, the natural microflora and beneficial bacteria are preserved.

[0042] • The use of bacteriophage-based films can reduce the reliance on additives used for ensuring food safety.

[0043] • To the best of current knowledge, no edible films incorporating bacteriophages have been produced worldwide.

[0044] The method for producing alginate-based edible films containing the bacteriophage of the present invention and the steps followed for evaluating their antibacterial efficacy are described in detail below.

[0045] Preparation of High-Titer Bacteriophage Solution

[0046] For the preparation of a high-titer phage solution, 100 pL of vB_EcoM-P34 phage samples and 300 pL of the active host bacterium Escherichia coli O157:H7 ATCC 35150 were transferred into 4 mL of nutrient soft agar (nutrient broth+0.6% agar) maintained at 45-50°C and mixed thoroughly to ensure homogeneity. The mixture was then poured and evenly spread onto Petri dishes containing nutrient agar (nutrient broth+1.5% agar). After solidification of the agar, the Petri dishes were incubated at 35-37°C for 24 hours. For phage recovery, sodium chloride-magnesium sulfate (SM) buffer was added to the Petri dishes, and the phage plaques were scraped off and transferred into sterile tubes. The composition of the SM buffer was as follows: 50 mM Tris-HCI (pH 7.5), 99 mM NaCI, 8 mM MgS04, and 0.01 % (w / v) gelatin.

[0047] After incubation in a shaking incubator at 25°C and 120 rpm for 2 hours, chloroform (50 pL / mL) was added to the tubes and mixed to induce lysis of the bacterial cells. This process allowed the release of phages formed within the bacterial cells. Following centrifugation at 4°C and 7000xg for 15 minutes, the supernatant was passed through a sterile membrane filter with a pore size of 0.45 pm to remove bacterial cells from the phage solution. Prepared phage samples were stored at 4°C to be used in film production after their titers were determined.

[0048] Determination of Phage Titer

[0049] The double-layer agar plate method was used to determine the phage titer. Serial dilutions were prepared from the previously obtained phage suspension samples. A 100 pL aliquot from each phage dilution was mixed with 300 pL of host cells in 4 mL of nutrient soft agar (nutrient broth + 0.6% agar) maintained at 45-50°C, and the mixture was poured onto Petri dishes containing solidified nutrient agar (nutrient broth+1.5% agar). After incubation at 37°C for 24 hours, plaques were counted, and the phage titer was expressed as plaque-forming units (PFU / mL).

[0050] Production of Sodium Alginate-Based Films

[0051] The film-forming solution was prepared by dissolving 1% (w / v) sodium alginate in distilled water and stirring at room temperature (350 rpm) for 18 hours. Glycerol was then added as a plasticizer at a concentration of 0.75% (v / v) and mixed at room temperature at 350 rpm. Subsequently, the phage solution (1O10PFU / mL) was incorporated into the film solution at a ratio of 1 :9 and homogenized by stirring for 30 minutes at room temperature. A 30 mL portion of the resulting film-forming solution was poured into a sterile Petri dish (9 cm in diameter). The films were dried and conditioned in a climatic chamber at 25°C and 53% relative humidity for 48 hours. The production volume can be scaled up according to the number of films required.

[0052] Phage Activity of the Films and Phage Release Profile from the Films

[0053] To determine the titer of phages incorporated into the films, film samples (2x2 cm2) were placed in sterile 15 mL tubes containing 2 mL of SM buffer. The tubes were stirred at 250 rpm for 45 minutes at room temperature to ensure complete release of the phages from the films. The number of released phage particles was determined using the double-layer agar plate method.

[0054] To evaluate the phage release profile from the films, film pieces (2x2 cm2) were aseptically transferred into 10 mL of SM buffer and continuously stirred at 4°C to facilitate phage release. At predetermined time intervals, 0.5 mL samples were withdrawn, and an equivalent volume of SM buffer was added to maintain a constant total volume. The number of released phage particles at each time point was determined using the double-layer agar plate method.

[0055] Determination of Color, Thickness, and Moisture Properties of the Films

[0056] Color analysis of the films was performed using a colorimeter (Minolta, CR-400, Japan) based on the CIE Lab* color system, where L* represents lightness, a* represents the red-green coordinate, and b* represents the yellow-blue coordinate. The measurement results were expressed as the average variations in L*, a*, and b* values (AL*, Aa*, Ab*). The thickness of the films was measured using a micrometer with a precision of 0.001 mm, and ten measurements were taken from different points of each film. Moisture content was determined gravimetrically by drying the film samples in an oven at 105°C until a constant weight was achieved.

[0057] Determination of Elongation at Break and Tensile Strength of the Films

[0058] The elongation at break and tensile strength analyses were performed using a texture analyzer (Stable Micro Systems TA-XT2I Texture Analyzer; Stable Microsystems, Godaiming, UK). Film samples with dimensions of 50 mm x 10 mm were tested at a speed of 2 mm / s and an extension distance of 20 mm. These measurements demonstrated the potential applicability of the produced films for wrapping food products.

[0059] Antibacterial Activity (in vitro and in vivo)

[0060] Microbiological analyses were performed under aseptic conditions. The antibacterial activity of the phage-loaded sodium alginate films was evaluated against Escherichia coli O157:H7 ATCC 35150.

