Ανάπτυξη καινοτόμου βιοδιυλιστηρίου για την παραγωγή βακτηριακής κυτταρίνης και προϊόντων διατροφής προστιθέμενης αξίας από υποπροϊόν τυποποίησης κορινθιακής σταφίδας
Authors: Πλειώνη Ίρις 
Published: September 2021

Corinthian currants are produced by the natural drying of Vitis vinifera L. var. Apyrena grapes and is a historic Greek product, cultivated since antiquity. It does not undergo any heat treatments, addition of sweeteners or other additives, and can be consumed as such, added to bakery, confectionery and cooking products, or it can be processed for the production of syrups or concentrates for use as sweetener or as raw material for alcohol and beverage production. Today, Corinthian currants are still one of the main export commodities of the country, with Vostitsa currants being the top quality. However, the amount of currants produced is steadily declining, mainly due to the high production costs and the lack of governmental subsidies. A Corinthian currant processing company produces large quantities of finishing side-stream (FSS) (accounting for 5-6% of the processed raw material), which differs from the marketable product mainly in the size of the berries (<4 mm, >8 mm), and the presence of seeds. The analysis of chemical composition and aroma (volatile) profile of FSS have not been studied and is one of the objectives of the thesis. Also, FSS is currently used almost exclusively for the production of vinegar by submerged fermentation, as defined by Greek law until recently. However, FSS has a great potential for the biotechnological exploitation as a raw material rich in fermentable sugar (glucose, fructose, and sucrose, ~70%). Thus, it can be used for the production of value-added products, which today constitutes a real requisite for the currants processing companies, especially after the repeal of the corresponding restrictive legislation on the use of FSS.The aim of this thesis was therefore, to upgrade FSS, within the “Biorefinery” concept, for the production of various added-value food products (sweeteners, vinegar, wines, microbial food cultures), as well as biotechnological products (bacterial cellulose). The thesis also aimed at developing novel production methods that could lead to products with good organoleptic and nutritional quality, preservability, and low-production cost, which are key prerequisites for their industrial application. The biorefinery is also one of the key strategies for achieving a “Circular Economy”, through which the raw material “Loops” can be closed, through utilization of wastes and side-streams to produce useful products, leading to multiple socio-economic and environmental benefits.Another aim was the chemical analysis of FSS and its products, with emphasis on the phenolic content, antioxidant capacity and aromatic profile (of the thesis food products), and physicochemical texture characteristics (of bacterial cellulose, which finds applications in both the food and materials sectors). The First Part of the thesis provides a comprehensive overview of the literature on the current situation regarding the generation and utilization of food industry wastes in the context of achieving a circular economy. Data on Corinthian currants (production, composition, legislation, industrial side-streams, and current situation in the corresponding agro-industrial sector) are also reported. Then, reference is made on products that can be produced from raisins or grapes, and which are the objectives of the thesis (syrups, wines, vinegars, bacterial cellulose, microbial biomass). Information on the current economic situation of these product markets worldwide, on their production methods, their chemical composition and their nutritional value is also provided, as well as on the new trends at scientific and industrial level. The Second Part provides a detailed description of the experimental design of the thesis. The raw materials, microorganisms, chemical reagents, and laboratory equipment used are listed, and all the analytical and experimental techniques, applied or developed in the thesis, are described in detail. In the Third Part, the results and conclusions of the thesis are presented. Initially, an analysis of the FSS composition and aroma profile was carried out and compared with the marketable product from which the FSS was generated (Vostitsa currants). The main difference between the two materials is, as mentioned above, the size of the raisin berries and the presence of seeds and stems. These differences were also reflected on the chemical composition of FSS. In particular, the results showed that total sugar concentration (~ 71% w/w), protein (2.6% w/w), ash, pH, total acidity and organic acids were at similar levels in both FSS and the commercial product, while the moisture content, fat (0.66% w/w), volatile acidity (0.2% as acetic acid), phenolic content and antioxidant capacity were increased in the FSS. These differences, apart from the different nature of FSS, can also be attributed to the different treatment of