Diversification for Tobacco Growing Farms by the alternative crop Stevia rebaudiana Bertoni

Project Information

DIVAS

Grant agreement ID: 232250

Closed project

Start date 1 October 2009

End date 31 January 2012

Funded under

• Specific Programme "Capacities": Research for the benefit of SMEs

Total cost € 1 496 858,20

EU contribution € 1 155 937,45

Coordinated by

UNIVERSITAET HOHENHEIM
Germany

Final ReportSummary - DIVAS (Diversification for Tobacco Growing Farms by the alternative crop Stevia rebaudiana Bertoni)

The CAP reform for the tobacco sector (EG No. 864/2004 from 29 April 2004, effective on 1 January 2006) has the aim to phase out the subsidy payment for tobacco cultivation. This will have a great impact on the tobacco growers in terms of income and employment. The project addresses a specific scientific and technological problem, as well as societal. It is exploring the possibilities to re-place partially the tobacco production by the production of Stevia rebaudiana. Therefore, the DIVAS proposal has the strategic objective to solve a problem of livelihood for EU tobacco farmers integrated in the DIVAS project (SME partner). The SME partners of the DIVAS proposal represent a total of 9.7 % of the tobacco farms in Greece, Italy, Portugal and Spain which cultivate together about 5 % of the tobacco cultivation area of the above mentioned countries.

This shall be done by setting up a consortium of seven SMEs of the agricultural tobacco sector and eight subcontracted RTD performers with complementary expertise responsible for the execution of the following work packages:

WP2 - Field trials: Evaluation of five Stevia rebaudiana varieties yield potential in selected tobacco growing region in EU
WP3 - Specification: Chemical characterisation of stevia leaves to develop an European standard for the stevia leaves for a novel food approach
WP4 - Consumer acceptance: Sensory evaluation, antioxidative potential examination and consumer expectations
WP5 - Market research: An up-to-date market potential assessment for Stevia rebaudiana and related products shall be compiled
WP6 - Dissemination: To develop a trans-European cooperation of EU tobacco farmers within the Stevia business
WP7 - Training: Set-up a network for vocational training on the needs of the tobacco farmers to match the impact of the tobacco reform on the educational level.

The results will transfer producers growing unhealthy plants to a new stage - the protection of human health.

Project context and objectives:

WP2 - Field trials: Evaluation of Stevia rebaudiana yield potential in selected tobacco growing regions in EU

The objective of this work package was to execute a set of field trials which will enable the SMEs to implement in future Stevia rebaudiana as an alternative crop to tobacco after the completion of the tobacco reform. The executed field tests were designed to bridge knowledge gaps collect data which are essential for quality issues and future farmer training on the crop. In the first reporting period, the variety trial was executed with four varieties and there was a delay in delivery. In the second reporting period, the variety trial was executed with five varieties in four locations and four varieties in another three locations. Additionally, five varieties have been tested in five locations. Four varieties have been produced from seeds (variety 3, 4, 5, and 6) and the other varieties by tissue culture. For the tissue culture, only a limited number of plants could be produced. Because of that, it was not possible to have plants in all locations. Variety 1 from the test in 2010 was not further available. It seems that the supply company disappeared. Variety 2 was excluded from testing as this variety brought up the sanitary problem in the first reporting period.

WP3 - Specification: Chemical characterisation of stevia leaves

WP 3 achieved a complete phytochemical characterisation of Stevia rebaudiana leaves. Around 60 secondary metabolites were identified. Among those present in significant quantities were chlorogenic acids, flavanoid glycosides, lipids and steviol glycosides with mono- and sesquiterpenes present as trace compounds. A full quantification of phenolics, and steviol glycosides and other chemical constituents was carried out providing detailed data on variances and compound profiles forming a scientific basis for a future EU specification for stevia as novel food.

WP4 - Consumer acceptance: Sensory evaluation, antioxidative potential examination and consumer expectations

The sensory quality, consumer acceptance and antioxidant properties of stevia leaves cultivated in different European countries in different stevia plant varieties and harvesting periods as well as the antioxidant activity of selected foods and beverages prepared with stevia leaves extract were evaluated. The sensory taste profile of the stevia leaves extracts showed generally a tendency to short lasting and low development of sweetness, flat to satisfying mouth feeling, perceptible herbal taste, low to perceptible bitter taste, low astringency, low liquorice, metallic, caramelic, sour and mentolic taste and slightly appearance of off-notes. The consumer acceptance was generally satisfying. There could be observed a significant antioxidant activity in all of the stevia leaves extracts. Generally, the extent of the antioxidant activity was rather depending on the origin of cultivation of stevia than on the stevia variety. The antioxidant properties of foods and beverages could be improved significantly by the application and incorporation of stevia leaves extracts.

WP5 - Market research

A market research was carried out in order to provide a list and characteristics of the large companies in all EU Member States as well as to present and analyze the world sugar and sweeteners' market and investigate the potential of stevia in the European market. The contacted research points out and sheds light on major concerns and views. In order to facilitate the introduction of stevia in the EU, tobacco cooperatives, along with F&B national associations, should consider familiarising the public with its characteristics and try to fill in the current knowledge gaps, mainly concentrated on issues like 'taste development', 'regulations' and 'health and safety'. According to the findings of the research, sugar gathers the most possibilities of being replaced by stevia , while the powdered extract is the leading choice on account of its desirable form. Without doubt, the future of stevia depends on the information provided to both companies and consumers. In addition, attention ought to be paid on how to best cope with competitive products, as this will be crucial for the future of stevia.

