Challenges in the Production Line of New-Generation Balsamic Vinegars

CHALLENGES IN THE PRODUCTION LINE OF NEW-GENERATION BALSAMIC VINEGARS

10

Sofia Lalou, Fani Th. Mantzouridou, Maria Z. Tsimidou Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece

10.1 Overview Technically, vinegar is a double fermented food product. Even if in the past it was the result of a spontaneous transformation of an alcoholic base by acetic bacteria, nowadays it belongs to a specific category of food items and is covered by specific legislation (Ho et al., 2017; RiosReina et  al., 2017; Samad et  al., 2016). Using as the only keyword the term “vinegar”, it is evident that the interest of the scientific community is steadily increasing since the 1970s (Fig.  10.1) (Scopus, last access: June 2017) for this type of products. Combination of the terms “balsamic” and “vinegar” indicates that the share of publications on balsamic vinegars is rather limited (~4%). These publications mainly cover aspects of the production line of the high added-value Italian vinegars, such as the Traditional Balsamic Vinegar of Modena (TBVM) and that of Reggio Emilia (TBVRE). The latter have been registered as products of Protected Designation of Origin (PDO) in 2000 and 2013 (Regulation (EC) No 813/2000; Regulation (EC) No 1279/2013), respectively. To our knowledge, the first publication on balsamic vinegar cited in Scopus dates back to 1998 and the interest since then is related to the registration process and the requirements derived from it as well as the general consumer interest in gourmet products. The European database DOOR includes the two above mentioned PDOs and the Balsamic Vinegar of Modena (BVM) that has been registered as Protected Geographical Indication (PGI) (Regulation (EC) No 583/2009). The market prices of PDOs differ to a great extent from that of the PGI balsamic vinegars, which in turn have higher ones compared to those of common vinegars. Biotechnological Progress and Beverage Consumption. https://doi.org/10.1016/B978-0-12-816678-9.00010-2 © 2020 Elsevier Inc. All rights reserved.

311

312  Chapter 10  PRODUCTION LINE OF NEW-GENERATION BALSAMIC VINEGARS

Vinegar

718

Balsamic vinegar

700 500

455

400 300

264 194

200

136

7 –1

5

7

16

–1

20

–1

24

20

0

5 06 20

01

00

20

–2 96

–0

0

5 –9

0 91

32

4

4

19

–9

5 86 19

–8

0

81

19

–8

5

55

21

12

7

76

–7

0 71 19

–7

5 66 19

–6

0 –6

61 19

5 56

–5 19

51 19

19

46

–5

0

0

10

1

19

6

11

100

19

No. of publications/5 years

700

Time period (years)

Fig. 10.1  Trends in vinegar (light gray) and balsamic vinegar (dark gray) number of publications in the period 1946–2017 (Scopus search of the respective terms in the title, abstract, keywords, carried out on June 2017).

In brief, TBVM and TBVRE are two traditional products that date back probably to the Middle Ages (Giudici et  al., 2015; Mattia, 2004). Their production line is illustrated in Fig. 10.2 and uses grapes typical in the provinces of Modena and Reggio Emilia, which “produce must with a saccharometer reading of 15°Brix or more” (GURI, No 124/2000). The must is then cooked in open containers until a minimum sugar content of 30°Brix is attained. Blended musts or musts containing additives or other substances are not permitted. Next, the cooked must undergoes double fermentation using traditional methods. The first step of the process is the spontaneous fermentation by autochthonous yeasts until a content of 5%–7% (v/v) alcohol is achieved. The latter is the minimum quantity that can guarantee the production of >5% w/v acetic acid at the second step of the process, that is, the acetic fermentation. Inoculation with mother (or seed) vinegar accelerates the start-up of the second step (Solieri and Giudici, 2009). The final step of the process, the aging period, is a long maturation period of at least 12 years. Aging takes place in a set

Fig. 10.2  Schematic presentation of the steps in the production of TBV and the “Rincalzo” (refilling) process.

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of barrels called a “battery.” Batteries usually consist of at least five barrels of different woods (e.g., oak, chestnut, mulberry, juniper, and cherry) and descending volume (usually between 60 and 20 L) (Giudici et  al., 2015; Mattia, 2004; Solieri and Giudici, 2009). A periodic, reciprocal, partial withdrawal and renewal of the content of each barrel occurs throughout the aging period. Barrel no 5 receives every year freshly cooked and fermented concentrated grape must (CGM). The final product is tested to satisfy the minimum regulated requirements. The tests are related to both analytical and organoleptic characteristics but are rather simple regarding the economic value of the product. Thus, the tests concern acidity expressed as g acetic acid/100 g product and density at 20°C. The sensory analysis of the PDOs is carried out by an expert panel trained by a competent authority and includes the evaluation of attributes related to visual, aroma, and texture parameters (Zeppa et al., 2013). The certified vinegar is transferred into “special round crystalline glass bottles with a square base and a capacity of 10–40 cl” (GURI, No 124/2000; EEC No 2081/92, 1992). Its production usually takes place in small-scale— mostly family—businesses and combines elements of art and skill of generations of artisans, who developed their own unique recipe. BVM is produced in a totally different way and thus possesses a very different physicochemical and sensorial profile. According to the article 4.5 of the Regulation (EC) No 583/2009, “BVM is obtained from grape must that is partially fermented and/or boiled and/or concentrated by adding a quantity of vinegar aged for at least 10  years and with the addition of at least 10% of vinegar produced from the acidification of wine only. The percentage of boiled and/or CGM should not be <20% of the volume sent for processing”. Maturation for at least 60 days in wood receptacles is obligatory. The wording “invecchiato” (aged) is used for products with a minimum period of 3 years’ storage. Addition of caramel for color stability is permitted up to 2% of the final product. No other additives are permitted. The bottles can be made out of different materials (e.g., glass, wood, and ceramic) in volumes of up to 5 L. Plastic containers up to 2 L can also be used but only for catering. No sensory panel judgment is required before their commercialization. As stated in the Regulation (EC) No 583/2009, the protection “is granted to the term ‘Aceto Balsamico di Modena’ as a whole.” “Individual non-geographical components of that term” may be used, “even jointly and also in translation, throughout the Community, provided the principles and rules applicable in the Community’s legal order are respected.” Currently, a new generation of products bearing the phrase “balsamic vinegar” in their legal name is commercialized in the EU member states and worldwide, which are regulated by national vinegar legislation (Giudici et al., 2015). In that last, the term “Balsamic Family” is introduced to describe a heterogeneous group of vinegars and condiments deriving from grapes (Table 10.1) and also the term Generic Balsamic Vinegars (BVs) to name the

