Periodic Report Summary 2 - SO2SAY (Replacement of sulphur dioxide (SO2) in food keeping the same quality and shelf-life of the products) Project context and objectives: The SO2SAY project will develop a strategy to replace the application of sulphur dioxide (SO2) or its releasing salts for the most relevant food products. Important aspects for food applications will be the maintenance of the sensory quality and shelf-life of the products produced without SO2 treatment. The main activity of SO2 is the prevention of enzymatic browning in foods, especially fruit and vegetable products. SO2 acts as inhibitor for Polyphenol oxidase (PPO), the main biocatalyst for oxidation of polyphenol to quinones, and parallel as reducing agent preventing further oxidation of the formed quinones. In addition, SO2 has strong antimicrobial power and preserves microbial spoilage. Three approaches will enable to dispense with SO2: i) inactivation of the enzyme PPO to prevent enzymatic browning; ii) development of plant extracts containing secondary plant metabolites with a high anti-oxidative capacity (e.g. plant polyphenols) and antimicrobial activity; iii) processing and packaging under an oxygen reduced atmosphere. These strategies are planned to be successful as stand-alone technologies, but more realistic are combinations of two or three technologies, depending on the food stuff. Tailored processes and technologies will be developed to meet the requirements of fluid and solid food products. SO2 is currently permitted for fruit and vegetable products, juices, beverages, wine, and seafood. The latter, however, is rarely produced using SO2. Instead, organic acids and modified atmosphere packaging are used to prevent browning. Thus, SO2SAY will focus on the most relevant food stuff currently treated with SO2: - fruits and vegetables; - wine and beverages; - snack and convenience products. To ensure the success of the SO2SAY project, all technological developments will be supported by an analytical work package taking care of sensorial analysis and of acceptable shelf-life of the novel developed products. In summary, the project will develop applicable technologies to replace SO2 in food. The SO2SAY approach will be based on fundamental scientific data and will enable the complete understanding of the molecular mechanism behind the novel technological applications to be developed in SO2SAY. Project results: The work initially started with the selection of model products to work within the project. Selection was done considering relevant consumption, amount SO2 used, as well as interest of the participating small and medium-sized enterprises (SME). Thus the following products were selected: dried apple rings, red wine grape juice (not for concentrate), apple fresh cuts, peeled and cut potatoes. For scientific work, it was decided to work with PPO derived from mushrooms and potato. The practical work follows three approaches; the first one is to reduce oxygen contact, the second one is the reduction of enzyme activity, and finally the application of reducing and antimicrobial agents. Thereby, each approach was followed alone and combined within the scale up and demonstration work at the end of the project. Afterwards, the most successful developments undergo shelf-life tests and consumer tests. Within the framework of reduced oxygen contact, several combinations of cover gases, packaging materials and modified atmosphere processing for apples and potatoes were tested. Therefore, potatoes or apples respectively were cut in a glove-box under an oxygen free atmosphere and packed in different plastics, metal, and ceramic bags. In addition these bags contained mixtures of cover gases such as nitrogen, argon, and carbon dioxide (CO2). Another approach was the application of edible coatings. Therefore, a large variety of carbohydrates and proteins were tested on fresh cut apples and potatoes. A unique approach was the application of CO2 saturated dip baths. Thereby, the CO2 is used to remove the oxygen from the water, to lower the pH value and the temperature. In order to reduce the enzyme activity ultrasound application was tested with cut potatoes. They were packed with a watery phase in plastic bags and treated with different ultrasound parameters. Grape must was tested in a continuous ultrasound flow cell. After ultrasound treatment the enzyme activity was measured with dopachrome assays. In addition, visual observation took place. In order to remove PPO grape must for red wine and grape juice was ultra-filtrated. Different numbers of membranes, in terms of pore size and surface were tested and a variety of pressures. Within the approaches to reduce enzyme activity several agents, based on a vast literature review, were tested towards their ability of forming complexes with the central copper ions of PPO and thus to inactivate the enzyme. To make sure that the effect is due to the formation of copper complexes Liquid chromatography - mass spectrometry (LC-MS) analysis was performed. With agents that form copper complexes practical trials on apple and potato fresh cuts was performed to test the in vivo effectiveness of these identified agents. The aim of this work was to establish the lowest dose necessary to inhibit enzymatic browning. Furthermore, detailed enzyme inhibition studies were performed. In cooperation with the University of Groningen, the crystal structure of mushroom derived PPO was revealed. Based on this work several modes of inhibition were suggested for the practical