For the implementation of Stevia rebaudiana as an alternative for tobacco growing regions the specific EU rules for rural development projects according to the requirements for the Rural Development Plans 2007-2013 are essential. In the actual rural development plan, there are specific rules for each country and each region. Every Member State has now the possibility to set up specific regulations by its own. The first step was to gather the necessary program information through the SME partners. For set up a business plan every SME partner filled a questionnaire on its properties and future investment plans. A business plan was developed

WP6 - Dissemination
It was finally possible to make the signature at the notary on 23 January 2012 in Stuttgart. It was decided to localise the EURO-STEVIA EEIG in Germany. It was decided that ones the EURO-STEVIA EEIG is registered the Greek association will be associated partner.

WP7 - Training
The developed training program combines specific training on Stevia rebaudiana with general vocational training on agricultural practise. The training shall be set up on modules to different aspects of agriculture practise. It is recommended to set up eight modules each for one week with different time scheduling for each course (full time, afternoon) depending on the thematic.

Project results:

Evaluation of yield potential of five Stevia rebaudiana varieties in selected tobacco growing regions

Objectives: Five different varieties, which are differing in their sweetener pattern and their origin, shall be tested on the yield potential in selected tobacco growing regions.
Results: The overall result shows that a cultivation of Stevia rebaudiana in the seven tobacco growing regions representing in the DIVAS project is a feasible undertaking.

A dry leaf yield of about 3000 kg/ha seems to be achievable with a plant density of 44 300 plants/ha (75 x 30 cm) and without fertilisation. An adequate fertilisation will allow to expect yields of about 4000 kg/ha.

One objective of WP2 was to identify one or two best suited stevia varieties. In that respect, the Steviolglycoside content variation for variety 1 was about 19 %, for variety 2 about 28 %, for variety 3 about 24 %, for variety 4 about 20 %, for variety 5 about 41 %, for variety 5 about 43 % and for variety 7 about 38 %. This is a preliminary result as some varieties have only been tested for one year.

It was shown that the varieties produced by tissue culture (variety 8 - 13) have a quite slow growth and a low homogeneity between the locations tested beside variety 13 (about 8 %). In general only one harvest was possible. It is advocated in the 'stevia world' to use vegetative propagated plant material for commercial plantation. This may be feasible in countries with very low labour costs like in China.

The overall productivity of all trial locations ranges between 400 kg/ha to about 500 kg/ha between the seven tested varieties. This is the result without fertilisation and with a relatively low plant density.

It should be possible to achieve as an average about 500 kg/ha and year of Steviolglycosides. The average Steviolglycoside content over all locations and varieties was 17,5% which requires 5,7 kg of Stevia leaves to produce on kilogram of Steviolglycosides. The Chinese factories calculate with about 20 kg Stevia leaves per one kilogram of Steviolglycosides. This brings a great competitive opportunity for the EU Tobacco growers.

Evaluation of soil matrix potential on growth and yield of Stevia rebaudiana

Results: The results confirmed the outcome of previous research (1987 - 1992, first irrigation trials in Spain by Dr Udo Kienle; 1998 - 2002 FAIR5-3751 with irrigation trials in Portugal at UALG and Spain by UHOH). Relatively low supply by irrigation or rainfall creates still considerable yields. No significant difference could be established between the highest and the lowest water supply in the FAIR5-3751 project using drip irrigation. The soil moisture was controlled by TDR and the irrigation was varied by using drippers with different outflows (2 - 8 l/h) and the irrigation time was controlled by timers. The lowest water supply was in the FAIR trial 176 mm where in the DIVAS trial it was 183 mm. The second confirmation of previous results is that the Steviolglycoside content of the Stevia leaves is mainly unaffected by the water supply. In the DIVAS trial, two significant differences in the second cut was found, which can be considered as weak. These two significant results occurred to irrigation stop because of the high wind speeds. It is obvious that a commercial cultivation in the EU irrigation is necessary (it is a myth that in other regions of the world a good stevia yield can be obtained without additional irrigation). To run successfully a stevia crop under irrigation two simple rules must be followed.

Influence of soil salinity on growth and yield of Stevia rebaudiana

Results: The salinity of the irrigation water (ECw) significantly reduced plant growth and yield, as well as the sweetener content of the leaves, reducing the sweetener yield from stevia crop. Considering the period until the first harvest, plants show a tolerance to ECw around 3,8 dS m -1, and a sensitivity around 7,5 %/ dS m -1. However, when analysing the whole cultural period the yield decreased from the lowest water salinity value (ECw of 0,4 dS m-1), and no tolerance or sensitivity could be determined by applying Maas and Hoffman model.

Crop rotation experiment

Results: The result from the crop rotation task shows that a legume before stevia cropping is beneficial for the stevia plant. For corn and what no problems in yield could be observed.

Evaluation and characterisation of diseases and pests

The task is dedicated to record and control diseases and pests that may affect Stevia rebaudiana varieties that will be evaluated in this project. Differences in the susceptibility of the tested varieties to any diseases that may appear will be also recorded. The task is dedicated to record and control diseases and pests that may affect Stevia rebaudiana varieties that will be evaluated in this project. Differences in the susceptibility of the tested varieties to any diseases that may appear will be also recorded.

