Development of active edible coating and biodegradable packaging for food application Monique Lacroix, Ph.D. Professor Research Laboratories in Sciences Applied to Food INRS-Institut Armand-Frappier 531 des Prairies blvd. Laval city, Québec, Canada H7V 1B7 Monique.lacroix@iaf.inrs.ca www.iaf.inrs.ca Tel: 1 450 687 5010 ext 4489 International Conference and Exhibition on  Biopolymers and Bioplastics  August 11,2015, San Francisco, USA ©INRS, 2014

Edible coating: definition Primary purpose of food coating is to provides a barrier to microorganisms, to moisture, to gas and to solute migration in food. Edible coating is normally applied on food surface and where a thin layer edible film is formed directly on food surfaces or between different layers.

Edible coating: potential Edible coatings can Extend the shelf life of the food by the inhibition of the microbial growth and by the improvement of the quality of food system Preservation of bioactive nutrients Inhibition of oxidation (inhibition of gaz transfert) Preservation of physico-chemical (ex: texture, color) and organoleptic properties of food Protection of probiotic bacteria viability

Biobased packaging Packaging containing raw materials originating from agricultural sources produced from renewable, biological raw materials such as starch, cellulose and bio-derived monomers

Global market of packaging $ 417 Billion 100 000 industries 5 Millions employees Food Packaging represent 65% of the market USA: $100 Billion Japan: $80 Billion Germany: $29 Billion France: $19 Billion

Driving in coating and packaging innovation Increasing consumer demand for ready to eat foods Environmental issue: recycling, biodegradability Request for fewer or no additive and preservation Change in retail and distribution practices associated with globalization Stricter requirements regarding consumer health and safety Shelf life extension needed Cost-efficiency

Post-process contamination 66% of the post-process contamination is caused by Product mishandling Faulty packaging

PROBLEMATIC ISSUES The Center for Disease Control and Prevention (CDC) estimates that 48 million people get sick due to foodborne diseases in USA annually. In Canada, the foodborne illness is estimated as more than 11 million episodes/year → Therefore, controlling of food pathogens in food products are very important. Listeria Salmonella E.coli Campylobacter

Post-processing protection by Active packaging Active coating Has been proposed as an Innovative approach that can be also applied to ready-to-eat products to minimize or prevent the growth of pathogenic microorganisms

Active edible coating and packaging refers to the incorporation of additives or extracts from natural sources into packaging or coating systems to increase the shelf life of foods and then to provide a high-quality products (fresh/safe).

Active Coating and Packaging Active coating and packaging allow interaction with food products and the environment and play a dynamic role in food protection The demand for minimally processed food and ready to eat food and the distribution from the centralized processing pose major problems. Recent food borne microbial out-brakes are driving a search for innovative ways to inhibit the growth of bacteria in the food while maintaining quality, freshness and safety. One option is to develop food packaging where natural antimicrobials will be incorporated. Natural antimicrobials can be included in the films (bacteriocins, essential oils from spices, organic acids). Active packaging allows to interact with food product and the environment and play a dynamic role in food protection Active packaging have led to advances in many areas including delayed oxydation, control the respiration of fruits for example, control the growth rate of bacteria, and moisture migration. Other active packaging can absorb CO2, odors, Remove ethylene and aroma emitters, Absorb drip (ex: meat industry) However, research is needed because the addition of antimicrobial compounds in the films can impart the color and opacity of the films, decrease the mechanical and barrier properties of the films, and may alter heat sealing strength, adhesion and printing properties.

Delay oxidation Delay microbial growth Assure innocuity of foods Control the respiration Delay moisture migration Absorb CO2 Remove ethylene and aroma emitters Absorb drip Better protection of the food quality and reduce the waste level Active packaging Active packaging have led to advances in many areas including delayed oxydation, control the respiration of fruits for example, control the growth rate of bacteria, and moisture migration. Other active packaging can absorb CO2, odors, Remove ethylene and aroma emitters, Absorb drip (ex: meat industry)

