Aim: This work describes the activities of new-generation enzymatic preparations in fruit-berry substrates engineered for use in the fruit-wine industry. The enzymes were produced after genetic modification and selection of fungi Penicillium verruculosum, which produce efficient cellulase and pectinase enzymatic complexes.
Methods and results: This paper covers the main characteristics of novel multi-enzyme complexes and the results of in-lab fruit-wine production with addition of enzymatic preparations, which could be used on an industrial scale. The juice yield and the content of suspended materials in the enzymatically treated samples were compared. Experiments included the sensory analysis of produced juices and fruit wines.
Conclusion: Results show a significant increase in juice yield from the fruit pulp processed with the enzymatic preparations, without any negative effect on the quality and organoleptic attributes of the final product.
Significance and impact of the study: The obtained data clearly show that the use of the new-generation enzymatic preparations in the fruit-wine industry is effective.
This paper addresses an important technological problem of the fruit-wine industry (Codex Alimentarius: wine made from fruit other than grapes): how to increase juice yield from raw material without compromising the quality of the final product. Fruit-wine consumption is significantly lower compared to traditional grape wines; however, in countries such as Great Britain, Poland or Russia, this type of beverage is well known and appreciated (Noller and Wilson, 2009; Kiselev et al., 2013). A vast range of raw materials can be used for fruit-wine production: apple, pear, pineapple, guava, kiwi (Soufleros et al., 2001), Chinese lychee, orange, cherry, cranberry, mango, passion fruit, papaya, peach, etc. . Selection of the raw material is mainly determined by traditional recipes existing in the country of origin.
The fruit-wine technology is characterized by the specificity of the raw materials, which vary in their chemical content and requirements for different processing conditions. The production of such types of wine is often confronted with numerous problems such as low juice yield, difficulties with pressing, slow juice clarification, clouding and color changes in the final product (Volchok et al., 2013).
Currently, preprocessing of fruits and berries with various enzymes prior to pressing and filtration is considered to be the most effective solution to these technological problems (Jayani et al., 2005; Liew Abdullah et al., 2007), ensuring better fiber maceration and juice clarification, prevention of colloidal hazes, and achievement of balanced and diverse flavors (Ageeva and Markosov, 2013). Selection of enzymes is based on their activities required for a particular fruit or berry.
The Enzyme Biotechnology Laboratory of the Bach Institute for Biochemistry, Russ. Acad. Sci. (INBI RAS) is developing new superior enzymes and enzymatic complexes with several activities at ratios allowing the efficient processing of various raw materials. This article describes the processing of several fruit substrates, containing cellulose and hemicellulose, with the new multi-enzymatic complexes BI_3-227.7 and BI_3-227.4, followed by the lab-scale production of fruit wines. Both enzyme preparations were derived from recombinant strains of Penicillium verruculosum. These complexes were selected in relation to earlier experiments processing fresh viburnum and strawberry juice yield from the pulp and larger content of reducing sugars (Volchok et al., 2013). The results of the organoleptic analysis and the comparisons of enzyme-processed juices and wines against the non-processed samples are presented to acknowledge the efficiency of the proposed method and multi-enzymatic complexes.
Materials and methods
Ash berry, plum and black currant provided by the Russian State Agrarian University named after K.A. Timiryazev were used as raw fruit and berry material. Table 1 shows the dates of harvest and sampling.
Table 1. Harvest and sampling dates of raw fruit and berry material.
|Materials||Harvest dates||sampling dates|
2. Enzyme preparations
Multi-enzyme preparations were obtained by cotransformation of the auxotrophic host strain P. verruculosum 537. Expression plasmid encoding P. canescens pectin lyase (PELA) and Aspergillus niger β-glucosidase (BG), and transforming plasmid pSTA 10 were used in transformation experiments. Details of the developed process for the recombinant strains and enzyme preparations are described in Bushina et al. (2012). Preparations are in the form of a light brown powder (easily soluble in water) obtained by lyophilization of culture filtrates (micro-filtrated and concentrated by ultrafiltration method) after fermentation of recombinant P. verruculosum strains, and they show stable, high enzymatic activity in the range of 25-50ºС for temperature and 4.0-5.0 for pH.