Results: Task leader surveyed all fields during 2011 vegetation period. During those surveys, the most serious disease recorded was a leaf spot disease mainly caused by Septoria sp. Almost all varieties were severely affected, except variety 7 that showed only a few symptoms. Also, the soilborn Sclerotium rolfsii was severely affecting plants, causing collar rot, wilt and plant death; however its incidence was restricted. Three Fusarium species (F. oxysporum, F. solani and F. semitectum) were isolated from a few plants with vascular diseases. While Verticilium sp and Alternaria alternata was isolated from one plant, however they were only of minor importance. None of tomato spotted wilt virus (TSWV) Potato virus Y (PVY), cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV), or tobacco mosaic virus (TMV) was detected in any of the trial sites, when testing plants (300 collected in 2010 and 1450 collected in 2011) with or without symptoms. The leaf borers Pieris endusa (Lepidoptera: Pieridae), Spondoptera sp and an unidentified larvae of the Geometridae family were identified; however, they were of minor importance. Slags and snails attacked Stevia, causing direct effect (leaves consumption) and indirect effect (top necrosis, stunting and reduced growth). Leafhoppers (Cicadellidae subfamily Deltocephalinae), leaf beetles (Coleoptera: Chryssomelidae) and shield or stink bugs (Hemiptera: Pentatomidae) were the possible cause of discoloration symptom observed in some trials. Cuscuta sp (Convolvulaceae) successfully infested a few stevia plants. Symptoms of high salinity, heavy soil and excess or lack of water were also observed. No differences among varieties were observed. Finally, when tested in the laboratory, stevia plants were successfully infested by the broad mite Polyphagotarsonemus latus (banks) (Acari: Tarsonemidae), the tobacco thrips Thrips tabaci Lindeman (Thysanoptera; Thripidae), and the silverleaf whitefly (Bemisia tabaci) (Homoptera: Aleyrodidae). The tobacco aphid Myzus nicotianae Blackman (Hemiptera: Aphididae), and the tomato leaf miner Tuta absoluta (Lepidoptera: Gelechiidae), failed to infest stevia.

Soil and plant analysis

Results: All used soils for the trial execution revealed a very low or low nutrition content by both macro and micro nutrients. Only very few nutrient sample content at some partner places showed a sufficient or a high content. Therefore it can be stated that the achieved yields reflects a certain 'zero' level which gives a hint on the natural productivity of the Stevia plant. It is expected that with a correct fertilisation of the stevia crop the productivity can be increased.

Common agricultural knowledge is that for soils with a pH > 6,0 a contamination of food by up-take of heavy metals by the root-system is unlikely if the soil serves with the following characteristics:

a) cadmium maximum 2 mg/kg soil (value measured after soil extraction with aqua regia);
b) plumb maximum 250 mg/kg soil (value measured after soil extraction with aqua regia).

None of the soils of the trial locations achieved such values. Anyhow, the possible contamination routes for heavy metals must be carefully considered and all actions avoiding any contamination must be incorporated in a HACCP manual.

From the harvests the nutrient content was analysed. The uptake of phosphorus and potassium are in the range of the findings of the FAIR5-3751 project. Demand of copper, manganese and zinc is low and deficits can be easily encountered with leave top spraying. The content of magnesium is very high and comparable with foods with a high content, like most vegetables and fruits.

Statistical analysis of field experiments

Plant mortality presented a strong variation according to the location, but in each location there was a relatively similar level of mortality of the tested varieties. Considering the main yield variable - leaves dry weight -three varieties reached more than 3 t ha-1.

WP3 - Specification: Chemical characterisation of stevia leaves

Five varieties with a specific sweetener pattern will be tested in WP2 at eight locations of Tobacco growing regions.

Results: Compound identification was in all cases carried out by LC-MS. On the mass spectrometrical side two experiments were carried out each, firstly a high resolution mass measurement (HR-MS) using an ESI-TOF-MS experiment that allowed determination of molecular formulae based on the accurate mass measurements. Molecular formulas were in general accepted if an error below 5 ppm was experimentally observed, as accepted by all peer reviewed chemistry journals. In a second experiment tandem MS experiments were carried out and the observed fragmentation patterns compared to those of authentic reference materials (either obtained commercially or from our own laboratory). After obtaining multi-dimensional information of four parameters on chromatographic retention times, UV-spectra (UV-VIS DAD detector coupled to LC-MS system), HR-MS and tandem-MS data comparison to authentic reference material did allow compound identification. For selected unknown compounds, spectroscopic MS rules developed in our laboratory was used to elucidate structures. All quantification method used in this project were prior to use subjected to a validation procedure unless stated otherwise. The validation procedure involved determination of limit of quantitation, determination of linear range for quantitation, repeatability studies including multiple injection and day to day repeatability. Additionally, inter sample repeatability experiments were carried out. Cross validation was carried out previous to this project on the chlorogenic acid quantification method.

Polyphenol analysis by LC-MS

Results: Polyphenols in stevia leaves were subjected to a detailed investigation. A total of forty polyphenolic secondary metabolites could be identified in stevia including twelve flavanoid glycosides, three hydroxycinnamic acids and twenty five chlorogenic acids (hydroxycinnamate esters of (-) quinic acids). The twenty five chlorogenic acids comprise mono-, di- and triacyl quinic acid derivatives, all known to be present in other dietary materials, most notably coffee beans. The work using stevia leaves not cultivated in this project has been published in the course of this project. The twelve flavanoid glycosides presents could be tentatively identified as such. All twelve derivatives were different to all available authentic reference standards. However, detailed tandem MS analysis combined with hydrolysis experiments clearly showed that the compounds in stevial are based on the aglycones quercetin, luteolin, apigenin and keampferol, bearing a series of hexose, deoxyhexose and pentose sugar moieties. From a toxicological point of view, it is well established that flavanoid glycosides undergo complete gut flora mediated hydrolysis to the aglycones, with all four aglycones present in Stevia, being toxicologically evaluated, since present in many edible fruits and vegetables.

Lipid analysis

Results: Firstly, total lipids were determined gravimetrically after extraction with a non-polar solvent. Secondly, the total lipid fraction was subjected to acid hydrolysis and derivatisation followed by GC-MS analysis to identify the individual fatty acid spectrum of all seven stevia varieties and of representative samples from all origins. Oleic acid was found to be the predominant fatty acid in stevia with an average ration of saturated to unsaturated fatty acids of 1:3.8. It should be noted that in plants fatty acid composition is reported to be a consequence of climatic conditions, in particular temperature, rather than botanical variation. This work was complemented by a MALDI-TOF-MS anaylsis of the lipid fraction allowing identification of selected intact lipids, mainly triacylglycerides of oleic acid. Finally, direct GC-MS analysis of the lipid fraction was carried out supplemented by a GC.MS analysis of a steam distillation extract of Stevia rebaudiana leaves. This analysis allowed positive identification of around twelve mono- and sesquiterpenes using a NIST library search.