FSS in relation to the marketable currant after their separation in the factory. In particular, the increased antioxidant capacity (av. 2.40 AAE/g) and phenolic content (av. 4.8 mg GAE/g) of FSS compared to the marketable raisins (av. 1,9 mg GAE/g and 2.40 AAE/g, respectively), can be attributed to the presence of seeds that are rich in antioxidant phenolics, and possibly to the chemical modification of the raisin phenolics during processing and storage of FSS.FSS also has a rich volatilome. In particular, 160 compounds were identified by SPME GC-MS, including (1) volatile compounds from the raw material (grapes or other parts of the vine plant), (2) sugar thermal breakdown products, such as Maillard and Strecker reaction products (e.g., diacetyl, furfural, phenylacetaldehyde, and Strecker aldehydes) formed during the dehydration of raisins, and (3) compounds of lipid metabolism (e.g., hexanal, heptanal, 2-heptenal, 2,4-decadienal, 2-nonanal), which probably derive from both from the metabolism of the vine and from the processing of the raisins (drying, storage, processing). The majority of these compounds were also identified in the products made from FSS. Finally, compounds not derived from grapes were also detected in both FSS and its products, but their presence can be attributed to impurities such as other parts of the vine plant (e.g., L-borneol from stems), or weeds (e.g., thujone). In addition to the above, the differences between FSS and the commercial product are also affected by soil and climatic conditions, the applied agricultural practices, the year of harvest, and the way the raisins are dried.However, the levels of acidity and the presence of specific volatile compounds (such as acetate esters, higher alcohols, aliphatic hydrocarbons, etc.) in FSS, are indicative of microbial spoilage. For this reason, it is suggested that attention should be given on the handling of FSS in the factory, after its separation from the marketable product, to reduce its microbial load and spoilage, especially when it is destined for the production of food products. The presence of mycotoxins in the FSS should also be investigated. As regards to the solid FSS residues (FSSR) left after the aqueous extraction of FSS for the production of the various products of the thesis, it was found to contain lower amounts of total nitrogen (protein) (av. 2.4%), while ash, fat, total and volatile acidity as well as pH were higher than those of FSS. On the contrary, the phenolic content (30.3±3.0 mg GAE/g) and antioxidant capacity (21.9±5.2 mg AAE/g) were higher, indicating its potential use as an additive in foods or beverages with high antioxidant activity, while it is also a good substrate for microbial processing to produce bio-enriched foods or animal feeds (e.g., "synbiotic" foods). From the FSS, 4 types of syrups were initially prepared (density 38-40oBe), two "brown" and two "blonde" syrups, with/without partial removal of tartrate. All syrups had similar glucose content (31.3-35.4%w/w), fructose (30.5-34.5%) and total sugars (65.1-68.8%), and in addition all contained traces of sucrose. The total acidity of the syrups was similar. They also had similar contents of total nitrogen, except for the blonde syrups that had lower content due to their production method (clarification and discoloration). The volatile acidity of all syrups was at low levels (0.08-0.27%), indicating that they did not suffer significant microbial alterations during their preparation. The blonde syrups had a higher pH value (~5.5-6.0) due to the partial precipitation of tartrate. The phenolic content of the brown syrups was higher (185-213 mg GAE/100 g), since in the blonde ones (134-143 mg GAE/100 g) part of the phenolic compounds and pigments were removed by activated carbon. The brown syrups, as expected, had greater antioxidant capacity (0.90-1.0 mg AAE/g) than the blonde ones (0.29-0.58 mg AAE/g). Regarding the SO2 addition process of the FSS extracts for the production of syrups, it was concluded that laboratory tests should be carried out in order to regulate the residual amount of SO2 in the final products to the desirable levels, since the stoichiometric calculation of the oxidant (e.g., H2O2) required to oxidize SO2 does not properly function due to the presence of other antioxidant compounds in the FSS. Improvements in this regard may also concern application of FSS sterilization techniques prior to the extraction and condensation in order to completely avoid SO2 addition in syrups production. In total, 144 volatile compounds were detected in the syrups by SPME GC-MS. From these compounds, carbonyls were found at higher levels in syrups compared to FSS, the major being 2- and 3-methylbutanal, acetone, furfural, 5-methyl-2-furfural and 1-ethyl-2-formylpyrrolle. In general, most of the identified compounds were either produced during the dehydration of raisins as lipid oxidation products or as products of the Maillard reaction (carbonyl compounds, furans, furfuryl compounds). On the other