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Table 10.1  Balsamic Family products According to Giudici et al. (2015) Type of Product

Group

Legal status

Balsamic condiments Balsamic vinegar of Modena Generic balsamic vinegars Traditional balsamic vinegar of Modena Traditional balsamic vinegar of Reggio Emilia

Condiment Vinegar Vinegar Condiment Condiment

General food regulation (national or international) Special protection-PGI Vinegar regulations (national or international) Special protection-PDO Special protection-PDO

above mentioned ones. These terms are adopted for the needs of the present chapter to avoid misunderstandings and confusion. To the best of our knowledge, generic BVs production conditions and characteristics are not included in the standards of Vinegars of Codex Alimentarius (FAO-WHO, CL 2000/18) or the recently amended European legislation for wine products (EC Regulation, EC 1308/2013), or in other national legislation for types of vinegar. Only Greece adopted a legal status for these products under an amendment on the vinegar types and their manufacturing practices that was added to the provisions of article 39 of the National Food Codex (OGGHR, No 270/2011 and No 231/2014). In this chapter, challenges in the production line of the generic BVs at the various stages of production line are highlighted and discussed. Principles of the methods followed for the production of registered products as well as contemporary industrial demands for standardized safe food products of high quality are taken into consideration. Emphasis is paid to generic BVs that can be produced by two-stage fermentation.

10.2  Production Line of Double Fermented Generic BVs The production of double fermented generic BVs is a procedure which can be perceived as quite perplexing for a new manufacturer. As illustrated in Fig. 10.3, the very first step in their production line is the selection of the proper starting materials. The next step is the production of CGM, which will be alcoholically fermented (step 3) until the desired degree at an industrial scale. The obtained product from step 3 is the base for the following step of acetic fermentation. The balsamic vinegar produced can be optionally aged before bottling and commercialization.

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Step 5 Step 4 Step 3 Step 2 Step 1 Starting material selection

Grape must concentration

Alcoholic fermentation

Aging

Acetic fermentation

Fig. 10.3  Schematic representation of the multistep production line of double fermented generic balsamic vinegar.

Each of these steps encounters critical points that should be considered carefully by the manufacturer in order to obtain a high quality product. The potential challenges at each step are examined from an industrial perspective aiming at ensuring a faster completion of all steps in a reproducible manner offering at the same time safe products of desirable sensorial profiles.

10.2.1  Selection of Starting Materials In TBVM, TBVRE, and BVM production, the grape varieties used are typical in the provinces of Modena and Reggio Emilia. More specifically, all the varieties and clones of Lambrusco and Trebbiano grapes as well as the varieties Ancellotta, Sauvignon, Sgavetta, Berzemino, and Occhio di Gatta are allowed for Traditional Balsamic Vinegars (TBV) production (GURI, No 124/2000). For BVM production the grape varieties used are Lambrusco, Sangiose, Trebbiano, Albana, Ancellotta, Fortana, and Montuni (Regulation (EC) No 583/2009). Among the aforementioned varieties, both red and white variety grapes are suitable for the production of CGM (Table 10.2). In the production of BVs, restriction in selection of grape variety does not exist. The prerequisite is to use well-ripened grapes in order to achieve high sugar content. During ripening, the increase in sugar content and the parallel decrease in certain acids (e.g., malate) leads to a shift from sour to sweet berries taste attributes (Ribéreau-Gayon et al., 2006). The selection of a red or a white variety or a combination of both is expected to affect the sensory and quality characteristics of the CGM due to differences in the composition and the content of responsible compounds (Zeppa et al., 2013). For example, in TBV production when red grape varieties are used, such as Lambrusco, a maceration procedure lasting 24 h is performed. This leads to the increase in the concentration of phenolic antioxidants due to the partial extraction

316  Chapter 10  PRODUCTION LINE OF NEW-GENERATION BALSAMIC VINEGARS

Table 10.2  Color of Grapes Used in Traditional Balsamic Vinegar (TBV) and Balsamic Vinegar of Modena (BVM) Production Variety

Grape Color

TBV

BVM

Albana Ancelotta Berzemino Fortana Lambrusco Montuni Occhio di Gatta Sangiovese Sauvignon Sgavetta Trebbiano

White Red-black Red Red-black Red White Red Red White Red White

− + + − + − + − + + +

+ + − + + + − + − − +

of these compounds from the red skin. Among them, anthocyanin, flavonol, and stilbene derivatives are three classes of compounds exhibiting unique properties, which affect the visual and organoleptic characteristics of grape must and the wine (Flamini et al., 2013). In the case of designing a new BV, grape selection should ensure— beyond color hues—the desirable traits of the final products, that is, high sugar content and high acidity. Giudici et  al. (2015) have proposed as desirable grape composition for TBV production, titratable acidity >7.5 g/L as tartatic acid, pH value lower than 3.2, sugar content >16°Brix and finally a sugar/acidity ratio between 1.2 and 2.5. In mature healthy grapes, the total sugar concentration ranges usually between 150 and 240 g/L with fructose being slightly predominant over glucose (glucose/fructose ratio (gluc/fruct) approximately 0.8–0.9) (RibéreauGayon et al., 2006; Antonelli et al., 2004). Another very important element for both wine making and BV production is the acidity of the must, which is essentially due to the presence of tartaric, malic, and citric acids. Their concentration in the grape depends on the cultivar, the climate, and maturity stage. Total acidity reaches up to 4.5–15 g/L in tartaric acid for healthy grapes (Ribéreau-Gayon et al., 2006). In Greek legislation for generic BVs, there are no restrictions in the selection of grape variety. The final product can indicate on the label the grape variety only if this is used exclusively in the production

Chapter 10  PRODUCTION LINE OF NEW-GENERATION BALSAMIC VINEGARS   317

of both the CGM and the wine vinegar. BVs from “partial alcoholic fermentation and subsequent acetic fermentation of concentrated juice of dried grapes (raisins)” are also permitted.