Sweetener analysis

Results: From each plot of the variety trial the Steviolglycoside content was analysed using HPLC. Additionally, sample for the irrigation trial and the salinity trial have been analysed. For the DIVAS project, the EU standard method for analysis was used. This was done in respect to generate data in line with the current EU legislation. However, the EU standard method is good for pure Steviolglycosides but difficult for plant samples. However, with a new sample preparation method improvements have been achieved.

Other stevia varieties composition

In this subtask task all other chemical composition parameters will be measured which are not mentioned in tasks 3.1.1. to 3.1.4.

Results: The composition of the stevia leaves was determined for those samples where all analysis values are already obtained between 74 to 90 % of the dry substance. Most of the samples have a composition determination of about 80 to 85 %. This is a very good result so far achieved. A final result can still not be drawn as not all analysis results have been obtained so far due to the delay in sample delivery and subsequent sample analysis.

WP4: Consumer acceptance: Sensory evaluation, antioxidative potential examination and consumer expectations

In this work package the expectations for consumer acceptance will be evaluated. This evaluation will cover two aspects and will therefore provide new knowledge:

- sensory evaluation of consumer expectations in respect of taste, flavour and sweetness characteristics of the stevia varieties tested in WP2;
- evaluation of the antioxidant activity of food prepared with the stevia varieties tested in WP2.

The objectives were to evaluate the sensory quality of stevia cultivated by the project partners (cooperatives) of the EU-project in different European countries in different plant varieties and harvesting periods with consumer sensory panel tests and to determine the consumer acceptance in this respect. Furthermore, the antioxidant properties of the stevia of the different varieties as well as selected foods and beverages prepared with stevia from the field trials of the project are evaluated. Additionally, the sensory profiles of stevia samples from other countries that were not cultivated in the field trials of the EU-project were evaluated in sensory panel tests. With respect to the antioxidant tests, additionally various commercially available food samples which were commercially prepared with stevia or Steviolglycosides as sweetening agent were examined for their antioxidant activity. Of all the different sensory taste parameters the term 'pleasant impact' is chosen as key parameter representing the highest informative sensory-related value and expressiveness and is most significant and synonymous in regard to the 'consumer acceptance'. Generally, in this regard all the stevia varieties showed throughout satisfying taste profiles with some outstanding taste parameters and exceptions depending on the stevia varieties and stevia cultivation origin.

Sensory profiles of stevia from other countries (not cultivated in the field trials of the EU-project)

Results: The sensory profiles from stevia from other countries (Turkey, India, Kenya, Crimea) which were not cultivated in the field trials of the EU-project show generally similar taste characteristics than the stevia from the field trials of the project. Within the obtained stevia samples the samples from India and Turkey show relatively higher herbal notes and off notes. Regarding the stevia samples from Turkey additionally a strong smell and taste of peppermint was noticed. The consumer acceptance scores are in the midrange. Compared with the stevia samples obtained in the first period the stevia from Argentina haves a significant higher pleasant impact than all others, whilst the stevia from Paraguay have a lower midrange score.

Antioxidant activity of stevia and stevia sweetened foods

Results: The antioxidant activity of the stevia obtained from field trials of the project DIVAS was determined by the DPPH radical scavenging assay. Analogous antioxidant tests were performed with various commercially available yogurt samples and beverages and teas with sucrose, stevia or Steviolglycosides as sweetening agent. Representative foods and beverages from different food categories were prepared in the laboratory by adding and incorporation of a selected and suitable stevia of the project DIVAS in appropriate concentration. Thereof, the antioxidant activity and radical scavenging properties were evaluated in comparison to analogous food and beverages prepared with sucrose as sweetening agent. Generally, the extent of the antioxidant activity of the different stevia samples is rather depending on the origin of cultivation than on the stevia variety. By comparing the antioxidant activities of the different stevia varieties there is a general distribution over the whole range of antioxidant activities. In the harvest year 2010, the stevia varieties 1, 4 and 3 show values from the upper to the lower range depending on the origin, whilst the stevia variety 2 is located in the very low range. In the harvest year 2011, the stevia varieties 3, 4, 5 and 6 show values from the upper to the lower range depending on the origin, whilst the stevia variety 1 and 7 is located in the very low range.

Antioxidant activity of stevia from other countries (not cultivated in the field trials of the EU-project)

Results: The antioxidant activity of stevia samples from other countries (Turkey, India, Crimea, Kenya) which were not cultivated in the field trials of the project is situated in the mid to lower range compared with the stevia samples from the project which means that they have minor antioxidant potency than most of the stevia samples of the field trials. In comparison to the stevia leaves, samples from Argentina and Paraguay which were analyzed in the first period the Stevia sample from Paraguay has similar antioxidant activity than the samples of Turkey and India, however the stevia sample from Argentina has a significant higher antioxidant activity than all other stevia samples which is even slightly higher than the most antioxidant active stevia samples from the field trials.

Antioxidant activity of commercially available foods and beverages

Results: Regarding the antioxidant capacity of selected commercially available yogurt samples no significant difference is observed between yogurts sweetened with sucrose or the Steviol glycoside Rebaudioside A. There are only slight differences between the different fruit versions which may be due to the different ingredients of these products.