Enzyme complexes contained pectin lyase A, cellobiohydrolase, endo-1,4-glucanase and β-glycosidase. Earlier activity of enzymes was tested on apple, citrus and beet pectin (Morozova et al., 2010; Bushina et al., 2012). Their main enzymatic activities are presented in Table 2.
Enzymatic activities towards polysaccharide substrates were determined from the initial rates of formation of reducing sugars by the Somogyi–Nelson method (Nelson, 1944; Somogyi, 1952). Activities against p-NP-derived substrates were determined at pH 5.0 and 40ºC by measuring p-nitrophenol release, as described elsewhere (Gusakov et al., 2005). All activities are expressed as international units per mg protein (U/mg) (one unit corresponds to the hydrolysis of 1 μmol of glycoside bonds from the substrate per minute). Methods of determination of enzymatic activities are described in detail in Bushina et al. (2012).
Table 2. Characteristics of multi-enzymatic preparations.
|Protein content, mg/g of preparation||854±39,77||503±33,6|
|Cellulase (CMCase) activity, U/g of preparation||3346±388,84||3194±310,6|
|Cellulase (Avicelase) activity, U/g of preparation||170±19,92||174±14,9|
|β-glycosidase activity, U/g of preparation||3999±388,11||395±25,49|
|Pectinlyase activity, U/g of preparation||2694±388,38||1164±113,2|
|Xylanase activity, U/g of preparation||3490±288,28||5310±334,9|
* - Sum of cellulase activities is equated to 1.
3. Scheme for producing wines
Fruit-wine materials were produced in the lab using the methodology presented in Table 3.
Table 3. In lab fruit-wine production schemes.
|Material||Technological scheme without enzymatic treatment of fruit pulp||Technological scheme with enzymatic treatment of fruit pulp|
|1. Washing and sorting of raw material|
|2. Crushing pulp, introduction of pulp water to obtain titrated acid content 5 g/1000 mL|
|3. Sulfitation of fruit pulp (K2S2O5, «Megahim», RU) up to 80 mg/1000 mL salt|
|4. Maceration of fruit pulp carried out in during 24 h at 50 ºС||4. Maceration of fruit pulp carried out in presence of enzyme preparation #3-227.7 (0,03% from pulp mass) during 24 h at 25 ºС|
|5. Pressing pulp (laboratory mechanical press capacity 1500 mL)|
|6. Introduction sugar up to 22 g/100 mL (sugar syrup 80%), sulfitation of fruit juice up to 120 mg/1000 mL|
|7. Sedimentation (24 h, 4 ºC), separation|
|8. Must fermentation process up to 1.5 g/100 mL sugar at 20 ºС. Adding commercial yeast «France universal» (France) 500 g/625 L, introduction in fruit must 0,6 g/1000 mL (NH4)H2PO4 (Sigma, USA)|
|9. Fermentation in during 15 days, 20 ºC|
|10. Sedimentation (24 h, 4 ºC), separation|
|11. Filtering wine materials (if necessary), storage in glass bottles for 4 ºС|
currant sweet wine
|1. Washing and sorting of raw material|
|2. Crushing pulp, introduction of pulp to obtain titr titretaed acid content 6,8 g/1000 mL|
|3. Sulfitation of fruit pulp (K2S2O5, «Megahim», RF) up to 80 mg/1000 mL|
|4. Maceration of fruit pulp carried out in during 24 h at 50 ºС||4. Maceration of fruit pulp carried out in presence of enzyme preparation #3-227.4 (0,03% from pulp mass) during 24 h at 25 ºС|
|5. Pressing pulp (laboratory mechanical press capacity 1500 mL )|
|6. Introduction sugar (sugar syrup 80%) up to 22 g/100 mL concentration, sulfitation of fruit must up to 120 mg/1000 mL concentration|
|7. Sedimentation (24 h, 4 , 4 ºC), separation|
|8. Must fermentation process up to 1,5 g/100 mL sugar at 20 ºС. Adding commercial yeast «France universal» (France) 500 g/625 L, introduction in fruit must 0,6 g/1000 mL (NH4)H2PO4 (Sigma, USA)|
|9. Introduction sugar (sugar syrup 80%) up to 15 g/100 mL sugar concentration, mash fermentation carried out in during 30 days, 20 ºC|
|10. Sedimentation (24 h, 4 ºC), separation|
|11. Filtering wine materials (if necessary), storage in glass bottles at 4 ºС|
4. Analytical methods
During the experiments, juice yield, viscosity and suspension content in fermented samples were compared.