10.2.2  Concentration of Grape Must Concentration of must is critical in geographical indication registered products as illustrated in Table 10.3. Concentration of grape must for PDO products is performed by boiling in open containers using a direct source of heat (GURI, No 124/2000; EEC No 2081/92, 1992). In the case of BVMs, the must can be either boiled or concentrated by other means. Interestingly, in contrast to the production of CGM in TBV production line, in certain national legislations direct heating is not permitted by law. The latter stands true for the Greek legislation. In practice, the degree of concentration depends on the subsequent use of CGM. It is expected to differ if generic BVs are manufactured by blending or by two-stage fermentation. For example, in Greek legislation, a generic BV should contain 100 g/L reducing sugars if manufactured by blending. The minimum quantity of CGM required is 10% (w/v). Based on simple calculations and taking into account that the presence of wine vinegar does not contribute to the final sugar concentration more than to 0.4–0.5 g/100 mL, this minimum amount does not seem to be realistic. As shown from the calculations in Fig.  10.4, to achieve the minimum sugar concentration in the BV a CGM concentrated by a factor of 6 must be used. According to our experience (Table 10.4), concentration of grape must by a factor higher than 4 is accompanied by undesirable characteristics, such as the extreme browning of the product and the increased bitterness. The safety aspects of the CGM are also influenced at high concentration factors as discussed below in the same subsection. Consequently, the regulated minimum quantity of 10% CGM for the production of generic BVs by blending needs revision. A manufacturer should use at least 15% (w/v) of a CGM concentrated by a factor of 4 to achieve 100 g/L reducing sugars in the BV and satisfy quality and safety standards. Thus, a reconsideration of the minimum quantity of CGM used for the production of generic BVs by blending will facilitate the compliance of the manufacturers with the law and the increase in the quality of these products. According to Greek legislation, a generic BV produced by two-stage fermentation of concentrated raisin extracts should contain at least 150 g/L reducing sugars. In literature, the alcoholic fermentation of CGM is expected to give a yield of 35%–40% (Landi et al., 2005) and the desired alcohol content is approximately 8% (w/v) (Giudici et al., 2015). If these two conditions apply, the raisin extract should be concentrated at least by a factor of 2 to attain a final concentration of 350 g/L reducing sugars.

Table 10.3  Physicochemical Composition of Concentrated Grape Must for Traditional Balsamic Vinegar Production as Reported in Literature Year

pH

2004 2007 2008

2.74–2.77 – –

2008 2011

2.69–2.96 –

2013

2.98–3.47

a b

Water (%)

Density (g/cm3)

Total Aciditya (%w/v)

Total Sugars (g/kg)

– 47–69 Prod A: 28–65 Prod B: 62 Prod C: 46–70 – Prod A: 39–45 Prod B: 54–69 Prod C: 66–67 –

– – Prod A: 1.14–1.32 Prod B: 1.15 Prod C: 1.07–1.14 1.13–1.23 Prod A: 1.26–1.28 Prod B: 1.15–1.18 Prod C: 1.14 –

– – Prod A: 0.87–1.88 Prod B: 0.48 Prod C: 0.6–1.03 – Prod A: 1.08–1.33 Prod B: 1.15–1.18 Prod C: 1.12–1.13 –

413 202–445 –

Expressed as acetic acid. Expressed as gallic acid.

– Prod A: 518–558 Prod B: 333–395 Prod C: 311–312 –

Glucose (g/kg)

Fructose (g/kg)

HMF (mg/ kg)

Furfural (mg/kg)

185–200 94–250 Prod A: 210–417 Prod B: 219 Prod C: 92–191 165–320 –

193–201 108–195 Prod A: 192–370 Prod B: 206 Prod C: 91–193 153–290 –

149–307

127.4–250

2283–3861 Trace-3100 Prod A: 94–7800 Prod B: 413 Prod C: 39–1102 216–592 Prod A: 621–6877 Prod B: 1107–2357 Prod C: 483–486 149.4–4021

– 2.94–7.80 Prod A: 3.3–31.0 Prod B: 0.49 Prod C: 0.09–3.30 – Prod A: 2.5–17.9 Prod B: 2.0–3.9 Prod C: ld–1.60 –

Total Phenolsb (mg/kg)

References

1144–1330 – –

Antonelli et al. (2004) Cocchi et al. (2007) Cocchi et al. (2008)

423–1259 –

Piva et al. (2008) Cocchi et al. (2011)

–

Tagliazucchi et al. (2013)

Chapter 10  PRODUCTION LINE OF NEW-GENERATION BALSAMIC VINEGARS   319

Fig. 10.4  Calculations for the production of a blended generic BV containing the minimum sugar amount according to the Greek legislation using the minimum provisioned amount of CGM (10%).

According to literature (Cocchi et  al., 2008), a concentration of 350–400 g sugar/kg of must represents “the suitable value for the starting raw material for TBVM production”. The degree of concentration is related to the subsequent use of the CGM as well as the desired sweetness of the end product and seems to be part of the recipe of each manufacturer. It is noteworthy to mention that in the last few years a consumer shift toward sweeter TBVs has been recorded. More specifically, the values of R factor that defines the ratio of total sugar concentration to titratable acidity used in the assessment of the balance between the sweet and the acid taste were increased 54% over a period of 22 years (1982–2004) (Giudici et al., 2009). The means and the degree of concentration are important not only for the achievement of the minimum sugar concentration but also for the control of the physicochemical phenomena that take place. During grape must concentration, apart from the loss of water, physicochemical changes concerning the two main sugars of grape must, that is, glucose and fructose, contribute significantly to the characteristics of the CGM. Among these characteristics, the final glucose/fructose ratio in the CGM is of great interest. During concentration, the more heat-sensitive fructose is degrading faster via the production of two intermediates, 1,2-endiol and 2,3-endiol (Kowalski et al., 2013; Antonelli et al., 2004), that modify the ratio of glucose to fructose. In musts produced from ripe grapes the glucose/fructose ratio is close to 0.8 and increases progressively to values higher than 1.0 in CGM (Antonelli et  al., 2004). The glucose/fructose ratio is considered as a quality indicator of the CGM, which can affect the stability of TBVM (Elmi, 2015). High concentration of glucose in TBVs and glucose/­fructose

320  Chapter 10  PRODUCTION LINE OF NEW-GENERATION BALSAMIC VINEGARS

Table 10.4  Sugar Concentration in Grape Must Before and After Boiling Using Direct Heat and the Corresponding Concentration Factor (Must From the Greek Red Variety Xinomavro and 4 Successive Harvest Years)