From the selected commercially available beverages sweetened with sugar or Steviol glycosides or stevia extract the tea beverages with natural stevia extract as sweetening agent have pronounced higher antioxidant activity compared with teas sweetened with sugar or Steviol glycosides. Although the highest antioxidant activity shows a green tea beverage sweetened with Steviolglycoside this fact can be referred to the exceptional high antioxidant capacity of green tea itself. From the commercially available orange drinks, the beverage sweetened with sugar shows lower antioxidant activity than its Rebaudioside A sweetened version which may be due to the different composition of both drinks.

Antioxidant activity of foods and beverages prepared with stevia from the field trials of the EU-project

Results: From different food categories the following selected and representative foods and beverages were prepared in the laboratory by adding and incorporating stevia for the evaluation of their antioxidant activity in comparison to analogous food and beverages containing sucrose as sweetening agent: black tea, lemonade drink, beer, yogurt and cake.

Generally, it can be stated that all of the selected foods and beverages that are sweetened with the stevia have higher antioxidant activity than their analogous prepared sample with sucrose as sweetening agent. This finding is underlining the fact that by application of stevia the antioxidant activity of foods and beverages could be improved.

Of all the selected foods and beverages black tea has highest antioxidant activity which is known to be due to the polyphenol ingredients of tea. By the addition of stevia, the antioxidant potential can be further improved significantly. The lemonade drink prepared with sucrose has no antioxidant activity at all. By the incorporation of stevia, a significant antioxidant activity of the beverage could be observed. Yogurt prepared with stevia shows slightly higher antioxidant activity than yogurt prepared with sucrose. In general, the obtained antioxidant activity values can be compared with the values obtained from the commercially available products. The baking product cake shows relatively low antioxidant activity at all. However, by addition of stevia as sweetening agent the antioxidant potency could be elevated in comparison to sucrose sweetened cake samples. Beer prepared with addition of stevia shows higher antioxidant activity than conventionally prepared beer.

WP5 - Assessment of potential market of stevia products in Europe

Results: ? three-stages survey was designed to understand the currently held perceptions and trends about stevia in the European Union, covering markets, technology, knowledge gaps, sweeteners' substitution by stevia, market drivers and limiters, and types of information needed in order to evaluate the dynamics of stevia and estimate its potential demand.

In order to measure the above-mentioned factors, a questionnaire was designed.

The selection of the participants in the survey was done, taking into account the objective of the project. For this reason, the research was concentrated on large companies from the food and beverage sector, which certainly have a big demand of food ingredients.

Participants were searched for through Internet, in specialised databases such as database for EU companies, EU-business and Europages, the Association of Chocolate, Biscuit and Confectionery Industries (CAOBISCO), the Association of German Confectionery Industry (BDSI), the Royal Belgian Association of the Biscuit, Chocolate, Pralines and Confectionery (CHOPRABISCO), the Spanish Professional Association of Biscuit Manufacturers, the British Soft Drink Association, the European Fruit Juice Association, the Union of European Beverages Association, the European Dairy Industry (EDA), the European Non-alcoholic Beverage Association (UNESDA), the Federation of European Union Manufacturers and Suppliers of Ingredients to the Bakery, Confectionery and Patisserie Industries (FEDIMA).

National associations of the European countries were also contacted, in order to help the collection of responses. For this reason, an invitation letter was sent by email to the associations, asking them to contact their members in order to collect more answers.

This paper presents the results of the third stage of the survey which was focused on 43 companies along European Union in order to assess the potential stevia market among European countries. The data were gathered by electronic or phone communication, by post communication and by personal on sight visits.

Statistical methods were used to summarise and describe a collection of data gathered from the respondents. Each item was analyzed separately or in some cases item responses were summed-up to create a score for a group of items. The questionnaire is formed by 10 questions and was designed to measure the market potentials of stevia products.

The first two questions determine the main sector of the participating companies' activity and the use of sugar and sweeteners in the production process. The next five questions were designed to measure perceptions regarding stevia. In particular, questions No. 3 and 4 explore the interest of the participants as far as the substitution of sugar and sweeteners by stevia is concerned. This will help determine if they have any pre-conceived notions about stevia glycoside. The following three questions (5 - 7) are closed ones, designed to estimate the factors that affect the subject's perception of stevia. They measure variables on a five point Likert scale applied to a group of items. The responses to these questions will determine the important drivers and limiters for stevia market. There is also a question about the preferred form of stevia's supply, and participants have to choose between dried leaves, liquid extract or powdered extract, the one that best suits in company's production. The last two questions measure the potential demand and the impact of different types of actions on stevia's market growth, such as advertising, trade shows and events, industry publications, guarantee offers etc.

All the survey's participants are using sugar in company's production process, while the 51 % of the companies are also using high intensity sweeteners, such as aspartame, sucralose, acesulfame K etc. The percentage of country of origin of the participated companies.

The majority of respondents (33 %) operate in the confectionery sector, followed by the soft drinks producing companies, representing the 21 % of the sample. Companies having as their main activity the production of biscuits, represent the 16 % of the sample, while companies operating in the dairy sector and those producing chocolate, represent 12 % and 9 %, respectively.

Companies were asked whether the replacement of the so far used high intensity sweeteners by stevia would be an option in the future, regarding the features of stevia. The interest of the sample companies in this replacement was measured using a five-point Likert scale, while answers range from 'not at all interested' to 'very interested'. The majority of respondents (54 %) showed interest in the suggested replacement. More specific, 35 % indicated an above average interest and an additional 19 % showed great interest. On the other hand, a sum of 25 % of sample companies, were not very interested or not at all interested in replacing an artificial sweetener by stevia.

Substitution of high intensity sweeteners by stevia is of a major concern from the perspective of the market and will certainly have a significant impact on the already well established sweeteners market. To measure the reaction of companies in the replacement of each single high intensity sweetener by stevia, participants were asked to define the percentage of each sweetener's market that could possibly be substituted by stevia in the production process.