hand, hydroxy-methylfurfural (HMF) or acrylamide were not detected in any of the syrups, since their production involved concentration under vacuum at low temperature (45οC). From the sensory evaluations, microbiological analyses, and volatile acidity levels, it was shown that the FSS syrups (with the proposed production methods) were good quality and easy to preserve products, while during their production and storage they did not develop microbial alterations. They were generally described as products with a strong aftertaste, good but not strong odor, good sweet/sour taste or similar to honey, and without a metallic feel. Their color was similar to that of honey for the brown syrups and golden-yellow for the blonde syrups. Finally, the FSS syrups can be considered products with a high nutritional value and suitable (in moderate amounts) for diabetics as they contain a high fructose content and no sucrose. Following, FSS jams were prepared with/without the addition of pectin and with/without preservatives. The jams generally had similar characteristics to the commercial grape jam used for comparisons. Some differences among them may be attributed to the different raw material and the different way of production. All jams had a similar phenolic content (2.2-3.2 mg GAE/g), while their antioxidant capacity was 1.16-0.96 mg AAE/g.From the microbiological analyses and volatile acidity levels (0.044-0.066%) it appeared that the products were easy to preserve. The phenolic content of the jams was at similar levels as that reported in the literature, and they did not contain HMF, although they were prepared by a more intense heat treatment than the syrups. Also, a total of 144 volatile compounds were identified by SPME GC-MS. Of these, certain groups such as alcohols, organic acids, lactones, and hydrocarbons, were greatly reduced in the jams compared to the raw material (due to dilution and loss due to evaporation during boiling). As in syrups, the percentages of carbonyl compounds (almost doubled) and terpenes (possible hydrolysis of their bound forms) increased in the jams. Of the Maillard reaction compounds (except carbonyl compounds), 2-acetylfuran appeared at particularly high levels in the jams. Also, very high was the level of 2-octenal, a substance found in grapes and raisins as a product of the oxidation of unsaturated fatty acids by grape lipoxygenase, and as a product of the Maillard reaction. Therefore, its increased concentration in the jams is justified. The differences in the volatile profile between jams and syrups obviously depend on the different temperature and time of their processing. Regarding taste and appearance, the jams were characterized as pleasant and were well accepted by the testers. Also, it was shown that the addition of preservatives or pectin was not necessary, as the jam made without additives had similar texture and shelf-life with the jams that contained additives. Finally, they can be considered products with a high nutritional value and suitable for diabetics (in moderate amounts) as they do not contain added sugar and their sweetness is by half attributed to fructose. The production of dry wine from FSS extracts at various temperatures (4, 10, 15, 22oC) was then studied, using free and immobilized cells of the cryotolerant and alcohol-resistant strain Saccharomyces cerevisiae AXAZ-1 which has been previously studied extensively in food and biofuel applications. The use of cell immobilization techniques can offer advantages over free cell systems, such as increased productivity, increased cell viability, operational stability, low fermentation capacity, and improved product quality. Especially, the combination of cell immobilization and fermentation at extremely low temperatures has been previously shown to lead to improved quality of both alcoholic and acetous fermentation products. The temperature significantly affected the wine fermentation times, while it did not significantly affect the levels of fermented sugar, ethanol, total and volatile acidity. The immobilized biocatalyst maintained its operational stability for more than 6 weeks, even at 4oC. The wines had better clarity than those made with free cells, they contained traces of sugar and the ethanol content was ~11.2%. Methanol was also not detected, which means that the grape pectin methylesterase was deactivated during the dehydration of the raisins. The low or no methanol content makes dry wines ideal raw materials for the production of distilled spirits, which can bring even higher added value to the FSS. Therefore, a future study for the production of spirits from FSS (“raisin brandy”) is strongly recommended. The pH of dry wines produced with immobilized cells was slightly lower, although total and volatile acidity were slightly higher in wines made with free cells. However, the observed changes were statistically insignificant. The same was observed for individual organic acids, except succinic, which was more abundant in wines produced by immobilized cells. The phenolic content