2012a 2012b 2013 2014 2015

Sugar Concentration in Grape Must (g/kg)

Sugar Concentration in Concentrated Grape Must (g/kg)

Concentration Factor

151.8

430.0 856.5 362.8 404.0 416.9

2.83 5.64 2.61 2.05 2.03

138.9 197.0 205.0

a,b

Two treatments of the same grape must.

ratio above a threshold increase the viscosity of the sample (Elmi, 2015; Falcone et al., 2011) but can jeopardize the stability of the final product in terms of jamming. More specifically, sugar concentration higher than 50% w/w in the final product in combination with high amounts of glucose can lead to the crystallization of glucose monohydrate molecules due to their decreased solubility in comparison with those of fructose (Falcone et  al., 2011). This phenomenon degrades the appearance of the final product. In BVs based on blending, using excess amounts of CGM with low glucose/fructose ratio in order to achieve a sweet product with high R ratio could lead to the formation of glucose crystals. In the case of double fermented generic BVs, the prediction of the glucose/fructose ratio in the final product can be more challenging because it is affected not only by the CGM used but also by the glucophilic or fructophilic nature of the yeast employed.

10.2.2.1  Safety Aspects of CGM Preparation The reactions that evolve during concentration affect also the safety of the product. Grape must is a favorable substrate for nonenzymatic reactions during heating. This is due to its high reducing sugar content and its weak acidic character. The multistep reactions taking place during concentration lead to the formation of furanic derivatives. Among these, 5-hydroxymethyl-2-furaldehyde (HMF) is

Chapter 10  PRODUCTION LINE OF NEW-GENERATION BALSAMIC VINEGARS   321

the ­major compound formed from both glucose and fructose under slightly different pathways (Antonelli et  al., 2004). HMF is a known cytotoxic, genotoxic, and tumorigenic compound. Recent studies of EFSA suggest a threshold of concern of 540 μg/person/day and a “Theoretical Added Maximum Daily Intake” of 1600 μg/person/day (EFSA, 2010). Despite the fact that this known health risk is compensated with the low consumption of BVs in the overall diet, HMF content is suggested as a potential marker for the chemical safety of the new-generation BVs and as a quality parameter regarding their production (Lalou et al., 2015a). In a market search in Greece, generic BVs produced by either blending of CGM with wine vinegar or double fermentation were bought and analyzed for their sugar and HMF content. CGM is not only the main contributor of sugar in blended BVs but also the major source of HMF. The HMF content of simple wine vinegar does not exceed 10 mg/L (Theobald et  al., 1998). In Table  10.5, the sugar and HMF content of the examined BVs as well as some basic information present in their labeling are presented. Overall, all the examined BVs produced by blending contained twofold higher sugar concentration than the minimum regulated amount. This finding is very promising regarding manufacturing process and knowledge gained by balancing legal provisions and desirable sensorial attributes of the new products. However, sugar contents is not the only concern in this production line as evidenced for samples 2 and 7 (Table 10.5). Sample 2 had 10 times lower HMF content than sample 7 despite the fact that the sugar concentration of the latter was only 2 times higher than that of the former. The quality of the starting commercial CGM is a critical point for the subsequent manufacturing steps. It can be, thus, suggested to authorities to examine whether a limit for HMF concentration should be set in the legislation of all products of the Balsamic Family, including registered ones. Indeed, in recent publications (Lalou and Tsimidou, 2015; Lalou et al., 2015a), high values for the HMF content were also found for BVMs (up to 4031 mg/kg) and TBV (~5000 mg/kg). Generic BVs produced by a two-stage fermentation of the concentrated raisin extract also seemed to comply with the specifications of their minimum sugar content in their respective category. The range of the sugar content in this case was rather limited in respect to the BVs produced by blending. Within this category of BVs, the HMF content exhibited extremely high values (up to ~14 g/kg) surpassing up to 7 times the highest HMF content of the blended BVs. The only exception was sample 2 most likely due to its white color. Milder condensation conditions are expected to prevail in this case in order to limit browning reactions. The high HMF content within this category can be attributed to the use of raisin. The latter is a starting material, which can contain considerable amounts of HMF (up to 57 mg/kg) according to studies (Çağlarirmak, 2006). The presence of HMF in the raisin extract can

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i­ nduce its formation in the subsequent concentration step resulting in the extremely high values reported in Table 10.5. These results suggest that authorities should also consider these points in a future revision of the legislation. Permission to double fermented products from grape must should be discussed in the near future regarding safety aspects.

Table 10.5  Labeling, Sugar and 5-Hydroxymethyl2-Furaldehyde (HMF) Content of Balsamic Vinegars (BVs) Present in the Greek Market Sample

Starting Materialb

Acidityb

Agingb

Sugarsc (g/kg)

HMFd (mg/kg)

7 6

− +

222.00 ± 8.29 240.80 ± 11.27

1236 ± 16 211 ± 28

6.2

+

384.60 ± 16.97

1047 ± 103

6

+

289.43 ± 8.43

342 ± 12.8

6

−

277.35 ± 9.86

1369 ± 16.1

6 −

− −

226.75 ± 8.69 517.93 ± 10.53

487 ± 24.8 2071 ± 70.7

6

+

243.13 ± 3.96

838 ± 31.7

239 ± 2.75 228 ± 8.60 231 ± 4.85 220 ± 2.68 194 ± 15.4

14,145 ± 625 12,030 ± 403 13,007 ± 842 202 ± 1.95 8756 ± 55.2

GENERIC BVS PRODUCED BY BLENDING

1 2 3 4 5

6 7

8

Wine and CGM Wine and concentrated grape, caramel color Wine and cooked grape must Vinegar from wine grapes, grape syrup Wine vinegar, grape must, antioxidant: E224, color: E150d BV, contains sulfides Wine vinegar and petimezi (cooked grape must) of organic farming Wine vinegar, CGM, caramel coloring

GENERIC BVS PRODUCED BY TWO-STAGE FERMENTATION

1Aa 1Ba 1Ca 2 3 a

Dry raisins Dry raisins Dry raisins Dry raisins Dry raisins of organic farming

6 6 6 6 6

− − − − −

Different capital letters refer to different lots of the same brand. According to the product label. c Mean values ± SD (n = 3) expressed as total content of glucose and fructose determined by HPLC (Lalou et al., 2015a). d Mean values ± SD (n = 3). −, Not stated/<6 months; +, >6 months. Mean values in the same column with the same online indicate that there are no significant differences between them (P < 0.05). b