Characteristics of fruit juices
Suspension content of fruit must was evaluated gravimetrically by centrifugation. 10-cm3 samples were put in pre-measured sedimentation tubes and centrifuged for 10 min at 3000 rpm. Supernatant was removed, leaving the tubes upside down for 1 min. Sediment content was calculated by the equation:
m2 – mass of sedimentation tube with sediment, g;
m1 – mass of empty sedimentation tube, g; and
V – sample volume, cm3.
For determination of relative viscosity, samples were centrifuged for 10 min at 8000 rpm. Then 5 cm3 of liquid was incubated in an Ostwald viscometer for 5 min at 20ºC (Ashapkin et al., 2005). Relative viscosity was calculated by the equation:
Тi – flow time of selected sample, sec; and
Т0 – flow time of water, sec.
5. Sensory analysis of fruit juices and wines
Ten people were recruited for participating in the sensory analysis of produced juices and fruit wines. The range of descriptors and reference terms allowing the complete organoleptic description of the juices and wines was selected previously (Baxter et al., 2005). 10-scale evaluation maps were developed using the following key terms: “weak”, “little” or “absent” for the left anchors and “strong” or “much” for the right anchors key words. During the week (5 days), panel members participated in training sessions to ensure an homogeneous interpretation of the terms and correct filling of the score cards (Laboissiere et al., 2007).
3 terms (2 for wine) were selected for color and turbidity, 5 for aroma, and 6 for flavor and aftertaste. Samples were served in 100-mL transparent plastic glasses coded with three-digit codes. Water and unsalted biscuits were provided for clearing the palate. Spider web plots were made for graphical representation of the tasting session results (Duarte et al., 2010).
6. Data analyses
Physical characteristics of the juices were measured in triplicate for each parameter. Identified Relative standard deviation (RSD%) and confidence interval were identified.
Student t-test capability on Microsoft Excel 2003 was used to determine the significance of the differences in between attributes (Doerffel, 1990). For data processing, a statistical significance level of P=0.05 was used.
Results and discussion
The yield of free-run juice in the course of in-lab production of fruit wine is presented in Figure 1 (data obtained were recalculated for 1 ton of pulp – raw weight). It is important to note that due to high acidity the substrate pulp used for the experiment was diluted with water. The must yield from untreated pulp was used as a control.
Figure 1. Volume of free-run juice from 1 ton of pulp, L.
It is clearly seen from the figure data that the use of enzymatic preparations significantly increases the yield of high quality free-run juice.
Table 4 shows the results of relative viscosity for the must produced from different raw materials and its sediment content.
Table 4. Physical attributes of fruit must.
|Tested samples, fresh must||Relative viscosity||%RSD||Mass fraction of suspended, g/100 mL||%RSD|
|Ash berry||Enzyme preparation||1,715±0,013||0,321||0,726±0,037||0,704|
|Black currant||Enzyme preparation||2,079±0,01||0,194||1,143±0,075||5,094|
The advantages of the enzymatic treatment of raw plant materials compared to non-processed samples are lower viscosity (lower biopolymer content - cellulose, hemicellulose, pectin - due to enzymatic destruction) and lower concentration of sediment in the fermented must.
Thus, enzyme preparation BI_3-227.4 was chosen for treatment of black currant and plum due to its β-glucosidase and pectin lyase activities leading to the rapid rarefaction bioconversion of pectin substrates. Another enzymatic preparation, BI_3-227.7, was chosen for the ash berry treatment due to its cellulase and hemicellulose activities. These results were expected as the preparation formula, either BI_3-227.4 or BI_3-227.7, correlates to the component composition of the cell wall of these plants.
Table 5 shows the results of the organoleptic analysis of the juices obtained with enzymes and the juices obtained by pressing after maceration. Participants in the sensory analysis especially noted more attractive color characteristics in the case of the enzyme-processed plum juice compared to the reference sample.