work. At the end, PPO from potatoes was isolated and purified. As SO2 possesses antimicrobial properties, as well as reducing capacities, a focus was set on plant extracts with these properties. In course of this, more than 50 plant extracts were screened towards their reducing and microbial activity. As these extracts consist of a large number of different compounds that can cause off flavour or off colouring, the most promising were fractionated to identify the most effective fractions and to apply these. Therefore, the extracts were autoclaved and tested in vitro assays and in vivo trials on selected model foods and in red wine. In addition the structure-function relationship of selected compounds was revealed. The most promising results were obtained for red wine with plant extracts and a scale up was performed at the participating winery for consumer tests. Also CO2 saturated dip baths were tested for apple fresh cuts on large scale. A third upscale was performed with extract treatment of potato fresh cuts at the participating SME beneficiary Gemuese Meyer production site. Potential impact: The SO2SAY project intends to establish procedures to be able to dispense with the application of SO2 and its releasing salts. This will lower the intake of sulphites for the European consumers and, thus, contribute to improved safety of foods and beverages. Microbial safety has to be safeguarded. The partners intend to develop plant extracts with antimicrobial property to meet the high quality standards of food producers and consumers. To reach the challenging objectives, highly scientific research is necessary. The research is divided into novel physical procedures such as oxygen exclusion in processing and packaging, ultrafiltration, high power ultrasound treatment, and novel chemical solution such as compounds from plant extract with reducing and antimicrobial activity, and novel biological alternatives such as targeted enzyme inhibition by copper chelation and biological available inhibitors of PPO. The project aims at a very broad application of the novel technologies. Depending on the nature, the form and the physical and chemical properties of the different foodstuffs different combinations of novel developed technologies will be applied. In order to demonstrate the high impact of this proposal all relevant food products that are currently produced under application of SO2 or its releasing salts. The products comprise different food products from fruits and vegetables including beverages and wine. Seafood is not included in the project because the current state of the art in seafood production does not include SO2 treatment anymore. But the SO2SAY project will have not only the direct impact on the fruit and vegetable processing industry. The technological developments can be easily transferred to other products as well. Exclusion of oxygen from the working environment, for example, might be applied in the pharmaceutical industry to prevent oxidation of active ingredients. The enzyme inhibition approach might also be applied for different undesired enzymatic procedures in food such as pectinase activity during fruit softening or even as medial approach for the treatment of diseases mediated by enzymatic activity. Reducing or antimicrobial plant extracts will have a potential in many different food applications, but also in pharmaceutical and cosmetic products. Suitable extracts will be able to replace synthetic food additives, which will increase the trust of the consumers in the whole food industry and will contribute to improved sustainability in the food production chain. The SO2SAY project will establish innovative processes and processing steps in traditional food processing. Many traditional fruit and vegetable products are processed on industrial scale using SO2 and sulphites to prevent enzymatic browning. SO2SAY will develop novel innovative processing steps that make the use sulphites unnecessary. Important aspects are the organoleptic properties of the products. The project can only be successful, if the products show the same organoleptic properties as untreated or SO2 treated products. The same counts of the shelf life. Only if a similar shelf life as SO2 treated products can be obtained, consumers will buy the novel products. The project also aims at improved sustainable processing of food and vegetables. Instead of using an inorganic additive such as SO2, novel reducing and antimicrobial plant extracts will be developed. These extracts will be mainly obtained from by-products of food processing and, therefore, contribute to on the ambitious way of total valorisation of food by-products. A third aspect considered in the SO2SAY project is improved packaging. Novel eco-efficient packaging technologies will be tested for the packaging of fruit and vegetable products. Aim is to use modified atmosphere and prevent permeation of oxygen through the package. In the same time, more eco-efficient packaging material will be tested in the SO2SAY project. In summary, the SO2SAY proposal will seriously contribute to improve the knowledge based bio-economy and, thus, strengthen the position of the European food industry on its way to establish competitive sustainable production processes and improve their position against competing economies from overseas. Project website: http://www.so2say.eu Other report summaries Periodic Report Summary 1 - SO2SAY (Replacement of sulphur dioxide (SO2) in food keeping the same quality and shelf-life of the products)