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To achieve a better control of HMF formation, it is necessary to examine the conditions that favor it. Relevant studies during grape must concentration indicated numerous factors affecting its content in the CGM. Among them, grape variety, sugar concentration, antioxidant content, pH, water activity, temperature, heating time and intensity, and pot characteristics have been thoroughly studied (Tagliazucchi et al., 2013; Cocchi et al., 2007, 2008; Piva et al., 2008). As mentioned earlier, the effect of the grape variety on the characteristics of the CGM is mainly related to the antioxidant capacity and sugar/organic acid profile of the grapes used. In the case of CGM for TBVM production, Tagliazucchi et al. (2013) identified red variety Lambrusco as a safer starting material compared with the white variety Trebbiano. The high antioxidant capacity and high polyphenol content of the former led to lower concentration of HMF in the CGM. As far as pot characteristics are concerned there is evidence for the effect of the type of material and dimensions to the kinetics of concentration and the characteristics of the CGM. For example, pots made from stainless steel seem to offer protection against polyphenol oxidation and improve the antioxidant activity of the nonphenolic fraction despite the increased values of HMF content in CGM compared to those obtained in a copper pot (Piva et al., 2008). Moreover, the use of a vessel of high surface/volume ratio has two positive effects (i) the required time for concentration was decreased due to the extended evaporation surface and (ii) lower HMF contents were found compared to those obtained using vessels of low surface/volume ratio (Cocchi et  al., 2007). High heating temperatures (above 90°C) in combination with low water content and prolonged heating times can increase the formation of furan derivatives (Cocchi et al., 2007, 2008, 2011). When direct heating is applied, the temperature should be higher in the beginning of the process and lower as evaporation of water progresses. Industrially, grape must concentrated to 55, 65, or 68°Brix is produced for prolonging the storage time and minimize the transfer costs. These CGMs are produced by evaporation or freeze concentration and are most often used to enhance the sweetness of other products (Bates et al., 2001). These or even more concentrated (up to 80°Brix) grape musts can be used in the production of new BVs depending on the employed method (i.e., blending or fermentation). There are different systems in the production of CGM by evaporation (e.g., rising film, falling film, plate, centrifugal, or conical evaporators) depending on the desired results. For example, plate evaporators are used to achieve >60°Brix in the CGM. In any case, the main components of ­evaporators are a heat transfer surface, a feed distribution device, a liquid-­vapor separator, and a condenser (Bates et al., 2001). These means offer short heating times, minimizing at the same time the effects on flavor,

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aroma, and sugar components. CGMs up to 50°Brix without appreciable loss of taste, aroma, color, or nutritive value can be achieved by freeze concentration. In this case, the physical phenomenon taking place is freezing point depression and the setup consists of a freezer or crystallizer, a centrifuge, a wash column or a filter press, and a refrigeration unit. Despite the fact that freeze-drying is producing high quality CGMs, its relatively high capital costs and low throughput makes them a less favorable choice. The concentration of grape must by freeze-drying as well as other indirect heating technologies such as reverse osmosis and nanofiltration membranes for balsamic products is—to the best of our knowledge—limited. This can be attributed to the fact that the latter two technologies are mostly used to improve the quality characteristics (e.g., enrich sugar content) of grape must in poor vintages by applying mild operating conditions and thus CGMs up to 280 g/L are obtained (Pati et al., 2014; Gurak et al., 2010). This is not applicable to the production of BVs.

10.2.3  Alcoholic Fermentation On alcoholic fermentation, the sugars present in the CGM are fermented by yeasts to produce ethanol and carbon dioxide. Scientific knowledge about alcoholic fermentation in wine production in general has grown at an exponential rate (Chambers and Pretorius, 2010). Industrially, pure cultures of selected strains of Saccharomyces cerevisiae are added to the grape must shortly after crushing. In traditional wine making several yeast strains and species that are part of the indigenous microflora of the grape ferment the grape juice sequentially (Ciani et  al., 2016, 2010; Mercado and Combina, 2010). Non Saccharomyces species that are predominant on grapes and have low fermentative ability dominate the initial stages of the fermentation. As the process progresses (i.e., ethanol concentration increase) S. cerevisiae species takes over and completes the fermentation (Ciani et al., 2010). In contemporary wine making the potential of the indigenous species for use as pure or mixed starter cultures is being evaluated to produce wines with specific aromatic character typical of the region and the grapes used. Targeting to the production of high quality wines with distinctive body, viscosity, color, flavor, and aroma, grape juice is fermented with different yeast strains and under different conditions to achieve the optimum combination (Capece et al., 2012). The experience of wine making can be used as a compass in BV production but CGM is a rather difficult substrate to ferment. It contains some inhibitory factors, which may counteract yeast survival and growth. Among them are the high sugar content, the low pH value, the presence of furanic derivatives, and the increasing concentration of ethanol. The combination of negative parameters can be detrimental to the

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yeast survival and growth rate since they interfere with the morphological and physiological characteristics, and metabolic activity of the cells (Field et al., 2015; Rantsiou et al., 2013; Lee et al., 2011; Liu et al., 2008). For many years, alcoholic and acetic fermentation of TBV were not studied separately obviously because they took place in the same barrel (Solieri and Giudici, 2009). However, alcoholic fermentation is a distinct step of the traditional production line taking place spontaneously by native microflora present in the barrel. This microflora is impregnated in the barrel containing the freshly prepared CGM either by “occasional contamination” when the barrel is left open or with a back-slopping technique (Solieri and Giudici, 2008). Old barrels are preferred over new ones. New barrels have to be sterilized with salt water and boiling vinegar and then to be filled with wine vinegar to be ready to accept the freshly cooked must. Nowadays, the role of yeasts and their influence to the final quality of the product is recognized and the first publications on their ecology started to appear (e.g., Solieri and Giudici, 2008). Most of the available publications on TBV production and quality pay special attention to the isolation and characterization of the yeast microflora and their succession during the alcoholic fermentation (Giudici et al., 2009; Solieri and Giudici, 2008). Talking about the alcoholic fermentation design of generic BVs, which should be lined with the demands of the modern industry, the need of suitable starter cultures becomes a prerequisite. Traditionally fermented products are usually associated with variable quality and composition. Modern food industry requires safe, high quality competitive products with stable composition using good manufacturing and hygiene practices (Petrović et al., 2011). The use of commercially available starters is expected to contribute to product standardization but a toll on maintaining the typical traits of the product is also expected (Quero et al., 2014; Petrović et al., 2011). Taking into consideration the unique characteristics of CGM as a fermentable substrate, it can be hardly expected that starters specific to wine production could possess all the desirable technological features that positively affect the performance of the bioprocess and the quality of the end product. Indigenous species, however, are well adapted to grow on specific substrates (Capece et al., 2012) similar to those that have been isolated from wine. The exploitation of indigenous species also occurs in the traditional method for TBV but these species are undefined. This situation complicates the control of the process and does not ensure the optimum performance of yeasts. Moreover, looking at the bigger picture, the exploitation of defined indigenous species with specific role in the alcoholic fermentation of BV production coincides with the current trend for modernization of fermentation processes and quality standardization of traditional fermented products (Modha et al., 2015; Song et al., 2015; Quero et al., 2014; Petrović et al., 2011).