Table 5. Fruit juices sensory attributes mean values.
|Attributes||Definitions||References||The results of the evaluation samples of juices (average)|
|Ash berry (enz.prep.)||Ash berry (control)||Black currant (enz.prep.)||Black currant (control)||Yellow plum (enz.prep.)||Yellow plum (control)|
|characteristic juice color||Color characteristic of juice (for ash berry – intense pink, for black currant – dark purple, for plum – light yellow)||Little: 0-1||8,84||8,93||9,7||9,85||9,53*||8,63*|
|presence of suspended particles||Presence of particles from fruit pulp||Absent: 0-1||4,83*||5,58*||2,95**||3,65**||4,353*||7,353*|
|turbidity||Non-limpid aspect related to the difficulty of light passing through juice||Absent: 0-2||4,6*||5,3*||2,55**||3,44**||5,753*||8,453*|
|natural||Characteristic aroma from natural juice||Weak: 0-1||8,69||8,84||9||8,95||7,553*||7,33*|
|acid||Aroma related to the presence of characteristic organic acids from fruit||Weak: 0-3||6,35||6,4||7,55**||8,2**||6,15||5,8|
|sweet||Aroma due to the presence of sucrose and other sugars from fruit||Weak: 0-3||5,25||5,25||5,12||4,95||4,2||4,05|
|cooked||Characteristic aroma from fruit submitted to thermal processing||Absent: 0-2||0,3||0,3||0,1||0,1||2,53*||3,453*|
|fermented||Characteristic aroma from fruit showing signs of early deterioration||Absent: 0||0,5||0,5||0,5||0,6||1||1|
|natural||Characteristic flavor from natural juice||Absent: 0-1||9||9,05||8,5||8,7||7,8||7,6|
|acid||Flavor stimulated by the presence of characteristic organic acids from fruit||Weak: 0-3||8,22||8,35||8,7||8,95||7,13*||7,653*|
|sweet||Flavor stimulated by the presence of sucrose and other sugars from fruit||Weak: 0-3||6,61||6,31||5,65||5,3||5,75||5,4|
|cooked||Characteristic flavor from fruit submitted to thermal processing||Absent: 0-2||0,1||0,2||0,4||0,4||2,8||3,8|
|fermented||Characteristic flavor from fruit showing signs of early deterioration||Absent: 0||0,3||0,5||0,1||0,5||1||1|
|astringency||Harsh sensation perceived in mouth and tongue characteristic of fruit||Weak: 0-4||7,32||7,72||5,3||5,5||3,65||3,25|
|consistency||Perception in mouth of juice dilution or concentration||Weak: 0-3||7,75||8,15||9,28||9,3||7,7||7,4|
* paired parameters ash berry juices (** for black currant, 3* for yellow plum) with t-test above the significance level P.
Figure 2 shows significant differences in aroma and appearance characteristics between the enzyme-processed samples and control.
Figure 2. Appearance and aroma attributes for simples of juices.
Juices that have undergone enzymatic treatment are characterized by lower amount of suspended particles and lower turbidity, which facilitates the subsequent clarification and filtration processes. Figure 3 depicts the differences in taste attributes of the juices. From this spider web plot illustrating the flavor attributes of the compared juices, we can draw the conclusion that the enzyme used had a minor influence on flavor consistency.
Figure 3. Flavor and consistency attributes for simples of juices.
Juices produced by traditional maceration are characterized by a stronger aroma. It happens as maceration done without multi-enzyme complexes takes significantly more time compared to enzymatic treatment. At the same time, the richness of the juice produced with enzymatic complexes can be corrected by adjusting the fermentation time.
The organoleptic study of the wines produced from analyzed juice samples included the determination of fruity and floral notes in aroma and main flavor parameters (Table 6).
Provided data show that the use of multi-enzyme complexes for fruit-wine production has a positive effect on appearance and aroma characteristics (especially noticeable in the case of plum juice) without affecting other organoleptic attributes.