was slightly lower than most white wines. Therefore, it is proposed to optimize the extraction of FSS in order to recover more phenolic components in the extracts. The phenolic content was also reduced at lower fermentation temperatures using immobilized cells. This can be a disadvantage in terms of the nutritional value of the wines, but not necessarily a technological disadvantage, since the phenolic components affect the taste, aroma and clarity of the wines, and the final product quality will depend on the winemaking practices that will be applied (fermentation temperature, use or not of immobilized cells, clarification, and other post-fermentation treatments). The individual phenolic components in the wines made using immobilized cells, as determined by RP-HPLC, were gallic, vanillic, p-coumaric and caffeic acids. A similar reduction at the lower fermentation temperatures was observed for these compounds. Vanillic acid was the major phenolic acid found in wines made at 22oC. A total of 160 volatile compounds were identified in the dry wines. In particular, the wines contained higher levels of terpenes than FSS (indicating hydrolysis of their bound forms in the wines), and a large number of volatile compounds (mainly esters) derived from fermentation. The effect of yeast immobilization on the wine volatile profile was as described in previous studies (higher ester levels by immobilized cells and at lower fermentation temperature). The wine prepared by immobilized cells had better clarity, without any sediments. Also, the intensity of the color was characterized as pale to moderate, and the shade was described as light, amber-like. As far as the aroma is concerned, a moderate aroma intensity was observed in both types of wines. They were generally characterized as dry, low-tannin, light-body and moderate intensity of flavor, acidity, and aftertaste. The alcohol sensation was medium for both wines tested.Subsequently, three types of sweet wines from FSS with alcohol content ~15.5% were prepared. One wine was made by addition of alcohol to stop the alcoholic fermentation. The other two wines were prepared by complete fermentation and then addition of FSS or brown syrup to achieve the desired sweetness. Most of their characteristics were similar to those of commercial sweet table wines. Total acidity ranged from ~7 g tartaric acid/l with higher acidity observed in the wines made by addition of FSS or syrup. The volatile acidity and pH of all wines were low. The main organic acids of the wines were tartaric and malic acid, while they contained much lower concentrations of citric, acetic, and succinic acids. All wines had similar phenolic content (244-325 mg GAE/l) and antioxidant capacity. In the sweet wines, 160 volatile compounds were identified, the same as those found in the dry wines. Regarding their appearance, a high clarity was observed for all three wines, without any sediments. The intensity of the color was characterized as intense with shades of light brown-orange to dark-amber. They were described as wines of high sweetness, moderate acidity, with a strong raisin taste and moderate aftertaste. The alcohol sensation was moderate to high for all three wines tested. In conclusion, the sweet wines prepared by the proposed methods are similar to sweet table wines with the main difference being the lower levels of phenolic content and antioxidant capacity (according to the literature, since no analysis of commercial wines was performed with the analytical methods applied in this thesis). To conclude, the proposed methods, led to wines of very good organoleptic quality, which could all be commercialized as raisin liquors, and of significantly reduced production cost since no ethanol addition is required. The production of vinegar from sweet FSS wine by immobilized and free cells of a wild vinegar culture, as well as a mixed culture of selected acetic acid bacteria, was then studied. Specifically, a fast fermenting species (Acetobacter aceti DSM 3508) and a species with high tolerance to acetic acid (Komagateibacter europaeus DSM 6160) were used. Also, the acetification capacity of the cultures after storage at low temperature for 2 months was studied. The results showed that in all cases the acetification time was slightly reduced from batch to batch (cell adaptation and operational stability of the system), and significantly reduced by the use of immobilized cells (increased productivity-reduced process cost). These results agree with previous studies on the increase of productivity by cell immobilization in other fermentation processes, such as alcoholic and lactic acid fermentation. Moreover, the storage of the cultures at low temperature in the refrigerator did not inactivate them but increased the time of the subsequent acetification batch. The use of the mixed culture of selected bacteria also resulted in a reduction of the acetification time, compared to the wild culture, thus ensuring better yields and evolution of the fermentation process. The phenolic