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Development of starter cultures for BV production can be very perplexing if all the desirable characteristics of the yeast strains are not considered. Among the traits reported for TBV starters (Solieri and Giudici, 2008), osmophilicity and glucophilicity are considered critical features to obtain a “well-fermented” cooked must. Yeast starters should be able to tolerate the high sugar concentration of the CGM in order to start the fermentation. A higher preference for glucose than for fructose metabolism is also required. Other desirable characteristics common with those for wine starters include high ethanol yield, high flocculation, and low foam abilities, certain enzymatic activities (e.g., esterase activity). These traits are expected to facilitate technological operation and ensure a high quality fermented product with desirable sensory characteristics (Lalou et  al., 2016; Styger et al., 2011; Chambers and Pretorius, 2010). A specific characteristic for yeast starters for BV productions that is contradictory with that of wine starters is the high acetic acid production. High acetic acid content (above 1.5% w/v) in the end of the alcoholic fermentation is undesirable for wines (since it provides a vinegar like character), but in the case of base wine production for BV a high acetic acid content will ease the successive step of acetification. Recent findings point out to another critical parameter for the selection of yeast starters for CGM fermentation, which is the HMF and furfural tolerance. Thus, it seems that tolerance of yeasts to both compounds is strain dependent with furfural being more toxic than HMF (Lalou et al., 2016). One could argue that CGM can contain low concentrations of furanic derivatives if good practices are followed. Still, their toxic effect can be harmful even at low concentrations as reported by Lalou et al. (2016) in experiments using yeasts isolated from CGM. The microflora exhibited growth inhibition even at low concentrations (e.g., 0.005 g/L furfural and 0.1 g/L for HMF) that are commonly found in CGMs. At the end of the alcoholic fermentation, the base wine should contain at least 8% w/v ethanol, 150 g/kg sugar content with fructose prevalence, and high acetic acid content. The ethanol content of 8% w/v can be considered low in terms of wine production but it ensures high residual sugar content (Giudici et al., 2015). As stressed above, in the fermented CGM, fructose prevalence over glucose is preferred. This is related to the stability of the product after its acetification and during aging. Otherwise, sedimentation of glucose crystals can occur degrading the appearance and the quality of the final product. In order to suppress the yeast activity and stop the fermentation at the right degree, the addition of base vinegar is suggested until a content of 2.5% w/v is reached (Giudici et al., 2015). Considering that this step slightly dilutes the base wine, high acetic acid content in the former is needed in order to limit the use of the base vinegar and thus the degree of dilution.

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10.2.4  Acetification Process After alcoholic fermentation, the base wine undergoes acetification, which refers to an aerobic biological process for the oxidation of ethanol to acetic acid via the metabolic activity of acetic acid bacteria (AAB). Individual enzymatic steps involved in this process have been described in detail previously (Gullo et al., 2014; Mamlouk and Gullo, 2013). The membrane-bound enzymes, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH) are the most important enzymes involved in the incomplete ethanol oxidation by AAB; the former catalyzes ethanol oxidation to acetaldehyde, which in turn is oxidized further to acetic acid by the latter. The complete oxidation of ethanol to acetic acid takes place in the cytoplasm by the action of a NAD(P+)dependent ADH and ALDH. When ethanol becomes depleted, acetic acid is further metabolized into CO2 and H2O via TCA cycle. This process goes through three distinct growth phases: ethanol oxidation phase, ­acetic-acid resistance phase, and acetate overoxidation-phase (Matsutani et al., 2013). From the technological point of view, acetification of base wine has been acknowledged as the most difficult step in TBV production. Typical failures of the procedure include unsuccessful start-up and acetate overoxidation (Giudici et al., 2015). As a result, the acidity value of the final product may not reach the minimum value required for TBV (minimum required acidity of 4.5%) and prevent microbial spoilage of the latter (Gullo et al., 2014; GURI, No 124/2000). CGM sugar content, temperature variation, and bacteriophage infection have been pointed out as the most important parameters for total or partial inactivation of AAB that negatively affect the initiation of the process for TBV production (Gullo et al., 2014, 2009; Gullo and Giudici, 2008). The slow fermentation process applied to TBV is accomplished by an undefined mixture of AAB present in the seed vinegar obtained from the previous batch processes (Gullo and Giudici, 2008). Although the production of acetic acid is a typical trait of AAB involved in vinegars, intra- and inter-species phenotypic and genetic variability is very high (Li et al., 2015). Given that the quality of vinegar is closely depended on the microbial diversity involved in the fermentation, in-depth analysis of the seed vinegar microflora and understanding of how AAB evolve during traditionally produced vinegars have been acknowledged to play an important role in improving the process control and the quality of the final product, and to select the most suitable strains as the potential starter cultures (Li et  al., 2015; Yetiman and Kesmen, 2015; Gullo et al., 2009, 2014; Gullo and Giudici, 2008). In this direction, quick and reliable culture-dependent and -independent methods have been developed and applied for the characterization of AAB at the species and strain level in traditionally produced