Table 6. Fruit wines sensory attributes mean values.
|Attributes||Definitions||References||The results of the evaluation samples of fruit-wines (average)|
|Ash berry (enz.prep.)||Ash berry (control)||Black currant (enz.prep.)||Black currant (control)||Yellow plum (enz.prep.)||Yellow plum (control)|
|characteristic color||Color characteristic of wine (for ash berry – intense pink, for black currant – dark purple, for plum – light yellow)||Little: 0-1||9||8,9||9,5||9,8||93*||7,93*|
|clarity||The degree of transparency||Absent: 0-2||9*||8,1*||9,5||9,2||8,73*||7,13*|
|fruity||Characteristic aroma from fruit||Weak: 0-1||8,8||8,71||9,5||9||8,23||7,45|
|sour||Aroma related to the presence of characteristic organic acids from fruit||Weak: 0-3||7,4||7||7,7||7,83||5,4||5,5|
|floral||Fresh floral notes in the aroma||Weak: 0-3||6,1||5,6||6,8||7,2||5,13*||4,33*|
|cooked||Characteristic aroma from fruit submitted to thermal processing||Absent: 0-2||0,5||0,5||0,2||0,2||23*||3,33*|
|fermentation||The aroma of the presence of yeast||Absent: 0-1||0,5||0,5||1||1||1,5||1,5|
|natural||Characteristic flavor from natural fruit||Absent: 0-1||9,5||9,3||8,75||8,7||7,83*||6,93*|
|acid||Flavor stimulated by the presence of characteristic organic acids from fruit||Weak: 0-3||7,8||7,7||9,1||8,8||5,5||5,6|
|sweet||Flavor stimulated by the presence of sucrose and other sugars from fruit||Weak: 0-3||6,6||6,25||6,1||5,9||6,2||6,5|
|cooked||Characteristic flavor from fruit submitted to thermal processing||Absent: 0-2||0,3||0,2||0,5||0,5||3,13*||4,23*|
|fermentation||The aroma of the presence of yeast||Absent: 0||0,3||0,3||0,2||0,2||0,5||0,5|
|astringency||Harsh sensation perceived in mouth and tongue characteristic of fruit||Weak: 0-4||6,2||6,4||4,5||4,3||2,1||1,8|
|consistency||Perception in mouth of fruit wine dilution or concentration||Weak: 0-3||8,8||8,6||9||9,1||7,1||6,96|
* paired parameters ash berry wines (** for black currant, 3* for yellow plum) with t-test above the significance level P.
Figure 4 depicts the results of the sensory analysis of produced fruit wines. In the case of yellow plum, significant positive difference can be observed. The sample produced with the developed enzyme complex is characterized by noticeably lower cooked odor and better clarity compared to the reference.
Figure 4. Appearance and aroma attributes for simples of fruit-wines.
Comparing flavor parameters of enzyme-processed wine and control showed no significant differences (Figure 5).
Figure 5. Flavor and consistency attributes for simples of fruit-wines.
In earlier research held in the Enzyme Biotechnology Laboratory INBI RAS, the organoleptic parameters of the red wine processed with the help of monitored enzymatic complexes were compared to the wine produced according to traditional technology. Cabernet Sauvignon and the local grape variety Tsimlyansky Black (provided by the «SARKEL» department of the «Tsimlyansky wines» company), a local grape variety, served as raw material for the wine. Results obtained during the tasting session are described in Volchok et al. (2014). These data show that the developed multi-enzymes applied to the grape-wine industry allow to achieve well balanced wines with rich fruity aromas.
Samples of fruit wines were produced in-lab, including a maceration stage with the use of new enzymatic preparations consisting of the target activities of cellulase, β-glucosidase and pectin lyase. Different ratios of the target activities allowed to apply specific enzymatic agents to a particular type of raw material. The juice yield was higher compared to the reference sample. And produced fruit-wines material were not inferior to the reference sample in quality attributes, showing lower viscosity, lower sediment content and higher color intensity. Lower sediment content was observed in the must treated by enzymatic preparations. Organoleptic analyses of juices and fruit wines showed a positive effect of multi-enzyme complexes on the sensory characteristics of products.
Obtained data clearly show the high efficiency of the new-generation enzymatic preparations in the fruit-wine industry.
Acknowledgements: this work was supported by FASIE, Russia (grant no. 346 GU1/2013).
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