content and antioxidant capacity were higher in the case of vinegars made by free cells (due to adsorption of phenolic compounds on the immobilization carrier), however it was at similar levels of common white wine vinegars. Comparing the different vinegar products regarding their volatile profiles, it can be observed that higher percentages of esters were contained in the products made by immobilized cells. Also, the esters in the products were increased by 8-fold compared to FSS. Larger percentages of alcohols were also contained in the vinegars made by the wild culture using free cells. The percentages of organic acids were similar in most samples, except for the vinegar made with free cells of the selected bacteria. Also, the products showed reduced percentages of terpenes, lactones and carbonyl compounds in relation to FSS. The results are encouraging for the production of vinegar from FSS using immobilized cells of selected acetic acid bacteria, provided that the application in the industry will involve specialized personnel, as well as the appropriate post-fermentation treatments. The thesis also proposes the use of FSS as raw material for the production of bacterial cellulose (BC), which, although is approached as a material with a variety of applications (hence its physicochemical properties were analyzed), it can also be used as food of high nutritional value (high in dietary fiber/prebiotic food). The results of the BC production optimization study with the microorganism Komagataeibacter sucrofermentans DSM 15973 in FSS extracts (FSSE) (using the Response Surface Methodology based on the Central Composite Design) show that the addition of N-sources to FSSE is necessary for optimal BC yield. The pH and incubation temperature also played an important role. Specifically, the ideal combination for optimal BC performance in FSSE was 28.07oC, pH 6.42, sugar concentration of 46.24 g/l and the addition of 2 N-sources, yielding 18.4-0.06 g/l BC. Using FSSE/whey mixture as a substrate, the ideal combination for optimal BC performance was pH 6.36 and addition of 1.7% yeast extract, leading to a production of 18.9±0.7 g/l BC. Therefore, whey can be used by K. sucrofermentans as a cheap N-source and source of other micronutrients required for the production of BC.Also, the method of BC drying after its production (thermal, freeze-drying) leads to various shapes and textures of the product (clear film with polymeric appearance and texture, with high permeability and ductility, or a texture similar to paper or cotton). The physicochemical texture analysis (porosimetry, SEM, FT-IR, XRD, TGA/TDA) showed that BC consists of cross-linked, but relatively highly oriented fibers (with nanoscale widths), which create a dense three-dimensional network structure, while the volume of its pores is similar to or greater than other types of plant delignified celluloses. Finally, the FSSEs of lower density remaining from the production of the above products, were used for the production of microbial biomass. Specifically, the production of baker’s or winemaking yeast (S. cerevisiae) was studied. The results showed that intensive air supply and the addition of N-source is necessary to increase the production efficiency of biomass in the FSSEs. The addition of N, P, S and Mg nutrients (in the form of inorganic salts) further increases the yields. The maximum cell mass yield achieved was 45.0±1.3 g /l and 40.9±2.5 g /l FSSE, for baker’s yeast and S. cerevisiae AXAZ-1, respectively. The viability of the baker’s yeast produced was examined over a period of 3 months and was found to be satisfactory, while its composition was similar to that of the commercial baker’s yeast used as inoculum. The technoeconomic analysis for a complete industrial wine production process (using immobilized cells) (780 m3 /year) and baker’s yeast (24.6 tn/year) with FSS as the raw material (7.5 m3/day, for 120 days/year) showed that the investment is realistic and worthwhile. For a Zero Net Present Value (NPV), the Minimum Sale Price (MSP) is estimated at $7.75/tn cumulatively for dry wine and baker’s yeast, or $0.08/kg of product.At the end of the thesis, the conclusions on the results are presented, which show that FSS is a raw material with high nutritional value, suitable for use as raw material for a variety of products such as wines, syrups, jams, vinegar, microbial metabolites and microbial biomass. The cell immobilization technique in all cases improved both the quality of the final products and in the efficiency of the processes. The novel methods used to produce sweet wines and sweeteners were suitable for the production of good quality products with low production costs. Finally, future actions are proposed in order to improve the quality of the proposed products and their manufacturing methods, the production of additional products from FSS, and the evaluation of their possible industrial application, in order to utilize the entire amount of FSS and create added value, within the biorefinery concept.