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vinegars (Yetiman and Kesmen, 2015; Gullo et al., 2009, 2014; Vegas et  al., 2013; De Vero et  al., 2006). In particular for TBV, the related studies highlighted the occurrence of Gluconacetobacter europaeus, G. xylinus as well as Acetobacter pasteurianus, A. aceti, and A.malorum (Gullo et al., 2014; De Vero et al., 2006). The limited composition of the TBV microflora reflects to the natural selection, according to which the members of AAB population fit better to specific characteristics of the base wine (Solieri and Giudici, 2009). Knowledge concerning the ecotypical strains of AAB involved in the TBV production was used as a solid base for the selection of proper starter cultures with desirable phenotypic traits. As stressed in the case of alcoholic fermentation, selected AAB can be a valuable tool to avoid many process imbalances by efficient control of the process. Taking into account the composition of the base wine (at least 150 g unfermented sugars/kg) and the desired characteristics of the final product (low acidity), tolerance to sugars (~25% by weight of sugars) is considered as the most distinguishing strain property for balsamic vinegar production (Gullo et  al., 2014; Gullo and Giudici, 2008). Among AAB species isolated from TBV, A. malorum, with a proven unique high sugar tolerance (30% of d-glucose) (Cleenwerck et  al., 2002), is an excellent candidate for use as a starter culture for BV production. However, selectable strains have then to be evaluated for other desirable technological traits, common for AAB suitable for industrial vinegar production that include (a) selective, highly efficient and rapid oxidation of ethanol to acetic acid, (b) tolerance to high concentration of acetic acid and low pH values, (c) no overoxidation, (d) resistance to bacteriophage infections, and (e) low production of exopolysaccharides (Gullo and Giudici, 2008). The ability of the selected strains to produce gluconic acid via the oxidation of glucose should also be considered as this compound protects the product against acidity loss due to acetic acid evaporation during the maturation and also contributes to its sweetness. Also, the glucose/fructose ratio decrease due to the selective catabolism of glucose via its transformation due to gluconic acid enhances the stability and the sensorial characteristics of the final product (Giudici et al., 2015). Because the acetification process is by far more exothermic than alcoholic fermentation (~8.4 MJ/L ethanol oxidized), previous findings concerning the isolation, identification, and characterization of thermotolerant AAB (Chen et al., 2016; Gullo et al., 2014; Seaki et al., 2014; Matsutani et al., 2013) may lead to the development of more efficient processes for BV production at higher temperatures that allow reduction of the cooling water expenses. Given this background, the selection of AAB for use as starter cultures in BV production is an important innovation, though this is not a straightforward process due to difficulty of cultivation of these bacteria on semisolid laboratory media

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and the genetic and phenotypic instability of the isolated strains during long-term storage and cultivation under industrial conditions (Hidalgo et al., 2012; Gullo et al., 2009). Combining all the above characteristics in one wild species seems difficult, thus, the use of multiple strain starters, each with its own specific effect, could be an interesting novelty (Vegas et  al., 2013; Gullo et al., 2009; Trček et al., 2007) This suggestion is in line with studies focusing on evolution of AAB during traditional wine vinegar (Vegas et al., 2013; Gullo et al., 2009). Innovations to improve process kinetics of the lengthy process for the industrial production of BV without changing the traditional mode of operations can be achieved by appropriate control of the fermentation system. As described previously, TBV production takes place in wooden barrels that allow for a limited access of oxygen. Modification of the barrel design must focus on the increase of the air-contact surface to enhance AAB growth. An appropriate choice of the barrel characteristics is wood type with increased porosity and a barrel shape with high surface/volume ratio (Hidalgo et  al., 2010). Future scenarios may be the submerged process of acetification in bioreactors with controlled stirring using inoculated cultures. Rigorous control of the fermentation conditions to avoid oxygen deficiency and temperature increase is required to have a successful process (Giudici et al., 2015; Gullo et al., 2014). However, different process conditions of submerged fermentation result in heterogeneity in the composition of the microflora involved when compared to surface fermentation. For example, in the work of Hidalgo et  al. (2010), in the highly aerated conditions Gluconacetobacter species were favored at the expense of A. pasteurianus, which predominated in surface fermentation in wooden barrels. This evidence confirms the need for appropriate selection of starters and setup of the new process by implementing them to the target technology. Titratable acidity, expressed as equivalents of acetic acid, is an important criterion in the official control of every type of vinegar. For example, values for these parameters presented in Table  10.6 show that most of the samples examined complied with regulatory limits for titratable acidity (i.e., 6% (w/w)) (OGGHR, No 270/2011). The titratable acidity of wine vinegar is attributed mainly to the presence of acetic acid. This is not the case for TBV, in which other minor organic acids contribute, too (Solieri and Giudici, 2009). This also applies for the acidity of geographical indication products but to a lesser degree (Lalou et al., 2015a). Acetic acid in all the examined BVs was the main organic acid, comprising >62% of the total acidity content. High acetic acid content can increase the pungency of the product, which becomes a negative attribute if not properly balanced by sugar content. According to Hillmann et al. (2012) organic acids beyond acetic

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Table 10.6  Titratable Acidity and Acetic Acid Content of Balsamic Vinegars (BVs) Present in the Greek Market Sample

Starting Materialb

Acidityb

Titratable acidityc (% w/v)

Acetic acidd (g/kg)

GENERIC BVS PRODUCED BY BLENDING

1 2

Wine and CGM Wine and concentrated grape, caramel color

7 6

6.77 ± 0.28 5.81 ± 0.34

63.57 ± 5.8 46.21 ± 2.23

3 4 5

Wine and cooked grape must Vinegar from wine grapes, grape syrup Wine vinegar, grape must, antioxidant:E224, color: E150d BV, contains sulfides

6.2 6 6

6.99 ± 0.49 5.50 ± 0.16 5.31 ± 0.12

43.44 ± 1.42 51.00 ± 2.32 45.98 ± 1.01

6

5.91 ± 0.10

49.47 ± 1.55

Wine vinegar and petimezi (cooked grape must) of organic farming Wine vinegar, CGM, caramel coloring

–

4.13 ± 0.13

28.03 ± 1.51

6

5.63 ± 0.06

49.94 ± 0.32

6 7 8

GENERIC BVS PRODUCED BY TWO-STAGE FERMENTATION

1Aa

Dry raisins

6

5.53 ± 0.43

46.08 ± 0.56

1Ba 1Ca

Dry raisins Dry raisins

6 6

5.57 ± 0.57 5.55 ± 0.35

44.87 ± 0.52 46.10 ± 0.45

2 3

Dry raisins Dry raisins of organic farming

6 6

6.52 ± 0.06 6.61 ± 0.18

62.26 ± 3.03 46.93 ± 3.02

a

Different capital letters refer to different lots of the same brand. According to the product label. c Mean values ± SD (n = 3) determined by titration with 0.5 M sodium hydroxide solution as proposed by AOAC. d Mean values ± SD (n = 3) determined by HPLC (Lalou et al., 2015a). b

(e.g., tartaric, citric, malic, etc.) can be considered the main sour ­tasting molecules in balsamic vinegars because their concentration exceeds their recognition thresholds. Thus, differences in the relative content of minor organic acids are expected to affect the sensorial balance of the product.