Five intensely - sweet sugar substitutes were proposed to be replaced by stevia products, based on their popularity of usage. These are aspartame, sucralose, acesulfame potassium, saccharin and cyclamate.

Sugar was also proposed to be an ingredient that could be substituted by stevia, proposed primarily for all those companies not using high intensity sweeteners in production process (49 %). Sugar indicated the utmost possibility of being replaced by stevia. Companies indicated that they could even substitute a small percentage of the already used sugar (10 - 30 %). It's worth mentioning that sugar is used in production process by the total amount of the respondents, in opposite to high intensity sweeteners that are used only by 51 % of the respondents. Aspartame and acesulfam K are two products, which showed highest possibility of getting replaced in high percentage (90 %).

To study the critical factors that make stevia a preferred option for the companies, participants were asked to determine the factor, between price and product features, that would make stevia a better option and finally lead to a substitution of the already used sweeteners. The participants had also the convenience to express themselves, by indicating any other possible factor, other than the ones already given.

It is remarkable that the 60 % of the respondents pointed out that product features are those to make stevia a preferred option and companies showed great interest in them. An additional 23 % states quite the same, by giving a positive answer, described as an 'above the average preference' in product features.

In order to understand what companies think about the replacement of already used sweeteners by stevia, and its introduction into their productive line, five factors were proposed to be a major concern for the companies. These are price, taste, reliability of the supply, regulations and consumer acceptance. The participants could also name any other factor they think is worth to mention as a concern.

The survey addressed these concerns as the major ones in stevia market at present:

- taste issues;
- the impact of regulation; and
- consumer acceptance.

In the sweetener industry, taste is the key to consumer acceptance, which is why it's not surprising to see respondents indicate that their top concern about stevia is its taste. This concern can emerge as a number one limiting factor for stevia's market growth in the future.

Additionally, regulations and the fact the stevia is not available at present for use as a sweetener, is also a major concern for companies. This may be as well the reason why companies are having second thoughts about its introduction in the production process.

Knowledge is what a company needs in order to overcome any difficulty that may come in its way and can always lead to its success. Knowledge gaps seem to hold back the development of a company, as no company is willing to risk anything without being well informed in the first place.

To evaluate the existing knowledge gaps that companies will have to face, participants had to rate each single knowledge gap and point out whether it is important to have this kind of information, in order to decide whether to use stevia.

Through literature search, the most important knowledge gaps for a company, in order to make its decision, are eight. These are:

- understanding the market
- regulations
- cultivation technology
- product development
- taste development
- extraction and refining technology
- economic issues
- health safety.

Respondents clearly indicated that taste development and health and safety issues are the two most important knowledge gaps followed by regulations. Respondents also indentified product development as a major knowledge gap, while economic issues, understanding the market, extraction and refining technology and cultivation technology don't seem to concern the companies so much.

As stevia comes in several forms (liquid extract, powdered extract, dried leaves), companies are now able to decide which one is more suitable for their product process.

It's remarkable, though not surprising, the fact that none of the respondents indicated the dried leaves as a form of stevia expected in the future. The reason may now seem more obvious. Its use into company's production isn't easy.

On the contrary, respondents indicating both liquid and powdered extract are over 25 %, a percentage showing their adaptability in using any of these two forms of stevia.

The interest in buying stevia now, if it were available, was also explored. For this reason, participants simply answered if they could probably buy and use stevia directly.

Not surprising, but certainly interesting to see through data, that companies knowing and expressing their interest in using stevia in the first place, are those who would like to use it right through. This demonstrates the great influence that stevia has in those companies that already know its existence and are looking forward in introducing this sweet ingredient in their production process.

Other than stevia itself, the respondents determine some key factors that would influence them on their decision in using it. The proposed factors were advertising, industry publications, trade shows and events, guarantee offers and supply chain opportunity.

The majority of respondents cited advertising, industry publications and supply chain opportunities as the three major factors influencing their decision in introducing stevia in company's production process. Of lower importance seem to be any guarantee offer given about stevia and the organisation of trade shows or events.

Task 5.2 Assessment on the legal status for Stevia rebaudiana products in the global market

The actual status is for EU: Steviolglycoside have been finally approved for the use in the EU on 2 December 2011 through the Commission Directive 1131/2011 from 12 November 2011. The approval is limited to only a few categories of foods and beverages and the maximum dose is limited to about 30 % sugar replacement. The purity is minimum 95 % on dry matter basis.

The actual status for the Codex Alimentarius:
The Codex Alimentarius Working Group on Steviolglycoside approved the list of maximum dose in the different food categories on 4 July 2011. The maximum use level is different to the EU list. Consequently, the EU Member States, Switzerland and Norway expressed their reservation on the Codex Alimentarius list. This was necessary due to the WTO regulations.

Global status:
With the decision of the Codex Alimentarius the approval all other countries in the world have legal basis on incorporation of the Steviolglycoside in their food laws. However, the status in different countries is not clear yet, but developing, e.g. India is studying the approval. It is probably too early to see a clear progress. May be until 2014 in all countries the legal implementation will be achieved.

Actual approvals:
Steviolglycoside are approved in the USA under GRAS notification where each applicant must apply separately, Switzerland under a preliminary approval, Canada as a food supplement, Australia and New Zealand as food additives and food supplements, China as food additive with much lower purity requirement than in the EU, Japan, Brazil, Argentina and Paraguay as food additives, Mexico as a food supplement and in the EU as a food additive with use limitations.

Potential impact:

The results of DIVAS project will strengthen the competitiveness of the SME participants to improving their industrial competitiveness across the European Union within the participating countries. The expected outcome demonstrates a clear economic impact for the SME participants, improving their competitiveness by creating new markets in the food sector. This new possibility will save the existence of the SME participants at local and European level.

