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Application of Grape Pomace as a Natural Food Preservative and Source of Biofuel

von Violeta Ivanova-Petropulos (Autor) Zorana Andonovic (Autor) Dusko Nedelkovski (Autor) Krste Tasev (Autor) Klime Beleski (Autor)

Forschungsarbeit 2014 54 Seiten




1.1 Grape pomace
1.2 Polyphenols
1.3 Yoghurt
1.4 Biofuel

2.1 Samples
2.2 Extraction of phenolic compounds from samples
2.3 Determination of total polyphenols
2.4 Determination of total anthocyanins
2.5 Concentration of extracts with rotoevaporation
2.6 Application of phenolic dry extracts in milk during fermentation
2.7 Application of phenolic dry extracts after fermentation i.e. in yoghurt
2.8 pH - Measurement of the new yoghurt

3.1 Spectrophotometric analysis
3.2 pH variation of the obtained yoghurt
3.3 Sensory analysis of the new product
3.4 Microbiological analysis of the new yoghurt products

4.1. Production of biodiesel from grape winery waste and fruit peels






Aim of the work

The aim of this work is utilization of the grape pomace piled up as a waste after winemaking and its application in producing biodiesel. Furthermore, a novel yoghurt product was produced, enriched with antioxidants, such as polyphenolics from the grape pomace and berries in order to improve its nutritional value, contributing to food preservation and significantly reducing the risk of diseases. In fact, for the first time we engaged these compounds in preparation of a new yoghurt product, that proved to be last - longer and more beneficial for human health than the traditional one, since the antioxidants play a great role in the prevention of cardiovascular disease, cancer etc. Furthermore, a cost- effective, easily made and environmentally friendly biodiesel was produced, that could represent an alternative to the old fossil petroleum to replace it and resolve the present energy crisis.

Samples and Procedures

Totally, 4 types of Macedonian grape pomace, (from Zupjanka, Prokupec, Kadinal and Vranec varieties) as well as blueberry and aronia, were used. For the extraction of polyphenolics liquid-liquid extraction with ethanol/water/acetic acid, followed by decantation and filtration was used. The phenolic content of the obtained extracts was determined by the Folin-Ciocalteu method (Ivanova et al. 2010); and the total anthocyanins were realized by the Di Stefano et al. (1989) method. Afterwards, three different volumes of the obtained extracts (10, 50 and 100 mL) from each sample were concentrated by rotoevaporation to dryness. The three different concentrates of each sample were applied on milk together with the lactic bacteria in order to study the influence of polyphenolics during the fermentation; as well as, applied on milk after the fermentation (into the obtained yoghurt). In meantime, the pH value of the newly generated yoghurts was observed by using a pH meter. In the second part the seeds were separated from the grape pomace and served as a new source of oil that was to be transformed into biodiesel. Additionally, blueberry and aronia peels were used for the same purpose. Six organic solutions (ethanol, hexane, benzene, diethyl ether, acetone, acetic acid) were added to the dried seeds and then filtration and distillation followed for obtaining the oil. For dividing the biodiesel transesterification reaction was applied and due to the combustion of the final product it proved to be biodiesel.


The aronia sample was the one containing the highest phenolic content (431 mg/L), while Zupjanka had the lowest content of polyphenols (67.9 mg/L). The grape pomace presenting best results was Prokupec (246 mg/L). All yoghurt samples containing polyphenolics applied before the fermentation, presented higher pH value compared to the control and samples with polyphenolics applied after fermentation. Furthermore, all yoghurt samples containing highest phenolic concentration, showed best results, presenting stable pH value. Zupjanka has shown the best results (pH-4.4 for 1-100) for all concentrations, that is even higher than aronia. After obtaining the yoghurt, sensory analysis was performed, stating that the new product has creamy texture; it tastes good, without unpleasant smell or bitterness. The colour of some samples turned into red-violet, excluding the yoghurt with dry extracts from: Zupjanka and Kardinal (white grapes). By microbiological analysis the presence of pathogen

and other harmful bacteria was proved to be negative, showing that this yoghurt could be a main basis for manufacturing a more beneficial dairy product that could be soon available on the market. Concerning the biodiesel production, the oil content, depending on the solvent used, differed from 0.11 g to 0.36 g. During the extraction acetone and acetic acid showed better results than ethanol, which proved to be a better extractor of antioxidands. Thus, acetic acid was the most proper solvent for obtaining impure biodiesel with a yield of 90.8%. Overall, the sample which showed the highest percentage when mixed with acetic acid was blueberry. On the other hand, from the grape pomace, the type comprising the largest amount of impure biodiesel was aronia with the average yield of 53.46% and then followed: blueberry, Prokupec, Kardinal, Zupjanka and Vranec, respectively.

Andonovic et al. Application of grape pomace as a natural food preservative and source of biofuel


The geographic location of Republic of Macedonia, the fertile soils and optimal climate conditions are exceptional for breeding vine and specific grape varieties.