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10.2.5 Aging The production of TBV requires a long aging period of at least 12 years that does not coincide with the industrial demands. The BVM is aged in wood receptacles for a period of 60 days after the raw materials are assembled and ready for processing. In Greek legislation for both types of BVs permitted, a minimum “maturing period” of 6 months in wood barrels is required for a product to use the wording “aged” on its label (OGGHR, No 270/2011). Generally, aging in wood barrels is necessary for vinegars in order to be of high quality (Cerezo et al., 2010). The influence of the wood type in the compositional and sensorial characteristics of the final products has been studied thoroughly for beverages such as wine, whiskey, and brandy (Basalekou et  al., 2017; Delgado-González et al., 2017; Herrero et al., 2016; Fernández de Simón et  al., 2014a,b; Soares et  al., 2012). The different pH and ethyl alcohol concentration of vinegars are expected to differentiate the extraction of the wood components providing different characteristics on the aged product (Delgado-González et al., 2017; Guerreiro et al., 2014; Tesfaye et al., 2002). Different woods have different porosities that influence not only the evaporation rate of water but the concentration of aroma compounds and the formation of esters (Basalekou et al., 2017; Rios-Reina et al., 2017; Tao et al., 2014; Del Signore, 2011). In TBV production, the barrels used for aging the product are made from different woods such as oak, chestnut, mulberry, ash, and cherry. The use of different woods is expected to increase the structure and persistence of the fragrance conveying a complex aroma to the final product (Mattia, 2004). The use of different woods seems to be associated with the size of the barrel as well. Specifically, larger barrels are made of wood, which allows good air permeability (such as mulberry), whereas wood types that are expected to contribute to the color of the final product (e.g., chestnut) are preferred for the intermediate barrels. The smallest barrels are from oak wood since it guarantees the best preservation of the aged product (Mattia, 2004). Generally, aging is expected to enhance flavor of the end product. In literature, aged vinegars have higher levels of aromatic aldehydes and their derivatives. The latter are produced by hydroalcoholysis of the lignin from wood and are released in the vinegar in the course of time (Cerezo et al., 2010). Beyond this process, during aging, the aroma compounds present in the vinegar are concentrated due to water evaporation through the wood pores (Callejón et  al., 2010). Extraction of phenolic compounds from wood also occurs as it is suggested by the increase in the total phenolic index and the dry matter of sherry vinegars aged in oak barrels (Tesfaye et  al., 2002).

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Fig. 10.5  Different woods used for aging of vinegars and wines and their potential contribution to the sensorial and compositional characteristics final aged product.

Each wood type is expected to provide different compositional and sensorial characteristics (Fig.  10.5). In studies about red wine vinegars (Ho et  al., 2017; Cerezo et  al., 2010), oak, the most commonly used wood for aging, infused woody, vanilla, and sweet notes in the final products whereas cherry wood intensified the perception of red fruit attributes. Acacia wood imparts sweeter and honey like tastes to wines compared to those aged in oak barrels (Tao et  al., 2014; Kozlovic et al., 2010). Aging periods shorter than 6 months and/or the use of bigger barrels do not add to the positive effects aimed, according to Del Signore (2011). Long aging times though do not agree with the industrial demands for fast production lines. In this view, the application of accelerated aging in BV production can be examined as a potential alternative as reported for wine vinegar (Cerezo et al., 2014). In that case accelerated aging was achieved using wood chips. Increasing surface/ volume ratio increases both the extraction rate and the quantities of extractable substances leading to shorter required aging periods compared to those needed in traditional methods. Which kind and quantity of wood chips will be added has a clear impact to the process. For example, large amounts of chips shorten the aging time but can also lead to very intense wood flavors that can be undesirable.

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10.2.6  Consumer Expectations From the NewGeneration BVs Consumer studies held at a wine exhibition (Thessaloniki, 2015) revealed that balsamic vinegar has become a part of Greek gastronomy. Approximately 52% of the consumers answered that they use balsamic vinegar in a week and another 30% at least once a month (data not shown). Generic BVs seem to be among the most popular products of the Balsamic Family as it is indicated by the 61% of the consumers who have used them. This popularity can reasonably be attributed to friendly prices and the array of Balsamic Family products present in the market. Consumers expect the maximum satisfaction from the new products. Most of them have not tried traditional ones although they probably know only BVM, which is also found in the Greek market. The properly trained panel (Hatzidimitriou et  al., 2015; Lalou et al., 2015a) judged as negative characteristics in BVs the high scores in the terms Pungency, Acidity, Bitterness, and Tannic. The terms Sweetness and Aftertaste were judged as positive ones in the BV profiling. Three BVs examined by the panel were also given to consumers, who had to assess the BVs for one aroma (Pungency) and one taste stimulus (Sweetness) using a 9-point hedonic scale (Lalou et al., 2015b). The majority of the consumers preferred the product of the lowest Acidity and highest Sweetness scores. This trend has to be considered by the manufacturers because it is in line with a preference for the production of sweeter TBVs in Italy (Giudici et al., 2009). Consumer judgements were related with the results obtained from the trained panel. For example, the less acceptable BV by consumers was also characterized by the assessors as the “most unbalanced with intense acidic character.” In any case, consumers were able to discriminate among the samples. Consumer views for these new products are important for the manufacturers, who want to establish them in the market.

10.3 Conclusion Changes in legislation and new market trends open new horizons in the production of products that get ideas from traditionally produced ones. These new generations of products should have their own legal and compositional identity. They are not meant to mislead consumers and should be produced by safe means. One such case is the category of generic BVs. Legislation can get feedback from scientists, the published work of whom supports consumer rights and “innovation that matters” in the international food market.

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