[0061] For the in vitro antibacterial activity test, a bacterial suspension of E. coli was prepared in Brain Heart Infusion (BHI) broth at a concentration of 105CFU / mL. A 100 pL aliquot of this bacterial suspension was evenly spread onto the surface of each film sample (2x2 cm2) using a sterile pipette tip. The inoculated film samples were placed in sterile Petri dishes and sealed with parafilm. Under the same conditions, alginate films without phages were inoculated with the same bacterial suspension and used as controls. Following incubation at 37°C for 24 hours, the films were transferred into a saline solution [0.9% (w / v) NaCI] and agitated at room temperature for 15 minutes at 250 rpm to facilitate bacterial detachment. The number of E. coli cells present on the films was determined using the spread plate method on MacConkey agar. For this purpose, 100 pL aliquots from serial dilutions of each sample were plated on the agar surface. After incubation under aerobic conditions at 37°C for 24-48 hours, bacterial colonies were enumerated.

[0062] For the analysis of the antibacterial activity of the films in a food matrix (in vivo), the surfaces of fresh Ka§ar cheese, used as a model food, were aseptically trimmed by 1 cm using a sterile knife. Cheese samples measuring 3 x 3 x 1 cm (length x width x thickness) were then cut, placed in sterile empty Petri dishes, and sterilized under UV light for 30 minutes. The upper surface of each cheese sample was inoculated with 100 pL of an E. coli suspension in BHI adjusted to 105CFU / mL and evenly spread over the surface using a sterile pipette tip. The inoculated samples were held at room temperature for 30 minutes to facilitate microbial adhesion to the cheese surface. Subsequently, the cheese samples were wrapped with phage-containing alginate films and stored at 4°C for 7 days. As a control, E. co / / -inoculated cheese samples were wrapped with phage-free alginate films and stored under the same conditions. On days 0, 1 , 3, 5, and 7, cheese samples were homogenized in sterile stomacher bags using saline solution [0.9% (w / v) NaCI] at a 1 :9 ratio. Serial dilutions were prepared from the homogenates. The number of E. coli cells was determined using the spread plate method on MacConkey agar. For this purpose, 100 pL aliquots from the serial dilutions were plated on the agar surface. After incubation under aerobic conditions at 37°C for 24-48 hours, bacterial colonies were counted.

[0063] Determination of Physicochemical Properties of Cheese Wrapped with Sodium Alginate-Based Films

[0064] The physicochemical properties of the cheese samples were analyzed on days 0, 1 , 3, 5, and 7 of storage. The color of all cheese samples was determined using a colorimeter (Konica Minolta, Japan) based on the CIE Lab* system, where L* represents lightness, a* represents the red-green coordinate, and b* represents the yellow-blue coordinate. The results are expressed as the average variations in L*, a*, and b* values (AL*, Aa*, Ab*). To determine the total acidity (% lactic acid), approximately 5 g of cheese was added to 25 mL of distilled water and thoroughly mixed. The volume was then adjusted to 50 mL with distilled water, and the samples were homogenized using a stomacher (BagMixer 400, Interscience, France). Following homogenization, the samples were filtered through filter paper, and the total acidity was determined by titration with 0.1 N NaOH in the presence of phenolphthalein as an indicator. The water activity (aw) of the cheese samples was measured using a water activity meter (Novasina, LabTouch-aw, Switzerland).

[0065] In the studies conducted within the scope of the invention:

[0066] • An average of 1 x 109PFU / cm2of phages was detected in the film samples, and phage activity was largely preserved.

[0067] • Phage release from the films was observed over a period of 15-240 minutes, ranging from 4 x 10sto 7 x 10sPFU / cm2.

[0068] • The thickness of the phage-containing films was measured to be 0.072 mm on average. The average dry matter content of the phage-containing films was found to be 60.97%.

[0069] • In preliminary studies, when the films were wrapped around Ka§ar cheese and stored, the lytic activity of the released phages against E. coli on the cheese surface was observed.

Claims

CLAIMS1. A sodium alginate-based film for use in food packaging, characterized by comprising the Escherichia co / / -specific bacteriophage vB_EcoM-P34.

2. The sodium alginate-based film according to claim 1, characterized in that it is used as packaging for Ka§ar cheese.

3. A method for producing a sodium alginate-based film for use in food packaging, characterized by comprising the steps of:• Transferring vB_EcoM-P34 phage samples and the active host bacterium E. coli O157:H7 ATCC 35150 into nutrient soft agar (nutrient broth + 0.6% agar) for the preparation of a high-titer phage solution;• Pouring the mixture onto Petri dishes containing nutrient agar and spreading evenly after ensuring homogeneous mixing;• Incubating the Petri dishes after solidification of the agar;• Adding a sodium chloride-magnesium sulfate (SM) buffer to the Petri dishes to recover phages, and collecting the phage plaques into sterile tubes;• Adding chloroform to the tubes after incubation in a shaking incubator and mixing to lyse bacterial cells and release the phages formed within the cells;• Centrifuging and filtering the supernatant through a sterile membrane to remove bacterial cells from the phage solution;• Preparing a film-forming solution by stirring sodium alginate in distilled water at room temperature;• Adding glycerol as a plasticizer and stirring at room temperature;• Incorporating the phage solution into the film solution and homogenizing by stirring at room temperature;• Subsequently drying and conditioning the film.