Example of edible coating:barrier properties Rancidity Chocolate  firm Fat bloom Rejection by the consumer Return the product to the producer We have here an example of an application that we have developped for a company where this company was looking to avoid the problematic of rancidification of chocolat, to improve the shelflife and the quality of its products. Rancidification is the hydrolysis and/or autooxidation of fats into short-chain aldehydes of ketones which result in undesirable odors or flavors. In the chocolate the firmness can also be affected

edible coating: transport limitation of unsaturated fatty acids Chocolate almond Oil DEVRAIS-JE ENLEVER CETTE DIAPO?? This kind of problem can also appears in almond cover of chocolate where the oil of the almond will migrate/diffuse to the surface

Diffusion of oil based on the addition of various polymers Results Diffusion of oil based on the addition of various polymers

Milk proteins have high nutritional value They are available in large amounts world-wide They have been extensively investigated as edible coatings and films

Edible coating: antioxydant properties Application against the browning of fresh fruits and vegetables  enzymatic browning Stabilizing the whiteness of the product Control Coated Edible coatings can be applied on food surfaces as a thin layer edible film. Edible coatings can potentially extend the shelf life and improve the quality of food by the control of mass transfer, moisture and oil diffusion, gas permeability (O2, CO2), and flavor and aroma losses and by maintaining mechanical, rheological characteristics, color and appearance of foods. Edible film as a solid sheet can be applied between food components or on the surface of the food system in order to inhibit migration moisture, oxygen, CO2, aromas and lipids. Edible films with adequate mechanical properties could also serve as edible packaging for selected foods. The selected proteins are originated from animal and plant sources consisting of caseins, whey proteins, collagen and gelatin, plasma proteins, myofibrillar proteins, egg white proteins, soy protein, wheat gluten and zein.

Edible coating:antimicrobial properties Application against the growth of molds on strawberries Protective barrier against moisture  shelf life of strawberries Another example of bio-coating to protect the emergence of moisture on strawberries Base: Base+PLS:

Chitosan Natural polysaccharides, the second most abundant after cellulose Poor mechanical properties, lack of water resistance High water permeability High gases barriers It has a broad antimicrobial spectrum Effective carriers of many active compounds

Chemical modification of chitosan N-acylation of chitosan Functionalization of chitosan with fatty acid derivatives allowed  hydrophobicity and emulsifiying properties Stabilization of active compounds in chitosan (encapsulation matrix) According to Han et al. (2008)

Modified chitosan-based coating on strawberries b) PLA-NCC-nisin film b) PLA-NCC-nisin film 3450-3150 3450-3150 3450-3150 Modified chitosan-based coating on strawberries In situ antimicrobial activity RT, PM EOs and LIM were the most efficient preservative agents in strawberries during storage. Efficient method to preserve the quality of strawberries up to 12 days Evolution of the decay level (%) in antimicrobial coated strawberries during storage.

Modified chitosan-based coating on strawberries b) PLA-NCC-nisin film b) PLA-NCC-nisin film 3450-3150 3450-3150 3450-3150 Modified chitosan-based coating on strawberries In situ antimicrobial activity Appearance of strawberries coated with modified chitosan-based formulation containing limonene and emulsifiers.

Encapsulation for the preservation of Nutrients and functional products using modified chitosan

Retention of -caroten (%) during storage at 45 ºC and 100% RH after encapsulation with modified chitosan

LAB Protection during gastro intestinal passage  encapsulation in polymer Based on modified chitosan, Modified alginate pH 1.5 -2.5  10 6-10 7 10 9 Bacteria polymer

Viability of L. rhamnosus RW-9595M * * * * * * * * * (FC: Free BAL; NA: native alg.; SA: modified alg. ; SC: modified chitosan; PA modified alg.).

The use of edible coating in combined treatment to increase the antimicrobial property

Coating application of modified chitosan-based coating on ready to eat vegetables Natural compounds have been formulated into coating to evaluate their efficiency in enhancing the shelf life and eliminating pathogens in brocoli Coating formulations containing active compounds at concentration that do not affect the sensorial properties were sprayed on the surface of precutted brocoli before irradiation treatment

Radiosensitization of E Radiosensitization of E. coli on green bean samples as affected by coating formulation under various atmospheres Radiosensitization of S. Typhimurium on green bean samples as affected by coating formulation under various atmospheres