The European added value

It is important in terms of maximising the impacts of the DIVAS project that the diversification alternative Stevia rebaudiana which was developed meets the needs of the SME participants which are the end-users of the project knowledge generated. This was achieved by close involvement of the users and stakeholders of all stages of the project. The close involvement of the SME administrators in the future implementation of the stevia diversification alternative resulted in the founding of a transnational cooperation EURO-STEVIA EEIG which will start work during 2012. This is a keystone in added value of European collaboration for a implementation of Stevia rebaudiana as a sustainable alternative for tobacco farmers. The DIVAS project has created this trans-national cooperation of the tobacco farmers in order to help them to be competitive in the future.

Contribution of the EURO-STEVIA project in addressing community societal objectives - Health aspects for the consumers

Fight against obesity (quality of life):

In humans, sweet taste exerts a profound influence on behaviour. Generally spoken, the more intense the sweet taste, the greater the pleasure response. The pleasure response to sweetness is assumed to serve a physiological need. A hungry organism is reputed to find sweetness attractive, while a satiated organism does not.

A hungry organism may also select foods that provide a maximum number of calories per unit weight. Energy density of the diet is often perceived through the sensation of taste. Sweetness, the traditional sensory indicator of both nutrients and calories, adds to the sensory appeal of a given food. According to recent reports, under ad-libitum conditions people tend to consume a fixed weight or volume of food.

When foods or diets differ in energy density, so do also daily energy intakes. But as a fact, as energy density for foods goes down, so does also palatability. However, only few consumers are willing to sacrifice palatability in the persuit of an energy dilute diet. The development of intense sweeteners represents a deliberate - and costly - strategy to separate the palatability from energy density in foods. Intense sweeteners cleanly separate sweet taste and calories.

Therefore, the development of a natural low calorie sweet food ingredient (novel food) supports the fight of the Commission against obesity and also the goals of the ETP 'Food for life' and the 'European Platform on Diet, Physical Activity and Health'. Go4STEVIA contributes by providing a low calorie sweet food ingredient which maintains all valuable plant ingredients as a natural sweet food. The results will transfer the tobacco farmers growing unhealthy plants to a new stage - the protection of human health.

Blood pressure and antioxidant status:

In a recent published study about the effects of fruit and vegetable consumption on plasma antioxidant concentrations and blood pressure, small effects have been observed (blood pressure fall 4,0 mm Hg systolic and 1,5 mm Hg diastolic). The authors concluded that these results produce only small clinical effects, but would substantially reduce cardiovascular disease at the population level. A reduction of 2 mm Hg in diastolic blood pressure results in a decrease of about 17 % in the incidence of hypertension, 6 % in the risk of coronary hart disease and 15 % in the risk of stroke and transient ischaemic attack. It was shown during the DIVAS project that Stevia rebaudiana develops a high anti-oxidative potential at all the locations the cultivations trials have been executed. It is like green tea.

Oral hygienic:

Paradontitis is a civilisation disease where 80 % of the elderly population (> 50 years) is affected and which has already a higher cost burden for the social insurance as caries. In the last few years, knowledge was built up that the status of oral hygienic is associated with chronic diseases:

- Parodontopathias are considered as a risk factor for coronary diseases. Gingivitis has a direct connection to cardiovascular and heart diseases.
- A large evidence base suggests that diabetes is associated with an increased prevalence, extent and severity of gingivitis and periodontitis.

It was shown that the metabolic activity of the microbiota in the mouth is involved not only on cariogenic and paradontitis phaenomens, but also on the development of tumours in the mouth. In that study was postulated that volatile sulphur compounds, which are metabolised by the paradonto pathogens, are improving the permeability for endotoxins. In the buccal ephitel cells, paradonto pathogens were identified (Actinobacillus actinomycetemcomitans, Porphyromas gingivalis and Tannerella forsythensis).The carcinoms of the mouth are on world scale on the sixth position and in developing countries on the third place. Every year 500 000 cases are diagnosed and 128 000 deaths are counted on a world scale. The five-year survival rate is below 50 %. The incidence is 1,8 (female, Japan) to 47 (male, Malaysia) for 100 000 persons per year. In e.g. Germany the incidence is 10 - 15 cases for male and 4 cases for female per 100 000 persons per year. 60 % of the patients show also metastases in the lymphic nodes. Stevia Sweeteners have been examined for beneficial effects on oral hygienic. In a human study executed at partner UFGRS, new formation of plaque was reduced by 58 %. Another study did not show any differences between a sodium-fluoride solution and a stevia solution. New plaque formations were reduced by 24 % after 3 weeks of application and by 39 % after 6 weeks. Interesting results however, was found in an in-vitro study conducted at the University of Basilea on various strains of buccal micro-organisms which are responsible for paradonitis and gingivitis. A strong suppressive effect was noted on the following strains: Actinobacillus actinomycetemcomitans ATCC43717, ATC43718, ZIB1001, ZIB1023, HK1651, Streptococcus mitis ATCC49456, ATCC2517, Streptococcus sobrinus OMZ176, Prevotella intermedia ATCC25611 and Porphyromonas gingivalis ATCC33277, ZIB3002, ZIB3003, ZIB3071, ZIB3251, ZIB3252. In vivo studies have not been conducted so far; however, it is likely that systemic effects of stevia novel food towards gingivitis disease will results in an increase of oral health.