35 000 hectares of vineyards with different grape varieties are located at the territory of Macedonia, producing grapes with high quality used for production of high quality wines, red and white. But, the wine industry waste in general is a problem in Macedonia, since it does not have any usage. In the European Union, there is approximately 14.5 million tons of wine industry waste produced from wineries, which is a lot of material that could be recycled and reused for other purposes. In fact, the wine industry waste contains primarily crushed grape skins and seeds rich in beneficial polyphenol compounds that act as antioxidants, antibacterial agents, anticarcinogenic agents, antiviral agents, etc.

1.1 Grape pomace

Grape pomace stands for the solid remains of grapes (skins, stalks and seeds), which are being discarded during winemaking (after grapes are pressed). The largest fraction of winery waste is pomace (Figure 1) which is thrown away ending up in landfills. Fruit processing industries generate tremendous amount of solid wastes which is almost 35-40% dry weight of the total produce used for the manufacturing of juices. During the management of these wastes there is a production of greenhouse gases (GHG) which must be taken into account. Winery leftovers pile up fast and can cause some serious environmental problems if not disposed properly.

Andonovic et al. Application of grape pomace as a natural food preservative and source of biofuel

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Figure 1. Samples of Macedonian grape pomace

1.2 Polyphenols

Polyphenols are secondary metabolites widely distributed in the plant kingdom and the most abundant micronutrients in our diet. Their beneficial effects have been ascribed to their strong antioxidant activity, their ability to scavenge oxygen radicals and other reactive species. These features make phenols a potentially interesting material for the development of functional foods or possible therapy for the prevention of some diseases. As metabolites and antioxidants, polyphenols may protect cell constituents against oxidative damage and, therefore, limit the risk of various degenerative illnesses associated to oxidative stress.

The main source of polyphenolic antioxidants is dietary, since they are found in a wide array of foods. For example, most legumes (fruit such as apples, blackberries, blueberries, cantaloupe, cherries, cranberries, grapes (Figure 2), pears, plums, raspberries, and strawberries; and vegetables such as broccoli, cabbage, celery, onion and parsley), are rich in polyphenolic antioxidants. Red Andonovic et al. Application of grape pomace as a natural food preservative and source of biofuel wine, chocolate, green tea, coffee, olive oil, fruit and plant-derived beverages, and many grains and pulses are also good sources.

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Figure 2. Polyphenols abundance in grapes (source: )

Polyphenols are divided into two groups: flavonoids and non-flavonoids (Ivanova et al. 2010; Ivanova et al. 2011). The flavonoids consist of two benzene rings (A and B) linked by an oxygen-containing pyrane ring (Figure 3). This group consists of anthocyanins, flavonols, flavan-3-ols, flavanols. The group of nonflavonoids is composed of phenolic acids (hydrozybenzoic and hydroxycinnamic acids and their derivatives) and stilbenes (resveratrol and resveratrol glucoside). The structure of resveratrol is presented in Fig. 4.

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Figure 3. Flavonoid structure

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Figure 4. Structure of resveratrol (R = OH)

Polyphenols can affect the appearance, taste, mouth-feel, fragrance, and antimicrobial properties of wine. The antioxidant potential of wine is largely attributable to its phenolic composition (Burns et al., 2000).

Since pomace is rich in polyphenols, these material could be used a potential source of antioxidants and their application at different areas (Pecket and Smal, 1980, Montealegre et al. 2006). Awareness is increasing among consumers about the food products that provide health benefits. The increased alertness leads to high acceptance levels and thereby increased demands for functional health products, especially probiotic dairy products. The aim is providing unique and innovative products that do not compromise on quality or performance.

There is a growing interest by the consumers in foods that are recognized as beneficial for human health because they are either low fat, or of greater nutritional value, or contain bioactive materials which are associated with reduced risk of disease. Ideally, these foods should be appealing, taste good, low in price and, most importantly for consumers’ acceptability, should contain all-natural ingredients, in a single, easily-accessible product (Petrotos et al. 2012).

1.3 Yoghurt

Yoghurt refers to a product obtained from fermentation of milk by means of cultures of bacteria (streptococcus thermophilus and lactobacillus). For yoghurt production, bacterial cultures are added to the milk to start the fermentation, and to transform the present sugar into lactic acid. The lactic acid then reacts with the proteins in the milk giving the yoghurt hickness and creamy texture. Many yoghurts (Figure 5) provide their benefits by adjusting the microflora (the natural balance of organisms) in the intestines, or by acting directly on body functions, such as digestion or immune function.

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Figure 5. Yoghurt

In recent years enormous research has been focused on the functionality of the polyphenols and their application in human health improvement (Mahdavi et al. 2010, Hii et al. 2009). Experimental studies strongly support a role of polyphenols in the prevention of cardiovascular disease, cancer, osteoporosis, diabetes mellitus and neurodegenerative disease (D’Archivio et al. 2007).

Andonovic et al. Application of grape pomace as a natural food preservative and source of biofuel Therefore, in this project, the natural source of polyphenols used is the grape pomace, which in Republic of Macedonia ends up as a waste disposal after winemaking (Figure 6a, 6b, 6c). Besides the 4 types of pomace; blueberry and aronia (both grown in Macedonia) were used as a material for the experiments; both serving as a “standard” for comparison in the amount of obtained polyphenols, since aronia and blueberry, resting on a scientific research, are regarded as fruit comprising a huge amount of antioxidants.