D10 values of selected pathogens and total microflora in broccoli florets coated with active coating Bacteria Control OA/LAB metabolites OA/FE OA/FE/SM OA/SE L. monocytogenes 0.4 0.29 0.3 0.27 E. coli 0.38 0.2* 0.16* 0.24 0.23 S. Typhimurium 0.50 0.29* 0.28* 0.25* Aerobic flora 0.57 0.36* 0.32* 0.33 OA: organic acid mixture; LAB: mixture of LAB ferment; FE: fruit extracts; SM: spice mixture; SE: spice extract Irradiation treatment from 0 to 3.3 kGy

on population of E. coli on green beans samples during storage at 4 °C Effect of bioactive coating containing carvacrol in combination with modified atmosphere packaging and gamma irradiation (0.25 kGy) on population of E. coli on green beans samples during storage at 4 °C   Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13 Control 2.98Aa 3.03Aa 3.10ABa 3.14ABa 3.18Ba 3.41Ca 3.95Da MAP 3.02Aa 3.19Aa 3.05ABa 3.01ABa 2.80Bb 2.98ABb 3.01ABb Coating (air) 2.45ABb 2.15Ab 2.57Bb 1.40Cb 1.25Cc ND Coating+MAP 2.64Ab 2.59ABc 2.30Bb 1.66Cb 1.19Dc γ (air) 1.71Ac 1.26Bd 1.18Bc γ +MAP 1.62Acd 1.45Be 1.19Cc γ+coating (air) 1.30Ad 1.35Ade 1.25Ac γ+coating+MAP The deposition of the bioactive coating on green bean samples caused an immediate reduction in E. coli population, which reached the value of 2.45 log CFU/g already on day 1 of storage. After 7 days of storage, E. coli population on coated samples was significantly lower (of about 1.7 log CFU/g) than in control samples at the same day. Remarkably, after 11 days of storage there were no detectable bacteria on coated samples, highlighting the strong bactericidal effect of the developed coating formulation, based on MC containing CN. The use of combined treatment of gamma irradiation and MAP did not significantly affect the effectiveness of gamma irradiation treatment alone: samples treated with gamma irradiation under MAP showed an E. coli population of 1.62 log CFU/g on day 1 of storage, and no detectable bacteria after 7 days of storage. Therefore, no significant difference can be noticed between irradiated samples packaged under air and irradiated samples packaged under MAP. The combined treatment of gamma irradiation and bioactive coating reduced E. coli population to 1.3 log CFU/g on day 1 of storage, with a significant reduction of 1.7 log CFU/g, as compared to control samples. This combined treatment also showed a strong residual antimicrobial effect, with no detectable bacteria after 7 days of refrigerated storage. The combined treatment of on green bean samples with gamma irradiation, bioactive coating and MAP exhibited the strongest antimicrobial effect against E. coli, with no detectable bacteria over the entire storage period. The combined treatment of MAP and bioactive coating showed a significant 1.5 log CFU/g reduction of E. coli population after 7 days of storage, as compared to control samples, with no detectable bacteria after 11 days of storage. Green bean samples treated with gamma irradiation doses of 0.25 kGy showed an E. coli population of 1.71 log CFU/g on day 1 of storage, with a significant reduction of 1.27 log CFU/g, as compared to control samples. Gamma irradiation treatment showed a strong residual antimicrobial effect already after 5 days of storage, with a microbial load reduction of 2 log CFU/g as compared to control samples, while after 7 days of refrigerated storage there were no detectable bacteria on treated samples. Values are means ± standard deviations. Means with different lowercase letters within the same column are significantly different (P ≤ 0.05), while means with different uppercase letters within each treatment lot are significantly different (P ≤ 0.05); MAP: (60% O2, 30% CO2, and 10% N2).

Bacterial population on refrigerated pizzas as affected by gamma irradiation and edible coating based on milk proteins 0 kGy 1 kGy 2 kGy 0 kGy 1 kGy 2 kGy C,3 days 2 kGy, 14D 1-2 kGy > 21 D 1 kGy,12D C,17D 1 kGy = shelf life extension of 9 days (from 3 to 12) 1 kGy = shelf life extension of 11 days (from 3 to 14) Coating + irradiation 1 kGy = shelf life extension of 18 days (from 3 to 21) Coating without irradiation shelf life extension of 14 days (from 3 to 17) Coating + irradiation at 2 kGy = shelf life extension more than 18 days (from 3 to > 21) Irradiation alone Irradiation + edible coating

The highly hydrophilic nature of protein coatings can limits their functional utilization Therefore, formations of cross- linked proteins can produce a strong, flexible film or coating.