The problem of diabetes in the world

Human studies at the University Clinic Ulm (Germany) showed already in 1981 that sweetened soft drinks have a high impact on the level of insulin over many hours after consumption. Each time after the consumption of a sugar sweetened beverage (one glass with 190 ml and 19 gr of sugar) rised the insulin level drastically. The beverage composition was mimicking a 10 % sugar solution Today soft drinks posses some less sugar (about 8 percent sugar solution which is 80 gr sugar per litre). Such a soft drink consumed today will give a similar result like the experiment in 1981. This easy test over four hours explains a typical consumption situation. Beverage consumption is frequent between two meals. As far as water is consumed nothing happens. However, is the beverage sweetened with sugar, like a sweetened coffee, tea or a soft drink, than the insulin level peaks as shown in the study. The use of high intense low calorie sweeteners does not show the same effect as sugar. This was also studied in detail for Steviolglycoside and botanical preparations of Stevia rebaudiana. Neither an increase of the insulin level was measured nor insulin peaks has been observed. The observed insulin peaks in the above described study will appear when drinking sugar sweetened soft drinks. The Uniclinic Ulm Research Group compared also the total insulin release within the two groups. The total insulin release of the group without drinking the sugar sweetened beverage was 28 % less during the test period of four hours than measured by the group drinking the sugar sweetened beverage. Hypothetical spoken a systematic exchange of sugar with high intense sweeteners, e.g. from Stevia rebaudiana would result in beverages with a much better metabolic profile. The physiological response of the human body would be not to release too much insulin. Each consumer can be profit from such a simple physiological effect saving insulin. The systematic effect of saving insulin by reducing such observed insulin peak releases will have a high desired preventive effect on health maintenance of the human body.

Time bomb diabetes for the world health system

The International Diabetes Federation estimates that 285 million people suffering from diabetes in 2009. In 1985, world statistics recorded only 30 million cases. In 2030, estimates speak about 435 million people suffering diabetes. 60 % of all cases are preventable. The figure below shows the estimates for each country in 2030.

It is estimated in the Green Paper of the Commission 'Promoting healthy diets and physical activity: a European dimension for the prevention of overweight, obesity and chronic diseases' that in 2025 about 10 % of the population of the European Union will suffer diabetes. A strong increase is forecasted in developing countries, but especially in Arabian countries.

The reason can be found in the trend for increased urban growth and the abandonment of traditional nutrition pattern. The access to hygienic drinking water is limited in a lot of urban centres for a great number of inhabitants. Due to that the consumption of sugar sweetened soft drinks is booming. The economic costs of the diabetes disease are estimated on USD 376 billion. This corresponds to about 11,6 % of the world expenses for the health system.

A very easy approach to overcome such health obstacles is the worldwide recognition of Stevia rebaudiana as a food and food ingredient. Until now only the sweetener from Stevia rebaudiana, the Steviolglycoside, are approved. However, Stevia rebaudiana novel food requires much less technical processing, but giving cost benefits to the food industry by providing a sensorical superior low calorie sweetener.

Potential impact for the tobacco farmers

By executing this EURO-STEVIA project the following key figures can be achieved:

a) A total of 10 500 family farms can survive after 2014.
b) In the seven concerned regions where the EURO-STEVIA project shall start seven factories shall be set-up which is a total investment of EUR 28,7 million involving the creation of specialised and qualified employment.
c) By implementing Stevia rebaudiana as a new cash crop a total revenue of EUR 45 500/ha can be expected on a mid-term range provided further research funding by the EU Commission.
d) The combined net sales volume of all STEVIA products can achieve about EUR 477 million whereas the former tobacco cultivation raised only about EUR 80 million for the same cooperatives and their members.
e) The combined income tax revenue will be about EUR 91 million during the first decade of project execution.
f) The EURO-STEVIA project keeps 10 500 family business in the regional rural economy and maintains them as contributors to the social system.
g) Reaching the maximum production capacity the EURO-STEVIA project will generate about EUR 90 million annual added value tax.

Dissemination activities

a) Dissemination towards German food industry: In this work package, an information day for the European food industry was foreseen. The coordinator UHOH organised an information day already on 22 October 2009. For that meeting only food industry was invited. A total of 136 persons attended from about 100 companies.
b) Dissemination towards the European food industry: In the course of the project several event on stevia have been launched by several organisations. It was then decided to get a better access to the European food industry by oral presentation on industry meetings. This charge was given to Dr Udo Kienle who was invited by several organisations.
c) Dissemination towards the civil society: During the course of the project the University of Hohenheim launched five press releases regarding the DIVAS project and the stevia sweetener. As a consequence in about 185 European periodicals, like newspapers and magazines reports appeared. During the life-time of the DIVAS project, about 20 times television from public or privet channels acceded to the University of Hohenheim to get information from the first hand about the DIVAS project and the stevia sweetener in general. Additionally, about 100 radio interviews have been given.

Exploitation of results

a) In the last steering committee meeting it was agreed that the tobacco cooperatives will do the exploitation in a common way. EURO-STEVIA EEIG will be the key organisation where the common exploitation will be executed.
b) An agreement of the use of the varieties will be concluded between EURO-STEVIA EEIG and the breeders once the register process for EURO STEVIA EEIG is finalised. When the agreement is concluded the registration process in the Community Plant Variety Office, Angers, France, shall be started.
c) The drafting of the specification is in progress however, not finalised yet completely. Details are still missing. The final draft specification will take some time more.
d) Once the specification is finalised the development of food and beverages can be started. In WP4, first tests have been done and it is shown that the use of the stevia novel food can improve the antioxidant status of the food and beverages. This will give a clear benefit for consumer and an advantage over Steviolglycoside which are sweet but do not have any anti-oxidative property by its own.
e) The drafting of the business plan is underway. It was decided to contact the European Investment Bank for a common loan arrangement within the cooperatives for the financing of the future commercial activities. The meeting with the European Investment Bank shall be attended by Mr Stelios Peletekiades (TAT), Dr Franco Bozzelin (TCV) and Dr Udo Kienle (UHOH).

Project website: https://stevia.uni-hohenheim.de/