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Figure 6. Grape pomace as winery waste disposal

1.4 Biofuel

From another point of view, transport is the third largest emitter of greenhouse gases (Figure 7) and biofuels can significantly reduce transport’s carbon footprint since it is dependent on finite fossil fuels such as oil and petroleum for its energy needs, so it is important that we move towards more renewable and sustainable fuels. Biofuels represent an immense growth area around the world and have an important role to play in displacing the types of fuels the world has used in the past.

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Figure 7. Transport as a greenhouse gases emitter (source: )

Added to this, biodiesel (Figure 8) can be used in existing engines, vehicles and infrastructure with practically no changes. Power and fuel economy using biodiesel is practically identical to petroleum diesel fuel, and year round operation can be achieved by blending with diesel fuel. Biodiesel provides significantly reduced emissions of carbon monoxide, particulate matter, Andonovic et al. Application of grape pomace as a natural food preservative and source of biofuel unburned hydrocarbons, and sulfates compared to petroleum diesel fuel. Additionally, biodiesel reduces emissions of carcinogenic compounds by as much as 85% compared with petrodiesel. When blended with petroleum diesel fuel, these emissions reductions are generally directly proportional to the amount of biodiesel in the blend. Biodiesel is made through a chemical process which converts oils and fats of natural origin into fatty acid methyl esters. The scarcity of known petroleum reserves will make renewable energy resources more attractive. The most feasible way to meet this growing demand is by utilizing alternative fuels. Biodiesel is the best candidate for diesel fuels in diesel engines. The biggest advantage that biodiesel has over gasoline and petroleum diesel is its environmental friendliness (Figure 9). Biodiesel burns similar to petroleum diesel as it concerns regulated pollutants. On the other hand, biodiesel probably has better efficiency than gasoline. One such fuel for compression-ignition engines that exhibit great potential is biodiesel. Diesel fuel can be replaced by biodiesel made from vegetable oils. The production and utilization of biodiesel is facilitated firstly through the agricultural policy of subsidizing the cultivation of (

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Figure 8. Samples of biodiesel fuel (source: )

non-food crops Andonovic et al. Application of grape pomace as a natural food preservative and source of biofuel

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Figure 9. The biodiesel cycle

(source: )


2.1 Samples

Pomace samples were obtained after maceration for the red grapes (Vranec, Kardinal and Prokupec) and after short pressing for the white grapes (Zupjanka) from winery located in Kumanovo region. Aronia and bluberry samples were taken from Kratovo region. All samples were from 2012 year. In order to prevent the samples from spoilage, the samples were kept frozen at -18 °C for a period of 2-3 weeks before the analysis.

2.2 Extraction of polyphenolic compounds from samples

Extraction of antioxidants from the 6 samples (grape pomace: Prokupec, Vranec, Kardinal, Zupjanka, as well as blueberry and aronia) was performed using the following materials:

Red grape pomace: Prokupec , Vranec and Kardinal, White grape pomace: Zupjanka, Berries: blueberry, aronia (chokeberry); Extraction solution (ethanol/water/acetic acid = 80/20/1, v/v/v); Distillated water.

Firstly, 10 g of each sample (Zupjanka, Kardinal, Prokupec, Vranec, Blueberry, Aronia) were measured on an analytical balance (Figure 10). The extraction was performed twice with 100 ml extraction solution (ethanol/water/acetic acid = 80/20/1, v/v/v) (Figure 11). Acetic acid was used to prevent the oxidation of polyphenolics during the extraction and to increase the efficiency of extraction. This mixture was transferred in an Erlenmeyer flask together with a magnetic stir Andonovic et al. Application of grape pomace as a natural food preservative and source of biofuel bar and the samples were stirred for 30 min on a magnetic stirrer (Figure 12). After the extraction, the both supernatants were collected together brought to a final volume of 250 ml with distilled water. All extractions for each grape sample were performed in triplicate. Table 1 presents the masses of the samples used in this experiment.

Table 1. Mass of the samples used for extraction

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In total 18 extracts were obtained. Before the spectrophotometric analysis, samples were filtrated (Figure 13) in order to disregard the solid leftovers of the samples used.

Then, the 18 extracts (200 ml) were pleased in 18 sealed bottles, which were immediately frozen, to avoid reduction of the antioxidant activity of the constituents of the extracts (oxidation of the polyphenols). Since the extraction was performed three times, all three extract of one sample (200 ml each) were combined together, obtaining one sample with total volume of 600 ml. This extract from each sample was concentrated with rotoevaporator. The extraction was repeated twice, obtaining two sets of 18 extracts, or 36 extracts in total. The first extract’s set (18) was applied in the milk before the fermentation, and the second set was applied after fermentation, in the obtained yoghurt.

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Figure 10. Measuring Prokupec sample

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Figure 11. Adding solution to Zupjanka 3



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Titel: Application of Grape Pomace as a Natural Food Preservative and Source of Biofuel