Formation of bityrosine in calcium caseinate films as a function of irradiation dose Bi-tyrosine was produced during irradiation and the amount is directly related to the dose of irradiation.

Fraction of insoluble matter in function of the irradiation dose Results are expressed as the percentage in solid yield after soaking the films 24 hours in water Films were immersed in boiled water for 30 minutes and then dried in oven for 7 days at 45’C to determine the insolyble matter. An increase of the recuperation of the films was observed by increasing the dose of irradiation. 70% of recuperation was observed in films containing casein + whey , however 88% of recuperation was observed for films based on casein only.

Effect of crosslinked films based on milk proteins containing essential oils on E.coli 0157:H7 growth on beef Beef without film film with pepper pepper + origano extract ---Films containing Oregano based films was evaluated for their antimicrobial properties on meat. Pimento based films was also added to the films for their antimicrobial properties but also for their high antioxidant activities. ---The growth of E.coli was evaluated during storage. Results showed that oregano based films was THE MOST EFFECTIVE AGAINST the growth of E.coli and were able to produce a 1.12 LOG REDUCTION OF E. COLI O157:H7 LEVEL on beef during storage. Origano extract

ADFs: New generation of antimicrobial device Trilayer film PCL/MC/PCL CNC filling in MC matrix Encapsulation of natural antimicrobials + Synthesis of Antimicrobial Diffusion Films (ADFs) (to get advantage from complementary functional properties of each component and process) Characterization and application

Preparation of trilayer ADFs as diffusion devices Principle scheme of compression molding process to prepare composite trilayer ADFs (MC film content = 30% w/w, dry basis).

ADFs on fresh broccoli Percentage of total phenolics (TP) release from ADFs during storage FTIR spectra of bioactive ADF internal layer in fingerprint area (1200-1800 cm-1) for the estimation of TP release (diffusion of volatiles). 1600 1515 1265  Day 0  Day 2  Day 6  Day 13 FTIR analysis of volatiles diffusivity of antimicrobials encapsulated in ADFs (from day 0 to day 14). Continue diffusion (controlled release) of volatiles can be monitored by quantification of FTIR bands: Aromatic stretching (1600 and 1515 cm-1) Ester antisym stretching (1265 cm-1)

ADFs on fresh broccoli Percentage of total phenolics (TP) release from ADFs during storage TP release (%) from bioactive ADFs during storage, deduced from TP availability in films by Folin-Ciocalteu‘s method. Slow diffusion of antimicrobial volatiles towards headspace environment Slight  of diffusion to 14-17% Good correlation obtained between the 2 methods (FTIR at 1600 cm-1 vs Folin-Ciocalteu)

ADFs on fresh broccoli Microbiolgical analysis Antimicrobial effect of trilayer ADFs on E. coli during storage of broccoli (12 days at 4°C). Total inhibition of E. coli at day 12 Stronger effect of formulation A at day 4

ADFs on fresh broccoli Microbiolgical analysis Antimicrobial effect of trilayer ADFs on S. Typhimurium during storage of broccoli (12 days at 4°C). Total inhibition of S. Typhimurium at day 7 Stronger antimicrobial efficiency against gram- negative bacteria

. Summary Edible coating and Biodegradable packaging based on Natural polymers can be used To protect food quality To carry natural antimicrobial compounds The functionalisation of the polymer can improve the protection and the release rate of the immobilized active compounds Crosslinking reaction of natural polymers can improve the physico-chemical properties of the films and their stability during storage time of the packaged food

. Summary ADFs (trilayer assembly) and encapsulation of natural antimicrobials showed strong inhibiting capacity against E. coli and S. Typhimurium over storage. These films could further be explored in food applications to prevent pathogenic contamination during storage of fresh food, based on a controlled release of volatiles into headspace of packaging.

Summary Edible active coating and packaging could be used in combination with modified packaging and pasteurization treatments to increase the bacterial sensitivity and to assure food safety

Research Laboratories in Sciences Applied to Food Monique Lacroix, Ph.D. Professor/Director Research Laboratories in Sciences Applied to Food Canadian Irradiation Centre INRS-Institut Armand-Frappier 531 des Prairies blvd. Laval city, Québec, Canada H7V 1B7 Monique.lacroix@iaf.inrs.ca www.iaf.inrs.ca Tel: 1 450 687 5010 ext 4489